mass spectrometry applications for comparative proteomics
TRANSCRIPT
Mass Spectrometry Applications for Comparative Proteomics and Peptidomic
Discovery
by
Robert Stewart Cunningham
A dissertation submitted in partial fulfillment of
the requirements for the degree of
Doctor of Philosophy
(Chemistry)
at the
UNIVERSITY OF WISCONSIN-MADISON
2012
Date of final oral examination 10412
The dissertation is approved by the following members of the Final Oral Committee
Lingjun Li Professor ChemistryPharmacy
Albee Messing Professor Comparative Biosciences
Lloyd Smith Professor Chemistry
Warren Heideman Professor Pharmacy
Tim Bugni Assistant Professor Pharmacy
i
Acknowledgements
I would like to acknowledge the support and guidance from professors colleagues
and friends at the University of Wisconsin-Madison who are indispensable to this thesis
First I would like to express my deep gratitude to my advisor Prof Lingjun Li for
allowing me the freedom to chase scientific endeavors all while offering her constant
guidance assistance and support through my PhD study Her constant energy and
enthusiasm in research have led by example in performing research and inspired me to
make the most of the time given to me Dr Li encouraged me to take on challenging
projects apply for awards travel and present my research to the larger scientific
community None of my work would be achieved without her and I want to thank Dr Li
for her support during these years
I would also like to thank the members of my committee Dr Lingjun Li Dr
Albee Messing Dr Lloyd Smith Dr Warren Heideman and Dr Tim Bugni I truly
appreciate the willingness of these professors to take time out of their busy schedules to
serve as members of my committee
I have benefited greatly from previous members of the Li Lab In particular I
would like to thank Dr James Dowell Dr Xin Wei Dr Robert Sturm and Dr Limei
Hui for their patient and valuable suggestions in my research and also teaching me
valuable experimental skills how to perform general shotgun proteomics and how to use
several instruments Specifically I would like to thank Daniel Wellner who has worked
with me on numerous projects over the past 2 years and has been a constant in my
research life I also want to thank my wonderful current colleagues Jingxin Wang Tyler
ii Greer Chris Lietz Chenxi Jia Dustin Frost Di Ma Hui (Vivian) Ye Nicole Woodards
and Claire Schmerberg for their collaboration in many challenging research projects and
fruitful discussions on various research areas There are too many people to thank each
one individually but every member of the Li lab has in some way contributed to my
learning experience Beyond research work their friendship also made my life here in
Madison much more enjoyable
I would also like to thank our collaborators Dr Albee Messing Dr Warren
Heideman Dr Xin Sun and Dr James Dowell It is my great pleasure to have the
opportunities to work with these amazing people and gain precious experience I have
learned so much from them and their achievements in the field have inspired me to strive
to do the best I could
Furthermore I would like to thank Gary Girdaukas and Dr Cameron Scarlett at
School of Pharmacy for the access of the MALDI-FTMS and Bruker amaZon ion trap
instruments
In particular I wish to thank my family my Mom and Step-Dad for raising me
and my Dad for always being there for me They all supported me in my decision to
pursue science and specifically a career in chemistry I would like to thank my Sister
who grew up with me and always led by example in academics Most importantly I
would like to thank my wife Na Liu for her constant support She has inspired and
helped me finish my PhD and always encouraged me to be the best I could be To them
I dedicate this thesis
iii
Table of Contents
Page
________________________________________________________________________
Acknowledgements i
Table of Contents iii
Abstract iv
Chapter 1 Introduction brief background and research summary 1
Chapter 2 Mass spectrometry-based proteomics and peptidomics for
biomarker discovery and the current state of the field 15
Chapter 3 Protein changes in immunodepleted cerebrospinal fluid from
transgenic mouse models of Alexander disease detected
using mass spectrometry 73
Chapter 4 Genomic and proteomic profiling of rat adapted scrapie 110
Chapter 5 Investigation of the differences in the phosphoproteome
between starved vs glucose fed Saccharomyces cerevisiae 139
Chapter 6 Use of electron transfer dissociation for neuropeptide
sequencing and identification 166
Chapter 7 Investigation and reduction of sub-microgram peptide loss
using molecular weight cut-off fractionation prior to
mass spectrometric analysis 187
Chapter 8 Conclusions and future directions 206
Appendix 1 Protocols for sample preparation for mass spectrometry
based proteomics and peptidomics 217
Appendix 2 Publications and presentations 233
_______________________________________________________________________
iv
Mass Spectrometry Applications for Comparative Proteomics and
Peptidomic Discovery
Robert Stewart Cunningham
Under the supervision of Professor Lingjun Li
At the University of Wisconsin-Madison
Abstract
In this thesis multiple biological samples from various diseases models or
treatments are investigated using shotgun proteomics and improved methods are
developed to enable extended characterization and detection of neuropeptides In general
this thesis aims to expand upon the rapidly evolving field of mass spectrometry (MS)-
based proteomics and peptidomics by primarily enhancing small scale sample analysis
A review of the current status and progress in the field of biomarker discovery in
peptidomics and proteomics is presented To this rapidly expanding body of literature
our critical review offers new insights into MS-based biomarker studies investigating
numerous biological samples methods for post-translational modifications quantitative
proteomics and biomarker validation Methods are developed and presented including
immunodepletion for small volume cerebrospinal fluid (CSF) samples for comparison of
the CSF proteomes between an Alexander disease transgenic mouse model with
overexpression of the glial fibrillary acidic protein and a control animal This thesis also
covers the application of the small scale immunodepletion of CSF for comparative
proteomic analysis of a novel rat adapted scrapie (RAS) model for prion disease and
v
compares the RAS CSF proteome to control rat CSF using MS Large scale
phosphoproteomics of starved vs glucose fed yeast is presented to better understand the
phosphoproteome changes that occur during glucose feeding Method development for
neuropeptide analysis is expanded upon using electron transfer dissociation (ETD)
fragmentation to successfully sequence for the first time the crustacean hyperglycemic
hormone precursor-related peptide (CPRP) from the blue crab Callinectes sapidus In
addition a method for ETD sequencing of sulfonated neuropeptides using a magnesium
salt adduct in an ion trap mass spectrometer is reported This thesis also reports on a
method for sub-microg peptide isolation when using a molecular weight cut-off filtration
device to improve sample recovery by over 2 orders of magnitude All the protocols used
throughout the work are provided in an easy to use step-by-step format in the Appendix
Collectively this body of work extends the capabilities of mass spectrometry as a
bioanalytical tool for shotgun proteomics and expands upon methods for neuropeptide
discovery and analysis
1
Chapter 1
Introduction Brief Background and Research Summary
2
Abstract
Mass spectrometry based comparative proteomics and improved methods for
neuropeptide discovery have been reported in this thesis A very brief introduction of crustacean
neuropeptidomics is covered in this chapter followed by Chapter 2 which covers in great detail
comparative proteomics using mass spectrometry with an emphasis on biomarker discovery
Chapter 3 reports a novel immunodepletion technique used for comparative proteomics between
glial fibrillary acidic protein (GFAP) overexpressor and control mouse cerebrospinal fluid (CSF)
Chapter 4 involves rat CSF comparative proteomics between rat adapted scrapie and control
animals Chapter 5 details comparisons of phosphoproteomes in Saccharomyces cerevisiae
(Bakerrsquos yeast) between starved vs glucose fed conditions Chapter 6 investigates the use of
electron transfer dissociation (ETD) for kDa sized neuropeptides and neuropeptides with tyrosine
sulfonation Chapter 7 presents a molecular weight cut-off (MWCO) protocol to allow sub-microg
peptides to be recovered Chapter 8 provides a conclusion to this thesis and outlines future
directions for certain projects
3
Background
Mass spectrometry (MS) requires gas phase ions for experimental measurement and
intact large nonvolatile biomolecules proved difficult to be analyzed by electron ionization or
chemical ionization until the invention of two soft ionization techniques matrix-assisted laser
desorptionionization (MALDI)1 and electrospray ionization (ESI)
2 ESI and MALDI are the
two most common soft ionization techniques for mass spectrometry Once ionized molecules
such as peptides or proteins can be separated by their mass to charge ratios (mz) using various
mass analyzers manipulating electric andor magnetic fields ESI and MALDI mass
spectrometric techniques have become central analytical methods in biological sciences because
they are suitable for nonvolatile thermally labile analytes in femtomole quantities3 ESI allows
the coupling of high pressure liquid chromatography and the constant flow of solvent is
electrically charged at the outlet to produce a Taylor cone4 During desolvation the Raleigh
limit is reached and a coulombic explosion occurs commonly producing multiply charged ions
A modification of ESI is nanoLC-ESI which operates at nLmin flow rates to analyze low sample
amounts such as proteins or peptides in biological samples5 NanoLC-ESI uses less solvent as
the source because of the reduced flow rate of standard LCMS with an ESI source and nanoLC-
ESI is the predominate inlet for shotgun proteomic ESI MS experiments6 Conversely MALDI
can use sub-microL volume of sample and be co-crystallized with an ultraviolet absorbing organic
matrix that is obliterated using a laser at appropriate wavelength to generate gas phase ions
Alternatively MALDI has the unique capability to work with tissue samples and ionize in the
solid state instead of liquid like ESI
4
Mass analyzers require an operating pressure between 10-4
-10-10
Torr to allow proper ion
transfer and separationisolation of the ion based upon its mz Numerous mass analyzers are
currently available and each have their own strengths and weaknesses as shown in Figure 1 The
biomolecules are separated by the mass analyzers and detected without fragmentation which is
termed MS analysis Following MS detection tandem mass spectrometry (MSMS) of the
original precursor ion can be performed to provide additional structural information such as a
ladder sequence of amino acids for peptides Numerous fragmentation techniques are available
for proteomics such as collision induced dissociation (CID) electron transfer dissociation (ETD)
or high energy collision induced dissociation (HCD) Each of these fragmentation techniques
have their own benefits in proteomics experiments and are discussed in depth in Chapter 2 The
background and current status for comparative proteomics with specific emphasis on biomarker
analysis are covered in Chapter 2
Neuropeptidomic Method Development in the Crustacean Model System
Utilizing Mass Spectrometry
Chapter 6 and 7 focus on sample preparation and ETD fragmentation techniques to
characterize neuropeptides in the neuroendocrine organs in the crustacean nervous system
Neuropeptides are short chains of amino acids which are a diverse class of cell-cell signaling
molecules in the nervous system Neuropeptides have been investigated for being involved in
numerous physiological processes such as memory7 learning
8 depression
9 pain
10 reward
11
reproduction12
sleep-wake cycles13
homeostasis14
and feeding15-17
Figure 2 depicts how
neuropeptides are synthesized as pre-prohormones in the rough endoplasmic reticulum and
5
packaged in the Golgi apparatus After being packaged these pre-prohormones are processed
into bioactive peptides within the vesicle which is occurring during vesicular transport down an
axon and released into the synaptic cleft Secreted neuropeptides stimulate the post synaptic
neurons by interacting with G-protein coupled receptors at the chemical synapse
The crustacean model nervous system is well-defined neural network which has been
used in neurobiology and neuroendocrine (neurosecretory) research and thus lends itself for
studying neuromodulation18-22
Figure 3 shows the locations of several neuroendocrine organs in
the crustacean Callinectes sapidus including the sinus gland (SG) which is studied in Chapter 6
The Lingjun Li lab has focused on neuropeptide discovery and function in crustacean
neuroendocrine organs using mass spectrometry23-25
The work presented in Chapters 6 and 7
expand on sample preparation and analytical tools to further investigate the neuropeptidome
Research Overview
Comparative Proteomics of Biological Samples
Chapter 2 provides a thorough review of comparative proteomics and biomarker analysis
using mass spectrometry The scientific community has shown great interest in the field of mass
spectrometry-based proteomics and peptidomics for its applications in biology Proteomics
technologies have evolved to generate large datasets of proteins or peptides involved in various
biological and disease progression processes producing testable hypotheses for complex
biological questions This chapter provides an introduction and insight into relevant topics in
proteomics and peptidomics including biological material selection sample preparation
separation techniques peptide fragmentation post-translational modifications quantification
6
bioinformatics and biomarker discovery and validation In addition current literature and
remaining challenges and emerging technologies for proteomics and peptidomics are discussed
Chapter 3 investigates comparative proteomics between a GFAP overexpressor mouse
model and a control mouses cerebrospinal fluid (CSF) CSF is a low protein content biological
fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in
direct contact with the brain but consist of very abundant proteins similar to serum which require
removal A modified IgY-14 immunodepletion treatment is presented to remove abundant
proteins such as albumin to enhance analysis of the low volumes of CSF that are obtainable
from mice These GFAP overexpressor mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we present the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates are performed to address animal variability as well as reproducibility in mass
spectrometric analysis Relative quantitation is performed using distributive normalized spectral
abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins with
significant changes in the CSF of GFAP transgenic mice are identified with validation from
ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
Chapter 4 explores the CSF proteomics of a novel prion model called rat adapted scrapie
(RAS) A similar IgY immunodepletion technique is presented as in Chapter 3 and is similarly
used to reduce the abundant proteins in CSF CSF from control and RAS (biological N=5
technical replicates N=3) were digested and separated using one dimensional reversed-phase
nanoLC separation In total 512 proteins 167 non-redundant protein groups and 1032 unique
peptides are identified with a 1 FDR Comparative analysis was done using dNSAF spectral
7
counting and 21 proteins were significantly up or down-regulated The proteins are compared to
the 1048 differentially regulated genes and additionally compared to previously published
proteins showing changes consistent with other prion animal models Of particular interest is
RAS specific proteins like RNAseT2 which is not up-regulated in mouse prion disease but is
designated as upregulated in both the genomic and proteomics data for RAS
Chapter 5 explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Previous work by the
Heideman lab investigated the transcriptional response to fresh glucose in yeast26
Kinases such
as protein kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose
response so we described a large scale phosphoproteomic MS based study in this chapter
Yeast cell extract was digested and phosphopeptides were enriched by immobilized metal
affinity chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase
(RP)-RP separation The low pH separation was infused directly into an ion trap mass
spectrometer with neutral loss triggered ETDfragmentation Specifically the CID fragmentation
can cause a neutral loss of 98z from the H3PO4 and can produce an incomplete fragmentation
pattern and lead to ambiguous identification so when the neutral loss is detected ETD MSMS
fragmentation is performed The neutral loss triggered ETD fragmentation is included in this
study to improve phosphopeptide identifications In total 477 phosphopeptides are identified
with 06 FDR and 669 phosphopeptides identified with 54 FDR Motif analysis of PKA and
phosphosite validation are performed as well
8
The future of comparative proteomics investigating small sample amounts or PTMs is
promising Further advances in enrichment separations science mass spectrometry
analyzersdetectors and bioinformatics will continue to create more powerful tools that enable
digging deeper in proteomics with both small (mouse CSF) and large (cell extracts) sample
amounts
Methods for Neuropeptide Analysis Using ETD fragmentation and Sample
Preparation
Chapter 6 investigates the utility of ETD fragmentation to sequence endogenous large
neuropeptides and labile tyrosine sulfation in the crustacean Callinectes sapidus The sinus
gland (SG) of the crustacean is a well-defined neuroendocrine site that produces numerous
hemolymph-borne agents including the most complex class of endocrine signaling moleculesmdash
neuropeptides One such peptide is the crustacean hyperglycemic hormone (CHH) precursor-
related peptide (CPRPs) which was not previously sequenced Electron transfer dissociation
(ETD) is used for sequencing of intact CPRPs due to their large size and high charge state In
addition ETD is used to sequence a sulfated peptide cholecystokinin (CCK)-like peptide in the
lobster Homarus americanus using a salt adduct Collectively this chapter presents two
examples of the utility of ETD in sequencing neuropeptides with large molecular weight or with
labile modifications
Chapter 7 reports a protocol on improving recovery of minute quantities of peptides by
adding methanol and a salt modifier (NaCl) to molecular weight cut-off membrane-based
centrifugal filters (MWCO) to enrich sub-microgram peptide quantities In some biological
9
fluids such as CSF the endogenous peptide content is very low and using pure water to perform
the MWCO separation produces too much sample loss Using a neuropeptide standard
bradykinin sample loss is reduced over two orders of magnitude with and without undigested
protein present The presence of bovine serum albumin (BSA) undigested protein and the
bradykinin standard lends evidence that sample loss occurs because of the MWCO and not the
presence of the protein Additionally a BSA tryptic digestion is presented where twenty-seven
tryptic peptides are identified from MALDI mass spectra after enriching with methanol while
only two tryptic peptides are identified after the standard MWCO protocol The strategy
presented in Chapter 7 greatly enhances recovery from MWCO separation for sub-microg peptide
samples
10
References
1 Tanaka K Waki H Ido Y Akita S Yoshida Y Yoshida T Matsuo T Protein and polymer analyses up to mz 100 000 by laser ionization time-of-flight mass spectrometry Rapid Communications in Mass Spectrometry 1988 2 (8) 151-153
2 Fenn J B Mann M Meng C K Wong S F Whitehouse C M Electrospray ionization for mass spectrometry of large biomolecules Science 1989 246 (4926) 64-71
3 Domon B Aebersold R Mass spectrometry and protein analysis Science 2006 312 (5771) 212-7
4 Whitehouse C M Dreyer R N Yamashita M Fenn J B Electrospray interface for liquid chromatographs and mass spectrometers Anal Chem 1985 57 (3) 675-9
5 Wilm M Mann M Analytical properties of the nanoelectrospray ion source Anal Chem 1996 68 (1) 1-8
6 Karas M Bahr U Dulcks T Nano-electrospray ionization mass spectrometry addressing analytical problems beyond routine Fresenius J Anal Chem 2000 366 (6-7) 669-76
7 Gulpinar M Yegen B The physiology of learning and memory Role of peptides and stress Curr Protein Pept Sci 2004 5 (6) 457-473
8 Ogren S O Kuteeva E Elvander-Tottie E Hokfelt T Neuropeptides in learning and memory processes with focus on galanin In Eur J Pharmacol 2010 Vol 626 pp 9-17
9 Strand F Neuropeptides general characteristics and neuropharmaceutical potential in treating CNS disorders Prog Drug Res 2003 61 1-37
10 Li W Chang M Peng Y-L Gao Y-H Zhang J-N Han R-W Wang R Neuropeptide S produces antinociceptive effects at the supraspinal level in mice Regul Pept 2009 156 (1-3) 90-95
11 Wu C-H Tao P-L Huang E Y-K Distribution of neuropeptide FF (NPFF) receptors in correlation with morphine-induced reward in the rat brain Peptides 2010 31 (7) 1374-1382
12 Tsutsui K Bentley G E Kriegsfeld L J Osugi T Seong J Y Vaudry H Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin new key neuropeptides controlling reproduction J Neuroendocrinol 2010 22 (7) 716-727
13 Piper D C Upton N Smith M I Hunter A J The novel brain neuropeptide orexin-A modulates the sleep-wake cycle of rats Eur J Neurosci 2000 12 (2) 726-730
14 Tsuneki H Wada T Sasaoka T Role of orexin in the regulation of glucose homeostasis - Tsuneki - 2009 - Acta Physiologica - Wiley Online Library Acta Physiol 2010
15 Kageyama H Takenoya F Shiba K Shioda S Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding Neuropeptides 2010 44 (2) 133-138
16 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide family of aplysia J Neurosci 2002 22 (17) 7797-7808
11
17 Jensen J Regulatory peptides and control of food intake in non-mammalian vertebrates Comp Biochem Physiol Part A Mol Integr Physiol 2001 128 (3) 469-477
18 Billimoria C P Li L Marder E Profiling of neuropeptides released at the stomatogastric ganglion of the crab Cancer borealis with mass spectrometry J Neurochem 2005 95 (1) 191-199
19 Cape S S Rehm K J Ma M Marder E Li L Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster Homarus americanus J Neurochem 2008 105 (3) 690-702
20 Cruz Bermuacutedez N D Fu Q Kutz Naber K K Christie A E Li L Marder E Mass
spectrometric characterization and physiological actions of GAHKNYLRFamide a novel FMRFamide‐like peptide from crabs of the genus Cancer J Neurochem 2006 97 (3) 784-799
21 Grashow R Brookings T Marder E Reliable neuromodulation from circuits with variable underlying structure Proc Natl Acad Sci USA 2009 106 (28) 11742-11746
22 Ma M Szabo T M Jia C Marder E Li L Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins Peptides 2009 30 (9) 1660-1668
23 Chen R Hui L Cape S S Wang J Li L Comparative Neuropeptidomic Analysis of Food Intake via a Multi-faceted Mass Spectrometric Approach ACS Chem Neurosci 2010 1 (3) 204-214
24 Li L Sweedler J V Peptides in the brain mass spectrometry-based measurement approaches and challenges Annu Rev Anal Chem 2008 1 451-483
25 Ma M Wang J Chen R Li L Expanding the crustacean neuropeptidome using a multifaceted mass spectrometric approach J Proteome Res 2009 8 (5) 2426-2437
26 Slattery M G Heideman W Coordinated regulation of growth genes in Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
12
Figure 1 Capabilities and performances of certain mass analyzers Check marks indicate
availability check marks in parentheses indicate optional + ++ and +++ indicate possible or
moderate goodhigh and excellentvery high respectively Adapted with permission from
reference 3
13
Figure 2 Synthesis processing and release of neuropeptides (a) Cartoon showing two
interacting neurons (b) The synthesis of neuropeptides in the neuronal organelles and their
transport down the axon to the presynaptic terminal is depicted (c) Shows neuropeptide release
and interaction with the dendrite of the post-synaptic cell (Adapted from PhD thesis by Dr
Stephanie Cape)
14
Figure 3 Schematic showing location of the neuronal tissues being analyzed in the MS studies
of neuropeptides in Callinectes sapidus The SGs (sinus glands located in the eyestalks of the
crab) and the POs (pericardial organs located in the chamber surrounding the heart) release
neurohormones into the hemolymph (crab circulatory fluid) The brain is near the STNS
(stomatogastric nervous system neural network that controls the motion of the gut and foregut)
which has direct connections to the STG (stomatogastric ganglion) The STG is located in an
artery so it is continually in contact with hemolymph (Adapted from PhD thesis by Dr Robert
Sturm)
15
Chapter 2
Mass Spectrometry-based Proteomics and Peptidomics for Biomarker
Discovery and the Current State of the Field
Adapted from ldquoMass spectrometry-based proteomics and peptidomics for systems biology and
biomarker discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
16
Abstract
The scientific community has shown great interest in the field of mass spectrometry-based
proteomics and peptidomics for its applications in biology Proteomics technologies have
evolved to produce large datasets of proteins or peptides involved in various biological and
disease progression processes producing testable hypothesis for complex biological questions
This review provides an introduction and insight to relevant topics in proteomics and
peptidomics including biological material selection sample preparation separation techniques
peptide fragmentation post-translation modifications quantification bioinformatics and
biomarker discovery and validation In addition current literature and remaining challenges and
emerging technologies for proteomics and peptidomics are presented
17
Introduction
The field of proteomics has seen a huge expansion in the last two decades Multiple factors have
contributed to the rapid expansion of this field including the ever evolving mass spectrometry
instrumentation new sample preparation methods genomic sequencing of numerous model
organisms allowing database searching of proteomes improved quantitation capabilities and
availability of bioinformatic tools The ability to investigate the proteomes of numerous
biological samples and the ability to generate future hypothesis driven experiments makes
proteomics and biomarker studies exceedingly popular in biological studies today In addition
the advances in post-translational modification (PTM) analysis and quantification ability further
enhance the utility of mass spectrometry (MS)-based proteomics A subset of proteomics
research is devoted to profiling and quantifying neurologically related proteins and endogenous
peptides which has progressed rapidly in the past decade This review provides a general
overview as outlined in Figure 1 of proteomics technology including methodological and
conceptual improvements with a focus on recent studies and neurological biomarker studies
Biological Material Selection
The choice of biological matrix is an important first step in any proteomics analysis The
ease of sample collection (eg urine plasma or saliva) versus usefulness or localization of
sample (eg specific tissue or proximity fluid) needs to be evaluated early on in a study design
Plasma derived by centrifugation of blood to remove whole cells is a very popular
choice in proteomics due to the high protein content (~65 mg ml1) and the ubiquitous nature of
blood in the body and the ability to obtain large sample amounts or various time points without
the need to sacrifice the animal or to perform invasive techniques Plasma is centrifuged
18
immediately after sample collection unlike serum where coagulation needs to occur first To
obtain plasma blood is collected in a tube with an anticoagulant added (ETDA heparin or
citrate) and centrifuged but previous reports have shown variable results when heparin has been
used as an anticoagulant2 Human Proteome Organization (HUPO) specifically recommends the
anticoagulants EDTA or citrate to treat plasma3 4
One of the primary concerns with plasma is
degradation of the protein content via endogenous proteases found in the sample5 One way to
address this problem is the use of protease inhibitors In addition freezethaw cycles need to be
minimized to prevent protein degradation and variability6 7
Plasma proteomics has seen
extensive coordinated efforts to start assessing the diagnostic needs using plasma8 HUPO also
has established a public human database for plasma and serum proteomics from 35 collaborating
labratories9 Large dynamic range studies have been performed on plasma with a starting sample
amount of 2625 microl (1575 mg) resulting in 3654 proteins identified with a sub 5 false
discovery rate10
The large dynamic range spanning across eleven orders of magnitude as visualized in
Figure 2 is one of the biggest obstacles in plasma proteomics Figure 2 also shows that as lower
abundance proteins are investigated the origins of those identified proteins are more diverse than
the most abundant proteins Recent mining of the plasma proteome showed an ability to search
for disease biomarker applications across seven orders of magnitude In addition the tissue of
origin for the identified plasma proteins were identified and its origin was more diverse as the
protein concentration decreased11
Plasma has been used as a source for biomarker studies such
as colorectal cancer12 13
cardiovascular disease14
and abdominal aortic aneurysm15
Even
though the blood brain barrier prevents direct blood to brain interaction neurological disorders
such as Alzheimerrsquos disease (AD) have had their proteomes studied using plasma16
19
An alternative sample derived from blood is serum which is plasma allowed to coagulate
instead of adding anti-coagulates The time for coagulation is usually 30 minutes and during that
time significant and random degradation from endogenous proteases can occur The additional
variability caused from the coagulation process can change the concentration of multiple
potentially valuable biomarkers As biodiversity between samples or organisms is a challenging
endeavor additional sample variability due to serum generation may be undesirable but serum is
still currently being used for biomarker disease studies17
Serum has been used to compare the
proteome differences in neurological diseases such as AD Parkinsonrsquos disease and amyotrophic
lateral sclerosis and a review can be found elsewhere discussing the subject18
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy of brain or spinal cord
in evaluating diseases of the central nervous system and has been used for studies in neurological
disorders due to being a rich source of neuro-related proteins and peptides19
The protein
composition of the most abundant proteins in CSF is well defined and numerous studies exist to
broaden the proteins identified20-22
CSF has an exceedingly low protein content (~04 μgμL)
which is ~100 times lower than serum or plasma and over 60 of the total protein content in
CSF consists of a single protein albumin23-25
In addition the variable concentrations of proteins
span up to twelve orders of magnitude further complicating analysis and masking biologically
relevant proteins to any given study26
One of the highest number of identified proteins is from
Schutzer et al with 2630 non-redundant proteins from 14 mL of pooled human CSF This study
involved the removal of highly abundant proteins by performing IgY-14 immunodepletion
followed by two dimensional (2D) liquid chromatography (LC) separation27
Studies have also
been performed to characterize individual biomarkers or complex patterns of biomarkers in
various diseases in the CSF28 29
One potential pitfall of CSF proteomic analysis is
20
contamination from blood which can be identified by counting red blood cells present or
examining surrogate markers from blood contamination other than hemoglobin such as
peroxiredoxin catalase and carbonic anhydrase30
A proof of principle CSF peptidomics study
identified numerous endogenous peptides associated with the central nervous system which can
be used as a bank for neurological disorder studies31
Numerous recent reports highlighted the
utility of CSF analysis for biomarker studies in AD32 33
medulloblastoma34
both post-mortem
and ante-mortem35
Cellular lysates offer the distinct advantage to work with a cell line yeast or bacteria
with large amounts of proteins available for analysis36 37
with Saccharomyces cerevisiae being
the most common cell lysate38 39
Other cell lines are also used including HeLa40
and E coli41
The ability to obtain milligrams of proteins easily to scale up experiments without animal
sacrifice offers a clear advantage in biological sample selection Current literature supports
cellular lysate as a valued and sought after source of proteins for large scale proteomics
experiments because of the ability to assess treatments conditions and testable hypotheses42-44
Cellular lysate from rat B104 neuroblastoma cell line was used as an in vitro model for cerebral
ischemia and showed abundance changes in multiple proteins involved in various neurological
disorders45
Other Sources of Biological Samples
Urine
The urine proteome appears to be another attractive reservoir for biomarker discovery
due to the relatively low complexity compared with the plasma proteome and the noninvasive
collection of urine Urine is often considered as an ideal source to identify biomarkers for renal
21
diseases due to the fact that in healthy adults approximately 70 of the urine proteome originate
from the kidney and the urinary tract 46
thus the use of urine to identify neurological disorders is
neglected However strong evidence have shown that proteins that are associated with
neurodegenerative diseases can be excreted in the urine47-49
indicating the application of urine
proteomics could be a useful approach to the discovery of biomarkers and development of
diagnostic assays for neurodegenerative diseases However the current view of urine proteome
is still limited by factors such as sample preparation techniques and sensitivity of the mass
spectrometers There has been a tremendous drive to increase the coverage of urine proteome
In a recent study Court et al compared and evaluated several different sample preparation
methods with the objective of developing a standardized robust and scalable protocol that could
be used in biomarkers development by shotgun proteomics50
In another study Marimuthu et al
reported the largest catalog of proteins in urine identified in a single study to date The
proteomic analysis of urine samples pooled from healthy individuals was conducted by using
high-resolution Fourier transform mass spectrometry A total of 1823 proteins were identified
of which 671 proteins have not been previously reported in urine 51
Saliva
For diagnosis purposes saliva collection has the advantage of being an easy and non-
invasive technique The recent studies on saliva proteins that are critically involved in AD and
Parkinsonrsquos diseases suggested that saliva could be a potentially important sample source to
identify biomarkers for neurodegenerative diseases Bermejo-Pareja et al reported the level of
salivary Aβ42 in patients with mild AD was noticeably increased compared to a group of
controls 52
In another study Devic et al identified two of the most important Parkinsons
22
disease related proteinsmdash α-synuclein (α-Syn) and DJ-1 in human saliva 53
They observed that
salivary α-Syn levels tended to decrease while DJ-1 levels tended to increase in Parkinsons
disease The published results from this study also suggest that α-Syn might correlate with the
severity of motor symptoms in Parkinsons disease Due in part to recent advancements in MS-
based proteomics has provided promising results in utilizing saliva to explore biomarkers for
both local and systemic diseases 54 55
the further profiling of saliva proteome will provide
valuable biomarker discovery source for neurodegenerative diseases
Tissue
Compared to body fluids such as plasma serum and urine where the proteomic analysis
is complicated by the wide dynamic range of protein concentration the analysis of tissue
homogenates using the well-established and conventional proteomic analysis techniques has the
advantage of reduced dynamic range However the homogenization and extraction process may
suffer from the caveat that spatial information is lost which would be inadequate for the
detection of biomarkers whose localization and distribution play important roles in disease
development and progression Matrix-assisted laser desorptionionization (MALDI) imaging
mass spectrometry (IMS) is a method that allows the investigation of a wide range of molecules
including proteins peptides lipids drugs and metabolites directly in thin slices of tissue 56-59
Because this technology allows for identification and simultaneous localization of biomolecules
of interests in tissue sections linking the spatial expression of molecules to histopathology
MALDI-IMS has been utilized as a powerful tool for the discovery of new cancer biomarker
candidates as well as other clinical applications60 61
The utilization of MALDI-IMS for human
or animal brain tissue to identify or map the distribution of molecules related to
neurodegenerative diseases were also recently reported62 63
23
Secretome
There has been an increasing interest in the study of proteins secreted by various cells
(the secretomes) from tissue-proximal fluids or conditioned media as a potential source of
biomarkers Cell secretomes mainly comprise proteins that are secreted or are shed from the cell
surface and these proteins can play important role in both physiological processes (eg cell
signaling communication and migration) and pathological processes including tumor
angiogenesis differentiation invasion and metastasis In particular the study of cancer cell
secretomes by MS based proteomics has offered new opportunities for cancer biomarker
discovery as tumor proteins may be secreted or shed into the bloodstream and could be used as
noninvasive biomarkers The latest advances and challenges of sample preparation sample
concentration and separation techniques used specifically for secretome analysis and its clinical
applications in the discovery of disease specific biomarkers have been comprehensively
reviewed64 65
Here we only highlight the proteomic profiling of neural cells secretome that has
been applied to neurosciences for a better understanding of the roles secreted proteins play in
response to brain injury and neurological diseases The LC-MS shotgun identification of
proteins released by astrocytes has been recently reported66-68
In these studies the changes
observed in the astrocyte secretomes induced by inflammatory cytokines or cholinergic
stimulation were investigated6667
Alternatively our group performed 2D-LC separation and
included cytoplasmic protein extract from astrocytes as a control to identify cytoplasmic protein
contaminants which are not actively secreted from cells68
Sample Preparation
24
Proteomic analysis and biomarker discovery research in biological samples such as body
fluids tissues and cells are often hampered by the vast complexity and large dynamic range of
the proteins Because disease identifying biomarkers are more likely to be low-abundance
proteins it is imperative to remove the high-abundance proteins or apply enrichment techniques
to allow detection and better coverage of the low-abundance proteins for MS analysis Several
strategies including depletion and protein equalizer approach have been used during sample
preparation to reduce sample complexity69 70
and the latest advances of these methods have been
reviewed by Selvaraju et al 71
Alternatively the complexity of biological samples can be
reduced by capturing a specific subproteome that may have the biological information of interest
The latter strategy is especially useful in the biomarker discovery where the changes in the
proteome are not solely reflected through the concentration level of specific proteins but also
through changes in the post-translational modifications (PTMs) Here we will mainly discuss
the enrichment of phosphoproteinpeptides glycoproteinpeptides and sample preparation for
peptidomics and membrane proteins
Phosphoproteomics
Phosphorylation can act as a molecular switch on a protein by turning it on or off within
the cell It is thought that up to 30 of the proteins can be phosphorylated72
and it plays
significant roles in such biological processes as the cell cycle and signal transduction73
Currently tens of thousands of phosphorylation sites can be proposed using analytical methods
available today74 75
The amino acids that are targeted for phosphorylation studies are serine
threonine and tyrosine with the abundance of detection decreasing typically in that order Other
25
amino acids have been reported to be phosphorylated but traditional phosphoproteomics
experiments ignore these rare events76
In a typical large-scale phosphoproteomics experiment the sample size is usually in
milligram amounts to account for the low stoichiometry of phosphorylated proteins The large
amount of protein is then digested typically with trypsin but alternatively experiments have
been performed with Lys-C digestion to produce large enzymatic peptides The larger peptides
produced from Lys-C render higher charged peptides during electrospray ionization (ESI) and
allow improved electron-based fragmentation to determine specific sites of phosphorylation77
From the pool of peptides phosphopeptides must be enriched otherwise they will be masked by
the vast number and higher ionization efficiency of non-phosphorylated peptides The two most
common enrichment techniques are immobilized metal ion affinity chromatography (IMAC) and
metal oxide affinity chromatography (MOAC) TiO2 being the most common oxide used for this
purpose A recent study reported that phosphorylation of neuronal intermediate filament proteins
in neurofibrillary tangles are involved in Alzheimerrsquos disease78
Glycoproteomics
Protein glycosylation is one of the most common and complicated forms of PTM Types
of protein glycosylation in eukaryotes are categorized as either N-linked where glycans are
attached to asparagine residues in a consensus sequence N-X-ST (X can be any amino acid
except proline) via an N-acetylglucosamine (N-GlcNAc) residue or the O-glycosylation where
the glycans are attached to serine or threonine Glycosylation plays a fundamental role in
numerous biological processes and aberrant alterations in protein glycosylation are associated
with neurodegenerative disease states such as Creutzfeld-Jakob Disease (CJD) and AD79 80
26
Due to the low abundance of glycosylated forms of proteins compared to non-glycosylated
proteins it is essential to enrich glycoproteins or glycopeptides in complex biological samples
prior to MS analysis Two of the most common enrichment methods used in glycoproteomics are
lectin affinity chromatography (LAC) and hydrazide chemistry The detailed methodologies of
LAC hydrazide chemistry and other enrichment methods in glycoproteomics have been
extensively reviewed in the past81 82
In particular LAC is of great interest in studies of
glycosylation alterations as markers of AD and other neurodegenerative diseases due to its recent
applications in brain glycoproteomics83
Our group has utilized multi-lectin affinity
chromatography containing concanavalin A (ConA) and wheat germ agglutinin (WGA) to enrich
N-linked glycoproteins in control and prion-infected mouse plasma84
This method enabled us to
identify a low-abundance glycoprotein serum amyloid P-component (SAP) PNGase F digestion
and Western blotting validation confirmed that the glycosylated form of SAP was significantly
elevated in mice with early prion infection and it could be potentially used as a diagnostic
biomarker for prion diseases
Membrane proteins
Membrane proteins play an indispensable role in maintaining cellular integrity of their
structure and perform many important functions including signaling transduction intercellular
communication vesicle trafficking ion transport and protein translocationintegration85
However due to being relatively insoluble in water and low abundance it is challenging to
analyze membrane proteins by traditional MS-based proteomics approaches Numerous efforts
have been made to improve the solubility and enrichment of membrane proteins during sample
preparation Several comprehensive studies recently covered the commonly used technologies in
27
membrane proteomics and different strategies that circumvent technical issues specific to the
membrane 86-90
Recently Sun et al reported using 1-butyl-3-methyl imidazolium
tetrafluoroborate (BMIM BF4) an ionic liquid (IL) as a sample preparation buffer for the
analysis of integral membrane proteins (IMPs) by microcolumn reversed phase liquid
chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MSMS)
The authors compared BMIM BF4 to the other commonly used solvents such as sodium dodecyl
sulfate methanol Rapigest and urea but they found that the number of identified IMPs from rat
brain extracted by ILs was significantly increased The improved identifications could be due to
the fact that BMIM BF4 has higher thermal stability and thus offered higher solubilizing ability
for IMPs which provided better compatibility for tryptic digestion than traditionally used solvent
systems38
In addition to characterization of membrane proteome the investigation of PTMs on
membrane proteins is equally important for characterization of disease markers and drug
treatment targets Phosphorylations and glycosylations are the two most important PTMs for
membrane proteins In many membrane protein receptors the cytoplasmic domains can be
phosphorylated reversibly and function as signal transducers whereas the receptor activities of
the extracellular domains are mediated via N-linked glycosylation Wiśniewski et al provides an
informative summary on recent advances in proteomic technology for the identification and
characterization of these modifications91
Our group has pioneered the development of detergent
assisted lectin affinity chromatography (DALAC) for the enrichment of hydrophobic
glycoproteins using mouse brain extract92
We compared the binding efficiency of lectin affinity
chromatography in the presence of four commonly used detergents and determined that under
certain concentrations detergents can minimize the nonspecific bindings and facilitate the
elution of hydrophobic glycoproteins In summary NP-40 was suggested as the most suitable
28
detergent for DALAC due to the higher membrane protein recovery glycoprotein recovery and
membranous glycoprotein identifications compared to other detergents tested In a different
study on mouse brain membrane proteome Zhang et al reported an optimized protocol using
electrostatic repulsion hydrophilic interaction chromatography (ERLIC) for the simultaneous
enrichment of glyco- and phosphopeptides from mouse brain membrane protein preparation93
Using this protocol they successfully identified 544 unique glycoproteins and 922 glycosylation
sites which were significantly higher than those using the hydrazide chemistry method
Additionally a total of 383 phosphoproteins and 915 phosphorylation sites were identified
suggesting that the ERLIC separation has the potential for simultaneous analysis of both glyco-
and phosphoproteomes
Peptidomics
Peptidomics can be loosely defined as the study of the low molecular weight fraction of
proteins encompassing biologically active endogenous peptides protein fragments from
endogenous protein degradation products or other small proteins such as cytokines and signaling
peptides Studies can involve endogenous peptides94
peptidomic profiling33
and de novo
sequencing of peptides95 96
Neuropeptidomics focuses on biologically active short segments of
peptides and have been investigated in numerous species including Rattus97 98
Mus musculus99
100 Bovine taurus
101 Japanese quail diencephalon
102 and invertebrates
103-106 The isolation of
peptides is typically performed through molecular weight cut-offs from either biofluids such as
CSF plasma or tissue extracts If the protein and peptide content is high such as for tissue or cell
lysates protein precipitation can be done via high organic solvents and the resulting supernatant
can be analyzed for extracted peptides where extraction solvent and conditions could have a
29
significant effect on what endogenous peptides are extracted from tissue107
A comparative
peptidomic study of human cell lines highlights the utility of finding peptide signatures as
potential biomarkers108
A thorough review of endogenous peptides and neuropeptides is beyond
the scope of this review and an excellent review on this topic is available elsewhere109
Fractionation and Separation
The mass spectrometer has a limited duty cycle and data dependent analysis can only
scan a limited number of mz peaks at any given time In addition significant ion suppression
can occur if there is a difference in concentration between co-eluting peptides or if too many
peptides co-elute Therefore one of the biggest challenges in biomarker discovery is the
complexity of the sample and the presence of high-abundance proteins in body fluids such as
CSF serum and plasma In addition to the removal of the most abundant proteins by
immunodepletion the reduction of the complexity of the sample by further fractionation is
indispensable to facilitate the characterization of unidentified biomarkers from the low
abundance proteins Traditionally used techniques for complex protein analysis include gel
based fractionation methods such as two-dimensional gel electrophoresis (2D-GE) and its
variation two-dimensional differential gel electrophoresis (2D-DIGE) or non-gel based such as
one- or multidimensional liquid chromatography (LC) and microscale separation techniques
such as capillary electrophoresis (CE)
2D-GE MS has been widely used as a powerful tool to separate proteins and identify
differentially expressed proteins ever since 2D gels were coupled to mass spectrometry In 2D-
GE MS thousands of proteins can be separated on a single gel according to pI and molecular
weight Individual protein spots that show differences in abundance between different samples
30
can then be excised from the gel digested into peptides and analyzed by MALDI MS or by
liquid chromatography tandem mass spectrometry (LC-MSMS) for protein identification The
introduction of 2D-DIGE adds a quantitative strategy to gel electrophoresis by enabling multiple
protein extracts to be separated on the same 2D gel thus providing comparative analysis of
proteomes in complex samples In 2D-DIGE protein extracts from two different conditions and
an internal standard can be labeled with fluorescent dyes for example Cy3 Cy5 and Cy2
respectively prior to two-dimensional gel electrophoresis Compared to traditional 2D-GE 2D-
DIGE provides the clear advantage of overcoming the inter-gel variation problem 110
Proteomic
profiling of CSF by 2D-GE and 2D-DIGE has led to the identification of putative biomarkers in
multiple neurological disorders For example Brechlin et al reported an optimized 2-DIGE
protocol profiled CSF from 36 CJD patients The applicability of their approach was proven by
the detection of known CJD biomarkers such as 14-3-3 protein neuron-specific enolase lactate
dehydrogenase and other proteins that are potentially relevant to CJD 111
In another study to
identify novel CSF biomarkers for multiple sclerosis CSF from 112 multiple sclerosis patients
and control individuals were analyzed by 2D-GE MS for comparative proteomics Ten potential
multiple sclerosis biomarkers were selected for validation by immunoassay 112
These
methodologies sample preparation techniques and applications of 2D-DIGE in
neuroproteomics were reviewed by Diez et al113
Although 2D gel provides excellent resolving
power and capability to visualize abundance changes there are some limitations to the method
For example gel based separation is not suitable for low abundance proteins extremely basic or
acidic proteins very small or large proteins and hydrophobic proteins114 115
Complementary to gel-based approaches shotgun proteomics coupled to LC have
become increasingly popular in proteomic research because they are reproducible highly
31
automated and capable of detecting low abundance proteins Furthermore another advantage of
LC-MS shotgun proteomics is the suitability for isotope labeling for protein quantification which
is reviewed in a later section In shotgun proteomics a protein mixture is digested and resulting
peptides are separated by LC prior to tandem MS fragmentation to identify different proteins by
peptide sequencing The most common separation for shotgun proteomics peptidomics or top-
down proteomics experiments use low-pH reversed phase (RP) C18 C8 or C4 columns RPLC
is well established which provides high resolution desalts the sample which can interfere with
ionization and the mobile phase is compatible with ESI Nanoscale C18 columns allow for
separation and introduction of sub microgram samples If larger amounts of sample are
available two dimensional separations are usually preferred to greatly enhance the coverage of
the investigated proteome which will be discussed in depth later It is preferable to have an
orthogonal separation method and since RP separates via hydrophobicity strong cation exchange
(SCX) was the original choice due to its separation by charge MudPIT (multidimensional
protein identification technology) usually refers to the use of SCX as the first phase of separation
and is a well-established platform116
SCX has the advantage over RP separation technologies to
effectively remove interfering detergents from the sample SCX separation is not based solely
off charge and hydrophobicity contributes to elution therefore a small amount of organic
modifier usually 10-15 is added to lessen the hydrophobicity effects117
The addition of
organic modifiers needs to be minimized otherwise binding to the C18 trap cartridge or C18
column will be reduced if performed on-line SCX can be used for PTMs and offers specific
applications for proteomic studies and an excellent current review is offered on this subject
elsewhere118
An alternative MudPIT separation scheme employing high pH RPLC as the first
phase of separation and low pH RPLC in the second dimension (RP-RP) has been successfully
32
applied to the proteomic analysis of complex biological samples119 120
The advantage of using
RP as the first dimension is the higher resolution for separation and better compatibility with
down-stream MS detection by eliminating salt Song et al reported a phosphoproteome analysis
based on this 2D RP-RP coupling scheme121
Hydrophilic interaction chromatography (HILIC) employs distinct separation modality
where the retention of peptides is increased with increasing polarity122
The loading of sample is
done by high organic and eluted by increasing the percentage of the aqueous phase or polarity of
the mobile phase opposite from RPLC thus establishing orthogonality of the two separation
modes123
HILIC has quickly become a very useful method and is actively used for proteomic
experiments124
for increased sensitivity125
phosphoproteomics126
glycoproteins127
and
quantification studies128
An alternative and modification to HILIC is ERLIC which adds an
additional mode of separation by electrostatic attraction An earlier study using ERLIC
demonstrated the ability to separate phosphopeptides from non-phosphorylated peptides at
pH=2129
A recent study looking into changes in the phosphoproteome of Marekrsquos Disease
applied ERLIC to chicken embryonic fibroblast lysate identifying only 13 phosphopeptides
out of all the identified peptides Due to the lack of isolation of phosphopeptides from ERLIC
the investigators performed immobilized metal affinity chromatography (IMAC) enrichment on
the fractions increasing identification of phosphopeptides over 50 fold130
A comparative study
of ERLIC to HILIC and SCX following TiO2 phospho-enrichment reported that
SCXgtERLICgtHILIC for phosphopeptide identifications126
Recent developments in instrumentation to combine LC with ion mobility spectrometry
(IMS) and MS (LC-IMS-MS) offered more advantages than conventional LC due to the rapid
high-resolution separations of analytes based on their charge mass and shape as reflected by
33
mobility in a given buffer gas The mobility of an ion in a buffer gas is determined by the ionrsquos
charge and its collision cross-section with the buffer gas The methodologies of IMS separations
and the application of LC-IMS-MS for the proteomics analysis of complex systems including
human plasma have been reviewed by Clemmerrsquos group131-133
They proposed a method that
employs intrinsic amino acid size parameters to obtain ion mobility predictions which can be
used to rank candidate peptide ion assignments and significantly improve peptide identification
134
Although 2D gel and LC are routinely used as separation techniques in MS-based
proteomics capillary electrophoresis (CE) has received increasing attention as a promising
alternative due to the fast and high-resolution separation it offers CE has a wide variety of
operation modes among which capillary zone electrophoresis (CZE) and capillary isoelectric
focusing (CIEF) have the greatest potential applications in MS-based proteomics thus will be
highlighted here CZE separates analytes by their charge-to-size ratios in buffers under a high
electrical field and is often used as the final dimension prior to MS analysis while the separation
feature of CIEF is based on isoelectric point and this technique is more suitable to be used as the
first dimension separation Detailed description of different CEndashMS interfaces sample
preconcentration and capillary coating to minimize analyte adsorption could be found in several
reviews135-141
CE technique is complementary to conventional LC in that it is suitable for the
analysis of polar and chargeable compounds Dovichirsquos group conducted proteomic analysis of
the secreted protein fraction of Mycobacterium marinum which has intermediate protein
complexity142
The tryptic digests were either analyzed by UPLC-ESI-MSMS in triplicates or
prefactionated by RPLC followed by CZE-ESI-MSMS It was demonstrated that the two
methods identified similar numbers of peptides and proteins within similar analysis times
34
However CZE-ESI-MSMS analysis of the prefractionated sample tended to identify more
peptides that are basic and have lower mz values than those identified by UPLC-ESI-MSMS
This analysis also presented the largest number of protein identifications by using CE-MSMS
suggesting the effectiveness of prefractionation of complex samples by LC method prior to CZE-
ESI-MSMS The use of CIEF as the first dimension of separation provides both sample
concentration and excellent resolving power The combination of CIEF and RPLC separation
has been applied to the proteomic analyses where the amount of protein sample is limited and
cannot meet the requirement of minimal load amount for 2D LC-MSMS143 144
So far CE-MS
has been widely applied to the proteomic analysis of various biological samples such as urine145
146 CSF
147 blood
148 frozen tissues
149 and the formalin-fixed and paraffin-embedded (FFPE)
tissue samples150
The recent CEndashMS applications to clinical proteomics have been summarized
in several reviews135 151 152
Protein Quantification
In 2D gel electrophoresis the quantitative analysis of protein mixtures is performed on
the gel by comparing the intensity of the protein stain The development of 2D-DIGE eliminated
the gel-to-gel variation and greatly improved the quantitative capability and reliability of 2D gel
methodology110
However the accuracy of 2D gel based protein quantification suffers from the
limitations that a seemingly single gel spot often contains multiple proteins and the difficulty of
detecting proteins with extreme molecular weights and pI values as well as highly hydrophobic
proteins such as membrane proteins Therefore non-gel based shotgun proteomics technology is
more suitable for accurate and large-scale protein identification and quantification in complex
samples Briefly the quantification in non-gel based shotgun proteomics can be categorized into
35
two major approaches stable isotope labeling-based and label-free methods The common
strategies for quantitative proteomic analysis are reviewed and summarized in Table 1
Isotope labeling methods
Because stable isotope-labeled peptides have the same chemical properties as their
unlabeled counterparts the two peptides within a mixture should exhibit identical behaviors in
MS ionization The mass difference introduced by isotope labeling enables the detection of a
pair of two distinct peptide masses by MS within the mixture and allowing for the measurement
of the relative abundance differences between two peptides Depending on how isotopes are
incorporated into the protein or peptide these labeling methods can be divided into two groups
In vitro chemical derivatization techniques which incorporate a label or tag into the peptide or
protein during sample preparation metabolic labeling techniques which introduce the isotope
label directly into the organism via isotope-enriched nutrients from food or media
1 In vitro derivatization techniques
There are multiple methods to introduce heavy isotopes into proteins or peptides in vitro
The commonly used strategies include 18
O 16
O enzymatic labeling Isotope-Coded Affinity Tag
(ICAT) Tandem Mass Tags (TMTs) and Isobaric Tags for Relative and Absolute Quantification
(iTRAQ) The 18
O labeling method enzymatically cleaves the peptide bond with trypsin in the
presence of 18
O-enriched H2O and introduces 4-Da mass shift in the tryptic peptides153
The
advantages of this method include 18
O-enriched water is extremely stable tryptic peptides will
be labeled with the same mass shift secondary reactions inherent to other chemical labeling can
be avoided Conversely widespread use of 18
O-labeling has been hindered due to the difficulty
of attaining complete 18
O incorporation and the lack of robustness154 155
Currently ICAT
36
TMTs and iTRAQ methods are extensively used in quantitative proteomics In ICAT cysteine
residues are specifically derivatized with a reagent containing either zero or eight deuterium
atoms as well as a biotin group for affinity purification of cysteine-containing peptides156 157
The advantage of ICAT is that the affinity purification via biotin moiety can facilitate the
detection of low-abundance cysteine-containing peptides In addition the mass difference
introduced by labeling increases mass spectral complexity with quantification from the different
precursor masses done by MS and peptide identification being achieved through tandem MS
(MSMS) This added complexity from different peptide masses was addressed by using isobaric
labeling methods such as TMTs and iTRAQ 158 159
where the same peptides in different samples
are isobaric after tagging and appear as single mz in MS scans thus enhancing the peptide limit
of detection and reducing the MS scan complexity Isobaric labeling reagents are composed of a
primary amine reactive group and an isotopic reporter group linked by an isotopic balancer group
for the normalization of the total mass of the tags The reporter group serves for quantification
purpose since it is cleaved during collision-induced dissociation (CID) to yield a characteristic
isotope-encoded fragment Moreover isobaric labeling methods allow the comparison of
multiple samples within a single experiment Recently a 6-plex version of TMTs was
reported160
and iTRAQ enables up to eight samples to be labeled and relatively quantified in a
single experiment161
8-plex iTRAQ reagents have been used for the comparison of complicated
biological samples such as CSF in the studies of neurodegenerative diseases 162
Recently our
group developed a novel N N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2)
tagging reagents with high quantitation efficacy DiLeu has the advantage of synthetic simplicity
and greatly reduced synthesis cost compared to TMTs and iTRAQ163
Xiang et al demonstrated
that DiLeu produced comparable iTRAQ ability for protein sequence coverage (~43) and
37
quantitation accuracy (lt15) for tryptically digested proteins More importantly DiLeu
reagents could promote enhanced fragmentation of labeled peptides thus allowing more
confident peptide and protein identifications
2 In Vivo Metabolic Labeling
Metabolic processes can also be employed for the incorporation of stable-isotope labels
into the proteins or organisms by enriching culture media or food with light or heavy versions of
isotope labels (2H
13C
15N) The advantage of in vivo labeling is that metabolic labeling does
not suffer from incomplete labeling which is an inherent drawback for in vitro derivatization
techniques In addition metabolic labeling occurs from the start of the experiment and proteins
with light or heavy labels are simultaneously extracted thus reducing the error and variability of
quantification introduced during sample preparation The most widely used strategy for
metabolic labeling is known as stable-isotope labeling of amino acids in cell culture (SILAC)
which was introduced by Mann and co-workers164 165
In SILAC one cell population is grown
in normal or light media while the other is grown in heavy media enriched with a heavy
isotope-encoded (typically 13
C or 15
N) amino acid such as arginine or leucine Cells from the
two populations are then combined proteins are extracted digested and analyzed by MS The
relative protein expression differences are then determined from the extracted ion
chromatograms from both the light and heavy peptide forms SILAC has been shown to be a
powerful tool for the study of intracellular signal transduction In addition this technique has
recently been applied to the quantitative analysis of phosphotyrosine (pTyr) proteomes to
characterize pTyr-dependent signaling pathways166 167
38
Labe-free quantification
Although various isotope labeling methods have provided powerful tools for quantitative
proteomics several limitations of these approaches are noted Labeling increases the cost and
complexity of sample preparation introduces potential errors during the labeling reaction It also
requires a higher sample concentration and complicates data processing and interpretation In
addition so far only TMTs and iTRAQ allow the comparison of multiple (up to eight) samples
simultaneously The comparison of more than eight samples in a single experiment cannot be
achieved by isotope labeling In order to address these concerns there has been significant
interest in the development of label-free quantitative approaches Current label-free
quantification methods for MS-based proteomics were developed based on the observation that
the chromatographic peak area of a peptide168 169
or frequency of MSMS spectra170
correlating
to the protein or peptide concentration Therefore the two most common label-free
quantification approaches are conducted by comparing (i) area under the curve (AUC) of any
given peptides171 172
or (ii) by frequency measurements of MSMS spectra assigned to a protein
commonly referred to as spectral counting173
Several recent reviews provided detailed and
comprehensive knowledge comparing label-free methods with labeling methods data processing
and commercially available software for label-free quantitative proteomics174-177
Dissociation Techniques
The vast majority of proteomic experiments have proteins or peptides being identified by
two critical pieces of data obtained from the mass spectrometer The first is the precursor ion
identified by its mz which is informative to the mass of the peptide being analyzed The second
is the use of tandem mass spectrometry to fragment or dissociate the precursor ion and record the
39
generated fragment ion pattern to discern the amino acid sequence The three most popular
dissociation or fragmentation techniques for peptides are CID electron-transfer dissociation
(ETD) and high-energy collision dissociation (HCD) A recent study on the human plasma
proteome demonstrated that combined fragmentation techniques enhance coverage by providing
complementary information for identifications CID enabled the greatest number of protein
identifications while HCD identified an additional 25 proteins and ETD contributed an
additional 13 protein identifications178
ETDECD
Electron capture dissociation (ECD) 179
preceded ETD but ECD was developed for use
in a Penning trap for Fourier transform ion cyclotron resonance (FTICR) mass spectrometers
ECD requires the ion of interest to be in contact with near-thermal electrons and for the electron
capture event to occur on the millisecond time scale but the time scale is inadequate for electron
trapping in Paul traps or quadrupoles in the majority of mass spectrometers180
ETD involves a
radical anion like fluoranthene with low electron affinity to be transferred to peptide cation
which results in more uniform cleavage along the peptide backbone The cation accepts an
electron and the newly formed odd-electron protonated peptide undergoes fragmentation by
cleavage of the N-Cα bond which results in fragmentation ions consisting of c- and zbull-type
product ions The uniformed cleavage results in reduced sequence discrimination to labile bonds
such as PTMs and also provides improved sequencing for larger peptides compared to CID181
The realization that larger peptides produced better MSMS quality spectra compared to CID led
to a decision tree analysis strategy where peptide charge states and size determined whether the
precursor peptide would be fragmented with CID or ETD182
One of the main benefits of
ETDECD is the ability to sequence peptides with labile PTMs such as phosphorylation77 183
40
sulfation184
glycosylation185
ubiquitination186
and histone modifications187
ETD also has the
benefit of providing better sequence information on larger neuropeptides when compared to
CID188
However a thorough analysis suggested that CID still yielded more peptideprotein
identifications than ETD in large scale proteoimcs189
HCD
High energy collision dissociation (HCD)190
is an emerging fragmentation technique that
offers improved detection of small reporter ions from iTRAQ-based studies191 192
HCD is
performed at a higher energy in a collision cell instead of an ion trap like CID thus HCD does
not suffer from the low-mass cutoff limitation Furthermore HCD offers enhanced
fragmentation efficiency assisting in MSMS spectra interpretation and protein identification193
A major drawback for HCD is that the spectral acquisition times are up to two-fold longer due to
increased ion requirement for Fourier transform detection in the orbitrap194
HCD has been
reported to increase phosphopeptide identifications over CID74
but in a different study CID was
reported to offer more phosphopeptide identifications over HCD194
Work has also been done to
transfer the decision tree analysis for HCD which basically switches CID with HCD claiming
better quality data determined by higher Mascot scores with more peptide identifications195
MSE
Data dependent acquisition (DDA) is the most commonly used ion selection process in
mass spectrometers for proteomic experiments An alternative process which does not have ion
selection nor switch between MS and MSMS modes is termed MSE MS
E is a data independent
mode and does not require precursor ions of a significant intensity to be selected for MSMS
analysis196
A data independent mode decouples the mass spectrometer choosing which
precursor ions to fragment and when the ions are fragmented MSE works by a low or high
41
energy scan and no ion isolation is occurring The low energy scan is where the precursor ion is
not fragmented and the high energy scan allows fragmentation The resulting mix of precursor
and fragmentation ions is then detected simultaneously197
The data will then need to be
deconvoluted using a proprietary time-aligned algorithm that is discussed elsewhere198
The
continuous data independent acquisition allows multiple MSMS spectra to be collected during
the natural analyte peak broadening observed in chromatography which provides more data
points for AUC label-free quantification In addition lower abundance peptides can be
sequenced as more MSMS spectra are collected throughout the elution of an LC peak allowing
better signal averaging for smaller analyte peak of interest during coelution and reducing
sampling bias in typical DDA experiments where only more abundant peaks can be selected for
fragmentation
A comparison of spiked internal protein standards into a complex protein digest provided
evidence that MSE was comparable to DDA analysis in LC-MS
199 MS
E has been used for label
free proteomics of immunodepleted serum in large scale proteomics samples200
In addition
MSE was performed for the characterization of human cerebellum and primary visual cortex
proteomes Hundreds of proteins were identified including many previously reported in
neurological disorders201
MSE is quickly becoming a versatile data acquisition method recently
used in such studies as cancer cells202
schizophrenia203
and pituitary proteome discovery204
The usefulness of MSE as an unbiased data acquisition method is being assimilated into multiple
proteomics studies including studies involving neurological disorders
Data Analysis
42
One of the major bottlenecks in non-targeted proteomic experiments is how to handle the
enormous amount of data obtained Database searches biostatistical analysis de novo
sequencing PTM validation all have their place and multiple available platforms are available
If the organism being studied has had its genome sequenced databases can be created
with a list of proteins in the FASTA format to be used in database searching There are
numerous database searching algorithms for sequence identification of MSMS data including
Mascot205
Sequest206
Xtandem207
OMSSA208
and PEAKS209
These searching algorithms are
performed by matching MSMS spectra and precursor mass to sequences found within proteins
How well the actual spectra match the theoretical spectra determines a score which is unique to
the searching algorithm and usually can be extrapolated to the probability of a random hit
Recently a database has been developed for PTM analysis by the use of the program SIMS210
Specifically for phosphopeptides Ascorersquos algorithm scans the MSMS data to determine the
likelihood of correct phosphosite identification from the presence of site identifying product
ions211
If the organism that is being analyzed has not had its genome sequenced and no (or very
limited) FASTA database is available a homology search can be performed using SPIDER212
available with PEAKS software Alternatively individual MSMS spectrum can be de novo
sequenced but software is available to perform automated de novo sequencing of numerous
spectra (PEAKS208
DeNovoX and PepSeq)
For large-scale protein identifications the false discovery rate (FDR) must be established
by the searching algorithm and that is accomplished by re-searching the data with a false
database created by reversing or scrambling the amino acid sequence of the original database
used for the protein search Any hits from the false database will contribute to the FDR and this
value can be adjusted usually around 1 An additional layer of confidence in the obtained data
43
can be achieved in shotgun proteomics experiments by removing all the proteins that are
identified by only one peptide
Once a set of confident proteins or peptides have been generated from database
searching bioinformatic analysis or biostatistical analysis is needed Numerous software
packages are available for different purposes FLEXIQuant is an example for absolute
quantitation of isotopically labeled protein or peptides of interest213
FDR analysis of
phosphopeptides or other specific PTMs can be adjusted with such software as Scaffold
providing data consisting only of a specific modification214
Bioinformatic tools such as
Scaffold or ProteoIQ also include gene ontology (GO) analysis which can classify identified
proteins by three categories cellular component molecular function or biological process
Custom bioinformatics programs can also be developed and are often useful in various proteomic
studies including biomarker discovery in neurological diseases215
More detailed review of
bioinformatics in peptidomics216
and proteomics217
can be found elsewhere
Validation of Biomarkers by Targeted Proteomics
The validation of putative biomarkers identified by MS-based proteomic analysis is often
required to provide orthogonal analysis to rule out a false positive by MS and providing
additional evidence for the biomarker candidate(s) from the study for future potential clinical
assays At present antibody-based assays such as Western blotting ELISA and
immunochemistry are the most widely used methods for biomarker validation Although accurate
and well established these methods rely on protein specific antibodies for the measurement of
the putative biomarker and could be difficult for large-scale validation of all or even a subset of a
long list of putative protein biomarkers typically obtained by MS-based comparative proteomic
44
analysis Large scale validation is impractical due to the cost for each antibody the labor to
develop a publishable Western blot or ELISA and the antibody availability for certain proteins
As an alternative strategy quantitative assays based on multiple-reaction monitoring (MRM) MS
using a triple quadrupole mass spectrometer have been employed in biomarker verification
MRM is the most common use of MSMS for absolute quantitation It is a hypothesis
driven experiment where the peptide of interest and its subsequent fragmentation pattern must be
known prior to the quantitative MRM experiments MRM involves selecting a specific mz (first
quadrupole) to be isolated for fragmentation (second quadrupole) followed by one or more of
the most intense fragment ions (third quadrupole) being monitored The ability to quantitate and
thus validate the proteins or peptides as potential biomarkers is achieved by performing MRM on
isotopically labeled reference peptide for targeted peptideprotein of interest The main obstacle
for quantification of peptides is interference and ion suppression effects from co-eluting
substances Since the isotopically labeled and native peptide will co-elute the same interference
and ion suppression will occur for both peptides and thus correcting these interfering effects
Peptides need to be systematically chosen for a highly sensitive and reproducible MRM
experiment to ensure proper validation of putative biomarkers Peptides require certain intrinsic
properties which include an mz within the practical mass detection range for the instrument and
high ionization efficiency If the desired peptide to be quantified is derived from a digestion
then peptides that have detectable incomplete digestion or missed cleavage site can be a major
source of variability Peptides with a methionine and to a lesser extent tryptophan are
traditionally removed from consideration from MRM quantitative experiments due to the
variable nature of the oxidation that can occur In addition if chromatographic separation is
performed the retention behavior of the peptide must be well behaved with little tailing effects
45
eluting late causing broadening of the peak and even irreversible binding to the column As an
example hydrophilic peptides being eluted off a C18 column may exhibit the previously
described concerns and a different chromatographic separation will need to be explored for
improved limits of detection quantitation and validation To determine consistent peptide
detection or usefulness of certain peptides databases such as Proteomics Database218
PRIDE219
PeptideAtlas220
have been developed to compile proteomic data repositories from initial
discovery experiments
After the peptide is selected for analysis the proper MRM transitions need to be selected
to optimize the sensitivity and selectivity of the experiment It is common for investigators to
select two or three of the most intense transitions for the proposed experiment It is imperative
that the same instrument is used for the determination of transition ions as different mass
spectrometers may have a bias towards different fragment ions
MRM experiments are still highly popular experiments for hypothesis directed
experiments221
biomarker analysis222
and validation223
Validation of putative biomarkers is
increasingly becoming a necessary step when performing large scale non-hypothesis driven
proteomics experiments The traditional validation techniques of ELISA Western blotting and
immunohistochemistry are still used but MRM experiments are becoming an attractive
alternative for validation of putative biomarkers due to its enhanced throughput and specificity
Current work is still being performed to both expand the linear dynamic range224
and
sensitivity225
of MRM A recent endeavor to increase the sensitivity for MRM experiments was
accomplished by ldquoPulsed MRMrdquo via the use of an ion funnel trap to enhance confinement and
accumulation of ions The authors claimed an increase by 5-fold for peak amplitude and a 2-3
fold reduction in chemical background225
46
Remaining Challenges and Emerging Technologies
Large sample numbers for mass spectrometry analysis
Multiple conventional studies in proteomics have been performed on a single or a few
biological samples As bio-variability can be exceedingly high the need for larger sample sizes
is currently being investigated Prentice et al used a starting point of 3200 patient samples
from the Womenrsquos Health Institute (WHI) to probe the plasma proteome using MS for
biomarkers The study did not test the 3200 patient samples by MS because even a simple one
hour one dimensional RP analysis on a mass spectrometer would take months of instrument time
for uninterrupted analysis Instead the authors pooled 100 samples together to bring the total
number of pooled samples to 32 To provide relevant plasma biomarkers the samples were then
subjected to immunodepletion 2-D protein separation (96 fractions total) and then 1-D RPLC of
tryptic peptide separation on-line interface to a mass spectrometer The large sample cohorts
help address bio-variability that can be a concern from small sample size proteomic experiments
and provide ample sample amounts to investigate the low abundance proteins226
Hemoglobin-derived neuropeptides and non-classical neuropeptides
Neuropeptides such as neuropeptide Y and enkephalin are short chains of amino acids
that are secreted from a range of neuronal cells that signal nearby cells In contrast non-classical
neuropeptides are termed as neuropeptides or ldquomicroproteinsrdquo which are derived from
intracellular protein fragments and synthesized from the cytosol227
MS was recently used to
determine that hemopressins which are hemoglobin-derived peptides are upregulated in Cpefatfat
mice brains Gelman et al designed an MS experiment to compare hemoglobin-derived
47
peptides comparing the brain blood and heart peptidome in mice The authors provided data
that specific hemoglobin peptides were produced in the brain and were not produced in the
blood Certain alpha and beta hemoglobin peptides were also up regulated in the brain for
Cpefatfat
mice and bind to CB1 cannabinoid receptors228
As discussed earlier in the review
peptidomics and specifically neuropeptidomics are popular fields of study utilizing MS and non-
classical neuropeptides is an exciting emerging area of research that could further expand the
diversity of cell-cell signaling molecules
Ultrasensitive mass spectrometry for single cell analysis
In addition to large scale analysis MS-based proteomics and peptidomics are making
progress into ultrasensitive single cell analysis The most successful MS-based techniques for
single cell analysis was performed with MALDI and studies that have been performed on
relatively large neurons are reviewed elsewhere229
The ultrasensitive MS analysis is currently
directed towards single cell analysis of smaller cells including cancer cells The first challenge
in single cell analysis is the isolation and further sample preparation to yield relevant data
Collection and isolation of a cell type can be accomplished using antibodies for fluorescence
activated cell sorting (FACS) and immune magnetic separation FACS works by flow cytometry
sorting cells by a laser that excites a fluorescent tag that is attached to an antibody Immune
magnetic separation allows separation by antibodies with magnetic properties such as
Dynabeads230
One exciting study combining FACS and MS termed mass cytometry This
technology works by infusing a droplet into an inductively coupled plasma mass spectrometer
(ICP-MS) containing a single cell bound to antibodies chelated to transition elements allowing a
quantifying response between single cells231
Clearly the future of single cell analysis for
48
biomarker analysis and proteomics is encouraging and has the potential to be an emerging field
in MS-based proteomics and peptidomics
Laserspray ionization (LSI)
Laserspray ionization (LSI) is an exciting new method to produce multiply charged mass
spectra from MALDI that is nearly identical to ESI232-234
Recently it has been reported that LSI
can be performed in lieu of matrix to produce a total solvent-free analysis234
The benefits of
being able to generate multiply charged peptides without any solvent may offer advantages
including MS analysis of insoluble membrane proteins or hydrophobic peptides avoidance of
chemical reactions while in solvents a reduction of sample loss due to liquid sample preparation
and ability to avoid diffusion effects from tissue imaging studies234
The multiply charged peptide and protein ions produced by LSI expand the mass range
for tissue imaging analysis More importantly the multiply-charged peptide ions are amenable
for electron-based fragmentation methods such as ETD or ECD which can be employed in
conjunction with tissue imaging experiments to yield in situ sequencing and identification of
peptides of interest235
Paper spray ionization
Paper spray (PS) is an ambient ionization method which was first reported using
chromatography paper allowing detection of metabolites from dried blood spots The original
method used a cut out piece of paper with a voltage clipped on the back while applying 10 microL of
methanolH2O236
Improvements have been made to this technology to enhance analysis
efficiency with a new solvent 91 dichloromethaneisopropanol (vv) and the use of silica paper
49
over chromatography paper237
Interesting applications or modifications have been made to PS
including direct analysis of biological tissue238
and leaf spray for direct analysis of plant
materials239
but both detect metabolites instead of proteins or peptides Paper spray ionization
was previously shown to enable detection of cytochrome c and bradykinin [2-9] standards in a
proof of principle study240
Clearly the utility of PS analysis in proteomics and peptidomics is
yet to be explored
niECD
New fragmentation techniques have been investigated for their utility in proteomics and
peptidomics including a recently reported negative-ion electron capture dissociation (niECD)
Acidic peptides which usually contain PTMs such as phosphorylation or sulfonation are often
difficult to be detected as multiply charged peptides in the positive ion mode As discussed
earlier multiply charged peptides are required for ECDETD fragmentation The fragmentation
of niECD is accomplished by a multiply negatively charged peptide adding an electron The
resulting fragmentation of multiply sulfated and phosphorylated peptide and protein standards
showed no sulfate loss and preserved phosphorylation site The resulting fragmentation pattern
from niECD was also improved in the peptide anions and provides a new strategy for de novo
sequencing with PTM localization241
Conclusions and Perspectives
Proteomics methodologies have produced large datasets of proteins involved in various
biological and disease progression processes Numerous mass spectrometry-based proteomics
and peptidomics tools have been developed and are continuously being improved in both
50
chromatographic or electrophoretic separation and MS hardware and software However several
important issues that remain to be addressed rely on further technical advances in proteomics
analysis When large proteomes consisting of thousands of proteins are analyzed and quantified
dynamic range is still limited with more abundant proteins being preferentially detected
Development and optimization of chemical tagging reagents that target specific protein classes
maybe necessary to help enrich important signaling proteins and assess cellular and molecular
heterogeneity of the proteome and peptidome Furthermore a significant bottleneck in
usefulness of proteomics research is the ability to validate the results and provide clear
significant biological relevance to the results The idea of P4 medicine242 243
is an attractive
concept where the four Prsquos stand for predictive preventive personalized and participatory
Proteomics is one of the critical ldquoomicsrdquo fields and has led to the development of enabling
innovative strategies to P4 medicine244
A goal of P4 medicine is to assess both early disease
detection and disease progression in a person A simplified example of how proteomics fits into
P4 medicine is that certain brain-specific proteins could be used for diagnosis with
presymptomatic prion disease244
The concept of proteomic experiments providing an individual
biomarker is becoming more obsolete with the revised vision being a biomolecular barcode that
could potentially be ldquoscannedrdquo or be a fingerprint for a specific disease or early onset to that
disease being closer to reality An excellent review on what biomarker analysis can do for true
patients is available245
Proteomics can also generate new hypothesis that can be tested by classical biochemical
approaches If a disease has an unknown pathogenesis proteomics is a good starting point to try
to assemble putative markers that can lead to further hypothesis for evaluation If a particular
protein or PTM is associated with a disease state either qualitatively or quantitatively potential
51
treatments could target that protein of interest or investigators could monitor that protein or
PTM during potential treatments of the disease Proteomics has expanded greatly over the last
few decades with the goal of providing revealing insights to some of the most complex
biological problems currently facing the scientific community
Acknowledgements
Preparation of this manuscript was supported in part by the University of Wisconsin Graduate
School Wisconsin Alzheimerrsquos Disease Research Center Pilot Grant and a Department of
Defense Pilot Award LL acknowledges an H I Romnes Faculty Fellowship
52
Figure 1 A summary of general workflows of biomarker discovery pipeline by MS-based
proteomic approaches
Biological sample (CSF blood urine saliva cell
lysate tissue homogenates secreted proteins etc)
Protein extraction Sample pretreatment
2D-GE2D-DIGE MS 1D or 2D LC-MSMS
MALDI-IMS
Identification of
differentially
expressed proteins
Protein identification
Potential biomarkers
Biomarker validation
- Antibody based immunoassays
- MRM
Quantitative analysis
- Isotope labeling
- Label free
Identification and
localization of
differentially expressed
biomolecules
Intact tissue
Sample preparation Slice frozen tissues
thaw-mounted on plate
Apply Matrix
53
Figure 2 Tissue expression and dynamic range of the human plasma proteome A pie chart
representing the tissue of origin for the high abundance proteins shows that the majority of
proteins come from the liver (A) Conversely the lower abundance plasma proteins have a much
more diverse tissue of origin (B and C) The large dynamic range of plasma proteins is presented
and the proteins can be grouped into three categories (classical plasma proteins tissue leakage
products interleukinscytokines) (D) Adapted from Zhang et al11
and Schiess et al246
with
permission
54
55
Table 1 A summary of the common strategies applied to MS-based quantitative proteomic
analysis
Gel based Stable isotope labeling Label free
2D-GE
2D-DIGE 110
In vitro derivatization
18O
16O
153
ICAT 156
TMT 159
iTRAQ 158
Formaldehyde 247
ICPL 248
In vivo metabolic labeling
14N
15N
249
SILAC 164
AUC measurement 169 172
Spectral counting 173
AUC Area Under Curve ICAT Isotope-Coded Affinity Tag TMT Tandem Mass Tags iTRAQ Isobaric Tags for
Relative and Absolute Quantification ICPL Isotope Coded Protein Labeling SILAC Stable Isotope Labeling by
Amino Acids in Cell Culture Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
56
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precursor and product ions from data independent LC-MS with data dependant LC-MSMS
Proteomics 2009 9 (6) 1683-95
200 Koutroukides T A Guest P C Leweke F M Bailey D M Rahmoune H Bahn
S Martins-de-Souza D Characterization of the human serum depletome by label-free shotgun
proteomics J Sep Sci 34 (13) 1621-6
201 Martins-de-Souza D Guest P C Guest F L Bauder C Rahmoune H Pietsch S
Roeber S Kretzschmar H Mann D Baborie A Bahn S Characterization of the human
primary visual cortex and cerebellum proteomes using shotgun mass spectrometry-data-
independent analyses Proteomics 12 (3) 500-4
202 Scatena R Bottoni P Pontoglio A Giardina B Revisiting the Warburg effect in
cancer cells with proteomics The emergence of new approaches to diagnosis prognosis and
therapy Proteomics Clin Appl 4 (2) 143-58
203 Herberth M Koethe D Cheng T M Krzyszton N D Schoeffmann S Guest P
C Rahmoune H Harris L W Kranaster L Leweke F M Bahn S Impaired glycolytic
response in peripheral blood mononuclear cells of first-onset antipsychotic-naive schizophrenia
patients Mol Psychiatry 16 (8) 848-59
204 Krishnamurthy D Levin Y Harris L W Umrania Y Bahn S Guest P C
Analysis of the human pituitary proteome by data independent label-free liquid chromatography
tandem mass spectrometry Proteomics 11 (3) 495-500
205 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
206 Eng J K McCormack A L Yates Iii J R An approach to correlate tandem mass
spectral data of peptides with amino acid sequences in a protein database Journal of the
American Society for Mass Spectrometry 1994 5 (11) 976-989
207 Craig R Beavis R C TANDEM matching proteins with tandem mass spectra
Bioinformatics 2004 20 (9) 1466-7
208 Geer L Y Markey S P Kowalak J A Wagner L Xu M Maynard D M Yang
X Shi W Bryant S H Open mass spectrometry search algorithm J Proteome Res 2004 3
(5) 958-64
209 Zhang J Xin L Shan B Chen W Xie M Yuen D Zhang W Zhang Z Lajoie
G A Ma B PEAKS DB De Novo sequencing assisted database search for sensitive and
accurate peptide identification Mol Cell Proteomics 2011
210 Liu J Erassov A Halina P Canete M Nguyen D V Chung C Cagney G
Ignatchenko A Fong V Emili A Sequential interval motif search unrestricted database
surveys of global MSMS data sets for detection of putative post-translational modifications
Anal Chem 2008 80 (20) 7846-54
70
211 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
approach for high-throughput protein phosphorylation analysis and site localization Nat
Biotechnol 2006 24 (10) 1285-92
212 Han Y Ma B Zhang K SPIDER software for protein identification from sequence
tags with de novo sequencing error J Bioinform Comput Biol 2005 3 (3) 697-716
213 Singh S Springer M Steen J Kirschner M W Steen H FLEXIQuant a novel tool
for the absolute quantification of proteins and the simultaneous identification and quantification
of potentially modified peptides J Proteome Res 2009 8 (5) 2201-10
214 Searle B C Scaffold a bioinformatic tool for validating MSMS-based proteomic
studies Proteomics 10 (6) 1265-9
215 Herbst A McIlwain S Schmidt J J Aiken J M Page C D Li L Prion disease
diagnosis by proteomic profiling J Proteome Res 2009 8 (2) 1030-6
216 Menschaert G Vandekerckhove T T Baggerman G Schoofs L Luyten W Van
Criekinge W Peptidomics coming of age a review of contributions from a bioinformatics
angle J Proteome Res 2010 9 (5) 2051-61
217 Kumar C Mann M Bioinformatics analysis of mass spectrometry-based proteomics
data sets FEBS Lett 2009 583 (11) 1703-12
218 Craig R Cortens J P Beavis R C Open source system for analyzing validating and
storing protein identification data J Proteome Res 2004 3 (6) 1234-42
219 Jones P Cote R G Cho S Y Klie S Martens L Quinn A F Thorneycroft D
Hermjakob H PRIDE new developments and new datasets Nucleic Acids Res 2008 36
(Database issue) D878-83
220 Deutsch E W Lam H Aebersold R PeptideAtlas a resource for target selection for
emerging targeted proteomics workflows EMBO Rep 2008 9 (5) 429-34
221 Miliotis T Ali L Palm J E Lundqvist A J Ahnoff M Andersson T B
Hilgendorf C Development of a highly sensitive method using liquid chromatography-multiple
reaction monitoring to quantify membrane P-glycoprotein in biological matrices and relationship
to transport function Drug Metab Dispos 2011 39 (12) 2440-9
222 Xiang Y Koomen J M Evaluation of Direct Infusion-Multiple Reaction Monitoring
Mass Spectrometry for Quantification of Heat Shock Proteins Anal Chem 2012
223 Ossola R Schiess R Picotti P Rinner O Reiter L Aebersold R Biomarker
validation in blood specimens by selected reaction monitoring mass spectrometry of N-
glycosites Methods Mol Biol 2011 728 179-94
224 Liu H Lam L Dasgupta P K Expanding the linear dynamic range for multiple
reaction monitoring in quantitative liquid chromatography-tandem mass spectrometry utilizing
natural isotopologue transitions Talanta 2011 87 307-10
225 Belov M E Prasad S Prior D C Danielson W F 3rd Weitz K Ibrahim Y M
Smith R D Pulsed multiple reaction monitoring approach to enhancing sensitivity of a tandem
quadrupole mass spectrometer Anal Chem 2011 83 (6) 2162-71
226 Prentice R L Paczesny S Aragaki A Amon L M Chen L Pitteri S J
McIntosh M Wang P Buson Busald T Hsia J Jackson R D Rossouw J E Manson J
E Johnson K Eaton C Hanash S M Novel proteins associated with risk for coronary heart
disease or stroke among postmenopausal women identified by in-depth plasma proteome
profiling Genome Med 2 (7) 48
227 Gelman J S Fricker L D Hemopressin and other bioactive peptides from cytosolic
proteins are these non-classical neuropeptides AAPS J 2010 12 (3) 279-89
71
228 Gelman J S Sironi J Castro L M Ferro E S Fricker L D Hemopressins and
other hemoglobin-derived peptides in mouse brain comparison between brain blood and heart
peptidome and regulation in Cpefatfat mice J Neurochem 2010 113 (4) 871-80
229 Li L Garden R W Sweedler J V Single-cell MALDI a new tool for direct peptide
profiling Trends Biotechnol 2000 18 (4) 151-60
230 Altelaar A M Heck A J Trends in ultrasensitive proteomics Curr Opin Chem Biol
231 Bandura D R Baranov V I Ornatsky O I Antonov A Kinach R Lou X
Pavlov S Vorobiev S Dick J E Tanner S D Mass cytometry technique for real time
single cell multitarget immunoassay based on inductively coupled plasma time-of-flight mass
spectrometry Anal Chem 2009 81 (16) 6813-22
232 Trimpin S Inutan E D Herath T N McEwen C N Laserspray ionization a new
atmospheric pressure MALDI method for producing highly charged gas-phase ions of peptides
and proteins directly from solid solutions Mol Cell Proteomics 2010 9 (2) 362-7
233 McEwen C N Larsen B S Trimpin S Laserspray ionization on a commercial
atmospheric pressure-MALDI mass spectrometer ion source selecting singly or multiply
charged ions Anal Chem 2010 82 (12) 4998-5001
234 Wang B Lietz C B Inutan E D Leach S M Trimpin S Producing highly
charged ions without solvent using laserspray ionization a total solvent-free analysis approach at
atmospheric pressure Anal Chem 2011 83 (11) 4076-84
235 Inutan E D Richards A L Wager-Miller J Mackie K McEwen C N Trimpin
S Laserspray ionization a new method for protein analysis directly from tissue at atmospheric
pressure with ultrahigh mass resolution and electron transfer dissociation Mol Cell Proteomics
2010 10 (2) M110 000760
236 Wang H Liu J Cooks R G Ouyang Z Paper spray for direct analysis of complex
mixtures using mass spectrometry Angew Chem Int Ed Engl 49 (5) 877-80
237 Zhang Z Xu W Manicke N E Cooks R G Ouyang Z Silica coated paper
substrate for paper-spray analysis of therapeutic drugs in dried blood spots Anal Chem 84 (2)
931-8
238 Wang H Manicke N E Yang Q Zheng L Shi R Cooks R G Ouyang Z
Direct analysis of biological tissue by paper spray mass spectrometry Anal Chem 83 (4) 1197-
201
239 Liu J Wang H Cooks R G Ouyang Z Leaf spray direct chemical analysis of plant
material and living plants by mass spectrometry Anal Chem 83 (20) 7608-13
240 Liu J Wang H Manicke N E Lin J M Cooks R G Ouyang Z Development
characterization and application of paper spray ionization Anal Chem 82 (6) 2463-71
241 Yoo H J Wang N Zhuang S Song H Hakansson K Negative-ion electron
capture dissociation radical-driven fragmentation of charge-increased gaseous peptide anions J
Am Chem Soc 2011 133 (42) 16790-3
242 Tian Q Price N D Hood L Systems cancer medicine towards realization of
predictive preventive personalized and participatory (P4) medicine J Intern Med 271 (2) 111-
21
243 Hood L Friend S H Predictive personalized preventive participatory (P4) cancer
medicine Nat Rev Clin Oncol 2011 8 (3) 184-7
244 Tian Q Price N D Hood L Systems cancer medicine towards realization of
predictive preventive personalized and participatory (P4) medicine J Intern Med 2012 271
(2) 111-21
72
245 Belda-Iniesta C de Castro J Perona R Translational proteomics what can you do for
true patients J Proteome Res 2011 10 (1) 101-4
246 Schiess R Wollscheid B Aebersold R Targeted proteomic strategy for clinical
biomarker discovery Mol Oncol 2009 3 (1) 33-44
247 Hsu J L Huang S Y Chow N H Chen S H Stable-isotope dimethyl labeling for
quantitative proteomics Anal Chem 2003 75 (24) 6843-52
248 Schmidt A Kellermann J Lottspeich F A novel strategy for quantitative proteomics
using isotope-coded protein labels Proteomics 2005 5 (1) 4-15
249 Wang Y K Ma Z Quinn D F Fu E W Inverse 15
N-metabolic labelingmass
spectrometry for comparative proteomics and rapid identification of protein markerstargets
Rapid Commun Mass Spectrom 2002 16 (14) 1389-97
73
Chapter 3
Protein changes in immunodepleted cerebrospinal fluid from transgenic
mouse models of Alexander disease detected using mass spectrometry
Adapted from ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse
models of Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P
Messing A Li L Submitted
74
ABSTRACT
Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range
spanning at least nine orders of magnitude in protein content and is in direct contact with the
brain A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the
low volumes of CSF that are obtainable from mice As a model system in which to test this
approach we utilized transgenic mice that over-express the intermediate filament glial fibrillary
acidic protein (GFAP) These mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we report the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates were performed to address animal variability as well as reproducibility in
mass spectrometric analysis Relative quantitation was performed using distributive normalized
spectral abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins
with significant changes in the CSF of GFAP transgenic mice has been identified with validation
from ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
75
INTRODUCTION
Alexander disease (AxD) is a fatal neurogenetic disorder caused by heterozygous point
mutations in the coding region of GFAP (encoding glial fibrillary acidic protein)1 The hallmark
diagnostic feature of this disease is the accumulation of astrocytic cytoplasmic inclusions known
as Rosenthal fibers containing GFAP Hsp27 αB-crystallin and other components2-5
Although
several potential treatment strategies6-8
are under investigation clinical trial design is hampered
by the absence of a standardized clinical scoring system or means to quantify lesions in MRI
that could serve to monitor severity and progression of disease One solution to this problem
would be the identification of biomarkers in readily sampled body fluids as indirect indicators of
disease
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy site for brain or spinal
cord in evaluating diseases of the central nervous system The protein composition of CSF is
well defined at least for the most abundant species of proteins and numerous studies exist that
characterize individual biomarkers or complex patterns of biomarkers in various diseases9 10
GFAP itself is present in CSF (albeit at much lower levels than in brain parenchyma) and in one
study of three Alexander disease patients its levels were markedly increased11
Whether an
increase in CSF GFAP will be a consistent finding in Alexander disease or whether other useful
biomarkers for this disease could be identified through an unbiased analysis of the CSF
proteome is not yet known
The rarity of Alexander disease makes analysis of human samples difficult However
mouse models exist that replicate key features of the disease such as formation of Rosenthal
fibers Unfortunately mouse CSF poses particular problems for proteomic studies and there is
76
an urgent need for technical improvements for dealing with this fluid For instance collection
from an adult mouse typically yields ~10 μL of CSF often with some contamination by blood12
To further complicate analysis CSF has an exceedingly low protein content (~04 μgμL) with
over 60 of the total protein content consisting of a single protein albumin13 14
A number of
techniques have been developed to remove albumin from biological samples including Cibacron
Blue15
IgG immunodepletion16
and IgY immunodepletion17-19
IgY which is avian in origin
offers reduced non-specific binding and increased avidity when compared to IgG antibodies from
rabbits goats and mice20-23
One widely used IgY cocktail is IgY-14 which contains fourteen
specific antibodies specific for albumin IgG transferrin fibrinogen α1-antitrypsin IgA IgM
α2-macroglobulin haptoglobin apolipoproteins A-I A-II and B complement C3 and α1-acid
glycoprotein Since existing protocols for IgY-14 depletion are optimized for use with large
volumes of serum new protocols must be developed to permit its use with the low volumes of a
low protein fluid represented by mouse CSF
Various improvements have also taken place in the field of proteomic analysis that could
facilitate analysis of mouse CSF Data dependent tandem mass spectrometry followed by
quantification of proteins is used in standard shotgun proteomics24-29
Several methods now exist
for introducing quantitation into mass spectrometry including stable isotope labeling30-32
isobaric tandem mass tags33 34
and spectral counting35 36
Spectral counting which is a
frequency measurement that uses MSMS counts of identified peptides as the metric to enable
protein quantitation is attractive because it is label-free and requires no additional sample
preparation Finally recent advances in spectral counting has produced a data refinement
strategy termed normalized spectral abundance factor (NSAF)37 38
and further developed into
distributive NSAF (dNSAF) to address issues with peptides shared by multiple proteins39
77
To identify potential biomarkers in AxD we report a novel scaled-down version of IgY
antibody depletion strategy to reduce the complexity and remove high abundance proteins in
mouse CSF samples The generated spectral counts data were then subjected to dNSAF natural
log data transformation and t-test analysis to determine which proteins differ in abundance when
comparing GFAP transgenics and controls with multiple biological and technical replicates
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) urea (gt99) sodium chloride (997) and ammonium
bicarbonate (certified) were purchased from Fisher Scientific (Fair Lawn NJ) Deionized water
(182 MΩcm) was prepared with a Milli-Q Millipore system (Billerica MA) Optima LCMS
grade acetonitrile and water were purchased from Fisher Scientific (Fair Lawn NJ) DL-
Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega
(Madison WI) Formic acid (ge98) was obtained from Fluka (Buchs Switzerland)
Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris ge999 ) ammonium formate
(ge99995) glycine (ge985) and IgY-14 antibodies were purchased from Sigma-Aldrich
(Saint Louis MO)
Mice
Transgenic mice over-expressing the human GFAP gene (Tg737 line) were maintained
as hemizygotes in the FVBN background and genotyped via PCR on DNA prepared from tail
samples as described previously40
The mice were housed on a 14-10 light-dark cycle with ad
libitum access to food and water All procedures were conducted using protocols approved by
the UW-Madison IACUC
78
CSF collection
CSF was collected from mice as described previously12
Briefly mice were anesthetized
with avertin (400-600 mgkg ip) A midline sagittal incision was made over the dorsal aspect
of the hindbrain and three muscle layers carefully peeled back to expose the cisterna magna The
membrane covering the cisterna magna was pierced with a 30 gauge needle and CSF was
collected immediately using a flexible plastic pipette Approximately ten microliters of CSF was
collected per animal All samples used for MS analysis showed no visible contamination of
blood
Enzyme-linked immunosorbent assay (ELISA)
A sandwich ELISA was used to quantify GFAP8 Briefly a microtiter plate was coated
with a cocktail of monoclonal antibodies (Covance SMI-26R) Plates were blocked with 5
milk before addition of sample or standards diluted in PBS with Triton-X and BSA A rabbit
polyclonal antibody (DAKO Z334) was used to detect the GFAP followed by a peroxidase
conjugated anti-rabbit IgG antibody (Sigma A6154) secondary antibody The peroxidase activity
was detected with SuperSignal ELISA Pico Chemiluminescent Substrate (PIERCE) and
quantified with a GloRunner Microplate Luminometer Values below the biological limit of
detection (16ngL) were given the value 16ngL before multiplying by the dilution factor
Immunodepletion of abundant proteins
Currently there are no commercial immunodepletion products available for use with CSF
and to address the low protein content of this fluid (~04 microgmicroL) Therefore 100 microL of
purchased IgY-14 resin was coupled with a Pierce Spin Cup using a paper filter from Thermo
Scientific (Waltham MA) CSF samples from either transgenics or controls were first pooled to
100 microL and then added to 100 microL of 20 mM Tris-HCl 300 mM pH 74 (2x dilution buffer) and
79
allowed to mix with the custom IgY-14 depletion spin column on an end-over-end rotor for 30
minutes at 4oC The sample was then centrifuged at 04 rcf for 45 seconds with an Eppendorf
Centrifuge 5415D (Hamburg Germany) The antibodies were then washed with 50 microL 1x
dilution buffer vortexed for 5 minutes centrifuged at 04 rcf for 45 seconds and the flow through
was collected for tryptic digestion The antibodies were then stripped of the bound proteins with
four 025 mL washes of 01 M glycine pH 25 neutralized with two 025 mL washes of 01 M
Tris-HCl pH 80 and washed once with 025mL of 1x dilution buffer The immunodepletion
protocol was repeated for each transgenic or control pooled 100 microL sample (N=3)
Preparation of tryptic digests
The immunodepleted pooled mouse CSF samples (200 microL total volume) were
concentrated to 10 microL using a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA)
To each sample 8 microL of 133 M urea and 1 microL of 050 M DTT were added and allowed to
incubate at 37oC for one hour After incubation 27 microL of 055 M IAA was added for
carboxymethylation and the sample was allowed to incubate for 15 minutes in the dark To
quench the IAA 1 microL of 050 M DTT was added and allowed to react for 10 minutes To
perform trypsin digestion 70 microL of 50 mM NH4HCO3 was then added along with 025 microg
trypsin which had previously been dissolved in 50 mM acetic acid at a concentration of 05
microgmicroL Digestion was performed overnight at 37oC and quenched by addition of 25 microL of 10
formic acid The tryptic peptides were then subjected to solid phase extraction using a Varian
Omix Tip C18 100 microL (Palo Alto CA) Peptides were eluted with 50 ACN in 01 formic
acid concentrated and reconstituted in 30 microL H2O in 01 formic acid
RP nanoLC separation
80
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto an Agilent
Technologies Zorbax 300 SB-C18 5 microm 5x03 mm trap cartridge (Santa Clara CA) at a flow
rate of 5 microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm
Atlantis dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B
at 250 nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
81
range of 300-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot41
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt mus musculus
(house mouse) database (version 575) False positive analyses42
were calculated using an
automatic decoy option of Mascot Results from the Mascot results were reported using
Proteinscape 21 and technical replicates were combined and reported as a protein compilation
using ProteinExtractor (Bruker Daltonics Bremen Germany)
Mascot search parameters were as follows Allowed missed cleavages 2 enzyme
trypsin variable modifications carboxymethylation (C) and oxidation (M) peptide tolerance
plusmn12 Da maximum number of 13
C 1 MSMS tolerance plusmn05 Da instrument type ESI-Trap
Reported proteins required a minimum of one peptide with a Mascot score ge300 with bold red
characterization Spectral counts were determined from the number of MSMS spectra identified
from accepted proteins A bold red peptide combines a bold peptide which represents the first
query result from a submitted MSMS spectrum with the red peptide which indicates the top
peptide for the identified protein Requiring one bold red peptide assists in removal of
homologous redundant proteins and further improves protein results In addition requiring one
82
peptide to be identified by a score gt300 removes the ability for proteins to be identified by
multiple low Mascot scoring peptides
Each immunodepleted biological replicate had technical triplicates performed and the
technical triplicates were summed together by ProteinExtractor Peptide spectral counts were
then summed for each protein and subjected to dNSAF analysis Details for this method can be
found elsewhere37 39
but briefly peptide spectral counts are summed per protein (SpC) based on
unique peptides and a weighted distribution of any shared peptides with homologous proteins
ProteinScape removed 83 homologous proteins found in the current study to bring the total
number of proteins identified to 266 but some non-unique homologous peptides which are
shared by multiple proteins are still present in the resulting 266 remaining proteins To address
these non-unique homologous peptides distributive spectral counting was performed as
described elsewhere39
The dSpC is divided by the proteinrsquos length (L) and then divided by the
summation of the dSpCL from all proteins in the experiment (N) to produce each proteinrsquos
specific dNSAF value
N
i
i
kk
LdSpC
LdSpCdNSAF
1
)(
)()(
The resulting data were then transformed by taking the natural log of the dNSAF value The
means for each protein were calculated using the ln(dNSAF) from N=3 biological replicates and
the whole data set was subjected to the Shapiro-Wilk test for normalityGaussian distribution
performed on the software PAST (Version 198 University of Oslo Norway Osla) The
Shapiro-Wilk test was repeated multiple times to determine a non-zero value for zero spectral
83
counts A non-zero value is required to alleviate the errors of dividing by zero which was
experimentally determined to be 043 The Gaussian data were then subjected to the t-test to
identify statistically significant changes in protein expression
RESULTS AND DISCUSSION
General workflow
Individual CSF samples were manually inspected and samples were only selected that
showed no visual blood contamination Preliminary experiments showed that the maximum
degree of blood contamination estimated from counts of red blood cells in the CSF that was not
visible to the eye was less than 005 by total mass Approximately 10 individual mouse CSF
samples were pooled to achieve the desired 100 μL volume for a single biological replicate The
CSF samples were IgY immunodepleted followed by digestion with trypsin The resulting
digested samples were then de-salted concentrated reconstituted in 30 μL of 01 formic acid
and 5 μL was loaded onto a reversed phase nanoflow HPLC column subjected to a 90 minute
gradient and infused into the amaZon ETD ion trap mass spectrometer The workflow for
mouse CSF analysis is shown in Figure 1 The remainder of the desalted sample was saved for
technical replicates
Immunodepletion for CSF
Currently there are no immunodepletion techniques specifically designed for CSF
Nonetheless the protein profiles between CSF and serum are similar enough to use currently
available immunodepletion techniques designed for serum as a starting point The smallest
commercially available IgY-14 immunodepletion kit is for 15 μL of serum which is equivalent in
protein content to ~15 mL of CSF Therefore for 100 μL CSF one fifteenth of the total IgY-14
84
beads would be used for the equivalent immunodepletion which is 66 μL of proprietary bead
slurry The potential for irreversible binding of abundant proteins to their respective IgY
antibody even after an extra stripping wash and low amounts of total beads made using 66 μL
of IgY-14 bead slurry unrealistic In practice almost doubling the amount of IgY-14 beads (100
μL) used was not sufficient and resulted in albumin and transferrin peptides to be observed in
high abundance (data not shown) The most important protein to immunodeplete is albumin and
it has been reported to be a greater percentage of total CSF protein content (~60) than serum
(~49) in humans14
The difference in albumin percentage supports the results that proprietary
blends of immunodepletion beads for high abundance proteins such as albumin cannot be
scaled down on a strict protein scale and further modifications to the serum immunodepletion
protocol need to be made
Since IgY-14 beads were developed for use with serum all of its protocols need to be
taken into account to modify the protocol for CSF Serum samples should be diluted fifty times
before mixing with the IgY-14 beads but CSF protein concentration is at least one hundred times
lower than serum Therefore CSF is below half the recommended diluted protein concentration
for IgY immunodepletion Consequently multiple steps have been devised to address this
limitation First the binding time between the proteins targeted for removal from the CSF and
IgY-14 beads was doubled during the end-over-end rotor depletion step from the recommended
15 minutes to 30 minutes Second to prevent non-specific protein binding to the antibodies the
CSF samples were diluted with an equal amount (100 μL) of 2X dilution buffer The dilution
buffer of NaCl and Tris-HCl was needed to assist in reducing non-specific binding of proteins to
the 14 antibodies and ensuring the sample is held at physiological pH In addition to these
modifications a doubling of the starting IgY-14 bead slurry to 200 μL provided the desired
85
results Overall this modified protocol results in effective depletion of CSF abundant proteins
using only one-fifth of the antibodies provided by the smallest commercially available platform
Data Analysis
Spectral counting technique for relative quantitation provides numerous benefits for the
study of mouse CSF due to its label-free advantage In contrast isotopic labeling method often
involves additional sample processing that could cause sample loss which is highly undesirable
for low protein content and low volume samples Labeling methods also require a mixing of two
sets of isotopically labeled samples which would effectively increase the sample complexity and
reduce the amount of sample that can be loaded onto the nanoLC column by half In addition
more than two sets of samples can be compared by label-free methods The use of label-free
spectral counting method does not lead to an increase in sample complexity or interference in
quantitation from peptides in the mz window selected for tandem MS Using spectral counting
for relative quantitation however is dependent on reproducible HPLC separation and careful
mass spectrometry operation to minimize technical variability during the study To address
concerns of analytical reliability and run to run deviations base peak chromatograms from two
transgenic IgY-14 immunodepleted biological replicates including two technical replicates of
each were shown to be highly reproducible (Figure 2)
Each biological sample was analyzed in triplicate with the same protocols on the amaZon
ETD with three control and three transgenic samples From the three technical replicates for
each biological replicate the spectral counts of the peptides for the proteins identified were
summed The results from these mouse CSF biological triplicates are shown in Figure 3A for
GFAP overexpressor and Figure 3B for control The summation of spectral counts for each
biological replicate was performed to remove the inherent bias related to data dependent analysis
86
for protein identification One concern in grouping technical replicates is a potential loss of
information regarding analytical variability Figure 4 provides a graphical representation of
variability of technical replicates illustrating the standard deviation of technical replicates with
error bars Figure 4 shows that a statistically changed protein creatine kinase M (A) and an
unchanged protein kininogen-1 (B) have similar variability within (technical replicates) and
between samples (biological replicates) for each protein In addition Figure 4B illustrates that
even with the variability of kininogen-1 the resulting mean shown by the dashed line of control
and transgenic samples were almost equal whereas Figure 4A shows significantly different
expression level of creatine kinase M Performing replicate analysis of each biological sample
(n=3) helps correct for systematic errors deriving from sample handling and pooling of samples
helps reduce random error during the CSF sample collection process
Protein Identification and Spectral Counting Analysis
The data for dNSAF analysis like any mass spectrometry proteomics experiment
requires multiple layers of verification to ensure reliable data Our initial protein identifications
were subjected to a database search using a decoy database from Mascot which resulted in an
average false positive rate below 1 for all the experimental data collected Representative
MSMS spectra between control and GFAP transgenic samples are illustrated in Figure 5
Overall 266 proteins were identified in a combination of control and transgenic samples
(Supplemental Table 1) A total of 349 proteins were initially identified but 83 proteins were
isoforms of previously identified proteins and automatically excluded by ProteinExtractor The
next level of quality control was to only include ln(dNSAF) values from proteins identified by 2
or more unique peptides having a Mascot score of ge300 and observed in two out of three
biological replicates These selection parameters resulted in 106 proteins remaining after
87
dNSAF analysis (Supplemental Table 2) The resulting spectral counts were then subjected to
dSpC in order to account and correct for the systematic error of peptides shared by multiple
proteins (Supplemental Table 3)
It is inevitable in large scale and complex proteomics experiments that some proteins will
be seen in some samples and not others In addition when controls were compared to transgenic
samples as shown in Figure 3C 127 proteins were unique to either control or GFAP transgenic
mice and 139 proteins were seen in both samples From dNSAF equation if the spectral count
is zero the numerator is zero and the value will not be normalized between runs In order to
circumvent the zero spectral counts in dNSAF analysis zero spectral counts must be replaced by
an experimentally determined non-zero value determined to be 043 The 043 spectral counts
for zero spectral counts was calculated by serial Shapiro-Wilk tests to determine the lowest value
(043) for zero spectral counts and maintain a Gaussian distribution for all data sets The 043
value for zero spectral counts in the current study was higher than the 016 reported value for
zero spectral counts in the original NSAF spectral counting study37
Our study may have a
higher zero spectral count value than the previous study because the spectral counting data were
an addition of three technical replicates and three times 016 is close to 043 The normalized
Gaussian data were then subjected to t-test analysis and P-values below 005 are considered as
statistically significant and are presented in Table 1 The proteins with significant up or down
regulation from Table 1 can be further evaluated as how close significant proteins were to a p-
value of 005 such as ganglioside GM2 activator serine protease inhibitor A3N and collagen
alpha-2(I) chain having t-test scores of 0045 0047 and 0043 respectively Proteins exhibiting
a P-value close to 005 were more likely to be highly variable proteins or have smaller fold
changes between control and transgenic samples and thus provide less biological relevancy to
88
future studies The modest change in antithrobin-III which is reduced by 14-fold in transgenic
is included due a low pooled standard deviation in spectral counts
Spectral counting has been analyzed with fold changes derived directly from the average
spectral counts from the technical replicates and then the average of the three biological
replicates We decided to perform additional analysis using fold changes to dig deeper into
proteins that statistically support the null hypothesis from the dNSAF analysis To only pull out
highly confident protein identifications we used the same strict cut-off of two unique peptides
identified per protein as in dNSAF analysis We only accepted proteins with greater than three-
fold change in total spectral counts listed in Table 2 Two proteins contactin-1 (cntn1) and
cathepsin B (CB) illustrate the potential biomarkers missed by dNSAF analysis Cntn1 had zero
spectral count in the transgenic sample and had an average spectral count of 41 in control
samples The lack of any spectral counts in one biological control for cntn1 resulted in a large
standard deviation in the ln(dNSAF) means for the control which resulted in the t-test supporting
the null hypothesis Another example is CB which was detected by numerous spectral counts in
every GFAP transgenic biological replicate with an average spectral count of 97 (Table 2) The
presence of CB in one biological control sample (23 average spectral counts) resulted in a high
standard deviation in the mean of the control samples These examples exhibit a limitation of
dNSAF analysis which could cause a loss of potentially useful information
Previously Identified Proteins with Expression Changes
Previously three proteins have been described as increased in CSF from individual(s)
suffering from AxD In one study a single patientrsquos CSF was found to have elevated levels of
αβ-crystallin and HSP2744
In a second study three patients were reported to have elevated
levels of GFAP with concentrations from 4760 ngL to 30000 ngL (compared to lt175 ngL for
89
controls)11
GFAP was detected in our current study however the other two proteins were not
detected One possibility is that αB-crystallin and Hsp-27 levels in mouse CSF are too low for
detection by MS analysis In addition while the transgenic mice display the hallmark
pathological feature of AxD in the form of Rosenthal fibers they do not have any evident
leukodystrophy and thus may not display the full range of changes in CSF as might be found in
human patients
Creatine Kinase M
Creatine kinase M (M-CK) is a 43 kDa protein whose role is to reversibly catalyze
phosphate transfer between ATP and energy storage compounds M-CK has been primarily
found in muscle tissue and for humans creatine kinase M is diagnostically analyzed in the blood
for myocardial infarction (heart attack) In addition M-CK is also found in Purkinje neurons of
the cerebellum45 46
A related protein creatine kinase B (B-CK) also exhibited an apparent 21
fold increase in transgenic CSF over control but this difference was not statistically different
B-CK concentration is known to be elevated in CSF following head trauma47
or cerebral
infarction48
but decreased in astrocytes in individuals affected by multiple sclerosis49
Cathepsin
The data showed multiple cathepsins were up regulated in the CSF of transgenic mice
when compared to control mice The up regulated cathepsins were S L1 and B isoforms which
are all cysteine proteases Cathepsin S (CS) was never observed in control samples but
observed with an average of 73 spectral counts per analysis Cathepsin L1 (CL1) was up
regulated by 94 times in transgenic mice These two cathepsins exhibited significant changes
using dNSAF and t-test statistics as shown in Table 1 and Cathepsin B (CB) showed a 42 fold
increase in transgenic CSF as shown in Table 2
90
Cathepsins regulate apoptosis in cells50
which is the major mechanism for elimination of
cells deemed by the organism to be dangerous damaged or expendable CL and CB are
redundant in the system as thiol proteases and inhibition of CB by CA-047 causes a diminished
apoptosis response in multiple cell lines51
Intriguingly increased levels of CB or CL are
correlated with poor prognosis for cancer patients and shorter disease-free intervals It is
believed that these proteases degrade the extracellular membrane which allows tumor cells to
invade adjacent tissue and metastasize52
With regards to AxD the up regulation of these
cathepsins may be indicative of the bodyrsquos natural defense and response to Rosenthal fibers
Thus stimulation of these cathepsins may provide a further protective stress response but the
positive correlation between these proteases and cancer highlights the multiple roles of these
proteins in pathological response Alternatively it has been shown that increased CB is involved
with the tumor necrosis factor α (TNFα) induced apoptosis cascade53
The activation of the
TNFα could produce oligodendrocyte toxicity54
with the expression of TNFα being elevated in
tissue samples from mouse models and AxD patients55
The potential for a positive or a negative
effect in increasing or decreasing cathepsins warrant future research with cathepsins and AxD
Contactin-1
Contactin-1 (Cntn1) is a 133 kDa cell surface protein that is highly glycosylated and
belongs to a family of immunoglobulin domain-containing cell adhesion molecues56
Table 2
shows that Cntn1 is down regulated in transgenic mice since spectral counts were only observed
in control mice Cntn1 is expressed in neurons and oligodendrocytes and higher levels were
observed during brain development57
In addition Cntn1 leads to activation of Notch1 which
mediates differentiation and maturation of oligodendrocyte precursor cells (OPC) Although the
mechanism leading to a reduction of Cntn1 in CSF is not known it may reflect a change in
91
astrocyte interactions with either neurons or oligodendrocytes to alter their expression of this
protein
Validation of putative biomarkers and MS proteomics data using ELISA and RNA
microarray data
To further validate the relative protein expression data obtained via MS-based spectral
counting techniques orthogonal immunological and molecular biological approaches have been
examined As GFAP was shown to be elevated in the CSF of transgenic animals we used a
well-defined GFAP ELISA protocol to test its CSF concentration CSF from 8 week old male
mice was collected from both transgenic and control animals Five samples of transgenic CSF
was prepared by pooling four to eight animals for the GFAP ELISA In the case of controls
each sample represents a single animal GFAP concentrations observed by both the MS and
ELISA showed significant increases of GFAP in the CSF when comparing transgenic to control
animals
Another validation of MS spectral counts is observed in a microarray analysis performed
on transgenic mouse olfactory bulb tissue 55
In this paper nine of the proteins found by MS
showed similar gene expression in the microarray (Table 3) It is not surprising that all the genes
observed in the microarray are not the same as the proteins observed by MS analysis Gene
expression and protein synthesis and expression are not always correlated but the similarities
and overlapping trends observed with these two assays are encouraging As shown in Table 3
gene expression analysis also revealed the up regulation of several cathepsin proteins GFAP
and down regulation of contactin-1 in CSF collected from transgenic mice corroborating the
MS-based proteomics results
92
CONCLUSIONS
In this study we have produced a panel of proteins with significant up or down regulation
in the CSF of transgenic GFAP overexpressor mice This mouse model for AxD was consistent
with the previous studies showing elevation of GFAP in CSF The development of a modified
IgY-14 immunodepletion technique for low amounts of CSF was presented This improved
protocol is useful for future investigations to deal with the unique challenges of mouse CSF
analysis Modified proteomics protocols were employed to profile mouse CSF with biological
and technical triplicates addressing the variability and providing quantitation with dNSAF
spectral counting Validation of the MS-based proteomics data were performed using both
ELISA and RNA microarray data to provide further confidence in the changes in the putative
protein biomarkers This study presents three classes of interesting targets for future study in
AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
93
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7 Tang G Yue Z Talloczy Z Hagemann T Cho W Messing A Sulzer D L
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55
8 Hagemann T L Boelens W C Wawrousek E F Messing A Suppression of GFAP
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9 Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C
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10 Sjodin M O Bergquist J Wetterhall M Mining ventricular cerebrospinal fluid from
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11 Kyllerman M Rosengren L Wiklund L M Holmberg E Increased levels of GFAP
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Neuropediatrics 2005 36 (5) 319-23
12 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M
Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta)
equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
13 Wong M Schlaggar B L Buller R S Storch G A Landt M Cerebrospinal fluid
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Pediatr Adolesc Med 2000 154 (8) 827-31
14 Roche S Gabelle A Lehmann S Clinical proteomics of the cerebrospinal fluid
Towards the discovery of new biomarkers PROTEOMICS ndash Clinical Applications 2008 2 (3)
428-436
15 Li C Lee K H Affinity depletion of albumin from human cerebrospinal fluid using
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Biochem 2004 333 (2) 381-8
94
16 Maccarrone G Milfay D Birg I Rosenhagen M Holsboer F Grimm R Bailey
J Zolotarjova N Turck C W Mining the human cerebrospinal fluid proteome by
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2412
17 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L
Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity
separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample
preparation and analysis Proteomics 2005 5 (13) 3314-28
18 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag
L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep
Biochem Biotechnol 2009 39 (3) 221-47
19 Huang L Fang X Immunoaffinity fractionation of plasma proteins by chicken IgY
antibodies Methods Mol Biol 2008 425 41-51
20 Greunke K Braren I Alpers I Blank S Sodenkamp J Bredehorst R Spillner E
Recombinant IgY for improvement of immunoglobulin-based analytical applications Clin
Biochem 2008 41 (14-15) 1237-44
21 Xiao Y Gao X Taratula O Treado S Urbas A Holbrook R D Cavicchi R E
Avedisian C T Mitra S Savla R Wagner P D Srivastava S He H Anti-HER2 IgY
antibody-functionalized single-walled carbon nanotubes for detection and selective destruction
of breast cancer cells BMC Cancer 2009 9 351
22 Liu T Qian W J Mottaz H M Gritsenko M A Norbeck A D Moore R J
Purvine S O Camp D G 2nd Smith R D Evaluation of multiprotein immunoaffinity
subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry
Mol Cell Proteomics 2006 5 (11) 2167-74
23 Hinerfeld D Innamorati D Pirro J Tam S W SerumPlasma depletion with
chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species
J Biomol Tech 2004 15 (3) 184-90
24 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D
Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in
the discovery of candidate protein biomarkers in a diabetes autoantibody standardization
program sample subset J Proteome Res 2008 7 (2) 698-707
25 Ru Q C Zhu L A Silberman J Shriver C D Label-free semiquantitative peptide
feature profiling of human breast cancer and breast disease sera via two-dimensional liquid
chromatography-mass spectrometry Mol Cell Proteomics 2006 5 (6) 1095-104
26 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S
Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-
dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of
Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66
27 Rao P V Reddy A P Lu X Dasari S Krishnaprasad A Biggs E Roberts C T
Nagalla S R Proteomic identification of salivary biomarkers of type-2 diabetes J Proteome
Res 2009 8 (1) 239-45
28 Yu K H Barry C G Austin D Busch C M Sangar V Rustgi A K Blair I A
Stable isotope dilution multidimensional liquid chromatography-tandem mass spectrometry for
pancreatic cancer serum biomarker discovery J Proteome Res 2009 8 (3) 1565-76
29 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422
(6928) 198-207
95
30 Ong S E Blagoev B Kratchmarova I Kristensen D B Steen H Pandey A
Mann M Stable isotope labeling by amino acids in cell culture SILAC as a simple and
accurate approach to expression proteomics Mol Cell Proteomics 2002 1 (5) 376-86
31 Hsu J L Huang S Y Chow N H Chen S H Stable-isotope dimethyl labeling for
quantitative proteomics Anal Chem 2003 75 (24) 6843-52
32 Liu H Sadygov R G Yates J R 3rd A model for random sampling and estimation
of relative protein abundance in shotgun proteomics Anal Chem 2004 76 (14) 4193-201
33 Xiang F Ye H Chen R Fu Q Li L NN-dimethyl leucines as novel isobaric
tandem mass tags for quantitative proteomics and peptidomics Anal Chem 82 (7) 2817-25
34 Ross P L Huang Y N Marchese J N Williamson B Parker K Hattan S
Khainovski N Pillai S Dey S Daniels S Purkayastha S Juhasz P Martin S Bartlet-
Jones M He F Jacobson A Pappin D J Multiplexed protein quantitation in
Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol Cell Proteomics
2004 3 (12) 1154-69
35 Zybailov B Coleman M K Florens L Washburn M P Correlation of relative
abundance ratios derived from peptide ion chromatograms and spectrum counting for
quantitative proteomic analysis using stable isotope labeling Anal Chem 2005 77 (19) 6218-
24
36 Old W M Meyer-Arendt K Aveline-Wolf L Pierce K G Mendoza A Sevinsky
J R Resing K A Ahn N G Comparison of label-free methods for quantifying human
proteins by shotgun proteomics Mol Cell Proteomics 2005 4 (10) 1487-502
37 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M
P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J
Proteome Res 2006 5 (9) 2339-47
38 Mosley A L Florens L Wen Z Washburn M P A label free quantitative
proteomic analysis of the Saccharomyces cerevisiae nucleus J Proteomics 2009 72 (1) 110-20
39 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome
quantitation how to deal with peptides shared by multiple proteins Anal Chem 82 (6) 2272-81
40 Messing A Head M W Galles K Galbreath E J Goldman J E Brenner M
Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice Am J Pathol 1998
152 (2) 391-8
41 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
42 Elias J E Gygi S P Target-decoy search strategy for increased confidence in large-
scale protein identifications by mass spectrometry Nat Methods 2007 4 (3) 207-14
43 You J S Gelfanova V Knierman M D Witzmann F A Wang M Hale J E The
impact of blood contamination on the proteome of cerebrospinal fluid Proteomics 2005 5 (1)
290-6
44 Takanashi J Sugita K Tanabe Y Niimi H Adolescent case of Alexander disease
MR imaging and MR spectroscopy Pediatr Neurol 1998 18 (1) 67-70
45 Hemmer W Wallimann T Functional Aspects of Creatine Kinase in Brain
Developmental Neuroscience 1993 15 (3-5) 249-260
46 Hemmer W Zanolla E Furter-Graves E M Eppenberger H M Wallimann T
Creatine kinase isoenzymes in chicken cerebellum specific localization of brain-type creatine
96
kinase in Bergmann glial cells and muscle-type creatine kinase in Purkinje neurons Eur J
Neurosci 1994 6 (4) 538-49
47 Nordby H K Tveit B Ruud I Creatine kinase and lactate dehydrogenase in the
cerebrospinal fluid in patients with head injuries Acta Neurochirurgica 1975 32 (3) 209-217
48 Bell R D Alexander G M Nguyen T Albin M S Quantification of cerebral
infarct size by creatine kinase BB isoenzyme Stroke 1986 17 (2) 254-60
49 Steen C Wilczak N Hoogduin J M Koch M De Keyser J Reduced Creatine
Kinase B Activity in Multiple Sclerosis Normal Appearing White Matter PLoS ONE 5 (5)
e10811
50 Chwieralski C Welte T Buumlhling F Cathepsin-regulated apoptosis Apoptosis 2006
11 (2) 143-149
51 Droga-Mazovec G Bojic L Petelin A Ivanova S Romih R Repnik U Salvesen
G S Stoka V Turk V Turk B Cysteine cathepsins trigger caspase-dependent cell death
through cleavage of bid and antiapoptotic Bcl-2 homologues J Biol Chem 2008 283 (27)
19140-50
52 Duffy M J Proteases as prognostic markers in cancer Clin Cancer Res 1996 2 (4)
613-8
53 Guicciardi M E Deussing J Miyoshi H Bronk S F Svingen P A Peters C
Kaufmann S H Gores G J Cathepsin B contributes to TNF-alpha-mediated hepatocyte
apoptosis by promoting mitochondrial release of cytochrome c J Clin Invest 2000 106 (9)
1127-37
54 Butt A M Jenkins H G Morphological changes in oligodendrocytes in the intact
mouse optic nerve following intravitreal injection of tumour necrosis factor J Neuroimmunol
1994 51 (1) 27-33
55 Hagemann T L Gaeta S A Smith M A Johnson D A Johnson J A Messing
A Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal
fibers reveals a stress response followed by glial activation and neuronal dysfunction Hum Mol
Genet 2005 14 (16) 2443-58
56 Falk J Bonnon C Girault J A Faivre-Sarrailh C F3contactin a neuronal cell
adhesion molecule implicated in axogenesis and myelination Biol Cell 2002 94 (6) 327-34
57 Eckerich C Zapf S Ulbricht U Muumlller S Fillbrandt R Westphal M Lamszus
K Contactin is expressed in human astrocytic gliomas and mediates repulsive effects Glia
2006 53 (1) 1-12
97
Table 1 Statistically changed proteins between transgenic and control mouse CSF using
dNSAF analysis
Accession Protein Pa SC
b Fold
Changec
Control
dSpCd
Transgenic
dSpCd
KCRM_MOUSE Creatine kinase M-type 0034 425 30 182 541
HEXB_MOUSE Βeta-hexosaminidase subunit β 0012 183 uarr 0 59
CMGA_MOUSE Chromogranin-A 000078 153 darr 44 0
ANT3_MOUSE Antithrombin-III 0017 176 -14 67 47
SAP3_MOUSE Ganglioside GM2 activator 0045 415 darr 28 0
SPA3N_MOUSE Serine protease inhibitor A3N 0047 170 42 10 42
CO1A2_MOUSE Collagen alpha-2(I) chain 0043 56 darr 19 0
BLVRB_MOUSE Flavin reductase 00054 204 uarr 0 12
CATS_MOUSE Cathepsin S 00032 232 uarr 0 73
GFAP_MOUSE Glial fibrillary acidic protein 0021 107 uarr 0 21
RET4_MOUSE Retinol-binding protein 4 0040 189 darr 13 0
CBPE_MOUSE Carboxypeptidase E 0043 139 darr 11 0
CATL1_MOUSE Cathepsin L1 0015 87 94 02 19
The statistics are performed using the t-test from the ln(dNSAF) Gaussian data
a P p-value of the t-test where the null hypothesis states that there was no change in expression between
control and transgenic GFAP overexpressor b SC percentage of sequence coverage of the listed protein from
sequenced tryptic peptides c Fold Change positive values are indicative of a fold change for transgenic CSF
negative values are fold changes for control CSF (ie down-regulated in transgenic CSF) uarr indicates the protein
was only detected in transgenic CSF and darr indicates the protein was only detected in control CSF d dSpC
distributive spectral counts which represent the average spectral counts observed per run analysis on the mass
spectrometer and corrected using distributive analysis for peptides shared by more than one protein
98
Table 2 Proteins showing greater than three-fold changes with at least two unique
peptides identified for each protein
Accession Protein SC ()a Fold
Change b
Control
dSpC c
Transgenic
dSpC c
MUG1_MOUSE Murinoglobulin-1 precursor 147 42 155 37
CO4B_MOUSE Complement C4-B 113 54 22 118
PRDX6_MOUSE Peroxiredoxin-6 576 46 14 64
CNTN1_MOUSE Contactin-1 65 darr 41 0
CATB_MOUSE Cathepsin B 263 42 23 97
CAH3_MOUSE Carbonic anhydrase 3 396 76 11 84
APOH_MOUSE Beta-2-glycoprotein 1 128 44 44 1
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
PGK1_MOUSE Phosphoglycerate kinase 1 355 36 17 61
NDKB_MOUSE Nucleoside diphosphate kinase B 408 34 13 44
FHL1_MOUSE
Four and a half LIM domains
protein 1 243 39 13 51
NELL2_MOUSE
Protein kinase C-binding protein
NELL2 45 -43 13 03
MDHM_MOUSE
Malate dehydrogenase
mitochondrial 385 41 12 49
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
a SC percentage of sequence coverage of the listed protein from sequenced tryptic peptides b Fold
Change positive values are indicative of a fold change for transgenic CSF negative values are fold changes for
control CSF and darr indicates the protein was only detected in control CSF c dSpC distributive spectral counts
which represent the average spectral counts observed per run analysis on the mass spectrometer and corrected using
distributive analysis for peptides shared by more than one protein
99
Table 3 Validation of changes in proteins revealed by MS-based spectral counting
consistent with previously published microarray data
Consistent changes in RNA and proteomic data
uarr regulated in transgenic darr regulated in transgenic
Cathepsin S Contactin-1
Cathepsin B Carboxypeptidase E
Cathepsin L1
Peroxiredoxin-6
Complement C4-B
Glial fibrillary acidic protein
Serine protease inhibitor A3N
Note Validation of putative biomarkers from the current proteomics dataset by previously
published RNA microarray data55
Both up and down regulated proteins were consistent with the
RNA microarray data
_
100
___________________________________________
SUPPLEMENTAL INFORMATION (Available upon request)
Table S1 Compilation list of proteins identified from all the control and transgenic biological
replicates
Table S2 Distributive spectral counting calculations performed for proteins observed to share
identified peptides
Table S3 Proteins with dNSAF spectral counting performed including t-test analysis for a
comparison between transgenic and control CSF
101
FIGURE LEGENDS
Figure 1 The general workflow indicating the major steps involved in sample collection sample
processing mass spectrometric data acquisition and analysis of mouse CSF samples
Figure 2 Assessment of run to run variability of the base peak chromatograms within and
between two biological and technical replicates The peak profile and intensity scale is
consistent between the four chromatograms The four panels show two biological replicates (Tg
4 and Tg5) with two technical replicates for each biological sample
Figure 3 A Venn-diagram of the biological triplicates (1 2 and 3) of transgenic mouse
CSF showing 78 proteins identified in all three experiments B Venn-diagram of the biological
triplicates (1 2 and 3) of control mouse CSF having 61 proteins identified by all three
replicates C The overlap between control and transgenic CSF proteomic analysis showing 139
proteins identified by both groups and 73 and 54 uniquely identified by respective groups
Figure 4 Assessment of technical replicate variability between biological replicates The error
bars in both A and B are the standard deviation derived from the technical triplicates for each
biological replicate Panel A shows creatine kinase M having more or equal variability in the
biological triplicates than each technical triplicate The means of the biological triplicates are
illustrated by the dashed line Panel B represents kininogen-1 which is unchanged between
control (Ctl) and transgenic (Tg) samples The biological variability coupled with the technical
replicates provides a barely noticeable difference in the pooled mean between control and
102
transgenic spectral counts The difference in means is contrasted with the three fold change
observed from creatine kinase M (A)
Figure 5 MSMS profile of the tryptic peptide LSVEALNSLTGEFK from creatine kinase M
(A) The top MSMS is from a control sample with an elution at 463 minutes (B) The bottom
MSMS is from the GFAP transgenic sample with an elution at 46 minutes These tandem MS
spectra show instrument reliability and consistent fragmentation patterns which are necessary for
spectral counting analysis
Figure 6 Validation of MS spectral counts using a GFAP ELISA GFAP content (ngL)
measured within mouse CSF from both transgenic and control animals The data represents the
average with standard deviation (n=5 each sample represents pooling of 1 to 8 animals) The
statistics are performed using a student t-test plt00001
103
Figure 1
104
Figure 2
105
Figure3
106
Figure 4
107
Figure 5
108
Figure 6
Ctl Tg
100
1000
10000
100000
Mouse CSF Sample
GF
AP
(n
gL
)
109
Table of Contents Summary
Cerebrospinal fluid (CSF) was obtained from transgenic mouse models of Alexander disease as
well as healthy controls We immunodepleted pooled CSF from mice by a modified IgY-14
protocol Shotgun proteomics utilizing trypsin digestion 1-D nanoRP separation CID tandem
mass spectrometry analysis Mascot database searching and relative quantitation via distributive
normalized spectral abundance factor resulted in the identification of 266 proteins and 27
putative biomarkers
110
Chapter 4
Genomic and proteomic profiling of rat adapted scrapie
Adapted from ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A
Cunningham R Wang J Wellner D Ma D Li L Aiken J In preparation
111
Abstract
A novel model of prion disease using rats called Rat Adapted Scrapie (RAS) was
developed with the genomics from brain and proteomics from cerebrospinal fluid (CSF) profiled
The CSF proteome from control and RAS (biological N=5 technical replicates N=3) were
digested and separated using one dimensional reversed-phase nanoLC coupled to data-
dependent tandem MS acquisition on an ion trap mass spectrometer In total 512 proteins 167
non-redundant protein groups and 1032 unique peptides were identified with a 1 false
discovery rate (FDR) Of the proteins identified 21 were statistically up-regulated (plt005) and
7 statistically down-regulated in the RAS CSF We also identified 1048 genes which were
differentially regulated in rat prion disease and upon mapping these changes to mouse gene
expression however only 22 of these genes were in common with mRNAs responding to
prion infection in mice suggesting that the molecular pathology observed in mice may not be
applicable to other species The proteins are compared to the differentially regulated genes as
well as to previously published proteins showing changes consistent with other prion animal
models
112
Introduction
Prion diseases are an unusual class of fatal transmissible neurodegenerative disorders
that affect the mammalian central nervous system They are caused by the accumulation of an
abnormal conformation of the normal host encoded cellular prion protein PrPC This
conformational rearrangement of PrPC is brought about by template directed misfolding wherein
seed molecules of the abnormal isoform PrPScrapie
PrPSc
convert PrPC into new PrP
Sc molecules
Scrapie in sheep and goats is the prototypic prion disease and is so named because clinically
affected animals scrape their wool off on pen and stall walls Laboratory investigation of prion
diseases typically relies upon rodents which can be infected with natural isolates of scrapie1
albeit with some difficulty as ovid scrapie isolates need time to adapt to rodents This adaptation
is characteristic of prion disease interspecies transmissions and properly reflects the molecular
adaptation that must occur to allow interaction between exogenous foreign PrPSc
and host PrPC
molecules selecting for conformers which exhibit template directed misfolding In some cases
no conformational solution is found reflecting a species barrier to disease transmission
In recent years advances in genomics and proteomics technologies have allowed
unprecedented examination of the biomolecules that are altered upon exposure to prion agents
These studies2 3
have relied upon analysis of gene and protein expression changes in response to
prion infection with the aim of trying to identify pathways that might underlie the mechanism of
prion-induced neurotoxicity A second important aim has been to identify signature molecules
that might act as surrogate biomarkers for these diseases as there are significant analytical
challenges associated with sensitively detecting and specifically distinguishing disease-induced
conformational changes (PrPSc
) of the prion protein from normal host conformations (PrPC)
113
Mass spectrometry (MS)-based proteomics has become a promising tool for biomarker
discovery from biological fluids such as CSF blood and urine4-6
Two-dimensional gel
electrophoresis MS (2D-GE MS) and two-dimensional differential gel electrophoresis (2D-DIGE
MS) are the most widely used methods for proteomic profiling of prion infected CSF so far due
to the advantage of ready separation and quantification of proteins in complex biological samples
Studies using 2D-GE MS or 2D-DIGE MS to profile proteins in CSF have led to the
identifications of currently accepted biomarker for sCJD 14-3-3 protein and other potential
biomarkers for prion diseases7-9
However the application of this method in biomarker
discovery is limited by insufficient sensitivity and potential bias against certain classes of
proteins as gel-based separation does not work well for the low abundance proteins very basic
or acidic proteins very small or large proteins and hydrophobic proteins 10 11
In contrast to 2D-
GE or 2D-DIGE shotgun proteomics employs enzymatic digestion of the protein samples
followed by chromatographic separation prior to introduction into a mass spectrometer for
tandem MS analyses Shotgun proteomics has become increasingly popular in proteomic
research because these methods are reproducible highly automated and have a greater
likelihood of detecting low abundance proteins12 13
Due to the sample complexity in CSF and
because albumin comprises over half of the protein content in CSF removal of high-abundance
proteins including albumin is necessary to improve proteomic coverage and identify low-
abundance proteins One method is IgY immunodepletion14 15
which is performed prior to LC-
MSMS and uses species specific antibodies from chicken yolk to remove abundant proteins in
biological samples such as CSF In the present work CSF from control and rat adapted scrapie
animal models (biological N=5 technical N=3 replicates) were analyzed by LC-MSMS and we
114
indentified 512 proteins and 167 non-redundant protein isoforms (referred to as protein groups)
with 21 proteins being statistically up-regulated (p lt 005) and 7 statistically down-regulated
By and large this work has been performed using laboratory mice for the gene
expression studies and human cerebrospinal fluid for proteomics mice do not provide sufficient
volumes of CSF for proteomic studies Both approaches are exceedingly valuable as the mouse
model allows cross-sectional time course experiments to be performed including the important
pre-clinical phase of disease Critically however the relevance and generalizability of mouse
prion responses to other prion diseases especially human disease is unknown Human proteomic
studies on CSF guarantee relevant protein identifications but are limited to the clinical phase of
the disease when apparent markers may reflect gross neurodegeneration covering up subtle but
more specific responses To address these issues we have adapted mouse RML prions into rats
with the aim of expanding the knowledge of prion disease responses addressing the limitations
of mice and opening up the possibility of preclinical proteomic profiling in a laboratory rodent
In the present work CSF samples from control and rat adapted scrapie were analyzed by system
biology approaches Here we show that Rat Adapted Scrapie (RAS) is a powerful tool for an -
omics based approach to decipher the molecular impact of prion disease in vivo with
applicability to the molecular mechanisms of disease and biomarker discovery We identified
1048 genes which were differentially regulated in rat prion disease Astonishingly on the whole
mouse orthologs of these genes did not change in mouse adapted scrapie and vice versa
questioning the universality of previous mouse gene expression profiles These RAS gene
expression changes were identified in the CSF proteome where we detected 512 proteins and 167
protein groups with 21 proteins being statistically up-regulated (plt005) and 7 statistically down-
115
regulated in the CSF of prion diseased rats Many of the proteins detected have previously been
observed in human CSF from CJD patients
Materials and Methods
Ethics Statement
This study was carried out in accordance with the recommendations in the NIH Guide for Care
and Use of Laboratory Animals and the guidelines of the Canadian Council on Animal Care The
protocols used were approved by the Institutional Animal Care and Use Committees at the
University of Wisconsin and University of Alberta
Chemicals
Acetonitrile (HPLC grade) and ammonium bicarbonate (certified) were purchased from
Fisher Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) Optima LCMS grade acetonitrile and water were purchased
from Fisher Scientific (Fair Lawn NJ) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris
ge999 ) ammonium formate (ge99995) glycine (ge985) and IgY-7 antibodies were
purchased from Sigma-Aldrich (Saint Louis MO)
Rat Transmission and Adaptation
Prion agents used were the rocky mountain lab RML strain of mouse adapted scrapie
Stetsonville transmissible mink encephalopathy16
(TME) Hyper (Hy) strain of Hamster TME 17
1st passage Skunk adapted TME prepared as described and C from genetically defined
transmissions18
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Brains from animals clinically affected with prion disease were aseptically removed and
prepared as 10 wv homogenates in sterile water 50 microL of 10 brain homogenate was
inoculated into weanling wistar rats intracranially After one year of incubation preclinical rats
from RML infections were euthanized by CO2 inhalation and the brain excised homogenized
and re-inoculated into naive animals Subsequent serial passages were from rats clinically
affected with rat adapted scrapie
Brains from rat passages were aseptically removed and bisected sagittally Brain halves
were reserved for immunohistochemistry (IHC) in formalin or frozen for immunoblotting RNA
isolation or subsequent passage For IHC tissues were first fixed in 10 buffered formalin
followed by antigen retrieval at 121 oC 21 bar in 10mM citrate buffer for 2 min After cooling
to room temperature sections were treated with 88 formic acid for 30 min and 4M guanidine
thiocyanate for 2 h Endogenous peroxidases were inactivated by 003 hydrogen peroxide and
tissue was blocked with 1 normal mouse serum Mouse anti-PrP mAB SAF83 (Cayman
Chemical) was biotinylated and applied at a 1250 dilution in blocking buffer overnight at 4 oC
Washes were performed in 10mM PBS with 005 tween-20 Streptavidin-peroxidase
(Invitrogen) was added for Diaminobenzidine (DAB) color reaction Anti-GFAP
immunohistochemistry was performed as above except that formic acid and guanidine treatment
steps were excluded Mouse anti-GFAP mab G-A-5 (Sigma) was used at a 1400 dilution
Brain samples for immunoblot were treated with or without Proteinase K (Roche) at a
ratio of 35 microg PK 100 ug protein at 50 microg mL for 30 minutes at 37 oC Phosphotungstenic acid
enrichments were performed as described14 19
Bis-Tris SDS-PAGE was performed on 12
polyacrylamide gels (Invitrogen) and transferred to PVDF Immunoblotting was performed using
117
mABs SAF83 (Cayman Chemical) 6H4 (Prionics) or 3F10 (a kind gift from Yong-Sun Kim) all
at a 120000 dilution
Gene Expression Profiling
RNA was extracted from frozen brain halves from clinically affected and control animals with
the QIAshredder and RNeasy mini kit (Qiagen Valencia CA) in accordance with the
manufacturerrsquos recommendations Due to the relatively large mass of rat brain an initial
homogenization was performed with a needle and syringe in 5mL of buffer RLT before further
diluting an aliquot in accordance with the manufacturers protocol Total RNA was amplified and
labeled in preparation for chemical fragmentation and hybridization with the MessageAmp
Premier RNA amplification and labeling kit (Life Technologies Grand Island NY) Amplified
and labeled cRNAs were hybridized on Affymetrix (Santa Clara CA) rat genome 230 20 high
density oligonucleotide arrays in accordance with the manufacturers recommendations
Differentially Expressed Genes were identified using Arraystar 50 (DNAStar Madison WI)
Robust multi-array normalization using the quantile approach was used to normalize all
microarray data A moderated T-test with a multiple comparison adjustment20
was used to reduce
the false discovery rate yet preserve a meaningful number of genes for pathway analysis
Pathway analysis was performed using the DAVID Bioinformatics database21
Comparative
analysis of genes induced by prions in mouse22
and rat disease was performed on genes
exhibiting at least a 2-fold change in expression Orthologs of rat and mouse genes were
identified using ENSEMBLE biomart release 6823
CSF Proteomic Profiling
118
CSF was collected from rats anaesthetized with isoflorane via puncture of the cisterna
magna A volume of 100-200 microL was routinely obtained CSF was touch spun for 30s at 2000xg
on a benchtop nano centrifuge to identify any blood contamination by the presence of a red
pellet CSF with blood contamination was not used for proteomic analysis CSF was prepared
for profiling by first depleting abundant proteins with an antibody based immunopartitioning
column IgY-R7 (Sigma-Aldrich Saint Louis MO) The manufacturers recommendations were
followed with a few deviations 40 microg of protein (~100 microL CSF) was bound to 100 microL of IgY
bead slurry in total volume of 600 microL formulated in 150mM ammonium bicarbonate The flow
through was collected and denatured by the addition of 10 microL of 8 M guanidine thiocyanate and
lyophilized in a speed-vac apparatus The sample was then reconstituted in 10 microL of water and 1
microL of 050 M DTT were added and allowed to incubate at 37oC for 30 minutes After incubation
27 microL of 055 M IAA was added for carboxymethylation and the sample was allowed to
incubate in the dark for 15 minutes After that 1 microL of 050 M DTT was added and allowed to
sit at room temperature for 10 minutes To perform trypsin digestion 70 microL of 50 mM
NH4HCO3 was then added along with 025 microg trypsin Digestion was performed overnight at
37oC and quenched by addition of 5 microL of 10 formic acid The tryptic peptides were then
subjected to solid phase extraction using an Agilent OMIX Tip C18 100 microL (Santa Clara CA)
Peptides were eluted with 50 ACN in 01 formic acid concentrated and reconstituted in 30
microL H2O with 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of a nanoAcquity (Milford MA) with a 10 microL injection
loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B consisted of
ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto a Waters 5 microm
119
Symmetry C18 180 microm x 20 mm trap cartridge (Milford MA) at a flow rate of 75 microLmin for 5
minutes at 95 A 5 B Separation was performed on a Waters 17 microm BEH130 C18 75 microm x
100 mm (Milford MA) with a gradient of 5 to 15 B over 10 minutes followed by 15 to
40 B over 80 minutes at room temperature
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with a Bruker CaptiveSpray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Waters Acquity console software to perform MS acquisitions for all experiments Smart
parameter setting (SPS) was set to 700 mz compound stability and trap drive level was set at
100 Optimization of the CaptiveSpray source resulted in dry gas temperature of 160oC dry
gas flow of 30 Lmin capillary voltage of -1325 V end plate offset of -500 V MSMS
fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
range of 350-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
120
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot24
(Version 24 Matrix
Science London UK) Database searching was performed against a forward and reversed
concatenated SwissProt Rattus rattus Mascot search parameters were as follows Allowed
missed cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) peptide tolerance plusmn12 Da maximum number of 13
C 1 MSMS
tolerance plusmn05 Da instrument type ESI-Trap Data was then transformed into Dat file formats
and transferred to ProteoIQ (NuSep Bogart GA) False positive analyses were calculated using
ProteoIQ and set at 1
Results
Development of Rat Adapted Scrapie
To develop a rat adapted strain of prion disease we introduced 6 different prion agents RML
TME Hy Skunk Adapted TME and Chronic Wasting Disease (CWD) from wild type 96G and
96S deer16-18
into 6 rats (Fig 1) Of these primary transmissions only RML induced the
accumulation of Proteinase K resistant PrP after one year of incubation as determined by western
blotting on 10 brain homogenates and PrPSc
enriched phoshotungstenic acid precipitated brain
homogenates Second passage of rat adapted RML scrapie resulted in clinically affected rats at
565 days post inoculation Serial passages of TME or CWD agents were not performed Clinical
symptoms consisted of ataxia lethargy wasting kyphosis and myoclonus Prion affected rats
121
also showed low level porphyrin staining around their head Subsequent serial passage decreased
incubation time to 215 days
Proteinase K resistant prion protein was observed from all clinically affected animals both by
immunoblot (Fig 2) and by immunohistochemistry (Fig 3) Di- and mono-glycosylated bands
were the most abundant isoforms of PrPSc
PrPSc
was extensively deposited in the cerebral cortex
hippocampus thalamus inferior colliculus and granular layer of the cerebellum GFAP
expressing activated astrocytes were found throughout the brain particularly in the white matter
of the hippocampus thalamus and cerebellum Spongiform change was a dominant feature of
clinical rat
Gene expression Profiling
In total 1048 genes were differentially regulated within a 95 confidence interval
(Supplementary Table 1) and 305 genes were differentially expressed by greater than 2-fold (Fig
4) The 1048 genes that were statistically significant were used for pathway analysis using
DAVID Pathway analysis suggested that the gene expression profile was consistent with
immune activation and maturation as well as inflammation (Supplementary Table 2) a likely
interpretation given the observable astrocytosis (Fig 3) and gliosis associated with prion disease
Other pathways highlighted by the analysis included increases in transcription of genes involved
in lysosomes and endosomes
To further probe the gene expression data we compared genes which were differentially
expressed in rat adapted scrapie against their orthologs expression in mouse scrapie and vice
versa (Figs 5 and 6) We did not observe a high degree of correlation between expression fold
changes For example GFAP a gene whose up-regulation in prion disease is well known was
122
increased 25 fold in rat adapted scrapie but up-regulated 93 fold in mouse scrapie A
qualitative analysis of expression of orthologs in prion disease suggests that many genes
deregulated at least 2-fold in mouse or rat do not have orthologs that are differentially expressed
For example the gene RNAseT2 was up-regulated greater than threefold in rat adapted scrapie
but was not significantly up-regulated in mouse Similarly three genes important in metals
homeostatsis metallothionein 1a and 2a and ceruloplasmin were up-regulated in rat 46 43 and
3 fold respectively but were not differentially expressed in mouse prion disease
CSF Proteomics
Each immunodepleted biological replicate (N=5 for each control and RAS) had technical
triplicates performed The triplicates were summed together using ProteoIQ Peptide spectral
counts were then summed for each protein and subjected to dNSAF analysis using ProteoIQ
internal algorithms Details for this method can be found elsewhere25 26
but briefly peptide
spectral counts are summed per protein (SpC) based on unique peptides and a weighted
distribution of any shared peptides with homologous proteins T-tests were used to identify
significant changes in protein expression 1032 unique peptides which identify 512 proteins and
167 protein groups were found Of these 512 proteins 437 were identified in both RAS and
control CSF samples (Fig 7) The complete list of all 512 proteins can be viewed in
Supplemental Table 3 Using the ProteoIQ the most likely isoform was selected such as 14-3-3
protein gamma
From Table 1 we observe five proteins that agree with the genomic data for up
regulations in RAS granulin macrophage colony-stimulating factor 1 receptor cathepsin D
complement factor H and ribonuclease T2 In addition serine protease inhibitor A3N was not
123
detected as up regulated in the RAS genomic data but was found to be up-regulated in previous
genomic profiling of the mouse prion model22
One interesting trend from the data in Table 1 is
that the majority of proteins found to be up-regulated in the RAS model were not detected in the
control samples The absence of the detection of those proteins such as ribonuclease T2 in the
control CSF does not necessarily suggest the absence of the protein nonetheless it is below the
detection limits for this current proteomics protocol and instrumentation
Discussion
Mice have been the preferred laboratory rodent for prion diseases research because they
can be inexpensively housed and are amenable to transgenesis which allows for short incubation
periods as well as hypothesis testing upon targeted pathways Pursuant to the determination of
the mouse genome and the development of high density transcriptional arrays for measurements
of gene expression profiling mice have been used extensively to examine the molecular
pathology of prion disease probing the impact of disease and animal strain In order to expand
upon this foundation we adapted mouse prions to infect rats We reasoned that by taking a
comparative approach to the molecular pathology of prion disease inferences could be obtained
into the variability of the molecular response to prion diseases and that understanding this
variability might suggest whether human prion disease responses are more or less similar to
mouse responses A second rationale is the desire to identify surrogate markers of prion disease
While this approach has been taken before using gene expression profiling a more direct
approach has been to use proteomic approaches on cerebral spinal fluid (CSF) identifying
proteins that are increase in abundance with disease A rat prion disease is valuable for this
because the rat proteome is established and rats allow for the collection of relatively large
volumes of CSF (~100 microL per animal) which allows for robust analytical separations enhancing
124
detection of biomarkers Finally rats unlike humans can be used in a time course study of prion
disease This allows for the identification of early transcriptional and proteomic responses to
prion infection responses which are particularly valuable for the identification of surrogate
disease biomarkers
To initiate the development of a rat prion disease we attempted to adapt six different
prion disease agents PrPres
molecules to rat via intracranial inoculation of weanling animals
(Figure 1) Of these six agents only mouse RML prions were able to surmount the species
barrier to prion disease transmission Mouse PrP differs from rat by eight amino acid changes
six in the mature protein and four in the N-terminal octa-peptide repeat region (Supplementary
Fig 1) As rat shares the most prion protein sequence homology with mouse it is perhaps not
surprising that it transmitted whereas the other did not confirming that the primary prion protein
sequence is the most important determinant for interspecies transmission We conclude that there
is a large molecular species barrier preventing conversion of rat PrPc into PrP
res
The transmission of mouse RML into rats was characterized by a shortening of the
incubation period following each passage This is indicative of agent adaption to the new host
and increases in the titer present in end-stage brain Overall our adaptation of mouse prion
disease into rats resulted in a similar agent to that observed by Kimberlin27
The differences in
incubation period at second passage are largely due to our collecting the animals at 365 days post
inoculation upon first passage whereas Kimberlin and Walker allowed the first passage animals
to reach end-stage clinical rats
Rat adapted scrapie is in many ways similar to other prion diseases The clinical period of
disease is marked by a progressive neurological dysfunction with prominent ataxia kyphosis and
125
wasting The protease resistant PrP observed is typical as is the widespread deposition of PrPSc
in
the brain Spongiosis and reactive astrogliosis are as expected of a prion disease
Gene expression profiles from rats clinically affected with prion disease revealed a strong
neuronal inflammation associated with proliferated and activated astrocytes This is perhaps best
observed through the up-regulation of GFAP GFAP positive astrocytes were omnipresent
throughout the brain and GFAP mRNA was up-regulated 25 fold The up-regulation of GFAP is
a hallmark of the molecular response to prion infection and has been routinely observed Our
comparative analysis of gene expression changes in mouse RML versus rat adapted scrapie
suggest substantial differences in gene expression in response to prion disease despite the fact
that the overall response is neuro-inflammatory This suggests that the potential overlap between
mouse expression profiles and a putative human CJD expression profile could be quite different
at the level of individual transcripts that might be expected to be changed
CSF Proteomics
CSF proteomics can be exceedingly challenging due to the small sample available large
dynamic range in proteins and abundant proteins limiting sample loading onto nanoscale
columns Dynamic range reduction in the CSF sample was achieved using a custom amount of
IgY-7 antibodies to abundant proteins such as albumin After immunodepletion the total
protein content was reduced by ~90 limiting the proteomics analysis to one dimensional
separation Label free quantitation spectral counting was performed because it requires less
protein and does not increase sample complexity The proteins identified from the affected and
control N=5 are shown in Supplemental Table 2 Consistent detection of proteins in CSF from
both control and infected rats was observed (Fig 7C) Only two proteins were identified in
126
controls that were not observed in RAS and only 10 proteins were only observed in RAS Some
of these proteins that were only identified in RAS are significantly changed (Supplemental Table
3) One concern in proteomics data is the variability from run to run and the possibility that
certain proteins are identified from different unique peptides Figure 7A shows that the vast
majority of the unique peptides detected (783) were identified with a 1 FDR in both RAS and
control CSF samples highlighting the analytical reproducibility of our methodology
Proteomic analysis of the infected rat CSF provides a reasonable approach to cross
validate biomarkers detected by gene expression profiling For example RNAseT2 a secreted
ribonuclease was up-regulated 3-fold at the mRNA level and was also enriched in CSF from
infected rats (Table 1) Similarly coordinated increases in detection of colony stimulating factor
1 receptor complement factor H granulin and cathepsin D were also observed Conversely
proteomic analysis of CSF also allows for the observation of post-transcriptional responses to
prion disease that might be absent in gene expression profiling The 14-3-3 proteins and neuron
specific enolase both known markers for CJD are only detected by proteomic analysis Thus
gene expression profiling and proteomic detection serve to increase confidence in the
observation of up-regulation enhancing the likelihood that proteins detected by both
methodologies are specific and perhaps may be more sensitive at earlier time points
Comparison to human CSF prion disease proteome
In our comparative analysis 21 proteins were found to be up-regulated while 7 proteins
down-regulated in prion infected rats Included in the up-regulated proteins were the 14-3-3
proteins epsilon and gamma 14-3-3 zetadelta was also up-regulated but not statistically
significant Enolase 2 or neuron specific enolase (NSE) was also up-regulated in CSF of infected
127
rats These proteins are all in agreement with results from previous proteomic profiling of human
CSF from patients with CJD8 9
The detection of 14-3-3 protein is included in the diagnostic
criteria approved by World Health Organization for the pre-mortem diagnosis of clinically
suspected cases of sCJD28
although its application in large-scale screening of CJD is still
debated due to high false positive rate where elevated 14-3-3 protein levels were also reported in
other conditions associated with acute neuronal damage29 30
It was suggested that other brain-
derived proteins that are also specific to CJD can be used in conjunction with 14-3-3 protein to
increase diagnosis accuracy and specificity31
NSE is present in high concentration in neurons
and in central and peripheral neuroendocrine cells NSE serves as another valuable biomarker in
diagnosis of CJD as the elevated level of NSE in CSF was found in early and middle stages of
CJD 32
Other proteins detected in CSF included cystatin C and serpina3N although both of
these were not statistically changed These proteins were both previously identified as being
putative biomarkers for CJD33 34
Utility for biomarker discovery with emphasis on onset of biomarker appearance in CSF
The investigation of the protein changes in CSF from RAS compared to control rats
provides a solid foundation when investigating potential biomarkers with prion disease onset
The cross-validation of the genomic and proteomics data further emphasizes the targets for
consideration during disease onset Biomarker discovery provides the potential to determine if
animals such as cows in bovine spongiform encephalopathy (BSE) are affected instead of
having to be euthanized when BSE is expected In addition CSF collection in mice or hamsters
Prion models is extremely difficult and limited alternatively with the advent of the RAS model
CSF can be collected in sufficient amounts for CSF proteomics CSF collection for mice or
hamsters can be ~10 microL and could be contaminated with blood further complicating proteomic
128
analysis unlike rats which over 10 times more CSF can be collected per animal35
Due to the
amount of CSF available time course studies analyzing CSF proteomics is possible in RAS due
to animal numbers that are manageable and reasonable The RAS model further allows
investigators to bypass working with highly infections CJD CSF samples to investigate the CSF
proteome changes
Conclusion
In this study we have described the gene and protein expression changes in brain and
spinal fluid from a transmission of mouse prions into rats We find that while the overall gene
expression profile in rats is similar to that in mice the specific genes that make up that profile
are different suggesting that genes that change in response to prion disease in different species
may not be as conserved Analysis of CSF from rat adapted scrapie identified similar protein
changes as known in human CJD The rat will be a useful model to identify surrogate markers
that appear prior to the onset of clinical disease and thus may be of higher specificity and
sensitivity
Supplemental Information Available Upon Request
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7 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66 8 Harrington M G Merril C R Asher D M Gajdusek D C Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease N Engl J Med 1986 315 (5) 279-83 9 Piubelli C Fiorini M Zanusso G Milli A Fasoli E Monaco S Righetti P G Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping Proteomics 2006 6 Suppl 1 S256-61 10 Lilley K S Razzaq A Dupree P Two-dimensional gel electrophoresis recent advances in sample preparation detection and quantitation Curr Opin Chem Biol 2002 6 (1) 46-50 11 Oh-Ishi M Maeda T Separation techniques for high-molecular-mass proteins J Chromatogr B Analyt Technol Biomed Life Sci 2002 771 (1-2) 49-66 12 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset J Proteome Res 2008 7 (2) 698-707 13 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422 (6928) 198-207 14 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample preparation and analysis Proteomics 2005 5 (13) 3314-28 15 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep Biochem Biotechnol 2009 39 (3) 221-47 16 Marsh R F Bessen R A Lehmann S Hartsough G R Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy J Gen Virol 1991 72 ( Pt 3) 589-94 17 Bessen R A Marsh R F Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent J Virol 1992 66 (4) 2096-101 18 Johnson C J Herbst A Duque-Velasquez C Vanderloo J P Bochsler P Chappell R McKenzie D Prion protein polymorphisms affect chronic wasting disease progression PLoS One 2011 6 (3) e17450 19 Safar J Wille H Itri V Groth D Serban H Torchia M Cohen F E Prusiner S B Eight prion strains have PrP(Sc) molecules with different conformations Nat Med 1998 4 (10) 1157-65 20 Benjamini Y Hochberg Y Controlling the False Discovery Rate A Practical and Powerful Approach to Multiple Testing Journal of the Royal Statistical Society Series B (Methodological) 1995 57 (1) 289-300 21 Huang da W Sherman B T Lempicki R A Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat Protoc 2009 4 (1) 44-57 22 Moody L R Herbst A J Aiken J M Upregulation of interferon-gamma-induced genes during prion infection J Toxicol Environ Health A 2009 74 (2-4) 146-53 23 Flicek P Amode M R Barrell D Beal K Brent S Carvalho-Silva D Clapham P Coates G Fairley S Fitzgerald S Gil L Gordon L Hendrix M Hourlier T Johnson N Kahari A K Keefe D Keenan S Kinsella R Komorowska M Koscielny G Kulesha E Larsson P Longden I McLaren W Muffato M Overduin B Pignatelli M Pritchard B Riat H S Ritchie G R Ruffier M Schuster M Sobral D Tang Y A Taylor K Trevanion S Vandrovcova J White S Wilson M Wilder S P Aken B L Birney E Cunningham F Dunham I Durbin R Fernandez-Suarez X M Harrow J Herrero J
130
Hubbard T J Parker A Proctor G Spudich G Vogel J Yates A Zadissa A Searle S M Ensembl 2012 Nucleic Acids Res 2012 40 (Database issue) D84-90 24 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 1999 20 (18) 3551-67 25 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J Proteome Res 2006 5 (9) 2339-47 26 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome quantitation how to deal with peptides shared by multiple proteins Anal Chem 2010 82 (6) 2272-81 27 Kimberlin R H Cole S Walker C A Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters J Gen Virol 1987 68 ( Pt 7) 1875-81 28 World Health Organization Global surveillance diagnosis and therapy of human transmissible spongiform encephalopathies report from a WHO consultation 1998 World Health Organization Geneva Switzerland 9-11 February 1998 1-26 29 Bartosik-Psujek H Archelos J J Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG J Neurol 2004 251 (4) 414-20 30 Saiz A Graus F Dalmau J Pifarre A Marin C Tolosa E Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders Ann Neurol 1999 46 (5) 774-7 31 Sanchez-Juan P Green A Ladogana A Cuadrado-Corrales N Saanchez-Valle R Mitrovaa E Stoeck K Sklaviadis T Kulczycki J Hess K Bodemer M Slivarichova D Saiz A Calero M Ingrosso L Knight R Janssens A C van Duijn C M Zerr I CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease Neurology 2006 67 (4) 637-43 32 Zerr I Bodemer M Racker S Grosche S Poser S Kretzschmar H A Weber T Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease Lancet 1995 345 (8965) 1609-10 33 Sanchez J C Guillaume E Lescuyer P Allard L Carrette O Scherl A Burgess J Corthals G L Burkhard P R Hochstrasser D F Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease Proteomics 2004 4 (8) 2229-33 34 Miele G Seeger H Marino D Eberhard R Heikenwalder M Stoeck K Basagni M Knight R Green A Chianini F Wuthrich R P Hock C Zerr I Aguzzi A Urinary alpha1-antichymotrypsin a biomarker of prion infection PLoS One 2008 3 (12) e3870 35 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
131
Figure 1 Interspecies transmission of prion disease to rats End-stage clinical brain homogenates
were used to passage prion disease After one year of incubation animals were euthanized to
determine the extent of PrPres
accumulation Protease resistance PrP was only observed in those
animals infected with RML scrapie prions This material was serially passaged for two more
incubations before becoming rat-adapted as indicated by the shortening of the incubation period
132
Table 1 A list of selected proteins and mRNAs up-regulated in RAS CSF and brain tissue If
the protein is detected in the RAS CSF but not the control CSF the CSF expression is reported
with a infin If there is no change or data on certain genes related to an up regulated protein nd is
noted The mouse genomic data presented here was previously published22
Gene Protein Symbol Accession CSF
Expression
Rat
GEX
Mouse
GEX
14-3-3 protein zetadelta Ywhaz NP_037143 infin nd nd
14-3-3 protein epsilon Ywhae NP_113791 infin nd nd
14-3-3 protein gamma Ywhag NP_062249 infin nd nd
serine protease inhibitor A3N Serpin A3N NP_113719 54 nd 975
enolase 2 (gamma neuronal) Eno2 NP_647541 infin nd nd
granulin GRN NP_058809 62 364 184
macrophage colony-stimulating
factor 1 receptor
Csf1r NP_001025072 infin 293 205
cathepsin D CTSD NP_599161 infin 255 299
complement factor H Cfh NP_569093 376 234 nd
ribonuclease T2 RNAset2 NP_001099680 infin 302 nd
133
Figure 2 Accumulation of PrPSc
in rat adapted scrapie First second and third passage brain
homogenates were assayed for the presence of protease resistant PrP by immunoblot PrPSc
was
observed following phosphotungstenic acid enrichment at first passage By clinical disease in 2nd
and 3rd
passage rats PrPSc
had substantially accumulated
134
Figure 3 Histological appearance of rat adapted scrapie in the hippocampus at clinical disease
Infected animals showed intense immuno-staining for deposits of PrPSc
and GFAP expressing
astrocytes Spongiform change is an abundant feature in rat adapted scrapie
135
Figure 4 Scatter plot of gene expression profiling of rat adapted scrapie The expression of
individual genes from uninfected and infected animals were plotted to display up and down
regulation The dashed green line is no change Solid green lines are 2-fold changes in gene
expression
136
Figure 5 Quantitative relationship between orthologous pairs of two-fold up-regulated genes in
mouse and rat scrapie Genes up-regulated more than two fold were mapped to their orthologs
and the fold change was plotted Expression is log2 transformed
137
Figure 6 Comparison of genes upregulated in rat and mouse prion diseases Genes up-regulated
two fold in rodent scrapie were identified and the expression of their orthologs was determined
138
Figure 7 Venn diagram comparison of the CSF proteomics data between rat adapted scrapie
(RAS) model and control rats (A) Unique peptides from tryptic peptides produced for the
proteins identified (B) The total proteins identified including all isoforms within the protein
groups (C) The protein groups comparing only the top protein hit of the protein isoforms
showing very consistent protein identifications between RAS and control
139
Chapter 5
Investigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiae
Adapted from ldquoInvestigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M
Heideman W Li L In preparation
140
Abstract
This work explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Kinases such as protein
kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose response
Thus performing a large scale phosphoproteomic MS study can be highly beneficial to map the
signaling networks and cascades involved in glucose-dependent stimulated yeast growth Yeast
cell extract was digested and phosphopeptides were enriched by immobilized metal affinity
chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase (RP)-RP
separation The low pH separation was infused directly into an ion trap mass spectrometer with
neutral loss electron-transfer dissociation (ETD)-triggered fragmentation to improve
phosphopeptide identifications In total 477 unique phosphopeptides are identified with 06
false discovery rate (FDR) and 669 unique phosphopeptides identified with 54 FDR This
study includes comparative phosphoproteins for one specific motif of PKA xxxKRKRxSxxxx
which is presented and differences between starved vs glucose fed are highlighted Phosphosite
validation is performed using a localization algorithm Ascore to provide more confident and
site-specific characterization of phosphopeptides
141
Introduction
Microorganisms such as Saccharomyces cerevisiae (yeast) cycle between growth when
nutrients are available and quiescence when nutrients are scarce When glucose is limited yeast
go into growth arrest state but when glucose is added growth quickly resumes Kinases such as
protein kinase A (PKA) and target of rapamycin (TOR) regulate growth in response to nutrient
conditions and have been well studied through transcriptional control1-4
Yeast execute large
transcriptome alterations in response to changing environmental growth conditions5 6
Gene
regulation by glucose introduction in yeast has been studied including genes used for growth on
alternative carbon sources and activation of genes coding for glucose transport and protein
synthesis7-10
Response to nutrients for survival is not limited to yeast biology and indeed all
living creatures both prokaryotes and multicellular eukaryotes like mammals require nutrient
responsiveness and coordinating cellular functions to survive
With regulation of certain genes well studied by glucose introduction the mechanism and
global protein modulation caused by glucose introduction remain unknown6 Large-scale
phosphorylation analysis has been previously performed in yeast using mass spectrometry11-14
Mapping the phosphoproteome in yeast transitioning from quiescence to growth conditions to
better understand the roles of phosphorylation in orchestrating growth is needed The
phosphorylation state during growth or starvation could be used to engineer cells to drive ectopic
activity (or inhibition) to promote growth and ethanol production on non-native sugars like
xylose
It has been reported that the phosphorylation state can be affected by the introduction of
glucose to carbon-starved yeast15
and phosphorylation plays a significant role in the cell cycle
and signal transduction16
Specifically O-Phosphorylation can function as a molecular switch by
142
changing the structure of a protein via alteration of the chemical nature of an amino acid for
serine threonine or tyrosine It has been reported that up to 30 of the proteome can undergo
phophorylation17
Mass spectrometry has evolved as a powerful tool to accomplish phosphosite
mapping using shotgun proteomics With available technology tens of thousands of
phosphorylation sites can be mapped to amino acids on thousands of proteins using shotgun
proteomics18-20
Mass spectrometry can offer sensitive automated non-targeted global analysis of
phosphorylation events in proteomic samples but in any large scale phosphoproteomic
investigation enrichment of phosphoproteinspeptides is required First phosphorylation is
usually a sub-stoichiometric process where only a percentage of all protein copies are
phosphorylated21
Various enrichment methods have been used for phosphopeptide enrichment
including metal oxide affinity chromatography (MOAC)22
such as TiO223
immobilized metal
affinity chromatography (IMAC)12 24 25
electrostatic repulsion-hydrophilic interaction
chromatography (ERLIC)26
and immunoaffinity of tyrosine phosphorylation27 28
After
enrichment MS analyses of phosphorylated peptides are still challenging due to ion suppression
from non-phosphorylated peptides
Even after phosphopeptide enrichment further sample preparation is needed for large
scale proteomic experiments Additional fractionation can increase protein coverage of a
sample by over ten fold such as MudPIT29
(multidimensional protein identification technology)
In online MudPIT a sample is injected onto a strong cation exchange (SCX) column coupled to
a RP column Successive salt bumps followed by low pH gradients provide the separation of
peptides in two dimensions Since the phosphate groups on phosphopeptides have a low pKa
value due to being more acidic then their unmodified counterparts they tend to elute earlier and
143
disproportionally in ion exchange chromatography like SCX Alternatively reversed-phase
reversed-phase (RP-RP) separation has been proposed as an alternative to MudPIT for offline
two dimensional (2D) separation30
One of the caveats of 2D separation is the potential for
wasted mass spectrometry time from early and late fractions having very few peptides present
all while having too much sample for middle fractions One simple method to reduce these
ldquowasted fractionsrdquo is to combine early or late fractions with the middle fractions to reduce MS
runs with little peptide content to analyze thus shortening the overall analysis time31
In addition the labile phosphorylation group has a large propensity to undergo cleavage
during collision induced dissociation (CID) producing a neutral loss The neutral loss can
produce insufficient backbone fragment ions for MSMS identification32
A solution to neutral
loss fragmentation in phosphopeptides is MS3 using CID to produce improved backbone
fragmentation13 14 33
An alternative fragmentation method to CID for fast sampling ion traps is
electron transfer dissociation (ETD)34-36
ETD produces a more uniform back-bone cleavage
where the cation peptide receives an electron from a low affinity radical anion37
The transferred
electron induces a dissociation at the N-Cα bond to produce c- and zbull-type product ions while
retaining the labile post-translational modifications (PTM) such as phosphorylation bound to the
product ions38
The Bruker amaZon ETD ion trap mass spectrometer has the potential to trigger
ETD fragmentation after a labile PTM produces a neutral loss from CID fragmentation This
method is termed neutral loss-triggered ETD fragmentation and provides a complementary
fragmentation pathway to labile poor fragmenting phosphorylated peptides
In this work we provide a qualitative comparative list of yeast phosphopeptides observed
in glucose fed vs glucose starved conditions
144
Experimental
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) ammonium hydroxide (ACS plus certified) urea (gt99)
sodium chloride (997) ammonium bicarbonate (certified) Optima LCMS grade acetonitrile
Optima LCMS grade water and EDTA disodium salt (99) were purchased from Fisher
Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) ammonium formate (ge99995) Trizma
hydrochloride (Tris HCl ge990) Triton X-100 (laboratory grade) and iron (III) chloride
hexahydrate (97) were purchased from Sigma-Aldrich (Saint Louis MO) Sodium dodecyl
sulfate (SDS) (998) was purchased from US Biological (Marblehead MA) Nickel
nitrilotriacetic acid (Ni-NTA) magnetic agarose beads were purchased from Qiagen (Valencia
CA) Autoclaved yeast peptone dextrose (YPD) media was prepared in 1 L of deionized water
using 10 g yeast extract 20 g Peptone from Becton Dickinson and Company (Sparks MD) and
20 g D-(+)-Glucose (ge995) purchased from Sigma-Aldrich (Saint Louis MO)
Modified Mary Miller Yeast Protein Isolation
The yeast culture was prepared and protein extraction was performed using a modified
Mary Miller protocol39
Briefly yeast strain s288c was inoculated with YPD media and shook
for 72 hours to allow the yeast to be glucose starved After the 72 hours the yeast culture was
partitioned into two flasks To one flask glucose was added at 2 of the final concentration and
allowed to incubate and shake for 10 minutes Then 100 optical density units (ODU) of the yeast
145
culture were collected from each flask Each yeast sample was spun down in a Beckman-Coulter
J6B centrifuge (Indianapolis IN) at 2500xg for three minutes The media was aspirated and the
tubes were flash frozen in liquid nitrogen and stored at -80oC The yeast pellets were thawed on
ice and resuspended in 1 mL of lysis buffer (50 mM Tris HCl 01 triton x-100 05 SDS
pH=80) and 1X protease and phosphatase inhibitor cocktail from Thermo Scientific (Rockford
IL) and transferred to an Eppendorf tube To the yeast 200 microL of glass beads were added and
amalgamated for 25 minutes at 4oC The sample was inverted and the Eppendorf tube was
pierced with a hot 23 gauge needle through the bottom and the tube was placed atop a 5 mL
culture tube and centrifuge in the Beckman-Coulter J6B at 2500xg for three minutes at 4oC to
collect the liquid containing the yeast cells while the glass beads remain trapped in the
Eppendorf tubes The protein lysate was then centrifuged at 3000xg for five minutes at 4oC and
the supernatant was collected and stored at -80oC
Preparation of tryptic digests
The Saccharomyces cerevisiae protein cell extract for fed or starved were analyzed with a
BCA protein assay kit (Thermo Scientific Rockford IL) and 2 mg of protein was added to four
parts -20oC acetone and allowed to precipitate the protein for 1 hour at -20
oC The samples were
then centrifuged at 14000xg for 5 minutes and the supernatant was removed and the protein
pellet was reconstituted in 360 microl of 8 M urea To each sample 20 microL of 050 M DTT was
added and allowed to incubate at 37oC for 45 minutes After incubation 54 microL of 055 M IAA
was added for carbamidomethyl of cysteine and the sample was allowed to incubate for 15
minutes in the dark To quench the IAA 20 microL of 050 M DTT was added and allowed to react
for 10 minutes To perform trypsin digestion 1400 microL of 50 mM NH4HCO3 was then added
along with 20 microg trypsin to each sample Digestion was performed overnight at 37oC and
146
quenched by addition of 25 microL of 10 formic acid For each sample the tryptic peptides were
then subjected to solid phase extraction using a Hypersep C18 100 mg solid phase extraction
(SPE) column (Thermo Scientific Bellefonte PA) Peptides were eluted with 50 ACN in
01 formic acid concentrated and reconstituted in 500 microL of 80 ACN and 01 formic acid
Phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC)
One ml of Ni-NTA was washed three times with 800 microL of H2O and then the Ni was
removed with 800 microL of 100 mM EDTA in 50 mM ammonium bicarbonate and vortexed for 30
minutes The supernatant was removed and the NTA was washed with 800 microL of H2O three
times followed by addition of 800 microL 10 mM FeCl3 hexahydrate and vortexed for 30 minutes
The Fe-NTA was then washed three times with 800 microL of H2O and once with 80 ACN in 01
formic acid before being combined with the cell extract for phosphopeptide enrichment and
vortexed for 40 minutes The sample was washed three times with 200 microL of 80 ACN in 01
formic acid before elution with two aliquots of 200 microL of 5 ammonium hydroxide in 5050
ACNH2O with 15 minutes of vortexing The enriched phosphopeptides were then dried down
with a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA) and reconstituted in 55 microL
25mM ammonium formate pH=75
First dimension neutral pH separation
Individually 50 microL of each yeast phosphopeptide enriched sample was injected onto a
Phenomenex Gemini-NX 3microm C18 110 Aring 150 x 2 mm column (Torrance CA) The Gemini
column was coupled to a Phenomenex SecurityGuard Gemini C18 2 mm guard cartridge
(Torrance CA) Mobile phase A consisted of 25 mM ammonium formate at pH=75 and mobile
phase B consisted of 25 mM ammonium formateACN with a 19 ratio respectively at pH=75
The column was operated at 50oC at a flow rate of 05 mLmin The flow profile was 2-30 B
147
over 65 minutes and then 5 min at 100 B A total of 22 fractions were collected every 3
minutes and the fractions were combined as follows 1 with 12 2 with 13 etc to 11 with 22
The combined fractions were dried down and reconstituted in 25 microL of 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL from each fraction onto an Agilent Technologies
Zorbax 300 SB-C18 5 microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5
microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm Atlantis
dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B at 250
nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) for MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
148
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions An additional mode of MSMS fragmentation electron transfer dissociation
(ETD) was triggered on the precursor ion when a neutral loss was observed in CID
fragmentation from phosphoric acid H3PO4 (Δmz of 49 and 327 for 2+ and 3+ charge states
respectively) or for metaphosphoric acid (HPO3) (Δmz of 40 and 267 for 2+ and 3+ charge
states respectively) For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms rolling average 2 acquisition range of 300-1500 mz
and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target
was set to 300000 maximum accumulation time 5000 ms two spectral averages acquisition
range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required to prevent artificial data
reduction Identification of peptides were performed using Mascot40
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt Saccharomyces
cerevisiae database (version 575) Mascot search parameters were as follows Allowed missed
cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) and phosphorylation (STY) peptide tolerance plusmn12 Da maximum
number of 13
C 1 MSMS tolerance peptide decoy (Mascot) checked plusmn05 Da instrument type
149
ESI-Trap Results from the Mascot search was exported as DAT files for analysis by Scaffold 3
and Scaffold PTM
Scaffold and Ascore data processing
Scaffold 3 (Proteome Software Portland OR)was used for MSMS proteomics data
comparison and false discovery rate analysis The DAT files were loaded into Scaffold 3 and
the fractions for the two dimensional fractionation were combined The resulting biological
triplicates for starved and glucose fed yeast were filtered with a 1 false discovery rate (FDR)
on the protein level (Supplemental Table 1) Due to the inherent poor fragmentation of
phosphopeptides a lenient FDR level may still yield biologically relevant data and thus a 54
FDR was generated for phosphopeptides (Supplemental Table 2) For a more conservative list of
phosphopeptides with a 06 FDR was generated from Scaffold 3 (Supplemental Table 3) FDR
analysis is sufficient at preventing poor data from being reported but does not prevent false
phosphosite identification in phosphopeptides To provide confidence in site identification
Scaffold PTM was used to perform Ascore41
analysis Ascore uses an algorithm to score the
probability of the phosphosite from a phosphopeptide identified by a database searching
algorithm such as Mascot Ascore can be freely accessed at httpascoremedharvardedu
Cell collection RNA isolation and microarray data analysis
All experiments were performed in biological duplicates Cell samples (10 ODU) were
taken at starved and fed states spun at 5000xg for 2 minutes at 30 oC the supernatant was
removed and the pellets snap frozen using liquid nitrogen RNA was isolated using the Epicentre
MasterPure Yeast Purification Kit (Epicentre Technologies) and quality was checked using gel
electrophoresis cRNA synthesis was carried out using the GeneChipreg Expression 3
Amplification One-Cycle Target Labeling and Control Reagents kit (Affymetrix) All
150
experiments followed the manufactures instructions cRNA samples were hybridized to
GeneChip Yeast Genome 20 Arrays for 16 hours Arrays were washed stained and scanned
according the manufactures recommendations Affymetrix CEL files were RMA normalized
with R and the Bioconductor Suite Data analysis was performed within TIGR Multiexperiment
Viewer v451 in-house Perl scripting R and Bioconductor
Results
Sample preparation for shotgun proteomics
As discussed in the introduction the purpose of this study is to provide an exploratory list
of qualitative differences in the phosphoproteome between starved vs glucose fed yeast After
yeast cell lysate production a substantial amount of sample preparation is performed to enhance
the depth of the phosphoproteome coverage A workflow of the steps following cell lysis is
outlined in Figure 1A The yeast proteins are isolated via acetone precipitation followed by
digestion with trypsin to produce peptides Phosphopeptides are intermixed with the entire
tryptic peptide sample and therefore Fe-NTA IMAC is used to enrich the phosphopeptides To
improve upon the number of phosphopeptides we then performed two dimensional separation
with high pH RP followed by low pH RP C18 and infused the eluent directly onto an ion trap
mass spectrometer Figure 1B show an improved technique for the first dimension of separation
to combine the early eluting and late eluting fractions from the first phase of separation to reduce
overall MS analysis time The combination of fractions 1 and 12 2 and 13 etc essentially
improves the duty cycle of the mass spectrometer by ensuring sufficient peptide content is
injected onto a low pH nanoLC RP C18 column
ETD-triggered mass spectrometry
151
In the present study labile phosphorylation can lead to non-informative neutral loss
During MS scanning mode the instrument will choose the 6 most abundant peaks with correct
isotopic distribution and select them for MSMS CID fragmentation During CID fragmentation
it is common for labile PTMs such as phosphorylation to undergo neutral loss and thus limited
informative b and y-type ions are formed Alternatively ETD fragmentation can be used on
specific MSMS scans which produce a neutral loss indicative of phosphorylation (98Daz or
80Daz) The subsequent ETD fragmentation on these putative phosphopeptides will lead to
uniform backbone cleavage resulting in confident identification of phosphopeptides with site-
specific localization during MSMS It is important to note that CID fragmentation still produces
very informative fragmentation for phosphorylation but ETD provides an orthogonal
fragmentation pathway to further increase the phosphoproteome coverage Additionally the
duty cycle for ETD fragmentation is roughly half that of CID therefore about half as many
potential peptides would be fragmented and sequenced if the instrument was using ETD
fragmentation exclusively
Protein Data
Even with phosphopeptide enrichment it is inevitable that non-phosphopeptides will also
be identified All data were searched with Mascot and in total over 1000 proteins were identified
with a 1 FDR rate and are listed in Supplemental Table 1 The proteins listed in Supplemental
Table 1 include both phosphoproteins and non-phosphorylated proteins In addition to the
proteins identified in the fed and starved states the unique peptides and spectral counts are also
listed for reference The phosphopeptides are specifically listed with a 54 FDR rate in
Supplemental Table 2 and comparisons between starved and fed with Ascore analysis are listed
for every phosphopeptide identified A higher confidence of phosphopeptide identification is
152
sometimes required before investing in time consuming biological experiments so a list of
phosphopeptides identified in starved and fed was taken from a setting directing Scaffold to
produce a 05 FDR which resulted in the generated FDR of 06 FDR and listed in
Supplemental Table 3
A summary of specific phosphorylation sites are listed in Table 1 with a 06 FDR and
Table 2 with a 54 FDR Each table gives totals for unique phosphopeptides identified having
an Ascore localization score ge80 without Ascore and phosphorylation events on each unique
peptides As expected the majority of phosphorylation events (over 50) occurred on serine
whereas fewer phosphorylation events (~10) occurred on tyrosine In addition the vast
majority of phosphorylation events were single phosphorylation (ge65) with very few
identifications having more than two phosphosites per peptide For specific phosphopeptide
identification and subsequent phosphoprotein identification refer to Supplemental Table 2 and 3
Discussion
Transcriptional response to glucose feeding
Yeast responds to the repletion of glucose after glucose-depletion by broad
transcriptional changes to induce growth Roughly one-third of the yeast genome is altered by at
least two fold when glucose is introduced as shown in Figure 2 Figure 2 is a heat map from a
microarray analysis showing 1601 genes up regulated and 1457 genes down regulated after
addition of glucose compared to the starved state The arbitrary cut-offs for these values were as
follows two fold induction after nutrient repletion and a false discovery rate (q-value) of 001
Red color in the heat map indicates a particular gene is up-regulated in the fed state compared to
the starved state Alternatively genes coded in green are less expressed in the fed state
compared to the starved condition The intensity of the green or red colors is indicative of the
153
intensity of the fold change in gene expression These large transcriptional changes after glucose
repletion drive and complement the current phosphoproteomic study
PKA motif analysis
One benefit of a large scale phosphoproteomics experiment is to examine the different
phosphopeptides from known motifs for specific kinases PKA and TOR are responsible for the
majority of the transcriptional response and thus PKA is a good target for motif analysis Figure
3 shows the PKA motif sequence xxxRKRKxSxxxxxx with the phosphorylation occurring on
the serine Other than F16P (fructose-16-bisphosphatase) all the proteins listed are unique to the
starved or fed samples A motif sequence will inevitably show up by random chance in any
analysis For this study the control for motif analysis uses the swissprot protein list for the
entire yeast proteome for the background Compared to background this specific PKA kinase
from Figure 3 is up-regulated by 264 fold when compared to the background One interesting
protein emerged from this motif analysis in the fed sample but not the starved sample is
Ssd1which is implicated in the control of the cell cycle in G1 phase42
Ssd1 also is
phosphorylated by Cbk1 and Cbk1 has been proposed to inhibit Ssd143
and provides an
intriguing target for future studies on starved vs glucose fed yeast growth
Localization of the phosphorylation sites
When a phosphopeptide contains any number of serine threonine or tyrosine amino
acids the localization of the phosphosite can sometimes be ambiguous Database searches used
to identify peptides like Mascot do not provide any probability for localization of correct
phosphorylation sites Ascore is an algorithm that uses the same MSMS spectra as Mascot but
instead of comparing theoretical peptide MSMS to experimental spectra Ascore searches for
informative a b or y-type peptide fragments giving evidence for phosphosite The Scaffold
154
program adds a localization probability to the Ascore values and the values are listed in
Supplemental Table 2 and 3 Figure 4 has a representative Ascore mass spectra assigning the
peaks identified and providing evidence that the phosphorylation site occurs at the threonine
instead of the serine Incorporating Ascore into this study provides a layer of validation for
putative phosphosite identification
Plasma Membrane 2-ATPase
A previous study identified and localized phosphorylation sites on plasma membrane 1-
ATPase after glucose was introduced to starved yeast15
In the current study PMA2 (plasma
membrane ATPase 2) was identified in glucose fed and not starved samples The doubly
threonine phosphorylated peptide identified in fed yeast is PMA2 with amino acid sequence
IKMLTGDAVGIAKETCR (583-599) whereas the homologous peptide in PMA1 has the exact
same amino acid sequence except for the first isoleucine substituted for valine
VKMLTGDAVGIAKETCR (554-570 not observed in data) PMA2 was observed in the 06
FDR list of phosphopeptides and has an Ascore localization probably of 100 A plant study
showed that PMA2 phosphorylation level was higher in early growth phase than when in
stationary phase44
In addition PMA2 expression in yeast permits the growth of yeast and
threonine phosphorylation has been reported on Thr-95545
The identification of PMA2 in the
fed glucose cell extract provides an interesting target for future study on the molecular
mechanisms involved in regulation growth in starved vs glucose fed yeast
Conclusion
In conclusion this work provides a qualitative comparison in the phosphoproteome
between starved and glucose fed yeast A large scale IMAC enrichment of yeast cell lysate
followed by 2-D separation provided a rich source of phosphopeptides for neutral loss triggered
155
ETD analysis using mass spectrometry A specific motif for PKA is highlighted to show the
differences in proteins identified between starved vs fed conditions In total 477 unique
phosphopeptides are identified with 06 FDR and 669 unique phosphopeptides identified with
54 FDR Phosphosite validation is performed using a localization algorithm Ascore to
provide further confidence on the site-specific characterization of these phosphopeptides The
proteins Ssd1 and PMA2 emerged as potential intriguing targets for more in-depth studies on
protein phosphorylation involved in glucose response
Supplemental Tables 1 2 and 3 are available upon request
References
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Rodriguez A L Aragon A D Quinones G A Allen C Werner-Washburne M Genomic
analysis of stationary-phase and exit in Saccharomyces cerevisiae gene expression and
identification of novel essential genes Mol Biol Cell 2004 15 (12) 5295-305
2 Radonjic M Andrau J C Lijnzaad P Kemmeren P Kockelkorn T T van Leenen
D van Berkum N L Holstege F C Genome-wide analyses reveal RNA polymerase II
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Cell 2005 18 (2) 171-83
3 Slattery M G Heideman W Coordinated regulation of growth genes in
Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
4 Wang Y Pierce M Schneper L GAtildefrac14ldal C G k e Zhang X Tavazoie S
Broach J R Ras and Gpa2 Mediate One Branch of a Redundant Glucose Signaling Pathway in
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5 Newcomb L L Hall D D Heideman W AZF1 is a glucose-dependent positive
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14
6 Newcomb L L Diderich J A Slattery M G Heideman W Glucose regulation of
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7 Carlson M Glucose repression in yeast Curr Opin Microbiol 1999 2 (2) 202-7
8 Gancedo J M Yeast carbon catabolite repression Microbiol Mol Biol Rev 1998 62
(2) 334-61
9 Johnston M Feasting fasting and fermenting Glucose sensing in yeast and other cells
Trends Genet 1999 15 (1) 29-33
156
10 Warner J R The economics of ribosome biosynthesis in yeast Trends Biochem Sci
1999 24 (11) 437-40
11 Li X Gerber S A Rudner A D Beausoleil S A Haas W Villen J Elias J E
Gygi S P Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces
cerevisiae J Proteome Res 2007 6 (3) 1190-7
12 Ficarro S B McCleland M L Stukenberg P T Burke D J Ross M M
Shabanowitz J Hunt D F White F M Phosphoproteome analysis by mass spectrometry and
its application to Saccharomyces cerevisiae Nat Biotechnol 2002 20 (3) 301-5
13 Gruhler A Olsen J V Mohammed S Mortensen P Faergeman N J Mann M
Jensen O N Quantitative phosphoproteomics applied to the yeast pheromone signaling
pathway Mol Cell Proteomics 2005 4 (3) 310-27
14 Peng J Schwartz D Elias J E Thoreen C C Cheng D Marsischky G Roelofs
J Finley D Gygi S P A proteomics approach to understanding protein ubiquitination Nat
Biotechnol 2003 21 (8) 921-6
15 Lecchi S Nelson C J Allen K E Swaney D L Thompson K L Coon J J
Sussman M R Slayman C W Tandem phosphorylation of Ser-911 and Thr-912 at the C
terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation J Biol
Chem 2007 282 (49) 35471-81
16 Cohen P The regulation of protein function by multisite phosphorylation--a 25 year
update Trends Biochem Sci 2000 25 (12) 596-601
17 Kalume D E Molina H Pandey A Tackling the phosphoproteome tools and
strategies Curr Opin Chem Biol 2003 7 (1) 64-9
18 Nagaraj N DSouza R C Cox J Olsen J V Mann M Feasibility of large-scale
phosphoproteomics with higher energy collisional dissociation fragmentation J Proteome Res
2010 9 (12) 6786-94
19 Olsen J V Vermeulen M Santamaria A Kumar C Miller M L Jensen L J
Gnad F Cox J Jensen T S Nigg E A Brunak S Mann M Quantitative
phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis Sci
Signal 2010 3 (104) ra3
20 Breitkopf S B Asara J M Determining In Vivo Phosphorylation Sites Using Mass
Spectrometry In Current Protocols in Molecular Biology John Wiley amp Sons Inc 2012
21 Steen H Jebanathirajah J A Rush J Morrice N Kirschner M W Phosphorylation
analysis by mass spectrometry myths facts and the consequences for qualitative and
quantitative measurements Mol Cell Proteomics 2006 5 (1) 172-81
22 Kweon H K Hakansson K Metal oxide-based enrichment combined with gas-phase
ion-electron reactions for improved mass spectrometric characterization of protein
phosphorylation J Proteome Res 2008 7 (2) 749-55
23 Larsen M R Thingholm T E Jensen O N Roepstorff P Jorgensen T J Highly
selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide
microcolumns Mol Cell Proteomics 2005 4 (7) 873-86
24 Kokubu M Ishihama Y Sato T Nagasu T Oda Y Specificity of immobilized
metal affinity-based IMACC18 tip enrichment of phosphopeptides for protein phosphorylation
analysis Anal Chem 2005 77 (16) 5144-54
25 Swaney D L Wenger C D Thomson J A Coon J J Human embryonic stem cell
phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry Proc
Natl Acad Sci U S A 2009 106 (4) 995-1000
157
26 Hao P Guo T Sze S K Simultaneous analysis of proteome phospho- and
glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction
chromatography PLoS One 2011 6 (2) e16884
27 Rush J Moritz A Lee K A Guo A Goss V L Spek E J Zhang H Zha X
M Polakiewicz R D Comb M J Immunoaffinity profiling of tyrosine phosphorylation in
cancer cells Nat Biotechnol 2005 23 (1) 94-101
28 Ficarro S Chertihin O Westbrook V A White F Jayes F Kalab P Marto J A
Shabanowitz J Herr J C Hunt D F Visconti P E Phosphoproteome analysis of
capacitated human sperm Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3
and valosin-containing proteinp97 during capacitation J Biol Chem 2003 278 (13) 11579-89
29 Washburn M P Wolters D Yates J R 3rd Large-scale analysis of the yeast
proteome by multidimensional protein identification technology Nat Biotechnol 2001 19 (3)
242-7
30 Dowell J A Frost D C Zhang J Li L Comparison of two-dimensional
fractionation techniques for shotgun proteomics Anal Chem 2008 80 (17) 6715-23
31 Song C Ye M Han G Jiang X Wang F Yu Z Chen R Zou H Reversed-
phase-reversed-phase liquid chromatography approach with high orthogonality for
multidimensional separation of phosphopeptides Anal Chem 2010 82 (1) 53-6
32 Palumbo A M Smith S A Kalcic C L Dantus M Stemmer P M Reid G E
Tandem mass spectrometry strategies for phosphoproteome analysis Mass Spectrom Rev 2011
30 (4) 600-25
33 Beausoleil S A Jedrychowski M Schwartz D Elias J E Villen J Li J Cohn M
A Cantley L C Gygi S P Large-scale characterization of HeLa cell nuclear
phosphoproteins Proc Natl Acad Sci U S A 2004 101 (33) 12130-5
34 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and
protein sequence analysis by electron transfer dissociation mass spectrometry Proc Natl Acad
Sci U S A 2004 101 (26) 9528-33
35 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion
dependence in the partitioning between proton and electron transfer in ionion reactions
International Journal of Mass Spectrometry 2004 236 (1acirceuroldquo3) 33-42
36 Hui L Cunningham R Zhang Z Cao W Jia C Li L Discovery and
characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP)
and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using
multiple tandem mass spectrometry techniques J Proteome Res 2011 10 (9) 4219-29
37 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-
site identity and position on electron-transfer dissociation of polypeptide cations J Am Chem Soc
2007 129 (40) 12232-43
38 Coon J J Collisions or electrons Protein sequence analysis in the 21st century Anal
Chem 2009 81 (9) 3208-15
39 Miller M E Cross F R Distinct subcellular localization patterns contribute to
functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae Mol Cell Biol
2000 20 (2) 542-55
40 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
158
41 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
approach for high-throughput protein phosphorylation analysis and site localization Nat
Biotechnol 2006 24 (10) 1285-92
42 Sutton A Immanuel D Arndt K T The SIT4 protein phosphatase functions in late
G1 for progression into S phase Mol Cell Biol 1991 11 (4) 2133-48
43 Jansen J M Wanless A G Seidel C W Weiss E L Cbk1 regulation of the RNA-
binding protein Ssd1 integrates cell fate with translational control Curr Biol 2009 19 (24)
2114-20
44 Kanczewska J Marco S Vandermeeren C Maudoux O Rigaud J L Boutry M
Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3
proteins converts a dimer into a hexamer Proc Natl Acad Sci U S A 2005 102 (33) 11675-80
45 Maudoux O Batoko H Oecking C Gevaert K Vandekerckhove J Boutry M
Morsomme P A plant plasma membrane H+-ATPase expressed in yeast is activated by
phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the
absence of fusicoccin J Biol Chem 2000 275 (23) 17762-70
159
Figure 1 The general workflow indicating the major steps involved in sample collection
sample processing mass spectrometric data acquisition and analysis of comparative
phosphoproteomics for starved vs glucose fed yeast (A) The high pH RP separation
procedure for combining fractions to reduce low peptide containing fractions from the
UV-VIS trace is also shown (B)
160
Figure 2 Heat map for the global transcriptional response to glucose fed vs starved samples
S288C cells starved for glucose until growth was arrested and subsequently glucose was added
161
Samples for microarray analysis were taken at the starved state and 60 minutes after glucose was
added The heat map shows the fed log2 fold change for each gene relative to the starved state
across the genome performed in biological replicate (A) Black indicates no change in
expression level while red indicates higher expression for the fed relative to the starved state
Green indicates higher expression for the starved state compared to the fed state (Adapted from
Dr Michael Conways Thesis)
162
Figure 3 The motif for protein kinase A (PKA) that was observed for fed vs starved which is
xxxRKRKxSxxxxxx with the phosphorylation occurring on the serine The motif occurred at a
rate 264 fold higher than the yeast proteome used for background In addition one protein was
observed in both starved and fed with accession identification of F16P (Fructose-16-
bisphosphatase)
163
06 FDR phosphopeptide analysis
Table 1 The number of phosphopeptides identified with a 06 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
Starved Fed All
Ascore ge80 score
unique
STY 164 153 317
S 87 (530) 82 (536) 169 (533)
T 60 (366) 55 (359) 115 (363)
Y 17 (104) 16 (105) 33 (104)
Unique no Ascore
STY 242 235 477
S 131 (541) 133 (566) 264 (553)
T 86 (355) 78 (332) 164 (344)
Y 25 (103) 24 (102) 49 (103)
Phosphorylation events
on each unique peptide
1 102 113 187
2 36 40 68
3 12 11 22
4 or more 8 3 11
164
54 FDR phosphopeptide analysis
Starved Fed All
Ascore ge80 score
unique
STY 217 217 434
S 115 (530) 113 (521) 228 (525)
T 78 (359) 78 (359) 156 (359)
Y 24 (111) 26 (120) 50 (115)
Unique no Ascore
STY 337 332 669
S 193 (573) 180 (542) 373 (558)
T 111 (329) 116 (349) 227 (339)
Y
Phosphorylation events
on each unique peptide
1
2
3
4 or more
33 (98)
135
56
16
11
36 (108)
169
55
14
3
69 (103)
280
100
27
13
Table 2 The number of phosphopeptides identified with a 54 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
165
Figure 4 Ascore validation of the phosphopeptide LSLAtKK from the protein SGM1 (slow
growth on galactose and mannose protein 1) with 100 localization probability observed
in only fed but not starved yeast samples Ascorersquos algorithm identifies a b and y-type
ions and looks to identify peaks that provide evidence for a specific phosphorylation site
For this example Ascore assigns this phosphosite to threonine (T5) instead of the serine
(S2) The intense peaks at 4024 mz and 5250 mz are not identified by any a b or y-
type ions From the ladder sequence of the peptide shown numerous ions indicate the
threonine is phosphorylated while the serine is not Among these ions used for
localization are b2 y2 y5+H2O y3 y4 and y5
166
Chapter 6
Use of electron transfer dissociation for neuropeptide sequencing and
identification
Adapted from ldquoDiscovery and Characterization of the Crustacean Hyperglycemic Hormone
Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue
Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniquesrdquo Hui L
Cunningham R Zhang Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
167
Abstract
The crustacean Callinectes sapidus sinus gland (SG) is a well-defined neuroendocrine site that
produces numerous hemolymph-borne agents including the most complex class of endocrine
signaling moleculesmdashneuropeptides One such peptide is the crustacean hyperglycemic hormone
(CHH) precursor-related peptide (CPRPs) which was not previously sequenced Electron
transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and
high charge state In addition ETD was used to sequence the sulfated peptide Cholecystokinin
CCK-like Homarus americanus using a salt adduct Collectively these two examples
demonstrated the utility of ETD in sequencing neuropeptides with large molecular weight or
with labile modifications
168
Introduction
Neuropeptides are the largest and most diverse group of endocrine signaling molecules in
the nervous system They are necessary and critical for initiation and regulation of numerous
physiological processes such as feeding reproduction and development1 2
Mass spectrometry
(MS) with unique advantages such as high sensitivity high throughput chemical specificity and
the capability of de novo sequencing with limited genomic information is well suited for the
detection and sequencing of neuropeptides in endocrine glands and simultaneously provides the
potential for information on post-translational modifications such as sulfonation3-6
The sinus glands (SG) are located between the medulla interna and medulla externa of the
eyestalk in Callinectes sapidus It is a well-known neuroendocrine site that synthesizes and
secretes peptide hormones regulating various physiological activities such as molting
hemolymph glucose levels integument color changes eye pigment movements and
hydro-mineral balance7 Our labrsquos previous neuropeptidomic studies of SG in several
crustacean species including Cancer borealis8-11
Carcinus maenas12
and Homarus americanus13
14 used multiple MS strategies combining MALDI-based high resolution accurate mass profiling
biochemical derivatization and nanoscale separation coupled to tandem MS for de novo
sequencing In the current study we explore the neuropeptidome of the SG in the blue crab
Callinectes sapidus a vital species of economic importance on the seafood market worldwide In
total 70 neuropeptides are identified including 27 novel neuropeptides and among them the
crustacean hyperglycemic hormone precursor-related peptides (CPRPs) and orcokinins represent
major neuropeptide families known in the SG
The crustacean hyperglycemic hormone (CHH) and its precursor-related peptide (CPRP) are
produced concurrently during the cleavage of CHH from the CHH preprohormone protein15
The
169
CPRP peptide is located between the signal peptide and the CHH sequence and is separated from
the CHH by a dibasic cleavage site The functions of CPRPs are currently unknown16
However
the complete characterization of CPRPs provides a foundation for future experiments elucidating
their functional roles in crustaceans The cDNA of CHH preprohormone in SG of Callinectes
sapidus has been characterized17
but the direct detection of CPRP has not been reported due to
its relatively large size and possible post-translational modifications While small fragments of
CPRPs can be identified using nanoLC-ESI-Q-TOF tandem mass spectrometry the intact
peptide is difficult to detect due to the large molecular weight of CPRPs
Peptidomics traditionally uses collision induced dissociation (CID) for tandem MS
experiments for de novo sequencing Recently an alternative peptide fragmentation method has
been developed using the transfer of an electron termed electron transfer dissociation (ETD)18 19
ETD involves a radical anion with low electron affinity to be transferred to peptide cation which
results in reduced sequence discrimination and thus provides improved sequencing for larger
peptides compared to CID20
Specifically for an ion trap ETD the fluoranthene radical anion is
allowed to react with peptide cations in the three dimensional trap The resulting dissociation
cleaves at the N-Cα bond to produce c and zbull type product ions The peptidome of model
organisms like Callinectes sapidus can be further explored and expanded utilizing ETD as a
complementary fragmentation technique to CID Previous peptidomic analysis has been
completed using ETD as an additional fragmentation method21
It was observed that
enzymatically produced peptides with a higher mz produced improved sequence coverage using
ETD This observation termed decision tree analysis determined that a charge state of ge6 all
peptides endogenous or enzymatic should be fragmented via ETD22
In the present study the
highly charged peptide CPRP with an intact molecular weight of 3837 readily produces plus six
170
charge states The higher charge state of CPRP is highly amenable to fragmentation by ETD
which produces remarkably improved fragmentation and thus increased sequence coverage when
compared to CID
Sulfonation of tyrosine is a post-translational modification (PTM) that is thought to occur on
trans-membrane spanning and secreted proteins23
Cholecystokinin-8 (CCK-8) is a sulfated
peptide which has been linked to satiety24-26
and a CCK-like peptide has been observed in
lobster27
Sulfonation is an extremely labile modification and is often lost during soft
ionization such as ESI or MALDI but can be avoided by optimizing the ionization process28
One potential strategy to identify sulfopeptides is to scan for a neutral loss of 80 Da after CID
but this method does not allow for identification of site of sulfonation and has the risk to be
mistaken for phosphorylation The sulfonation PTM is a negatively charged modification on
the peptide which allows for negative ion scanning in the mass spectrometer but provides
minimal MSMS sequence coverage during CID due to preferential loss of the sulfate group
Alternatively electron-based dissociation technique enables more rapid radical driven
fragmentation where the cleavage pattern is more uniform along the peptide backbone without
initially cleaving the labile sulfated group thus preserving the site of modification These types
of dissociation techniques only work for multiply-charged ions thus a method to install a
multiply-charged cation on the target peptide is desirable It has been shown that the electron
capture dissociation (ECD) can be used to sequence sulfated peptides when a multi-charged
cation is added to the solution29
We use a similar multi-charge cation solution technique to
sequence a sulfated peptide from Homarus americanus via ETD on an ion trap mass
spectrometer Here we presented the use of the ETD fragmentation technique for the analysis
of large peptides and peptides containing labile post-translational modification
171
Experimental Section
Chemical and materials
Acetonitrile methanol magnesium chloride (ACS grade) potassium chloride (ACS grade) and
calcium chloride (ACS grade) were purchased from Fisher Scientific (Pittsburgh PA) Formic
acid was from Sigma-Aldrich (St Louis MO) Human sulfated CCK-8 and CCK-like peptide
(E(pyro)FDEY(Sulfo)GHMRFamide) were purchased from American Peptide (Sunnyvale CA)
Fused-silica capillary with 75 μm id and 360 μm od was purchased from Polymicro
Technologies (Phoenix AZ) Millipore C18 Ziptip column was used for sample cleaning and all
water used in this study was deionized water (182 MΩcm) prepared from a Milli-Q Millipore
system (Billerica MA) The physiological saline consisted of 440 mM NaCl 11mM KCl 26
mM MgCl2 13 mM CaCl2 11 mM Trizma base and 5 mM maleic acid in pH 745
Animals and dissection
Callinectes sapidus (blue crab) were obtained from commercial food market and maintained
without food in artificial sea water at 10-12 ordmC Animals were cold-anesthetized by packing on
ice for 15-30min before dissection The optic ganglia with the SG attached were dissected in
chilled (~4ordmC) physiological saline and individual SGs were isolated from the optic ganglia by
micro-dissection and immediately placed in acidified methanol (90 methanol 9 glacial acetic
acid and 1 water) and stored at -80ordmC until tissue extraction
Tissue homogenization
Acidified methanol was used during the homogenization of SGs The homogenized SGs were
immediately centrifuged at 16100 g using an Eppendorf 5415D tabletop centrifuge (Eppendorf
172
AG) The pellet was washed using acidified methanol and combined with the supernatant and
further dried in a Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation) The
resulting dried sample was re-suspended with a minimum amount (20 microL) of 01 formic acid
Fractionation of homogenates using reversed phase (RP)-HPLC
The re-suspended extracts were then vortexed and briefly centrifuged The resulting supernatants
were subsequently fractionated via high performance liquid chromatography (HPLC) HPLC
separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax
UV-D II absorbance detector (Rainin Instrument Inc Woburn MA) The mobile phases included
Solution A (deionized water containing 01 formic acid) and Solution B (acetonitrile containing
01 formic acid) About 20 μl of extract was injected onto a Macrosphere C18 column (21 mm
id x 250 mm length 5 microm particle size Alltech Assoc Inc Deerfield IL) The separation
consisted of a 120 minute gradient of 5-95 Solution B Fractions were automatically collected
every two minutes using a Rainin Dynamax FC-4 fraction collector (Rainin Instrument Inc
Woburn MA) The fractions were then concentrated using in Savant SC 110 SpeedVac
concentrator (Thermo Electron Corporation) and re-suspended with minimum amount of 01
formic acid
Nano-LC-ESI-Q-TOF MSMS
Nanoscale LC-ESI-Q-TOF MSMS was performed using a Waters nanoAcquity UPLC system
coupled to a Q-TOF Micro mass spectrometer (Waters Corp Milford MA)
Chromatographic separations were performed on a homemade C18 reversed phase capillary
column (75 microm internal diameter x100 mm length 3 microm particle size 100 Aring) The mobile phases
173
used were 01 formic acid in deionized water (A) 01 formic acid in acetonitrile (B) An
aliquot of 50 microl of a tissue extract or HPLC fraction was injected and loaded onto the trap
column (Zorbax 300SB-C18 Nano trapping column Agilent Technologies Santa Clara CA)
using 95 mobile phase A and 5 mobile phase Bat a flow rate of 10 microlmin for 10 minutes
Following this the stream select module was switched to a position at which the trap column
came in line with the analytical capillary column and a linear gradient of mobile phases A and B
was initiated For neuropeptides the linear gradient was from 5 buffer B to 45 buffer B over
90 min The nanoflow ESI source conditions were set as follows capillary voltage 3200 V
sample cone voltage 35 V extraction cone voltage 1 V source temperature 100ordmC A data
dependent acquisition was employed for the MS survey scan and the selection of three precursor
ions and subsequent MSMS of the selected parent ions The MS scan range was from mz
400-1800 and the MSMS scan was from mz 50-1800
Peptide Prediction De Novo Sequencing and Database Searching
De novo sequencing was performed using a combination of MassLynxTM
41 PepSeq software
(Waters) and manual sequencing Tandem mass spectra acquired from the Q-TOF were first
deconvoluted using MaxEnt 3 software (Waters) to convert multiply charged ions into their
singly charged forms The resulting spectra were pasted into the PepSeq window for sequencing
analysis The candidate sequences generated by the PepSeq software were compared and
evaluated for homology with previous known peptides The online program blastp (National
Center for Biotechnology Information Bethesda MD httpwwwncbinlmnihgovBLAST)
was used to search the existing NCBI crustacean protein database using the candidate peptide
sequences as queries For all searches the blastp database was set to non-redundant protein
174
sequences (ie nr) and restricted to crustacean sequences (ie taxid 6657) For each of the
proteins putatively identified via blastp the BLAST score and BLAST-generated E-value for
significant alignment are provided in the appropriate subsection of the results Peptides with
partial sequence homology were selected for further examination by comparing theoretical
MSMS fragmentation spectra generated by PepSeq with the raw MSMS spectra If the
fragmentation patterns did not match well manual sequencing was performed
NanoLC Coupled to MSMS by CID and ETD
Setup for RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN (Fisher Scientific Optima LCMS grade Fair Lawn NJ) Injections
consisted of 5 microL of prepared tissue extract onto an Agilent Technologies Zorbax 300 SB-C18 5
microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5 microLmin for 5 minutes at 95
A5 B Separation was performed on a Waters HPLC column with 3 microm Atlantis dC18 75 microm
x 150 mm (Milford MA) on a gradient from 5 to 45 mobile phase B at 250 nLmin over 90
minutes at room temperature Emitter tips were pulled from 75 microm inner diameter 360 microm
outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using a commercial
laser puller model P-2000 (Sutter Instrument Co Novato CA)
Mass Spectrometry Data Acquisitions for Collision Induced Dissociation (CID)
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany) equipped
with an on-line nanospray source was used for mass spectrometry data acquisition Hystar
(Version 32 Bruker Daltonics Bremen Germany) was used to couple and control Eksigent
175
nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for all
experiments Smart parameter setting (SPS) was set to mz 700 compound stability and trap
drive level were set at 100 Optimization of the nanospray source resulted in dry gas
temperature 125oC dry gas 40 Lmin capillary voltage -1300 V end plate offset -500 V
MSMS fragmentation amplitude 10V and SmartFragmentation set at 30-300
Data was generated in data dependent mode with strict active exclusion set after two spectra and
released after one minute MSMS was obtained via CID fragmentation for the six most
abundant MS peaks with a preference for doubly charged ions and excluded singly charged ions
For MS generation the ion charge control (ICC) target was set to 200000 maximum
accumulation time 5000 ms four spectral average acquisition range of mz 300-1500 and scan
speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target was set to
200000 maximum accumulation time 5000 ms three spectral averages acquisition range of
mz 100-2000 and scan speed (Ultrascan) of 32000 mz s-1
Mass Spectrometry Data Acquisitions for Electron Transfer Dissociation (ETD)
The amaZon ETD ion-trap mass spectrometer was operated in electron transfer dissociation for
MSMS fragmentation with the same optimized settings as reported for CID unless otherwise
stated Smart parameter setting (SPS) was set to 500 mz compound stability and trap drive
level were set at 100 MSMS was obtained via ETD fragmentation for the four most
abundant MS peaks with no preference for specifically charged ions except to exclude singly
charged ions The ETD reagent parameters were set to 400000 target ICC for the fluoranthene
radical anion The fluoranthene radical anion has an mz of 210 and therefore the 210 mz value
was removed from the resulting MSMS spectra and 160 mz was set for the MSMS low mz
cut-off
176
Direct Infusion of Sulfated Peptide Standards into the Mass Spectrometer using ETD and
CID Fragmentation
The sulfated peptide standards were infused into the mass spectrometer at a flow rate of 300
nlmin using a fused-silica capillary with 75 μm id and 360 μm od coupled to a custom pulled
tip The CCK-8 human peptide (20 microM) was infused in 5050 ACNH2O and detected in
negative ionization mode with an ICC of 70000 and fragmented with CID using the same
settings as the previous CID experiments Alternatively the CCK-like lobster sulfated peptide
(2 microM) was mixed with 30 microM of different salts (MgCl2 KCl or CaCl2) and 01 formic acid in
5050 ACNH2O The plus three charge of the CCK-like lobster peptide was selected for ETD
fragmentation in positive mode with the same setting as the previous ETD experiments The
data were then de novo sequenced for coverage and localization of the sulfation site
Data Analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen Germany)
Deviations in parameters from the default Protein Analysis in DataAnalysis were as follows
intensity threshold 1000 maximum number of compounds 1E7 and retention time window 05
minutes These parameter changes assisted in providing better quality spectra for sequencing
Identification of peptides was performed using Mascot (Version 23 Matrix Science London
UK) Searches were performed against a custom crustacean database none enzyme allow up to
1 missed cleavage amidated C-terminal as variable modifications peptide tolerance mass error
12 Da MSMS mass error tolerance is 06 Da
Results and Discussion
177
Identification and Characterization of Intact CPRPs Using ETD
Using ESI-Q-TOF 9 truncated CPRPs fragments were sequenced to cover the amino acid
sequence of the intact CPRP peptide RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE
A representative CID MSMS spectrum of ASLKSDTVTPLR is shown in Figure 1 (a) CID
using ESI-Q-TOF mainly produces fragmentation for doubly and triply charge molecules which
is adequate to sequence peptides with relatively small molecular weight (less than 2000 Da)
However CID fragmentation is insufficient to sequence the larger intact CPRP in a complex
sample whose molecular mass is 3837 Da and therefore it is extremely difficult to directly
sequence by traditional CID ESI-Q-TOF ETD is an attractive fragmentation alternative to
sequence the highly charged CPRP peptide In ETD a protonated peptide reacts with an anion
(fluoranthene radical) where the anion transfers an electron to the peptide cation and the resulting
fragmentation occurs along the N-Cα bond by free-radical-driven-cleavage The large size of
CPRP offers multiple basic amino acid sites to sequester charge and reduce the sequence
coverage from collision induced dissociate by preventing random backbone cleavage whereas
ETD does not cleave the weakest bond and provides the alternative fragmentation pathway to
obtain superior sequence coverage to larger peptides As shown in Figure 1 (cd) the
fragmentation of ETD is highly amenable to ETD compared to the CID fragmentation in Figure
1 (b) As evident from comparison ETD offers more extensive fragmentation than CID thus
providing enhanced coverage for large intact peptides such as CPRPs Specifically we observe
125 of the b- and y- cleavages for CID as compared to an average of 535 of c- and zbull-
fragments More than a four-fold increase in fragments using ETD suggests the utility of this
relatively new tandem MS fragmentation method as complementary techniques for de novo
sequencing of large intact endogenous peptides and novel neuropeptide discovery endeavors
178
Negative Mode Sulfated Peptide Identification
An accepted method for identification and quantification for sulfated peptides is negative
ionization30
CCK-8 sulfated peptide standards show intense signal in negative ionization mode
without needing to have additives added such as salts Figure 2 shows the CID MSMS
spectrum from the negative ionization and produces an intense b6 ion at 430 mz The transition
from the doubly negatively charged MS ion of 570 mz to the b6 ion provides a selected reaction
monitoring transition for quantification studies but the sequence information is limited and the
presence of the methionine produces variable oxidation In addition Figure 2 shows the
MSMS product ions with the loss of the sulfate group thus making site-specific location of
sulfation more difficult
Investigation of Cation Salt Adducts to Produce Multiply Charged Sulfated Peptides
Sulfated peptides can be isolated in positive scanning mode but usually only in a plus one
state with low signal intensity If ETD is performed on the singly charged peptide cation a
neutral is formed and is lost in the mass spectrometer and thus no sequence information can be
obtained In order to remedy this situation a technique that adding metal salts to peptides to
increase charge state for ECD used in Fourier transform ion cyclotron resonance mass
spectrometry (FTICR-MS)29
inspired the investigation of increasing charge state of targeted
peptide via metal salt adduction followed by ETD analysis in a Bruker amaZon ion trap
Various salts were investigated including MgCl2 CaCl2 and KCl each used at a concentration of
30 microM and mixed with a sulfated peptide standard at 2 microM The addition of KCl produced
mostly doubly charged ion of the lobster CCK-like peptide standard K adduct which produced
insufficient sequence information from ETD fragmentation (data not shown) In comparison
the MgCl2 and CaCl2 produced intense triply charged peptide ions but CaCl2 produced lower
179
signal intensity compared to MgCl2 (data not shown)
Cation Assisted ETD Fragmentation of CCK-like Lobster Sulfated Peptide and Future
Directions
The addition of MgCl2 to the lobster CCK-like sulfated peptide is shown in Figure 3
Except for z1 the complete z-series is obtained including the z7 ion with and without the
sulfation PTM The spectrum is complicated by the additional Mg adducts but several peaks
are shown without any Mg adduct such as z2 z3 z4 etc Clearly the method of cation
assisted ETD fragmentation can be useful using ion trap instruments and can properly sequence
sulfated peptides that are prone to neutral loss from the labile PTM One concern about future
direction in sulfopeptide isolation in non-genome sequenced species is isolation of sulfopeptides
Currently antibodies to specific sulfopeptides is the only commonly used enrichment technique
for sulfopeptides Also since metal cations are needed for this method direct infusion into an
ESI mass spectrometer is preferred to prevent running large amounts of adduct forming salts
through the LC system With direct infusion the lack of separation confounds the problem in
sulfopeptide detection because any co-eluting peptides could have ionization efficiency and thus
reduce the signal of the sulfopeptide Once discovered and sequenced a selected reaction
monitoring (SRM) method could be developed using LC retention coupled with negative
ionization mode followed by CID MSMS or possibly MSMSMS to perform quantitative
studies for sulfopeptides
Conclusions
In this study ETD was performed to improve the sequence coverage of large endogenous
neuropeptides with higher charge and bigger size The intact CPRP from Callinectes sapidus was
identified and characterized with 68 sequence coverage by MS for the first time Cation
180
assisted ETD fragmentation was also shown to be a powerful tool in de novo sequencing of
sulfated neuropeptides These endeavors into using ETD for certain neuropeptides will assist in
future analysis of large neuropeptides and PTM containing neuropeptides
181
Reference
1 Schwartz M W Woods S C Porte D Jr Seeley R J Baskin D G Central nervous system control of
food intake Nature 2000 404 (6778) 661-71
2 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R
Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide
family of aplysia J Neurosci 2002 22 (17) 7797-808
3 Baggerman G Cerstiaens A De Loof A Schoofs L Peptidomics of the larval Drosophila melanogaster
central nervous system Journal of Biological Chemistry 2002 277 (43) 40368-40374
4 Desiderio D M Mass spectrometric analysis of neuropeptidergic systems in the human pituitary and
cerebrospinal fluid Journal of Chromatography B 1999 731 (1) 3-22
5 Li L J Kelley W P Billimoria C P Christie A E Pulver S R Sweedler J V Marder E Mass
spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab Cancer
borealis Journal of Neurochemistry 2003 87 (3) 642-656
6 Li L J Moroz T P Garden R W Floyd P D Weiss K R Sweedler J V Mass spectrometric survey of
interganglionically transported peptides in Aplysia Peptides 1998 19 (8) 1425-1433
7 Strand F L Neuropeptides regulators of physiological processes 1st ed ed MIT Press Cambridge Mass
1999 p 658 p
8 Fu Q Goy M F Li L J Identification of neuropeptides from the decapod crustacean sinus glands using
nanoscale liquid chromatography tandem mass spectrometry Biochemical and Biophysical Research
Communications 2005 337 (3) 765-778
9 Fu Q Christie A E Li L J Mass spectrometric characterization of crustacean hyperglycemic hormone
precursor-related peptides (CPRPs) from the sinus gland of the crab Cancer productus Peptides 2005 26 (11)
2137-2150
10 Ma M M Chen R B Ge Y He H Marshall A G Li L J Combining Bottom-Up and Top-Down Mass
Spectrometric Strategies for De Novo Sequencing of the Crustacean Hyperglycemic Hormone from Cancer borealis
Analytical Chemistry 2009 81 (1) 240-247
11 Ma M M Sturm R M Kutz-Naber K K Fu Q Li L J Immunoaffinity-based mass spectrometric
characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis Biochemical
and Biophysical Research Communications 2009 390 (2) 325-330
12 Ma M M Bors E K Dickinson E S Kwiatkowski M A Sousa G L Henry R P Smith C M Towle
D W Christie A E Li L J Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and
functional genomics General and Comparative Endocrinology 2009 161 (3) 320-334
13 Chen R B Jiang X Y Conaway M C P Mohtashemi I Hui L M Viner R Li L J Mass Spectral
Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus
Journal of Proteome Research 2010 9 (2) 818-832
14 Ma M M Chen R B Sousa G L Bors E K Kwiatkowski M A Goiney C C Goy M F Christie A
E Li L J Mass spectral characterization of peptide transmittershormones in the nervous system and
neuroendocrine organs of the American lobster Homarus americanus General and Comparative Endocrinology
2008 156 (2) 395-409
15 Ollivaux C Gallois D Amiche M Boscameric M Soyez D Molecular and cellular specificity of
post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family FEBS J 2009 276
(17) 4790-802
16 Wilcockson D C Chung J S Webster S G Is crustacean hyperglycaemic hormone precursor-related
peptide a circulating neurohormone in crabs Cell and Tissue Research 2002 307 (1) 129-138
17 Choi C Y Zheng J Watson R D Molecular cloning of a cDNA encoding a crustacean hyperglycemic
hormone from eyestalk ganglia of the blue crab Callinectes sapidus General and Comparative Endocrinology 2006
148 (3) 383-387
18 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and protein sequence analysis
by electron transfer dissociation mass spectrometry Proc Natl Acad Sci U S A 2004 101 (26) 9528-33
19 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion dependence in the partitioning
between proton and electron transfer in ionion reactions International Journal of Mass Spectrometry 2004 236
(1-3) 33-42
20 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-site identity and
position on electron-transfer dissociation of polypeptide cations J Am Chem Soc 2007 129 (40) 12232-43
182
21 Altelaar A F Mohammed S Brans M A Adan R A Heck A J Improved identification of endogenous
peptides from murine nervous tissue by multiplexed peptide extraction methods and multiplexed mass spectrometric
analysis J Proteome Res 2009 8 (2) 870-6
22 Swaney D L McAlister G C Coon J J Decision tree-driven tandem mass spectrometry for shotgun
proteomics Nat Methods 2008 5 (11) 959-64
23 Monigatti F Hekking B Steen H Protein sulfation analysis--A primer Biochim Biophys Acta 2006 1764
(12) 1904-13
24 Chandler P C Wauford P K Oswald K D Maldonado C R Hagan M M Change in CCK-8 response
after diet-induced obesity and MC34-receptor blockade Peptides 2004 25 (2) 299-306
25 Little T J Feltrin K L Horowitz M Meyer J H Wishart J Chapman I M Feinle-Bisset C A
high-fat diet raises fasting plasma CCK but does not affect upper gut motility PYY and ghrelin or energy intake
during CCK-8 infusion in lean men Am J Physiol Regul Integr Comp Physiol 2008 294 (1) R45-51
26 Blevins J E Morton G J Williams D L Caldwell D W Bastian L S Wisse B E Schwartz M W
Baskin D G Forebrain melanocortin signaling enhances the hindbrain satiety response to CCK-8 Am J Physiol
Regul Integr Comp Physiol 2009 296 (3) R476-84
27 Turrigiano G G Selverston A I A cholecystokinin-like hormone activates a feeding-related neural circuit in
lobster Nature 1990 344 (6269) 866-8
28 Wolfender J L Chu F Ball H Wolfender F Fainzilber M Baldwin M A Burlingame A L
Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB further investigation
of labile sulfo- and phosphopeptides by electrospray matrix-assisted laser desorptionionization (MALDI) and
atmospheric pressure MALDI mass spectrometry J Mass Spectrom 1999 34 (4) 447-54
29 Liu H Hakansson K Electron capture dissociation of tyrosine O-sulfated peptides complexed with divalent
metal cations Anal Chem 2006 78 (21) 7570-6
30 Young S A Julka S Bartley G Gilbert J R Wendelburg B M Hung S C Anderson W H
Yokoyama W H Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using
immunoprecipitation liquid chromatography-mass spectrometrymass spectrometry Anal Chem 2009 81 (21)
9120-8
183
Figure 1 Comparison of tandem MS spectra of truncated CPRP (ASLKSDTVTPL mz 128766)
by ESI-Q-TOF (a) and intact CPRP (MW 3837) by the amaZon ETD for both CID and ETD
fragmentation (a) MSn of precursor ion with mz 640 with charge state +6 by CID represent
loss of NH3 ordm represent loss of H2O (b) MS+6
of precursor ion with mz 640 with charge state +6
by ETD at z represent z+1 z represent z+2 (c) MS+5
of precursor ion with mz 768 with charge
state +5 by ETD z represent z+1 z represent z+2 For all labels in Figure 1 charge state +1 is
not specified
184
185
Figure 2 Negative CID spectrum of CCK-8 peptide standard (20 microM) via direct infusion show
the doubly charged b6 ion provides the most intense MSMS transition
186
Figure 3 ETD spectrum of E(pyro)FDEY(Sulfo)GHMRF-amide (2 microM) obtained on the
amaZon ETD via direct infusion with 30 microM MgCl2 The sulfated peptide can be identified
with a visible z-series of z2 to z9 and identified sulfate loss
187
Chapter 7
Investigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysis
Adapted from ldquoInvestigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J
Wellner D Li L Journal of Mass Spectrometry In Press
188
ABSTRACT
This work investigates the introduction of methanol and a salt modifier to molecular
weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide
quantities Using a neuropeptide standard bradykinin sample loss was reduced over two orders
of magnitude with and without undigested protein present Additionally a bovine serum
albumin (BSA) tryptic digestion was investigated Twenty-seven tryptic peptides were identified
from MALDI mass spectra after enriching with methanol while only two tryptic peptides were
identified after the standard MWCO protocol The strategy presented here enhances recovery
from MWCO separation for sub-microg peptide samples
INTRODUCTION
Molecular weight cut-off membrane-based centrifugal filter devices (MWCO) are
commonly used to desalt and concentrate large molecular weight proteins [1] Greeing and
Simpson recently investigated various MWCO membranes for large amounts of starting material
(~6 mg) focusing on optimal conditions for the sub 25 kDa protein fraction [2] The authors
recovered 200 μg to 29 mg of protein from multiple MWCO experiments and demonstrated that
a 1090 acetonitrile (ACN)H2O elution solvent produced optimal results [3] In addition Manza
et al provided an alternative approach to isolate proteins with a 5 kDa MWCO by using
NH4HCO3 and recovering the retained proteins [4] Alternatively the elution from MWCOs can
be collected to recover only low molecular weight peptides Multiple peptidomic studies have
utilized MWCOs for peptide isolation during the first few steps of sample preparation [5 6]
When sample amount is limited or peptide content is below 1 microg sample loss is a significant
concern when using MWCOs to isolate endogenous peptides Optimized protocols have been
189
investigated using ACN [3 7] salt [4 8] SDS [5] or native sample [6 9 10] but these
experiments primarily focused on large sample amounts rather than sub-microgram peptide
quantities
MWCOs separate large molecules from small molecules The small molecule fraction
may be rich with signaling peptides (SP) cytokines and other small molecules involved in cell-
cell signaling Signaling peptides perform various functions in the body including cell growth
cell survival and hormonal signaling between organs [11] Individual SP contribute to different
aspects of behavior such as pain (enkephalins) [12] feeding (neuropeptide Y) [13] and blood
pressure (bradykinin) [14] MWCO separations can be used to enrich biologically important SP
and explore the peptide content from biological fluids with relatively low peptide content like
blood or cerebrospinal fluid (CSF) In a recent investigation the detection of neuropeptides and
standards in crustacean hemolymph was improved when methanol and protease inhibitors were
present before performing MWCO neuropeptide isolation The impact of methanol on MWCO
sample loss was not investigated in the study [15] In another study a large-scale mass
fingerprinting protocol of endogenous peptides from CSF used a combination of salts before
MWCO fractionation but the impact of adding salts was not discussed [16] The most
commonly used brand of MWCO in the publications and in peptidomic studies is Millipore
Therefore Millipore MWCOs (using regenerated cellulose as the membrane) were used in the
present study The purpose of this work is to provide an optimized sample preparation technique
for MWCO filtering to reduce sample loss and allow sub-microg detection of peptides using MALDI
mass spectrometry
MATERIALS AND METHODS
190
Materials and Chemicals
Water acetonitrile methanol (optima LCMS grade) and sodium chloride (995) were
purchased from Fisher Scientific (Fair Lawn NJ) The α-cyano-4-hydroxy-cinnamic acid (99)
formic acid (FA) (ge98) and bovine serum albumin (ge96) were purchased from Sigma-
Aldrich (St Louis MO) Amicon Ultra 05 mL 10000 MWCO centrifugal filters and ZipTips
packed with C18 reversed-phase resin were purchased from Millipore (Billerica MA) Trypsin-
digested bovine serum albumin (BSA) was purchased from Waters (Milford MA) Bradykinin
was purchased from American Peptide Company (Sunnyvale CA)
MALDI MS Instrumentation
An AutoFLEX III MALDI TOFTOF mass spectrometer (Bruker Daltonics Billerica
MA) was operated in positive ion reflectron mode The MALDI MS instrument is equipped with
a proprietary smart beam (Bruker Daltonics Billerica MA) laser operating at 200 Hz The
instrument was internally calibrated over the mass range of mz 500minus2500 using a standard
peptide mix Two thousand laser shots were collected per sample spot at an accelerating voltage
of 19 kV and a constant laser power using random shot selection The acquired data were
analyzed using FlexAnalysis software (Bruker Daltonics Billerica MA) Mass spectrometry
data acquisition was obtained by averaging 2000 laser shots
Molecular weight cut off separation procedure
The MWCO separations were performed using Amicon Ultra 05 mL 10000 MWCO
centrifugal filters (Billerica MA) Before MWCO separation three washing steps were
performed to remove contaminants from the filter The three washes were 500 μL of 5050
H2OMeOH 500 μL of H2O and 400 μL of the solution used for MWCO separation For the
191
100 H2O solution 1 microg of BSA or bradykinin was used for separation All the other MWCO
separation experiments used 500 ng of BSA or 100 ng or less of bradykinin The MWCO filter
was then centrifuged at 14000 rcf for 5 min at room temperature in an Eppendorf 5415 D
microcentrifuge (Brinkmann Instruments Inc Westbury NY) The filtrate was concentrated in a
Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation West Palm Beach FL)
and acidified The resulting sample was desalted according to the manufacturer using C18
ZipTips from Millipore (Billerica MA) by washing the ZipTip with three times 100 ACN
three aqueous washes of 01 FA binding the peptides from the solution and one aqueous wash
of 01 FA Peptides were then eluted from the ZipTips using 15 microL of 50 ACN in 01 FA
Matrix deposition
Equal volumes of 05 μL from the 15 μL sample solution (including standards not subject
to MWCO filtering) and α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution in 50
ACN50 H2O were mixed using a dried-droplet method and spotted on a MALDI target The
resulting droplets were allowed to air dry prior to mass spectrometry acquisition
RESULTS AND DISCUSSION
Analysis of two orders of magnitude increase for bradykinin standard
Bradykinin was selected to assess the potential peptide loss in the flow-through after
performing MWCO separation As shown in Figure 1 1 microg of bradykinin standard does not
produce a detectable signal by MALDI mass spectrometry analysis after a 10 kDa MWCO
separation in water (performed in triplicate) For comparison 1 ng of bradykinin standard
diluted to 15 microL produced an intense signal on the MALDI mass spectrometer suggesting
192
significant sample loss occurs when the target analyte is low in quantity (data not shown
performed in triplicate) Figure 1 shows that the addition of a salt in this case NaCl improves
the limits of detection and decreases sample loss when 7030 watermethanol was compared to
7030 aqueous 1 M NaClmethanol The reproducible results gave a relative standard deviation
(RSD) of 6 for peak intensity Figure 1 shows that even when starting with 1 μg of bradykinin
too much sample is lost during the MWCO separation in water to detect the remainder
However an intense signal is observed for 10 ng of bradykinin after the MWCO separation when
7030 aqueous 1 M NaClmethanol is used as an elution solvent Sample loss from zip-tipping
was estimated in triplicate by calculating the decreases in peak intensity When 10 ng of
bradykinin was used sample loss was ~41 In comparison the calculated yield for 10 ng of
bradykinin after the 7030 aqueous 1 M NaClmethanol MWCO separation and zip-tip desalting
showed an estimated sample loss of 63 meaning more loss can be attributed to sample clean-
up than MWCO filtration
A series of experiments were performed to determine if 7030 aqueous 1 M
NaClmethanol is an optimal solution to recover peptides during a MWCO separation (data not
shown) A 5050 aqueous 1 M NaClmethanol and a 5050 watermethanol elution were
performed in duplicate but signal intensity of the resulting bradykinin was poor and numerous
polymer peaks were detected in the flow through A lower salt concentration 01 M NaCl was
used but also produced greatly reduced signal when compared to aqueous 1 M NaCl To assess
the optimized methodrsquos compatibility with lower sample amounts the 7030 aqueous 1 M
NaClmethanol solution was added to 1 ng of bradykinin for MWCO separation but no signal
was obtained (data not shown) Using a neuropeptide standard the addition of methanol and
NaCl salt significantly improved the sample recovery in sub-microg amounts
193
BSA tryptic peptide mixture analysis
After demonstrating the importance of using an optimized solution for MWCO
separations with an individual peptide the new method was applied to 500 ng of BSA tryptic
digest to investigate its utility with more complex peptide mixtures Table 1 lists the BSA
tryptic peptides identified in the MALDI MS analysis from different solution conditions
processed by MWCO separation As shown in Table 1 a directly spotted BSA tryptic peptide
standard in the absence of any MWCO filtration enabled identification of 39 tryptic peptides by
accurate peptide mass measurements Once again when using 100 H2O for MWCO
separations the starting amount was doubled to 1 microg (also done with 500 ng data not shown)
However many tryptic peptides were not detected due to low signal intensities and non-optimal
elution conditions Instead of H2O a 1 M NaCl solution was used for the MWCO elution but
only two tryptic peptides were identified (Table 1) The addition of 30 methanol into the
sample before MWCO filtration produced the first increase in identified BSA tryptic peptides
The remaining data from Table 1 shows improved BSA tryptic peptide identifications as the
sample (elution) conditions were further optimized Figure 2 shows the actual mass spectra
associated with the three most promising elution solutions along with 100 H2O
The BSA tryptic peptide intensities are shown in Figure 2A and the most intense tryptic
peptide YLYEIAR mz 92749 was observed in the four different solutions shown in Figure 2B
but not in the 100 H2O or 100 1 M NaCl solutions (data not shown) In Figure 2A all mass
spectra are normalized to an intensity of 7 x 104 to illustrate two points First the MWCO
filtering step still produced sample loss regardless of the solvent conditions chosen Second
there is a noticeable increase in peptide peak intensity using the optimized solvent 6040
194
aqueous 1 M NaClmethanol (Figure 2A) Figure 2C displays a zoomed-in view of a BSA
tryptic peptide signal LKECC
DKPLLEK mz 153266 (
carbamidomethyl) observed only in
the optimized solvent The detection of the mz 153266 peptide in Figure 2C highlights the
potential gain in sample and detectable peptides by using an optimized saltMeOH combination
A non-optimized saltMeOH combination will still reduce sample loss but further minimizing
sample loss during sample preparation will always be desirable in any analytical protocol
MWCO composition
The purpose of this application note is to provide evidence of sub-microg sample loss during
MWCO separations of peptide samples and a solution to overcome this limitation The
explanation of why adding MeOH and NaCl to the sample solution provides a significant
reduction in sample loss is beyond the scope of this application note Regardless Supplemental
Table S1 is an expanded version of Table 1 showing the amino acid sequence hydrophobicity
calculated using GRAVY scores and pI of the identified peptides in this study No discernible
trend was obtained from the data The membrane of commonly used MWCO in peptidomics and
for this study is comprised of chemically treated (regenerated) cellulose which is a
polysaccharide containing β (1rarr4) linked D-glucose Glucose has numerous free hydroxyl
groups which could non-specifically adsorb peptides flowing through the MWCO The addition
of MeOH has the most significant effect on signal which could be due to disrupting the
interaction between peptides and hydroxyl groups from glucose NaCl has a less significant
effect on sample recovery compared to MeOH but a detectable reduction in sample loss is noted
This improvement in sample recovery could be analogous to the use of NaCl in
195
immunodepletion protocols to reduce non-specific binding which is accomplished by adding
150 mM NaCl [17]
Analysis of bradykinin in the presence of undigested BSA
When using MWCO for peptide isolation proteins are typically present in the samples
usually in larger amounts Figure 3 shows the effect that adding BSA protein to a 10 ng
bradykinin solution before MWCO fractionation has on the resulting recovery of bradykinin
Adding 10 μg of BSA to the optimized 7030 aqueous 1 M NaClmethanol solution slightly
decreased bradykininrsquos signal with a RSD of 10 More severe signal reduction occurred after
adding 100 microg BSA with a RSD of 2 (N=2) It is not unexpected that more signal reduction
due to sample loss would occur especially in the 100 microg BSA sample because the BSA protein
has a molar ratio of 160 BSA protein molecules to one bradykinin peptide Figure 3 shows the
usefulness of the MWCO method with samples containing large amounts of proteins
RecommendationConclusion
The present work provides solutions to reduce sample loss from the use of MWCO for
sub-microg peptide isolation with or without non-digested proteins in the sample Despite its
widespread utility significant sample loss often occurs during the MWCO fractionation step
which is particularly problematic when analyzing low-abundance peptides from limited starting
material This application note aims to reduce sample loss during MWCO separations
specifically for sub-microg peptide isolation If complex samples are processed with MWCO
separation the authors recommend eluting the sample with 6040 aqueous 1 M NaClmethanol
solution as a starting point to minimize sample loss This application note provides a viable
196
alternative for sub-microg peptide MWCO separation circumventing the need to increase the starting
material by minimizing sample loss from using a MWCO membrane-based centrifugal filter
device
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[2] DW Greening RJ Simpson Low-molecular weight plasma proteome analysis using
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[3] DW Greening RJ Simpson A centrifugal ultrafiltration strategy for isolating the low-
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637
[4] LL Manza SL Stamer AJ Ham SG Codreanu DC Liebler Sample preparation and
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[5] D Theodorescu D Fliser S Wittke H Mischak R Krebs M Walden M Ross E Eltze O
Bettendorf C Wulfing A Semjonow Pilot study of capillary electrophoresis coupled to mass
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2005 26 2797
[6] K Antwi G Hostetter MJ Demeure BA Katchman GA Decker Y Ruiz TD Sielaff LJ
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4722
[7] LP Aristoteli MP Molloy MS Baker Evaluation of endogenous plasma peptide extraction
methods for mass spectrometric biomarker discovery J Proteome Res 2007 6 571
[8] A Zougman B Pilch A Podtelejnikov M Kiehntopf C Schnabel C Kumar M Mann
Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7
386
[9] X Yuan DM Desiderio Human cerebrospinal fluid peptidomics J Mass Spectrom 2005 40
176
[10] X Zheng H Baker WS Hancock Analysis of the low molecular weight serum peptidome
using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer J Chromatogr A
2006 1120 173
[11] L Li JV Sweedler Peptides in the brain mass spectrometry-based measurement approaches
and challenges Annu Rev Anal Chem 2008 1 451
[12] GB Stefano G Fricchione Y Goumon T Esch Pain immunity opiate and opioid
compounds and health Med Sci Monit 2005 11 MS47
[13] J Jensen Regulatory peptides and control of food intake in non-mammalian vertebrates Comp
Biochem Physiol A Mol Integr Physiol 2001 128 471
197
[14] A Kuoppala KA Lindstedt J Saarinen PT Kovanen JO Kokkonen Inactivation of
bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma Am
J Physiol Heart Circ Physiol 2000 278 H1069
[15] R Chen M Ma L Hui J Zhang L Li Measurement of neuropeptides in crustacean
hemolymph via MALDI mass spectrometry J Am Soc Mass Spectrom 2009 20 708
[16] H Jahn S Wittke P Zurbig TJ Raedler S Arlt M Kellmann W Mullen M Eichenlaub H
Mischak K Wiedemann Peptide fingerprinting of Alzheimers disease in cerebrospinal fluid
identification and prospective evaluation of new synaptic biomarkers PLoS One 2011 6
e26540
[17] NA Cellar AS Karnoup DR Albers ML Langhorst SA Young Immunodepletion of high
abundance proteins coupled on-line with reversed-phase liquid chromatography a two-
dimensional LC sample enrichment and fractionation technique for mammalian proteomics J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877 79
198
Table 1 Identified BSA tryptic peptides from various MWCO separation conditions
BSA tryptic
peptide (MH+)
100
H2O 1microg
100
1 M NaCl
70
H2O
80
1 M NaCl
70
1 M NaCl
60
H2O
60
1 M NaCl
5083
5453
6894
7124
8985
9275
10345
10725
11385
11636
12496
12837
13057
13997
14157
14197
14398
14636
14798
15026
15118
15328
15547
15677
15768
16399
16678
16738
17248
17408
17477
17497
18809
18890
19019
19079
20450
21139
22479
Total 39 2 2 6 8 15 15 27
199
Figure 1 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard showing improvement over two orders of magnitude in detection limits Each MWCO
separation was performed at minimum in triplicate with representative spectrum selected for
each with a calculated RSD from the peak heights Three different amounts of bradykinin were
tested to assess the magnitude of sample loss under different MWCO solvent conditions The
top panel shows 1 microg of bradykinin standard after MWCO separation with 100 H2O elution
produced no signal The addition of 40 or 30 MeOH produced very low bradykinin signals
for both 100 ng (RSD of 28) and 10 ng (SNlt3 no RSD calculated) respectively In the
bottom two spectra each showed very large intensity but the 7030 aqueous 1 M NaClmethanol
10 ng bradykinin was processed with a 10kDa MWCO and zip-tipped and was reproducible with
200
a RSD of 6 The last spectrum was from 10 ng bradykinin (RSD of 3) which was diluted to
an equivalent volume as all the other experiments and directly spotted onto the MALDI plate
201
Figure 2 Representative MALDI mass spectra from MWCO separation of a BSA tryptic
peptide standard showing sample loss Stacked mass spectra from mz range 875-2150
normalized to 7 x 104 intensity representing the detection difference from a BSA tryptic peptide
standard from different MWCO separation conditions (A) It should be noted that when the
solvent for MWCO elution was 100 H2O 1 microg of BSA tryptic peptides was processed instead
of 500 ng A zoomed in view of the most abundant BSA tryptic peptide detected YLYEIAR
mz 92749 (B) Various percentages of MeOH produced significant signal but addition of a salt
(1 M NaCl) increases the signal which is closest to a directly spotted BSA tryptic peptide
standard A zoomed in view of a representative low intensity BSA tryptic peptide detected
LKECC
DKPLLEK mz 153266 (C) The optimized solution to be used for MWCO filtration
202
6040 aqueous 1 M NaClmethanol was the only procedure that enabled the detection of the
tryptic peptide in Figure 2C which was also detected in the directly spotted BSA tryptic peptide
standard All experiments were performed a minimum of two times with nearly identical results
) Carbamidomethyl amino acid modification
ordm) Tryptic peptide identified in three of the spectra in Figure 2A
dagger) Tryptic peptide identified in two of the spectra in Figure 2A
) Tryptic peptide identified in a single spectrum in Figure 2A
203
Figure 3 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard with a BSA protein present showing optimized solvent conditions minimized samples
losses Each experiment was performed in duplicate Two different amounts of BSA protein
were tested to assess the magnitude of sample loss caused by the presence of a protein The top
panel shows 10 microg of BSA protein and the second panel shows 100 microg of BSA protein added
while only 10 ng of bradykinin was added Detectable sample loss was observed when the BSA
protein was added but panel two shows that the amount of BSA protein was 1 x 104 greater
(equivalent to 160 fold molar excess) than bradykinin The last two spectra were controls using
a MWCO with the optimized solution in panel 3 and panel 4 using 10 ng bradykinin which was
diluted to an equivalent volume as all the other experiments and directly spotted onto the
MALDI plate
204
Supplemental Table 1 Expanded Table 1 including grand average of hydropathicity (GRAVY)
score theoretical pI and the sequence from the underlying amino acid sequence for the peptides
identified in the BSA digest The GRAVY and pI scores were obtained from ExPASy
Bioinformatics and modifications were not taken into consideration
(httpwebexpasyorgprotparam) Peptide assignments to the recorded peaks were done by
BSA
tryptic
peptide
(MH+)
GRAVY
score
Theoretical
pI
Sequence 100
H2O
1microg
100
1 M
NaCl
70
H2O
80
1 M
NaCl
70
1 M
NaCl
60
H2O
60
1 M
NaCl
5083 NA NA FGER
5453 0900 972 VASLR
6894 0267 979 AWSVAR
7124 -0950 647 SEIAHR
8985 0529 674 LcVLHEK
9275 -0071 600 YLYEIAR
10345 -0725 674 NEcFLSHK
10725 -0211 538 SHcIAEVEK
11385 0 599 ccTESLVNR
11636 0130 453 LVNELTEFAK
12496 -1250 545 FKDLGEEHFK
12837 0264 675 HPEYAVSVLLR
13057 -0582 532 HLVDEPQNLIK
13997 0567 437 TVMENFVAFVDK
14157 0567 437 TVmENFVAFVDK
14197 0058 530 SLHTLFGDELcK
14398 -0133 875 RHPEYAVSVLLR
14636 -0515 465 TcVADESHAGcEK
14798 0292 600 LGEYGFQNALIVR
15026 -0625 409 EYEATLEEccAK
15118 0207 597 VPQVSTPTLVEVSR
15328 -0617 617 LKEccDKPLLEK
15547 -0823 441 DDPHAcYSTVFDK
15677 -0085 437 DAFLGSFLYEYSR
15768 -0985 456 LKPDPNTLcDEFK
16399 -0067 875 KVPQVSTPTLVEVSR
16678 0064 437 MPCTEDYLSLILNR
16738 -1723 550 QEPERNEcFLSHK
17248 0064 437 MPcTEDYLSLILNR
17408 0064 437 mPcTEDYLSLILNR
17477 -0914 414 YNGVFQEccQAEDK
17497 -0621 410 EccHGDLLEcADDR
18809 -0537 606 RPcFSALTPDETYVPK
18890 -0567 674 HPYFYAPELLYYANK
19019 -1275 466 NEcFLSHKDDSPDLPK
19079 0044 454 LFTFHADIcTLPDTEK
20450 -0812 839 RHPYFYAPELLYYANK
21139 -0682 480 VHKEccHGDLLEcADDR
22479 -0458 423 EccHGDLLEcADDRADLAK
Total 39 2 2 6 8 15 15 27
205
mass matching with no tandem mass spectrometry performed Lower case amino acids indicate
a modification present in the peptide of carbamidomethyl (c) or oxidation (m)
206
Chapter 8
Conclusions and Future Directions
207
Summary
Comparative shotgun proteomics investigating numerous biological changes in various
species and sample media and peptidomic method development have been reported The
developed comparative shotgun proteomics based on label-free spectral counting with nanoLC
MSMS platform have been employed in mouse CSF rat CSF yeast culture and other biological
specimens Mass spectrometry (MS)-based peptidomic enhancements using ETD and improved
sample preparation methods for molecular weight cut-offs have been reported Together these
studies add to the Li Labs capabilities in comparative shotgun proteomics and expand available
methods for peptidomic research
Immunodepletion of CSF for comparative proteomics
Chapters 3 and 4 used similar methods to generate a list of differentially expressed
proteins providing insight into how Alexander disease (AxD) perturbs the proteome and how the
new prion model rat adapted scrapie (RAS) proteome varies from control rats In the GFAP
overexpressor mouse CSF study in Chapter 3 we have produced a panel of proteins with
significant up or down regulation in the CSF of transgenic GFAP overexpressor mice MS-based
proteomic study of this mouse model for AxD was consistent with the previous studies showing
elevation of GFAP in CSF The development of a modified IgY-14 immunodepletion technique
for low amounts of CSF with recommendations for future antibody depletion techniques to deal
with the unique challenges of mouse CSF was presented Modified proteomics protocols were
employed to profile mouse CSF with biological and technical triplicates addressing the
variability and providing quantitation with dNSAF spectral counting Validation of the data was
performed using both ELISA and RNA microarray data to provide corroboration with the
208
changes in the putative biomarkers This work presents numerous interesting targets for future
study in AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
Using the protocol developed in Chapter 3 we performed a similar study in RAS CSF
compared to control rat CSF Two differences in sample preparation for the rat CSF compared
to the mouse CSF are noted (1) rat IgY-7 immunodepletion was performed and (2) each rat
CSF sample was collected from one animal due to sufficient volume instead of pooling from
multiple animals for the mouse CSF sample After immunodepletion the CSF samples from
control and RAS (biological N=5 technical replicates N=3) were digested and separated using
one dimensional RP nanoLC separation We were able to identify 167 proteins with redundant
isoforms removed which was derived from total 512 proteins with a 1 FDR from all the CSF
samples Comparative analysis was performed using dNSAF spectral counting and 21 proteins
were significantly changed Our data were consistent with previous prion CSF studies showing
14-3-3 protein increased in CSF of prion infected animals Genomic analysis was also
performed and was used to cross-validate our proteomic data and numerous proteins were found
to be consistent including a novel marker related to prion disease RNAset2 (ribonuclease T2)
In summary this work provides a foundation for investigation of the perturbed proteome of a
new prion model RAS
Yeast Phosphoproteomic Comparison between Starved and Glucose Fed Conditions
This work presented a qualitative comparison of the phosphoproteome between starved
and glucose fed Saccharomyces cerevisiae (bakers yeast) A large scale IMAC enrichment of
yeast cell lysate followed by 2-D separations provided a rich source of phosphopeptides for CID
MSMS and neutral loss triggered ETD analysis using mass spectrometry A specific motif for
PKA was highlighted to show the differences in proteins identified between starved and glucose
209
fed conditions In total 477 unique phosphopeptides were identified with 06 FDR and 669
unique phosphopeptides identified with 54 FDR Phosphosite validation was performed using
a localization algorithm Ascore to provide further confidence on the site-specific
characterization of these phosphopeptides Two proteins Ssd1 and PMA2 emerged as potential
intriguing targets for more in-depth studies on protein phosphorylation involved in glucose
response
Methods for Peptide Sample Preparation and Sequencing
In this study ETD was performed to improve the sequence coverage of endogenous large
neuropeptides and labile tyrosine sulfation CCK-like neuropeptides found in the blue crab
Callinectes sapidus The intact CPRP from Callinectes sapidus was identified and characterized
with 68 sequence coverage by MS for the first time Cation assisted ETD fragmentation using
MgCl2 was also shown to be a powerful tool in de novo sequencing of sulfated neuropeptides
These endeavors into using ETD for certain neuropeptides will assist in future analysis of large
neuropeptides and PTM containing neuropeptides
In addition to ETD sequencing I presented a protocol on improving recovery of minute
quantities of peptides by adding methanol and a salt modifier (NaCl) to molecular weight cut-off
membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities
Despite its widespread utility significant sample loss often occurs during the MWCO
fractionation step which is particularly problematic when analyzing low-abundance peptides
from limited starting material This work presented a method to reduce sample loss during
MWCO separations specifically for sub-microg peptide isolation Using a neuropeptide standard
bradykinin sample loss was reduced by over two orders of magnitude with and without
210
undigested protein present The presence of bovine serum albumin (BSA) undigested protein
and the bradykinin standard lends evidence that sample loss occurs because of the MWCO and
not the presence of the protein Additionally a BSA tryptic digestion is presented where twenty-
seven tryptic peptides are identified from MALDI mass spectra after enriching with methanol
while only two tryptic peptides are identified after the standard MWCO protocol
Ongoing Projects and Future Directions
CSF Projects
Rat Adapted Scrapie and Time Course Study of Rat CSF
In ongoing experiments from the work described in Chapter 4 related to rat adapted
scrapie (RAS) we are working towards more complete proteome coverage of CSF and a time
course study of RAS After the promising results of the 1-D proteomic comparison between
RAS and control CSF we want to perform IgY immunodepletion on 1 mL of rat CSF followed
by 2-D high pH-low pH RP-RP LC-MSMS IgY immunodepletion is still required and
afterwards approximately 40 microg of low abundance protein would remain Following traditional
urea tryptic digestion (see Appendix 1) and solid phase extraction (SPE) clean-up the sample
would be dried down and reconstituted in mobile phase A (25 mM ammonium formate basic
pH) for high pH RP separation Fractions would be dried and subjected to MS analysis similar to
the work described in Chapter 4 The purpose of this work would be to increase the proteome
coverage from a few hundred to a thousand CSF proteins identified A time course study of RAS
is also desirable to gain insight into disease progression Rats at different stages will be
sacrificed and CSF will be collected The goal is to perform isobaric labeling between the time
courses with spectral counting being an alternative for relative protein expression We will use
the targets identified in Chapter 4 to track certain proteins for time course analysis Overall
211
these future projects will dig deeper into the proteome and how this novel prion model RAS
perturbs the proteins expressed in rats over time
Glycoproteomics and Peptidomic Analysis of CSF Samples from Individuals with
Alzheimerrsquos Disease
Alzheimerrsquos disease is an incurable progressive neurodegenerative disorder which results
in cognitive impairment Our aim is to provide additional biomarkers andor targets for drug
treatment Currently we used 500 microL of human CSF for non-membrane glycoprotein
enrichment (see Appendix 1) followed by 2-D high-pH-low-pH RP-RP amaZon ion trap LC-
MSMS analysis The initial work was a failure due to low amount of signal and significant
sample loss (data not shown) Through a comparison with Dr Xin Weirsquos PhD thesis we
estimated roughly 20 microg of proteins was obtained after glycoprotein enrichment and 1-D analysis
produced over 100 protein identifications (data not shown) but the additional off-line 2-D
separation and sample clean up resulted in low number of protein identifications for each fraction
analyzed by MS (data not shown) We have recently obtained 1 mL of human CSF samples
from healthy controls and individuals suffering from Alzheimerrsquos disease and plan to perform
the same experiments with double the starting amount and reduce the fractions collected from 2-
D separation from 11 to 6 In addition to the glycoproteomics anything not enriched will be
subjected to MWCO separation using the method presented in Chapter 7 The obtained peptide
sample will be subjected to Q-TOF nanoLC MSE data acquisition followed by de novo
sequencing using various programs including PEAKS and Mascot Collectively we feel this
project has great potential to lead to interesting targets and further expand the proteomic
knowledge of Alzheimerrsquos disease
GFAP Knock-in Mouse CSF
212
In a direct follow-up to Chapter 3 we aim to perform comparative proteomics on control
vs glial fibrillary acidic protein (GFAP) knock-in mouse CSF samples The sample preparation
protocol as presented in Chapter 3 will be used For quantitative proteomics we plan on
performing isobaric labeling followed by performing high energy collision induced dissociation
(HCD) on the Orbitrap Elite The MS acquisition will also perform a top ten scan where the top
ten MS peaks will be selected for tandem MS analysis In addition targeted quantitation of
specific proteins using multiple reaction monitoring (MRM) can be performed on potential
biomarkers and identified in this follow-up study and the work presented in Chapter 3 Also any
CSF samples with noticeable blood content cannot be used for the exploratory proteomics
experiments but can potentially be used for the MRM analysis and should be kept for such
experiments in the future
Large Scale Proteomics
Proteomics of Mouse Amniotic Fluid for Lung Maturation
The overall goal of this project is to determine what proteins are present in amniotic fluid
when the embryo is 175 weeks compared to amniotic fluid at 155 weeks The rationale behind
why these two time points matter was investigated through a lung explant culture where amniotic
fluid was added at 155 weeks and 175 weeks Visual maturation of lungs occurred with the
175 week amniotic fluid and lung maturation genes were up regulated in the mRNA in the lung
explant culture when compared to the 155 week amniotic fluid The compound which is
causing the maturation of the lungs is unknown and search for a secreted protein might provide a
clue to this process In order to test this hypothesis we carried out discovery proteomics
experiments The workflow involves tryptic digestion SPE and 1-D low pH nanoLC separation
coupled to an amaZon ETD ion trap for tandem MS analysis The resulting mass spectrometric
213
acquisition identified less than 100 proteins with a 1 FDR (data not published) To address the
lack of depth in the proteome coverage we purchased an IgY immunodepletion column to
remove the most abundant proteins in amniotic fluid similarly to Chapter 3 and 4 but on a larger
scale One abundant protein alpha-fetoprotein is present in amniotic fluid but absent or present
in very low concentration in CSF serum or plasma Alpha-fetoprotein is similar to albumin and
thus we ran amniotic fluid on an IgY immunodepletion column and observed significant
reduction in alpha-fetoprotein amount (data not published) After immunodepletion 2-D high
pH-low pH RP-RP will be performed followed by nanoLCMSMS on the amaZon ETD ion trap
We will perform mass spectrometry experiments using the 155 week amniotic fluid and 175
week amniotic fluid which have average protein contents ~2 mgmL We anticipate a minimum
of tenfold increase in the proteome coverage of amniotic fluid and provide meaningful
hypothesis driven biological experiments from this work
Phosphoproteomics of JNK Activation
c-Jun N-terminal kinase (JNK) is an important cellular mediator of stress activated
signaling Under conditions of oxidative stress JNK is activated resulting in the downstream
phosphorylation of a large number of proteins including c-Jun However the cellular response
to JNK activation is extremely complex and JNK activation can result in extremely different
physiological outcomes For example JNK is at the crossroads of cellular death and survival
and exhibits both pro-death and pro-survival activities These highly disparate outcomes of JNK
activation are highly contextual and depend on the type of stressor and duration of stress In the
brain JNK has been shown to be activated in models of Alzheimerrsquos disease Parkinsonrsquos
disease amyotrophic lateral sclerosis and stroke It is thought that JNK is activated in these
diseases as a result of oxidative stress and it is unknown whether this activation is pro-survival or
214
pro-death To elucidate JNK signaling in the brain we chose to analyze primary cortical
astrocytes under conditions of oxidative stress Since hydrogen peroxide is a physiologically
relevant oxidant in the brain and known to activate JNK we chose to use this to treat astrocytes
and then analyze changes to the phosphoproteome by mass spectrometry By doing this we
hope to elucidate important JNK-dependent targets involved in stress signaling in the brain and
that identifying these targets could lead to the identification of novel disease mechanisms and
potentially new therapeutic targets for neurodegeneration Specifically we plan on performing
stable isotope labeling by amino acids (SILAC) of astrocytes for control hydrogen peroxide
treated and hydrogen peroxide treated with JNK inhibitor The SILAC labeled astrocyte cell
lysate will be digested enriched using Fe-NTA IMAC and separated using 2-D high pH-low pH
RP-RP and infused into the amaZon ETD ion trap The protocol is similar to the yeast
comparative phosphoproteomics from Chapter 5 After analysis we plan on processing the data
using ProteoIQ to identify phosphoproteins with significant changes
Immunoprecipitation Followed by Mass Spectrometry
Stb3 Mass Spectrometry Analysis
Stb3 (Sin3-binding protein) has previously been shown to change location depending on
the presence or absence of glucose (Heideman Lab Dr Michael Conways Thesis) An
immunoprecipitation of Stb3 was performed in parallel to the work described in Chapter 5 Two
separate experiments were performed one with wild type Stb3 and another tagged with myc for
improved protein immunoprecipitation Myc tagging is a polypeptide tag which can be
recognized by an antibody and provide a better enrichment compared to using a Stb3 antibody
alone The myc tagging was done because of the low abundance of Stb3 and the limited amount
of protein that was pulled down by the antibody for Stb3 Immunopreciptation experiments were
215
performed for both starved and glucose fed samples All samples were tryptically digested
followed by low pH RP nanoLCMSMS Both CID and ETD were used for fragmentation
analysis of each sample (n=2) The Stb3 wild type had two peptide hits in one MS run which is
not surprising due to low Stb3 antibody affinity In contrast a significant amount of Stb3 was
pulled down from Myc tagged starved and glucose fed samples For the glucose starved
samples 22 unique peptides from Stb3 were identified whereas glucose fed samples yielded 21
unique Stb3 peptides identified (unpublished data) The identified Stb3 in the myc samples
allowed us to investigate what other proteins were pulled down that are not present in the wild
type samples
From previous work by our collaborator Dr Heideman it had been suggested that Stb3
forms a complex with Sin3 and Rpd3 Our proteomic data shows multiple significant peptide
hits for Sin3 and Rpd3 in myc tagged but not in wild type where Stb3 is only observed once
with a low Mascot score When looking at the unique proteins identified in myc tagged glucose
fed sample but not included in the wild type samples the myc fed sample contained eight unique
ribosomal proteins with numerous unique peptides for each The ribosomal proteins identified in
myc fed sample supports the novel hypothesis that in the glucose fed state the complex of Stb3
Rpd3 and Sin3 interact with ribosomal proteins Other proteins unique to myc tagged glucose
starved samples are glyceraldehyde-3-phosphate dehydrogenase 2 and transcriptional regulatory
protein UME6 Also three proteins were observed in both myc fed and starved but not in the
wild type samples including transposon Ty1-DR1 Gag-Pol polyprotein(YD11B) SWIRM
domain-containing protein (FUN19) and RNA polymerase II degradation factor 1 (DEF1) Our
proteomics data for the Stb3 immunoprecipitation experiments with starved vs glucose fed
216
samples provide exciting evidence to support previous observation made by the Heideman group
and highlight the utility of MS-based approach to deciphering protein-protein interactions
Conclusions
The majority of the work described in this dissertation revolves around sample
preparation for proteomics and peptidomics with a focus on generating biologically testable
hypotheses In the area of neuropeptides CPRP was fully sequenced analytical methods were
transformed for use in an ETD ion trap and sub-microg peptides can now be analyzed by mass
spectrometry after MWCO separation In the field of comparative proteomics comparisons
between mouse CSF in GFAP overexpressors and control or rat adapted scrapie model and
control CSF yeast fed vs glucose starved cell extract have all been presented Collectively this
thesis has developed new techniques for neuropeptide sample preparation and presented
numerous comparative proteomic analyses of various biological samples and how the proteomes
are dynamically perturbed by various treatments and disease conditions
217
Appendix 1
Protocols for sample preparation for mass spectrometry based
proteomics and peptidomics
218
Small Scale Urea Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 8 μL of urea solution
(400mg05mL) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Allow to digest overnight in 37degC water bath
10 Acidify with 10μL 10 formic acid
11 Perform solid phase extraction using tips dependent of sample amount
a Sub-5μg amounts ndash Millipore Ziptips
b 5-75μg amounts ndash Bond-Elut tips from Agilent (OMIX tips)
12 Dry down in Speedvac as needed for experiment
219
Small Scale ProteaseMAX Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 2 μL of 1 of
ProtesaeMAX (Promega) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Add 1 μL ProteaseMAX and let sit for 3-4 hours
10 Acidify with 2μL 10 formic acid
11 Dry down in Speedvac as needed for experiment
220
Large Scale Urea Tryptic Digestion (mg of proteins)
1 All solutions are in a 50 mM NH4HCO3 buffer (395 mgmL)
2 Add 20μL of 05M Dithiothreitol (DTT) (77mg100μL) and 160μL of urea solution
(400mg05mL) to sample
3 Allow sample to denature 45-60 minutes in a 37degC water bath
4 Add 54μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
5 Quench reaction with 20μL of DTT solution
6 Dilute with 14mL of NH4HCO3 solution
7 Add 100μg of trypsin
8 Allow to digest overnight in 37degC water bath
9 Acidify sample with 100μL of 10 formic acid
10 Perform solid phase extraction with Hypersep C18 Cartridges with 100 mg of C18
bead volume (Thermo)
11 Dry down with the Speedvac as needed for experiment
221
Fe-NTA Preparation from Ni-NTA Beads
1 Centrifuge (5 min at 140 rcf) and use a magnet to keep beads in place as supernatant
is removed
2 Wash 3 times with 800μL of H2O (using same technique of centrifuging and using
magnet to keep beads in places as supernatant is removed)
3 Add 800μL of 100mM EDTA (372mgmL) in a 50mM NH4HCO3 (395mgmL)
buffered solution (pH around 8) and vortex for 30 minutes to chelate the Ni
centrifuge and remove supernatant
4 Wash 3 times with 800μL of H2O
5 Add 800μL of 10mM FeCl3 hexahydrate (27mgmL) and vortex for 30 minutes to
bind Fe to beads centrifuge and remove supernatant
6 Wash 3 times with 800μL H2O
7 Resuspend in a storage solution of 111 ACNMeOH001 Acetic Acid (1mL total)
222
Fe-NTA IMAC Phospho-enrichment
1 Use wash solution (80ACN 01Formic Acid) to rinse beads vortex 1 minute
centrifuge and remove supernatant
2 Add sample (around 500μL in 80ACN 01Formic Acid) and vortex 40 minutes to
allow sample to bind dispose of supernatant after centrifuging
3 Wash 3 times with 200μL of wash solution discard supernatant
4 Add 200μL of elution solution (5050 ACN5Ammonium Hydroxide) vortex 15
minutes and save supernatant
5 Add 200μL of elution solution vortex 10 minutes and save supernatant
6 Wash 2 time with wash solution (collect supernatant of first wash)
7 If reusing store in 1mL solution of 111 ACNMeOH001 Acetic Acid
223
High pH Off-line Separation
1) In general a minimum of 20 microg of peptides are needed to gain any benefit
from off-line 2D fractionation It is better to inject 100 microg of peptides on
column
2) Use a Gemini column or a similar column that can handle pH=10 and for this
protocol a 21 mm x 150 mm column was used
3) Prepare ldquobuffer Ardquo 25 mM NH4 formate pH=10 Also prepare ldquobuffer Brdquo
4) Dry down desired sample and reconstitute in buffer A
5) Inject based off sample loop (current Alliance HPLC has a 50 microL sample
loop)
6) Run gradient at bottom of the page collecting fractions every 3 minutes except
for the 1st minute which is the void volume
7) Optional If you want to reduce instrument time you can combine fractions 1
with 12 2 with 13 etc until 11 with 22
Time Mobile phase A Mobile phase B Flow Rate
05mlmin
0 98 2 05 mLmin
65rsquo 70 30 05 mLmin
65rsquo1rdquo 5 95 05 mLmin
70 5 95 05 mLmin
224
Non Membrane Glycoprotein Enrichment
1 For protocols involving membrane glycoprotein enrichment see Dr Xin Weirsquos
thesis
2 Add 75 microL of Wheat germ agglutinin (WGA) bound to agarose beads and 150 microL
of Concanavalin A (ConA) to a Handee spin cup (Thermo) Wash 2 times with
lectin affinity chromatography (LAC) binding buffer (015 M NaCl 002 M Tris-
HCl 1 mM CaCl2 pH=74) centrifuging at 400 g for 30 seconds
3 Place stopper on bottom of spin cup and add 200 microg of sample (500 microL for CSF)
Bring up to 300 microL using lectin LAC binding buffer
4 Incubate for 1 hour with continuous mixing at room temperature
5 Centrifuge at 400 g for 30 seconds
6 Perform two more 300 microL LAC binding washes followed by centrifugation
7 Add 300 microL LAC eluting buffer (0075 M NaCl 001 M Tris-HCl 02 M alpha-
methyl mannoside 02 M alpha-methyl glucoside and 05 M acetyl-D-
glucosamine) vortex for 10 minutes (have stopper in place while vortexing)
centrifuge and collect
7 Add another 300 microL LAC eluting buffer centrifuge and collect
225
MWCO separation for Sub-microg peptide concentrations
1 Wash molecular weight cut-off (MWCO) with 5050 waterMeOH centrifuge at
14000 g for 5 minutes for 10 kDa filter (time will vary Millipore Amicon Ultra
filters)
2 Wash with 100 water centrifuge at 14000 g for 5 minutes
3 Add methanol to the sample to get the concentration to 30 methanol and add
salt to achieve a roughly 05 M NaCl before adding the sample to the MWCO
4 Centrifuge at 14000 for 10 minutes collect flow through
226
Immunoprecipitation
Modified from Thermo Fisher Scientific Classic IP Kit
1 10 microL Polyclonal antibody added to 1 microg protein standard in a Handee spin cup
(Thermo) diluted with 1x TBS buffer to 400 microL and incubated overnight at on
shakerend-over-end rotator
2 Wash Protein GA beads with 2x 200microL TBS buffer and added to the
antibodysample for 15 hours at 4oC
3 Centrifuge at 400 g for 30 seconds and discard flow through
4 Wash 3x with 100microL TBS buffer centrifuge at 400 g for 30 seconds and discard
flow through
5 Wash with 1x conditioning solution (neutral pH buffer) centrifuge at 400 g for 30
seconds and discard flow through
6 Eluted with elution buffer (2x100microL) centrifuge at 400 g for 30 seconds and
collect flow through
227
C18 Solid Phase Extraction (SPE)
1 Determine amount of peptides for SPE ge5 microg use Ziptips from Millipore If
between 5-75 microg use OMIX from Agilent 100 microL version and if ge75 microg use SPE
cartridges such as 100 mg Hypersep from Thermo
2 Ensure no detergents are in the sample and it is acidified
3 The three SPE procedures all use the same sets of solutions only volumes vary
4 3x Wetting solution (100 ACN) for 60 microL OMIX (20 microL ziptip and 15 mL for
100 mg cartridge)
5 3x Wash solution (01 formic acid in 100 H2O) same volumes as 4
6 Draw up the sample about 10 times minimum (generally 30-40 is preferred)
without letting the bead volume dry out
7 1X Wash solution same volumes as 4
8 Back load the eluting solution 01 formic acid in 5050 H2OACN into the
Ziptips (15 microL) or OMIX (50 microL) tips For the SPE cartridge add 700 microL of
eluting solution
9 Dry down and prepare for next step in sample preparation
228
Laser Puller Programs and Protocols
1) Obtain about two feet of Fused-silica capillary with 75 μm id and 360 μm od
2) Wash with methanol and then air dry using the bomb
3) Cut into one foot or desired length
4) Use a lighter to burn the middle portion (about an inch in length) of the tubing
5) Remove the ash by rinsing with methanol and rubbing with a Kimwipe
6) Make sure the laser puller has been on for at least 30 minutes before use to allow
for the instrument to warm up
7) Place capillary in instrument with the burnedexposed portion in the center
making sure that the length of the capillary is pulled taut
8) Enter desired program (next page) and press pull
229
Laser Puller Programs
Program 99 (default lab program)
Heat Filament Velocity Delay Pull
250 0 25 150 15
240 0 25 150 15
235 0 25 150 15
245 0 25 150 15
Program 97 (developed for larger inner diameter tips)
Heat Filament Velocity Delay Pull
230 - 25 150 -
220 - 25 150 -
215 - 25 150 8
230
On column Immunodepletion (serum plasma CSF amniotic fluid)
1) Prepare buffer A ldquoDilution Bufferrdquo 10 mM Tris-HCl pH=74 150 mM NaCl
2) Prepare buffer B ldquoStripping Bufferrdquo 01 M Glycine-HCl pH=25
3) Prepare buffer C ldquoNeutralization Bufferrdquo 100 mM Tris-HCl pH=80
4) Determine loading amount (LC1=~1000 ug of serum or plasma less for CSF due
to the increased amount of albumin percentage in CSF)
5) Add Dilution buffer to sample before injection and ensure the sample is proper
pH (~7)
6) Use gradient below
Time A B C Flow Rate
(mLmin)
0rsquo 100 0 0 02
4rsquo59rdquo 100 0 0 02
5rsquo 100 0 0 05
8rsquo59rdquo 100 0 0 05
9rsquo 0 100 0 05
22rsquo 0 100 0 05
22rsquo1rdquo 0 0 100 05
39rsquo 0 0 100 05
7) Store the column in 1x dilution buffer until next use
231
Small Scale Immunodepletion (100 microL of CSF)
1) Use 75 microg (per 100 microL CSF) of Sigma Seppro IgY-14 or IgY-7 bead slurry
2) Add an equal volume of 2x dilution buffer (20 mM Tris-HCl pH=74 300 mM
NaCl) to the starting amount of CSF
3) Add to a spin cup with a filter and allow to mix for 30 minutes
4) Centrifuge at 400 g for 30 seconds and collect the flow through
5) Add 50 microL of 1x dilution buffer mix for 5 minutes centrifuge at 400 g for 30
seconds and collect the flow through
6) Use 1x stripping washes (01 M Glycine-HCl pH=25) centrifuge as before and
discard Repeat four times
7) Use 1x neutralization buffer (100 mM Tris-HCl pH=80) centrifuge as before
and discard Repeat two times
8) Store the beads in the spin column in 1x dilution buffer until next use
232
Alliance Maintenance Protocol
Seal Wash
10 methanol no acetonitrile This wash cleans behind the pump-head seals to
ensure proper lubrication Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime SealWsh gt Start
2 Press Stop after 5 minutes
Prime Injector
10 methanol for maintenance high organic solvent for dirty runs (eg 95
acetonitrile) Done before injecting any real samples to ensure no bubbles are
present in the injector Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime NdlWsh gt Start
2 After completion press Close
Purge Injector
Solvent is dependent on run Run this protocol at beginning of experiments each day
Minimum once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Navigate Direct Function gt 4 Purge Injector gt OK
3 Set Sample loop volumes 60 leave Compression Check unchecked gt OK
Prime Solvent Pumps
Solvent is dependent on run If solvents are changed run this protocol Minimum
once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys choose composition A type 100 Enter x4
3 Navigate Direct Function gt 3 Wet Prime gt OK
4 Set Flow Rate 7000 mLmin Time 100 min gt OK
5 Repeat for all changedactive solvent pumps
Condition Column
Dependent on user Use starting conditions for run
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys type starting solvent compositions for run
3 Navigate Direct Function gt 6 Condition Column gt OK
4 Set Time as desired
233
Appendix 2
List of Publications and Presentations
234
PUBLICATIONS
ldquoDiscovery and characterization of the crustacean hyperglycemic hormone precursor related
peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes
sapidus using multiple tandem mass spectrometry techniquesrdquo Hui L Cunningham R Zhang
Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
ldquoInvestigation and reduction of sub-microgram peptide loss using molecular weight cut-off
fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J Wellner D Li L
Journal of Mass Spectrometry In Press
ldquoMass spectrometry-based proteomics and peptidomics for systems biology and biomarker
discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse models of
Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P Messing A Li
L Journal of Proteome Research Submitted
ldquoInvestigation of the differences in the phosphoproteome between starved vs glucose fed
Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M Heideman
W Li L In preparation
ldquoIdentification of astrocytic JNK targets by phosphoproteomics using mass spectrometryrdquo
Cunningham R Dowell J Wang J Wellner D Li L Milhelm M In preparation
ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A Cunningham R Wellner
D Wang J Ma D Li L Aiken J In preparation
235
INVITED SEMINARS AND CONFERENCE PRESENTATIONS
Robert Cunningham Davenne Mayour and Shoshanna Coon ldquoMorphology and Thermal
Stability of Monolayers on Porous Siliconrdquo The 231th
ACS National Meeting 2006 Atlanta
GA
Robert Cunningham Xin Wei Paige Jany Albee Messing and Lingjun Li ldquoMass
Spectrometry-Based Analysis of Cerebrospinal Fluid Peptidome and Proteome for Biomarker
Discovery in Alexander Diseaserdquo The 57th
ASMS Conference 2009 Philadelphia PA
Robert Cunningham ldquoWhat to Expect in a PhD Graduate Schoolrdquo Invited seminar University
of Northern Iowa 2010 Cedar Falls IA
Robert Cunningham Dustin Frost Albee Messing and Lingjun Li ldquoInvestigation of an
Optimized ProteaseMAX Assisted Trypsin Digestion of Human CSF for Pseudo-SRM
Quantification of GFAP and Identification of Biomarkersrdquo The 58th
ASMS Conference 2010
Salt Lake City UT
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo Oral presentation at Pittcon Conference and Expo 2011 2011 Atlanta
GA
Robert Cunningham Michael Conway Daniel Wellner Douglas Grunwald Warren
Heideman and Lingjun Li ldquoDevelopment of optimized phosphopeptide enrichment methods for
comparison of starved and glucose fed yeast Saccharomyces cerevisiaerdquo The 59th
ASMS
Conference 2011 Denver CO
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo 2011 Wisconsin Human Proteomics Symposium 2011 Madison WI
i
Acknowledgements
I would like to acknowledge the support and guidance from professors colleagues
and friends at the University of Wisconsin-Madison who are indispensable to this thesis
First I would like to express my deep gratitude to my advisor Prof Lingjun Li for
allowing me the freedom to chase scientific endeavors all while offering her constant
guidance assistance and support through my PhD study Her constant energy and
enthusiasm in research have led by example in performing research and inspired me to
make the most of the time given to me Dr Li encouraged me to take on challenging
projects apply for awards travel and present my research to the larger scientific
community None of my work would be achieved without her and I want to thank Dr Li
for her support during these years
I would also like to thank the members of my committee Dr Lingjun Li Dr
Albee Messing Dr Lloyd Smith Dr Warren Heideman and Dr Tim Bugni I truly
appreciate the willingness of these professors to take time out of their busy schedules to
serve as members of my committee
I have benefited greatly from previous members of the Li Lab In particular I
would like to thank Dr James Dowell Dr Xin Wei Dr Robert Sturm and Dr Limei
Hui for their patient and valuable suggestions in my research and also teaching me
valuable experimental skills how to perform general shotgun proteomics and how to use
several instruments Specifically I would like to thank Daniel Wellner who has worked
with me on numerous projects over the past 2 years and has been a constant in my
research life I also want to thank my wonderful current colleagues Jingxin Wang Tyler
ii Greer Chris Lietz Chenxi Jia Dustin Frost Di Ma Hui (Vivian) Ye Nicole Woodards
and Claire Schmerberg for their collaboration in many challenging research projects and
fruitful discussions on various research areas There are too many people to thank each
one individually but every member of the Li lab has in some way contributed to my
learning experience Beyond research work their friendship also made my life here in
Madison much more enjoyable
I would also like to thank our collaborators Dr Albee Messing Dr Warren
Heideman Dr Xin Sun and Dr James Dowell It is my great pleasure to have the
opportunities to work with these amazing people and gain precious experience I have
learned so much from them and their achievements in the field have inspired me to strive
to do the best I could
Furthermore I would like to thank Gary Girdaukas and Dr Cameron Scarlett at
School of Pharmacy for the access of the MALDI-FTMS and Bruker amaZon ion trap
instruments
In particular I wish to thank my family my Mom and Step-Dad for raising me
and my Dad for always being there for me They all supported me in my decision to
pursue science and specifically a career in chemistry I would like to thank my Sister
who grew up with me and always led by example in academics Most importantly I
would like to thank my wife Na Liu for her constant support She has inspired and
helped me finish my PhD and always encouraged me to be the best I could be To them
I dedicate this thesis
iii
Table of Contents
Page
________________________________________________________________________
Acknowledgements i
Table of Contents iii
Abstract iv
Chapter 1 Introduction brief background and research summary 1
Chapter 2 Mass spectrometry-based proteomics and peptidomics for
biomarker discovery and the current state of the field 15
Chapter 3 Protein changes in immunodepleted cerebrospinal fluid from
transgenic mouse models of Alexander disease detected
using mass spectrometry 73
Chapter 4 Genomic and proteomic profiling of rat adapted scrapie 110
Chapter 5 Investigation of the differences in the phosphoproteome
between starved vs glucose fed Saccharomyces cerevisiae 139
Chapter 6 Use of electron transfer dissociation for neuropeptide
sequencing and identification 166
Chapter 7 Investigation and reduction of sub-microgram peptide loss
using molecular weight cut-off fractionation prior to
mass spectrometric analysis 187
Chapter 8 Conclusions and future directions 206
Appendix 1 Protocols for sample preparation for mass spectrometry
based proteomics and peptidomics 217
Appendix 2 Publications and presentations 233
_______________________________________________________________________
iv
Mass Spectrometry Applications for Comparative Proteomics and
Peptidomic Discovery
Robert Stewart Cunningham
Under the supervision of Professor Lingjun Li
At the University of Wisconsin-Madison
Abstract
In this thesis multiple biological samples from various diseases models or
treatments are investigated using shotgun proteomics and improved methods are
developed to enable extended characterization and detection of neuropeptides In general
this thesis aims to expand upon the rapidly evolving field of mass spectrometry (MS)-
based proteomics and peptidomics by primarily enhancing small scale sample analysis
A review of the current status and progress in the field of biomarker discovery in
peptidomics and proteomics is presented To this rapidly expanding body of literature
our critical review offers new insights into MS-based biomarker studies investigating
numerous biological samples methods for post-translational modifications quantitative
proteomics and biomarker validation Methods are developed and presented including
immunodepletion for small volume cerebrospinal fluid (CSF) samples for comparison of
the CSF proteomes between an Alexander disease transgenic mouse model with
overexpression of the glial fibrillary acidic protein and a control animal This thesis also
covers the application of the small scale immunodepletion of CSF for comparative
proteomic analysis of a novel rat adapted scrapie (RAS) model for prion disease and
v
compares the RAS CSF proteome to control rat CSF using MS Large scale
phosphoproteomics of starved vs glucose fed yeast is presented to better understand the
phosphoproteome changes that occur during glucose feeding Method development for
neuropeptide analysis is expanded upon using electron transfer dissociation (ETD)
fragmentation to successfully sequence for the first time the crustacean hyperglycemic
hormone precursor-related peptide (CPRP) from the blue crab Callinectes sapidus In
addition a method for ETD sequencing of sulfonated neuropeptides using a magnesium
salt adduct in an ion trap mass spectrometer is reported This thesis also reports on a
method for sub-microg peptide isolation when using a molecular weight cut-off filtration
device to improve sample recovery by over 2 orders of magnitude All the protocols used
throughout the work are provided in an easy to use step-by-step format in the Appendix
Collectively this body of work extends the capabilities of mass spectrometry as a
bioanalytical tool for shotgun proteomics and expands upon methods for neuropeptide
discovery and analysis
1
Chapter 1
Introduction Brief Background and Research Summary
2
Abstract
Mass spectrometry based comparative proteomics and improved methods for
neuropeptide discovery have been reported in this thesis A very brief introduction of crustacean
neuropeptidomics is covered in this chapter followed by Chapter 2 which covers in great detail
comparative proteomics using mass spectrometry with an emphasis on biomarker discovery
Chapter 3 reports a novel immunodepletion technique used for comparative proteomics between
glial fibrillary acidic protein (GFAP) overexpressor and control mouse cerebrospinal fluid (CSF)
Chapter 4 involves rat CSF comparative proteomics between rat adapted scrapie and control
animals Chapter 5 details comparisons of phosphoproteomes in Saccharomyces cerevisiae
(Bakerrsquos yeast) between starved vs glucose fed conditions Chapter 6 investigates the use of
electron transfer dissociation (ETD) for kDa sized neuropeptides and neuropeptides with tyrosine
sulfonation Chapter 7 presents a molecular weight cut-off (MWCO) protocol to allow sub-microg
peptides to be recovered Chapter 8 provides a conclusion to this thesis and outlines future
directions for certain projects
3
Background
Mass spectrometry (MS) requires gas phase ions for experimental measurement and
intact large nonvolatile biomolecules proved difficult to be analyzed by electron ionization or
chemical ionization until the invention of two soft ionization techniques matrix-assisted laser
desorptionionization (MALDI)1 and electrospray ionization (ESI)
2 ESI and MALDI are the
two most common soft ionization techniques for mass spectrometry Once ionized molecules
such as peptides or proteins can be separated by their mass to charge ratios (mz) using various
mass analyzers manipulating electric andor magnetic fields ESI and MALDI mass
spectrometric techniques have become central analytical methods in biological sciences because
they are suitable for nonvolatile thermally labile analytes in femtomole quantities3 ESI allows
the coupling of high pressure liquid chromatography and the constant flow of solvent is
electrically charged at the outlet to produce a Taylor cone4 During desolvation the Raleigh
limit is reached and a coulombic explosion occurs commonly producing multiply charged ions
A modification of ESI is nanoLC-ESI which operates at nLmin flow rates to analyze low sample
amounts such as proteins or peptides in biological samples5 NanoLC-ESI uses less solvent as
the source because of the reduced flow rate of standard LCMS with an ESI source and nanoLC-
ESI is the predominate inlet for shotgun proteomic ESI MS experiments6 Conversely MALDI
can use sub-microL volume of sample and be co-crystallized with an ultraviolet absorbing organic
matrix that is obliterated using a laser at appropriate wavelength to generate gas phase ions
Alternatively MALDI has the unique capability to work with tissue samples and ionize in the
solid state instead of liquid like ESI
4
Mass analyzers require an operating pressure between 10-4
-10-10
Torr to allow proper ion
transfer and separationisolation of the ion based upon its mz Numerous mass analyzers are
currently available and each have their own strengths and weaknesses as shown in Figure 1 The
biomolecules are separated by the mass analyzers and detected without fragmentation which is
termed MS analysis Following MS detection tandem mass spectrometry (MSMS) of the
original precursor ion can be performed to provide additional structural information such as a
ladder sequence of amino acids for peptides Numerous fragmentation techniques are available
for proteomics such as collision induced dissociation (CID) electron transfer dissociation (ETD)
or high energy collision induced dissociation (HCD) Each of these fragmentation techniques
have their own benefits in proteomics experiments and are discussed in depth in Chapter 2 The
background and current status for comparative proteomics with specific emphasis on biomarker
analysis are covered in Chapter 2
Neuropeptidomic Method Development in the Crustacean Model System
Utilizing Mass Spectrometry
Chapter 6 and 7 focus on sample preparation and ETD fragmentation techniques to
characterize neuropeptides in the neuroendocrine organs in the crustacean nervous system
Neuropeptides are short chains of amino acids which are a diverse class of cell-cell signaling
molecules in the nervous system Neuropeptides have been investigated for being involved in
numerous physiological processes such as memory7 learning
8 depression
9 pain
10 reward
11
reproduction12
sleep-wake cycles13
homeostasis14
and feeding15-17
Figure 2 depicts how
neuropeptides are synthesized as pre-prohormones in the rough endoplasmic reticulum and
5
packaged in the Golgi apparatus After being packaged these pre-prohormones are processed
into bioactive peptides within the vesicle which is occurring during vesicular transport down an
axon and released into the synaptic cleft Secreted neuropeptides stimulate the post synaptic
neurons by interacting with G-protein coupled receptors at the chemical synapse
The crustacean model nervous system is well-defined neural network which has been
used in neurobiology and neuroendocrine (neurosecretory) research and thus lends itself for
studying neuromodulation18-22
Figure 3 shows the locations of several neuroendocrine organs in
the crustacean Callinectes sapidus including the sinus gland (SG) which is studied in Chapter 6
The Lingjun Li lab has focused on neuropeptide discovery and function in crustacean
neuroendocrine organs using mass spectrometry23-25
The work presented in Chapters 6 and 7
expand on sample preparation and analytical tools to further investigate the neuropeptidome
Research Overview
Comparative Proteomics of Biological Samples
Chapter 2 provides a thorough review of comparative proteomics and biomarker analysis
using mass spectrometry The scientific community has shown great interest in the field of mass
spectrometry-based proteomics and peptidomics for its applications in biology Proteomics
technologies have evolved to generate large datasets of proteins or peptides involved in various
biological and disease progression processes producing testable hypotheses for complex
biological questions This chapter provides an introduction and insight into relevant topics in
proteomics and peptidomics including biological material selection sample preparation
separation techniques peptide fragmentation post-translational modifications quantification
6
bioinformatics and biomarker discovery and validation In addition current literature and
remaining challenges and emerging technologies for proteomics and peptidomics are discussed
Chapter 3 investigates comparative proteomics between a GFAP overexpressor mouse
model and a control mouses cerebrospinal fluid (CSF) CSF is a low protein content biological
fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in
direct contact with the brain but consist of very abundant proteins similar to serum which require
removal A modified IgY-14 immunodepletion treatment is presented to remove abundant
proteins such as albumin to enhance analysis of the low volumes of CSF that are obtainable
from mice These GFAP overexpressor mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we present the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates are performed to address animal variability as well as reproducibility in mass
spectrometric analysis Relative quantitation is performed using distributive normalized spectral
abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins with
significant changes in the CSF of GFAP transgenic mice are identified with validation from
ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
Chapter 4 explores the CSF proteomics of a novel prion model called rat adapted scrapie
(RAS) A similar IgY immunodepletion technique is presented as in Chapter 3 and is similarly
used to reduce the abundant proteins in CSF CSF from control and RAS (biological N=5
technical replicates N=3) were digested and separated using one dimensional reversed-phase
nanoLC separation In total 512 proteins 167 non-redundant protein groups and 1032 unique
peptides are identified with a 1 FDR Comparative analysis was done using dNSAF spectral
7
counting and 21 proteins were significantly up or down-regulated The proteins are compared to
the 1048 differentially regulated genes and additionally compared to previously published
proteins showing changes consistent with other prion animal models Of particular interest is
RAS specific proteins like RNAseT2 which is not up-regulated in mouse prion disease but is
designated as upregulated in both the genomic and proteomics data for RAS
Chapter 5 explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Previous work by the
Heideman lab investigated the transcriptional response to fresh glucose in yeast26
Kinases such
as protein kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose
response so we described a large scale phosphoproteomic MS based study in this chapter
Yeast cell extract was digested and phosphopeptides were enriched by immobilized metal
affinity chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase
(RP)-RP separation The low pH separation was infused directly into an ion trap mass
spectrometer with neutral loss triggered ETDfragmentation Specifically the CID fragmentation
can cause a neutral loss of 98z from the H3PO4 and can produce an incomplete fragmentation
pattern and lead to ambiguous identification so when the neutral loss is detected ETD MSMS
fragmentation is performed The neutral loss triggered ETD fragmentation is included in this
study to improve phosphopeptide identifications In total 477 phosphopeptides are identified
with 06 FDR and 669 phosphopeptides identified with 54 FDR Motif analysis of PKA and
phosphosite validation are performed as well
8
The future of comparative proteomics investigating small sample amounts or PTMs is
promising Further advances in enrichment separations science mass spectrometry
analyzersdetectors and bioinformatics will continue to create more powerful tools that enable
digging deeper in proteomics with both small (mouse CSF) and large (cell extracts) sample
amounts
Methods for Neuropeptide Analysis Using ETD fragmentation and Sample
Preparation
Chapter 6 investigates the utility of ETD fragmentation to sequence endogenous large
neuropeptides and labile tyrosine sulfation in the crustacean Callinectes sapidus The sinus
gland (SG) of the crustacean is a well-defined neuroendocrine site that produces numerous
hemolymph-borne agents including the most complex class of endocrine signaling moleculesmdash
neuropeptides One such peptide is the crustacean hyperglycemic hormone (CHH) precursor-
related peptide (CPRPs) which was not previously sequenced Electron transfer dissociation
(ETD) is used for sequencing of intact CPRPs due to their large size and high charge state In
addition ETD is used to sequence a sulfated peptide cholecystokinin (CCK)-like peptide in the
lobster Homarus americanus using a salt adduct Collectively this chapter presents two
examples of the utility of ETD in sequencing neuropeptides with large molecular weight or with
labile modifications
Chapter 7 reports a protocol on improving recovery of minute quantities of peptides by
adding methanol and a salt modifier (NaCl) to molecular weight cut-off membrane-based
centrifugal filters (MWCO) to enrich sub-microgram peptide quantities In some biological
9
fluids such as CSF the endogenous peptide content is very low and using pure water to perform
the MWCO separation produces too much sample loss Using a neuropeptide standard
bradykinin sample loss is reduced over two orders of magnitude with and without undigested
protein present The presence of bovine serum albumin (BSA) undigested protein and the
bradykinin standard lends evidence that sample loss occurs because of the MWCO and not the
presence of the protein Additionally a BSA tryptic digestion is presented where twenty-seven
tryptic peptides are identified from MALDI mass spectra after enriching with methanol while
only two tryptic peptides are identified after the standard MWCO protocol The strategy
presented in Chapter 7 greatly enhances recovery from MWCO separation for sub-microg peptide
samples
10
References
1 Tanaka K Waki H Ido Y Akita S Yoshida Y Yoshida T Matsuo T Protein and polymer analyses up to mz 100 000 by laser ionization time-of-flight mass spectrometry Rapid Communications in Mass Spectrometry 1988 2 (8) 151-153
2 Fenn J B Mann M Meng C K Wong S F Whitehouse C M Electrospray ionization for mass spectrometry of large biomolecules Science 1989 246 (4926) 64-71
3 Domon B Aebersold R Mass spectrometry and protein analysis Science 2006 312 (5771) 212-7
4 Whitehouse C M Dreyer R N Yamashita M Fenn J B Electrospray interface for liquid chromatographs and mass spectrometers Anal Chem 1985 57 (3) 675-9
5 Wilm M Mann M Analytical properties of the nanoelectrospray ion source Anal Chem 1996 68 (1) 1-8
6 Karas M Bahr U Dulcks T Nano-electrospray ionization mass spectrometry addressing analytical problems beyond routine Fresenius J Anal Chem 2000 366 (6-7) 669-76
7 Gulpinar M Yegen B The physiology of learning and memory Role of peptides and stress Curr Protein Pept Sci 2004 5 (6) 457-473
8 Ogren S O Kuteeva E Elvander-Tottie E Hokfelt T Neuropeptides in learning and memory processes with focus on galanin In Eur J Pharmacol 2010 Vol 626 pp 9-17
9 Strand F Neuropeptides general characteristics and neuropharmaceutical potential in treating CNS disorders Prog Drug Res 2003 61 1-37
10 Li W Chang M Peng Y-L Gao Y-H Zhang J-N Han R-W Wang R Neuropeptide S produces antinociceptive effects at the supraspinal level in mice Regul Pept 2009 156 (1-3) 90-95
11 Wu C-H Tao P-L Huang E Y-K Distribution of neuropeptide FF (NPFF) receptors in correlation with morphine-induced reward in the rat brain Peptides 2010 31 (7) 1374-1382
12 Tsutsui K Bentley G E Kriegsfeld L J Osugi T Seong J Y Vaudry H Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin new key neuropeptides controlling reproduction J Neuroendocrinol 2010 22 (7) 716-727
13 Piper D C Upton N Smith M I Hunter A J The novel brain neuropeptide orexin-A modulates the sleep-wake cycle of rats Eur J Neurosci 2000 12 (2) 726-730
14 Tsuneki H Wada T Sasaoka T Role of orexin in the regulation of glucose homeostasis - Tsuneki - 2009 - Acta Physiologica - Wiley Online Library Acta Physiol 2010
15 Kageyama H Takenoya F Shiba K Shioda S Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding Neuropeptides 2010 44 (2) 133-138
16 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide family of aplysia J Neurosci 2002 22 (17) 7797-7808
11
17 Jensen J Regulatory peptides and control of food intake in non-mammalian vertebrates Comp Biochem Physiol Part A Mol Integr Physiol 2001 128 (3) 469-477
18 Billimoria C P Li L Marder E Profiling of neuropeptides released at the stomatogastric ganglion of the crab Cancer borealis with mass spectrometry J Neurochem 2005 95 (1) 191-199
19 Cape S S Rehm K J Ma M Marder E Li L Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster Homarus americanus J Neurochem 2008 105 (3) 690-702
20 Cruz Bermuacutedez N D Fu Q Kutz Naber K K Christie A E Li L Marder E Mass
spectrometric characterization and physiological actions of GAHKNYLRFamide a novel FMRFamide‐like peptide from crabs of the genus Cancer J Neurochem 2006 97 (3) 784-799
21 Grashow R Brookings T Marder E Reliable neuromodulation from circuits with variable underlying structure Proc Natl Acad Sci USA 2009 106 (28) 11742-11746
22 Ma M Szabo T M Jia C Marder E Li L Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins Peptides 2009 30 (9) 1660-1668
23 Chen R Hui L Cape S S Wang J Li L Comparative Neuropeptidomic Analysis of Food Intake via a Multi-faceted Mass Spectrometric Approach ACS Chem Neurosci 2010 1 (3) 204-214
24 Li L Sweedler J V Peptides in the brain mass spectrometry-based measurement approaches and challenges Annu Rev Anal Chem 2008 1 451-483
25 Ma M Wang J Chen R Li L Expanding the crustacean neuropeptidome using a multifaceted mass spectrometric approach J Proteome Res 2009 8 (5) 2426-2437
26 Slattery M G Heideman W Coordinated regulation of growth genes in Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
12
Figure 1 Capabilities and performances of certain mass analyzers Check marks indicate
availability check marks in parentheses indicate optional + ++ and +++ indicate possible or
moderate goodhigh and excellentvery high respectively Adapted with permission from
reference 3
13
Figure 2 Synthesis processing and release of neuropeptides (a) Cartoon showing two
interacting neurons (b) The synthesis of neuropeptides in the neuronal organelles and their
transport down the axon to the presynaptic terminal is depicted (c) Shows neuropeptide release
and interaction with the dendrite of the post-synaptic cell (Adapted from PhD thesis by Dr
Stephanie Cape)
14
Figure 3 Schematic showing location of the neuronal tissues being analyzed in the MS studies
of neuropeptides in Callinectes sapidus The SGs (sinus glands located in the eyestalks of the
crab) and the POs (pericardial organs located in the chamber surrounding the heart) release
neurohormones into the hemolymph (crab circulatory fluid) The brain is near the STNS
(stomatogastric nervous system neural network that controls the motion of the gut and foregut)
which has direct connections to the STG (stomatogastric ganglion) The STG is located in an
artery so it is continually in contact with hemolymph (Adapted from PhD thesis by Dr Robert
Sturm)
15
Chapter 2
Mass Spectrometry-based Proteomics and Peptidomics for Biomarker
Discovery and the Current State of the Field
Adapted from ldquoMass spectrometry-based proteomics and peptidomics for systems biology and
biomarker discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
16
Abstract
The scientific community has shown great interest in the field of mass spectrometry-based
proteomics and peptidomics for its applications in biology Proteomics technologies have
evolved to produce large datasets of proteins or peptides involved in various biological and
disease progression processes producing testable hypothesis for complex biological questions
This review provides an introduction and insight to relevant topics in proteomics and
peptidomics including biological material selection sample preparation separation techniques
peptide fragmentation post-translation modifications quantification bioinformatics and
biomarker discovery and validation In addition current literature and remaining challenges and
emerging technologies for proteomics and peptidomics are presented
17
Introduction
The field of proteomics has seen a huge expansion in the last two decades Multiple factors have
contributed to the rapid expansion of this field including the ever evolving mass spectrometry
instrumentation new sample preparation methods genomic sequencing of numerous model
organisms allowing database searching of proteomes improved quantitation capabilities and
availability of bioinformatic tools The ability to investigate the proteomes of numerous
biological samples and the ability to generate future hypothesis driven experiments makes
proteomics and biomarker studies exceedingly popular in biological studies today In addition
the advances in post-translational modification (PTM) analysis and quantification ability further
enhance the utility of mass spectrometry (MS)-based proteomics A subset of proteomics
research is devoted to profiling and quantifying neurologically related proteins and endogenous
peptides which has progressed rapidly in the past decade This review provides a general
overview as outlined in Figure 1 of proteomics technology including methodological and
conceptual improvements with a focus on recent studies and neurological biomarker studies
Biological Material Selection
The choice of biological matrix is an important first step in any proteomics analysis The
ease of sample collection (eg urine plasma or saliva) versus usefulness or localization of
sample (eg specific tissue or proximity fluid) needs to be evaluated early on in a study design
Plasma derived by centrifugation of blood to remove whole cells is a very popular
choice in proteomics due to the high protein content (~65 mg ml1) and the ubiquitous nature of
blood in the body and the ability to obtain large sample amounts or various time points without
the need to sacrifice the animal or to perform invasive techniques Plasma is centrifuged
18
immediately after sample collection unlike serum where coagulation needs to occur first To
obtain plasma blood is collected in a tube with an anticoagulant added (ETDA heparin or
citrate) and centrifuged but previous reports have shown variable results when heparin has been
used as an anticoagulant2 Human Proteome Organization (HUPO) specifically recommends the
anticoagulants EDTA or citrate to treat plasma3 4
One of the primary concerns with plasma is
degradation of the protein content via endogenous proteases found in the sample5 One way to
address this problem is the use of protease inhibitors In addition freezethaw cycles need to be
minimized to prevent protein degradation and variability6 7
Plasma proteomics has seen
extensive coordinated efforts to start assessing the diagnostic needs using plasma8 HUPO also
has established a public human database for plasma and serum proteomics from 35 collaborating
labratories9 Large dynamic range studies have been performed on plasma with a starting sample
amount of 2625 microl (1575 mg) resulting in 3654 proteins identified with a sub 5 false
discovery rate10
The large dynamic range spanning across eleven orders of magnitude as visualized in
Figure 2 is one of the biggest obstacles in plasma proteomics Figure 2 also shows that as lower
abundance proteins are investigated the origins of those identified proteins are more diverse than
the most abundant proteins Recent mining of the plasma proteome showed an ability to search
for disease biomarker applications across seven orders of magnitude In addition the tissue of
origin for the identified plasma proteins were identified and its origin was more diverse as the
protein concentration decreased11
Plasma has been used as a source for biomarker studies such
as colorectal cancer12 13
cardiovascular disease14
and abdominal aortic aneurysm15
Even
though the blood brain barrier prevents direct blood to brain interaction neurological disorders
such as Alzheimerrsquos disease (AD) have had their proteomes studied using plasma16
19
An alternative sample derived from blood is serum which is plasma allowed to coagulate
instead of adding anti-coagulates The time for coagulation is usually 30 minutes and during that
time significant and random degradation from endogenous proteases can occur The additional
variability caused from the coagulation process can change the concentration of multiple
potentially valuable biomarkers As biodiversity between samples or organisms is a challenging
endeavor additional sample variability due to serum generation may be undesirable but serum is
still currently being used for biomarker disease studies17
Serum has been used to compare the
proteome differences in neurological diseases such as AD Parkinsonrsquos disease and amyotrophic
lateral sclerosis and a review can be found elsewhere discussing the subject18
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy of brain or spinal cord
in evaluating diseases of the central nervous system and has been used for studies in neurological
disorders due to being a rich source of neuro-related proteins and peptides19
The protein
composition of the most abundant proteins in CSF is well defined and numerous studies exist to
broaden the proteins identified20-22
CSF has an exceedingly low protein content (~04 μgμL)
which is ~100 times lower than serum or plasma and over 60 of the total protein content in
CSF consists of a single protein albumin23-25
In addition the variable concentrations of proteins
span up to twelve orders of magnitude further complicating analysis and masking biologically
relevant proteins to any given study26
One of the highest number of identified proteins is from
Schutzer et al with 2630 non-redundant proteins from 14 mL of pooled human CSF This study
involved the removal of highly abundant proteins by performing IgY-14 immunodepletion
followed by two dimensional (2D) liquid chromatography (LC) separation27
Studies have also
been performed to characterize individual biomarkers or complex patterns of biomarkers in
various diseases in the CSF28 29
One potential pitfall of CSF proteomic analysis is
20
contamination from blood which can be identified by counting red blood cells present or
examining surrogate markers from blood contamination other than hemoglobin such as
peroxiredoxin catalase and carbonic anhydrase30
A proof of principle CSF peptidomics study
identified numerous endogenous peptides associated with the central nervous system which can
be used as a bank for neurological disorder studies31
Numerous recent reports highlighted the
utility of CSF analysis for biomarker studies in AD32 33
medulloblastoma34
both post-mortem
and ante-mortem35
Cellular lysates offer the distinct advantage to work with a cell line yeast or bacteria
with large amounts of proteins available for analysis36 37
with Saccharomyces cerevisiae being
the most common cell lysate38 39
Other cell lines are also used including HeLa40
and E coli41
The ability to obtain milligrams of proteins easily to scale up experiments without animal
sacrifice offers a clear advantage in biological sample selection Current literature supports
cellular lysate as a valued and sought after source of proteins for large scale proteomics
experiments because of the ability to assess treatments conditions and testable hypotheses42-44
Cellular lysate from rat B104 neuroblastoma cell line was used as an in vitro model for cerebral
ischemia and showed abundance changes in multiple proteins involved in various neurological
disorders45
Other Sources of Biological Samples
Urine
The urine proteome appears to be another attractive reservoir for biomarker discovery
due to the relatively low complexity compared with the plasma proteome and the noninvasive
collection of urine Urine is often considered as an ideal source to identify biomarkers for renal
21
diseases due to the fact that in healthy adults approximately 70 of the urine proteome originate
from the kidney and the urinary tract 46
thus the use of urine to identify neurological disorders is
neglected However strong evidence have shown that proteins that are associated with
neurodegenerative diseases can be excreted in the urine47-49
indicating the application of urine
proteomics could be a useful approach to the discovery of biomarkers and development of
diagnostic assays for neurodegenerative diseases However the current view of urine proteome
is still limited by factors such as sample preparation techniques and sensitivity of the mass
spectrometers There has been a tremendous drive to increase the coverage of urine proteome
In a recent study Court et al compared and evaluated several different sample preparation
methods with the objective of developing a standardized robust and scalable protocol that could
be used in biomarkers development by shotgun proteomics50
In another study Marimuthu et al
reported the largest catalog of proteins in urine identified in a single study to date The
proteomic analysis of urine samples pooled from healthy individuals was conducted by using
high-resolution Fourier transform mass spectrometry A total of 1823 proteins were identified
of which 671 proteins have not been previously reported in urine 51
Saliva
For diagnosis purposes saliva collection has the advantage of being an easy and non-
invasive technique The recent studies on saliva proteins that are critically involved in AD and
Parkinsonrsquos diseases suggested that saliva could be a potentially important sample source to
identify biomarkers for neurodegenerative diseases Bermejo-Pareja et al reported the level of
salivary Aβ42 in patients with mild AD was noticeably increased compared to a group of
controls 52
In another study Devic et al identified two of the most important Parkinsons
22
disease related proteinsmdash α-synuclein (α-Syn) and DJ-1 in human saliva 53
They observed that
salivary α-Syn levels tended to decrease while DJ-1 levels tended to increase in Parkinsons
disease The published results from this study also suggest that α-Syn might correlate with the
severity of motor symptoms in Parkinsons disease Due in part to recent advancements in MS-
based proteomics has provided promising results in utilizing saliva to explore biomarkers for
both local and systemic diseases 54 55
the further profiling of saliva proteome will provide
valuable biomarker discovery source for neurodegenerative diseases
Tissue
Compared to body fluids such as plasma serum and urine where the proteomic analysis
is complicated by the wide dynamic range of protein concentration the analysis of tissue
homogenates using the well-established and conventional proteomic analysis techniques has the
advantage of reduced dynamic range However the homogenization and extraction process may
suffer from the caveat that spatial information is lost which would be inadequate for the
detection of biomarkers whose localization and distribution play important roles in disease
development and progression Matrix-assisted laser desorptionionization (MALDI) imaging
mass spectrometry (IMS) is a method that allows the investigation of a wide range of molecules
including proteins peptides lipids drugs and metabolites directly in thin slices of tissue 56-59
Because this technology allows for identification and simultaneous localization of biomolecules
of interests in tissue sections linking the spatial expression of molecules to histopathology
MALDI-IMS has been utilized as a powerful tool for the discovery of new cancer biomarker
candidates as well as other clinical applications60 61
The utilization of MALDI-IMS for human
or animal brain tissue to identify or map the distribution of molecules related to
neurodegenerative diseases were also recently reported62 63
23
Secretome
There has been an increasing interest in the study of proteins secreted by various cells
(the secretomes) from tissue-proximal fluids or conditioned media as a potential source of
biomarkers Cell secretomes mainly comprise proteins that are secreted or are shed from the cell
surface and these proteins can play important role in both physiological processes (eg cell
signaling communication and migration) and pathological processes including tumor
angiogenesis differentiation invasion and metastasis In particular the study of cancer cell
secretomes by MS based proteomics has offered new opportunities for cancer biomarker
discovery as tumor proteins may be secreted or shed into the bloodstream and could be used as
noninvasive biomarkers The latest advances and challenges of sample preparation sample
concentration and separation techniques used specifically for secretome analysis and its clinical
applications in the discovery of disease specific biomarkers have been comprehensively
reviewed64 65
Here we only highlight the proteomic profiling of neural cells secretome that has
been applied to neurosciences for a better understanding of the roles secreted proteins play in
response to brain injury and neurological diseases The LC-MS shotgun identification of
proteins released by astrocytes has been recently reported66-68
In these studies the changes
observed in the astrocyte secretomes induced by inflammatory cytokines or cholinergic
stimulation were investigated6667
Alternatively our group performed 2D-LC separation and
included cytoplasmic protein extract from astrocytes as a control to identify cytoplasmic protein
contaminants which are not actively secreted from cells68
Sample Preparation
24
Proteomic analysis and biomarker discovery research in biological samples such as body
fluids tissues and cells are often hampered by the vast complexity and large dynamic range of
the proteins Because disease identifying biomarkers are more likely to be low-abundance
proteins it is imperative to remove the high-abundance proteins or apply enrichment techniques
to allow detection and better coverage of the low-abundance proteins for MS analysis Several
strategies including depletion and protein equalizer approach have been used during sample
preparation to reduce sample complexity69 70
and the latest advances of these methods have been
reviewed by Selvaraju et al 71
Alternatively the complexity of biological samples can be
reduced by capturing a specific subproteome that may have the biological information of interest
The latter strategy is especially useful in the biomarker discovery where the changes in the
proteome are not solely reflected through the concentration level of specific proteins but also
through changes in the post-translational modifications (PTMs) Here we will mainly discuss
the enrichment of phosphoproteinpeptides glycoproteinpeptides and sample preparation for
peptidomics and membrane proteins
Phosphoproteomics
Phosphorylation can act as a molecular switch on a protein by turning it on or off within
the cell It is thought that up to 30 of the proteins can be phosphorylated72
and it plays
significant roles in such biological processes as the cell cycle and signal transduction73
Currently tens of thousands of phosphorylation sites can be proposed using analytical methods
available today74 75
The amino acids that are targeted for phosphorylation studies are serine
threonine and tyrosine with the abundance of detection decreasing typically in that order Other
25
amino acids have been reported to be phosphorylated but traditional phosphoproteomics
experiments ignore these rare events76
In a typical large-scale phosphoproteomics experiment the sample size is usually in
milligram amounts to account for the low stoichiometry of phosphorylated proteins The large
amount of protein is then digested typically with trypsin but alternatively experiments have
been performed with Lys-C digestion to produce large enzymatic peptides The larger peptides
produced from Lys-C render higher charged peptides during electrospray ionization (ESI) and
allow improved electron-based fragmentation to determine specific sites of phosphorylation77
From the pool of peptides phosphopeptides must be enriched otherwise they will be masked by
the vast number and higher ionization efficiency of non-phosphorylated peptides The two most
common enrichment techniques are immobilized metal ion affinity chromatography (IMAC) and
metal oxide affinity chromatography (MOAC) TiO2 being the most common oxide used for this
purpose A recent study reported that phosphorylation of neuronal intermediate filament proteins
in neurofibrillary tangles are involved in Alzheimerrsquos disease78
Glycoproteomics
Protein glycosylation is one of the most common and complicated forms of PTM Types
of protein glycosylation in eukaryotes are categorized as either N-linked where glycans are
attached to asparagine residues in a consensus sequence N-X-ST (X can be any amino acid
except proline) via an N-acetylglucosamine (N-GlcNAc) residue or the O-glycosylation where
the glycans are attached to serine or threonine Glycosylation plays a fundamental role in
numerous biological processes and aberrant alterations in protein glycosylation are associated
with neurodegenerative disease states such as Creutzfeld-Jakob Disease (CJD) and AD79 80
26
Due to the low abundance of glycosylated forms of proteins compared to non-glycosylated
proteins it is essential to enrich glycoproteins or glycopeptides in complex biological samples
prior to MS analysis Two of the most common enrichment methods used in glycoproteomics are
lectin affinity chromatography (LAC) and hydrazide chemistry The detailed methodologies of
LAC hydrazide chemistry and other enrichment methods in glycoproteomics have been
extensively reviewed in the past81 82
In particular LAC is of great interest in studies of
glycosylation alterations as markers of AD and other neurodegenerative diseases due to its recent
applications in brain glycoproteomics83
Our group has utilized multi-lectin affinity
chromatography containing concanavalin A (ConA) and wheat germ agglutinin (WGA) to enrich
N-linked glycoproteins in control and prion-infected mouse plasma84
This method enabled us to
identify a low-abundance glycoprotein serum amyloid P-component (SAP) PNGase F digestion
and Western blotting validation confirmed that the glycosylated form of SAP was significantly
elevated in mice with early prion infection and it could be potentially used as a diagnostic
biomarker for prion diseases
Membrane proteins
Membrane proteins play an indispensable role in maintaining cellular integrity of their
structure and perform many important functions including signaling transduction intercellular
communication vesicle trafficking ion transport and protein translocationintegration85
However due to being relatively insoluble in water and low abundance it is challenging to
analyze membrane proteins by traditional MS-based proteomics approaches Numerous efforts
have been made to improve the solubility and enrichment of membrane proteins during sample
preparation Several comprehensive studies recently covered the commonly used technologies in
27
membrane proteomics and different strategies that circumvent technical issues specific to the
membrane 86-90
Recently Sun et al reported using 1-butyl-3-methyl imidazolium
tetrafluoroborate (BMIM BF4) an ionic liquid (IL) as a sample preparation buffer for the
analysis of integral membrane proteins (IMPs) by microcolumn reversed phase liquid
chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MSMS)
The authors compared BMIM BF4 to the other commonly used solvents such as sodium dodecyl
sulfate methanol Rapigest and urea but they found that the number of identified IMPs from rat
brain extracted by ILs was significantly increased The improved identifications could be due to
the fact that BMIM BF4 has higher thermal stability and thus offered higher solubilizing ability
for IMPs which provided better compatibility for tryptic digestion than traditionally used solvent
systems38
In addition to characterization of membrane proteome the investigation of PTMs on
membrane proteins is equally important for characterization of disease markers and drug
treatment targets Phosphorylations and glycosylations are the two most important PTMs for
membrane proteins In many membrane protein receptors the cytoplasmic domains can be
phosphorylated reversibly and function as signal transducers whereas the receptor activities of
the extracellular domains are mediated via N-linked glycosylation Wiśniewski et al provides an
informative summary on recent advances in proteomic technology for the identification and
characterization of these modifications91
Our group has pioneered the development of detergent
assisted lectin affinity chromatography (DALAC) for the enrichment of hydrophobic
glycoproteins using mouse brain extract92
We compared the binding efficiency of lectin affinity
chromatography in the presence of four commonly used detergents and determined that under
certain concentrations detergents can minimize the nonspecific bindings and facilitate the
elution of hydrophobic glycoproteins In summary NP-40 was suggested as the most suitable
28
detergent for DALAC due to the higher membrane protein recovery glycoprotein recovery and
membranous glycoprotein identifications compared to other detergents tested In a different
study on mouse brain membrane proteome Zhang et al reported an optimized protocol using
electrostatic repulsion hydrophilic interaction chromatography (ERLIC) for the simultaneous
enrichment of glyco- and phosphopeptides from mouse brain membrane protein preparation93
Using this protocol they successfully identified 544 unique glycoproteins and 922 glycosylation
sites which were significantly higher than those using the hydrazide chemistry method
Additionally a total of 383 phosphoproteins and 915 phosphorylation sites were identified
suggesting that the ERLIC separation has the potential for simultaneous analysis of both glyco-
and phosphoproteomes
Peptidomics
Peptidomics can be loosely defined as the study of the low molecular weight fraction of
proteins encompassing biologically active endogenous peptides protein fragments from
endogenous protein degradation products or other small proteins such as cytokines and signaling
peptides Studies can involve endogenous peptides94
peptidomic profiling33
and de novo
sequencing of peptides95 96
Neuropeptidomics focuses on biologically active short segments of
peptides and have been investigated in numerous species including Rattus97 98
Mus musculus99
100 Bovine taurus
101 Japanese quail diencephalon
102 and invertebrates
103-106 The isolation of
peptides is typically performed through molecular weight cut-offs from either biofluids such as
CSF plasma or tissue extracts If the protein and peptide content is high such as for tissue or cell
lysates protein precipitation can be done via high organic solvents and the resulting supernatant
can be analyzed for extracted peptides where extraction solvent and conditions could have a
29
significant effect on what endogenous peptides are extracted from tissue107
A comparative
peptidomic study of human cell lines highlights the utility of finding peptide signatures as
potential biomarkers108
A thorough review of endogenous peptides and neuropeptides is beyond
the scope of this review and an excellent review on this topic is available elsewhere109
Fractionation and Separation
The mass spectrometer has a limited duty cycle and data dependent analysis can only
scan a limited number of mz peaks at any given time In addition significant ion suppression
can occur if there is a difference in concentration between co-eluting peptides or if too many
peptides co-elute Therefore one of the biggest challenges in biomarker discovery is the
complexity of the sample and the presence of high-abundance proteins in body fluids such as
CSF serum and plasma In addition to the removal of the most abundant proteins by
immunodepletion the reduction of the complexity of the sample by further fractionation is
indispensable to facilitate the characterization of unidentified biomarkers from the low
abundance proteins Traditionally used techniques for complex protein analysis include gel
based fractionation methods such as two-dimensional gel electrophoresis (2D-GE) and its
variation two-dimensional differential gel electrophoresis (2D-DIGE) or non-gel based such as
one- or multidimensional liquid chromatography (LC) and microscale separation techniques
such as capillary electrophoresis (CE)
2D-GE MS has been widely used as a powerful tool to separate proteins and identify
differentially expressed proteins ever since 2D gels were coupled to mass spectrometry In 2D-
GE MS thousands of proteins can be separated on a single gel according to pI and molecular
weight Individual protein spots that show differences in abundance between different samples
30
can then be excised from the gel digested into peptides and analyzed by MALDI MS or by
liquid chromatography tandem mass spectrometry (LC-MSMS) for protein identification The
introduction of 2D-DIGE adds a quantitative strategy to gel electrophoresis by enabling multiple
protein extracts to be separated on the same 2D gel thus providing comparative analysis of
proteomes in complex samples In 2D-DIGE protein extracts from two different conditions and
an internal standard can be labeled with fluorescent dyes for example Cy3 Cy5 and Cy2
respectively prior to two-dimensional gel electrophoresis Compared to traditional 2D-GE 2D-
DIGE provides the clear advantage of overcoming the inter-gel variation problem 110
Proteomic
profiling of CSF by 2D-GE and 2D-DIGE has led to the identification of putative biomarkers in
multiple neurological disorders For example Brechlin et al reported an optimized 2-DIGE
protocol profiled CSF from 36 CJD patients The applicability of their approach was proven by
the detection of known CJD biomarkers such as 14-3-3 protein neuron-specific enolase lactate
dehydrogenase and other proteins that are potentially relevant to CJD 111
In another study to
identify novel CSF biomarkers for multiple sclerosis CSF from 112 multiple sclerosis patients
and control individuals were analyzed by 2D-GE MS for comparative proteomics Ten potential
multiple sclerosis biomarkers were selected for validation by immunoassay 112
These
methodologies sample preparation techniques and applications of 2D-DIGE in
neuroproteomics were reviewed by Diez et al113
Although 2D gel provides excellent resolving
power and capability to visualize abundance changes there are some limitations to the method
For example gel based separation is not suitable for low abundance proteins extremely basic or
acidic proteins very small or large proteins and hydrophobic proteins114 115
Complementary to gel-based approaches shotgun proteomics coupled to LC have
become increasingly popular in proteomic research because they are reproducible highly
31
automated and capable of detecting low abundance proteins Furthermore another advantage of
LC-MS shotgun proteomics is the suitability for isotope labeling for protein quantification which
is reviewed in a later section In shotgun proteomics a protein mixture is digested and resulting
peptides are separated by LC prior to tandem MS fragmentation to identify different proteins by
peptide sequencing The most common separation for shotgun proteomics peptidomics or top-
down proteomics experiments use low-pH reversed phase (RP) C18 C8 or C4 columns RPLC
is well established which provides high resolution desalts the sample which can interfere with
ionization and the mobile phase is compatible with ESI Nanoscale C18 columns allow for
separation and introduction of sub microgram samples If larger amounts of sample are
available two dimensional separations are usually preferred to greatly enhance the coverage of
the investigated proteome which will be discussed in depth later It is preferable to have an
orthogonal separation method and since RP separates via hydrophobicity strong cation exchange
(SCX) was the original choice due to its separation by charge MudPIT (multidimensional
protein identification technology) usually refers to the use of SCX as the first phase of separation
and is a well-established platform116
SCX has the advantage over RP separation technologies to
effectively remove interfering detergents from the sample SCX separation is not based solely
off charge and hydrophobicity contributes to elution therefore a small amount of organic
modifier usually 10-15 is added to lessen the hydrophobicity effects117
The addition of
organic modifiers needs to be minimized otherwise binding to the C18 trap cartridge or C18
column will be reduced if performed on-line SCX can be used for PTMs and offers specific
applications for proteomic studies and an excellent current review is offered on this subject
elsewhere118
An alternative MudPIT separation scheme employing high pH RPLC as the first
phase of separation and low pH RPLC in the second dimension (RP-RP) has been successfully
32
applied to the proteomic analysis of complex biological samples119 120
The advantage of using
RP as the first dimension is the higher resolution for separation and better compatibility with
down-stream MS detection by eliminating salt Song et al reported a phosphoproteome analysis
based on this 2D RP-RP coupling scheme121
Hydrophilic interaction chromatography (HILIC) employs distinct separation modality
where the retention of peptides is increased with increasing polarity122
The loading of sample is
done by high organic and eluted by increasing the percentage of the aqueous phase or polarity of
the mobile phase opposite from RPLC thus establishing orthogonality of the two separation
modes123
HILIC has quickly become a very useful method and is actively used for proteomic
experiments124
for increased sensitivity125
phosphoproteomics126
glycoproteins127
and
quantification studies128
An alternative and modification to HILIC is ERLIC which adds an
additional mode of separation by electrostatic attraction An earlier study using ERLIC
demonstrated the ability to separate phosphopeptides from non-phosphorylated peptides at
pH=2129
A recent study looking into changes in the phosphoproteome of Marekrsquos Disease
applied ERLIC to chicken embryonic fibroblast lysate identifying only 13 phosphopeptides
out of all the identified peptides Due to the lack of isolation of phosphopeptides from ERLIC
the investigators performed immobilized metal affinity chromatography (IMAC) enrichment on
the fractions increasing identification of phosphopeptides over 50 fold130
A comparative study
of ERLIC to HILIC and SCX following TiO2 phospho-enrichment reported that
SCXgtERLICgtHILIC for phosphopeptide identifications126
Recent developments in instrumentation to combine LC with ion mobility spectrometry
(IMS) and MS (LC-IMS-MS) offered more advantages than conventional LC due to the rapid
high-resolution separations of analytes based on their charge mass and shape as reflected by
33
mobility in a given buffer gas The mobility of an ion in a buffer gas is determined by the ionrsquos
charge and its collision cross-section with the buffer gas The methodologies of IMS separations
and the application of LC-IMS-MS for the proteomics analysis of complex systems including
human plasma have been reviewed by Clemmerrsquos group131-133
They proposed a method that
employs intrinsic amino acid size parameters to obtain ion mobility predictions which can be
used to rank candidate peptide ion assignments and significantly improve peptide identification
134
Although 2D gel and LC are routinely used as separation techniques in MS-based
proteomics capillary electrophoresis (CE) has received increasing attention as a promising
alternative due to the fast and high-resolution separation it offers CE has a wide variety of
operation modes among which capillary zone electrophoresis (CZE) and capillary isoelectric
focusing (CIEF) have the greatest potential applications in MS-based proteomics thus will be
highlighted here CZE separates analytes by their charge-to-size ratios in buffers under a high
electrical field and is often used as the final dimension prior to MS analysis while the separation
feature of CIEF is based on isoelectric point and this technique is more suitable to be used as the
first dimension separation Detailed description of different CEndashMS interfaces sample
preconcentration and capillary coating to minimize analyte adsorption could be found in several
reviews135-141
CE technique is complementary to conventional LC in that it is suitable for the
analysis of polar and chargeable compounds Dovichirsquos group conducted proteomic analysis of
the secreted protein fraction of Mycobacterium marinum which has intermediate protein
complexity142
The tryptic digests were either analyzed by UPLC-ESI-MSMS in triplicates or
prefactionated by RPLC followed by CZE-ESI-MSMS It was demonstrated that the two
methods identified similar numbers of peptides and proteins within similar analysis times
34
However CZE-ESI-MSMS analysis of the prefractionated sample tended to identify more
peptides that are basic and have lower mz values than those identified by UPLC-ESI-MSMS
This analysis also presented the largest number of protein identifications by using CE-MSMS
suggesting the effectiveness of prefractionation of complex samples by LC method prior to CZE-
ESI-MSMS The use of CIEF as the first dimension of separation provides both sample
concentration and excellent resolving power The combination of CIEF and RPLC separation
has been applied to the proteomic analyses where the amount of protein sample is limited and
cannot meet the requirement of minimal load amount for 2D LC-MSMS143 144
So far CE-MS
has been widely applied to the proteomic analysis of various biological samples such as urine145
146 CSF
147 blood
148 frozen tissues
149 and the formalin-fixed and paraffin-embedded (FFPE)
tissue samples150
The recent CEndashMS applications to clinical proteomics have been summarized
in several reviews135 151 152
Protein Quantification
In 2D gel electrophoresis the quantitative analysis of protein mixtures is performed on
the gel by comparing the intensity of the protein stain The development of 2D-DIGE eliminated
the gel-to-gel variation and greatly improved the quantitative capability and reliability of 2D gel
methodology110
However the accuracy of 2D gel based protein quantification suffers from the
limitations that a seemingly single gel spot often contains multiple proteins and the difficulty of
detecting proteins with extreme molecular weights and pI values as well as highly hydrophobic
proteins such as membrane proteins Therefore non-gel based shotgun proteomics technology is
more suitable for accurate and large-scale protein identification and quantification in complex
samples Briefly the quantification in non-gel based shotgun proteomics can be categorized into
35
two major approaches stable isotope labeling-based and label-free methods The common
strategies for quantitative proteomic analysis are reviewed and summarized in Table 1
Isotope labeling methods
Because stable isotope-labeled peptides have the same chemical properties as their
unlabeled counterparts the two peptides within a mixture should exhibit identical behaviors in
MS ionization The mass difference introduced by isotope labeling enables the detection of a
pair of two distinct peptide masses by MS within the mixture and allowing for the measurement
of the relative abundance differences between two peptides Depending on how isotopes are
incorporated into the protein or peptide these labeling methods can be divided into two groups
In vitro chemical derivatization techniques which incorporate a label or tag into the peptide or
protein during sample preparation metabolic labeling techniques which introduce the isotope
label directly into the organism via isotope-enriched nutrients from food or media
1 In vitro derivatization techniques
There are multiple methods to introduce heavy isotopes into proteins or peptides in vitro
The commonly used strategies include 18
O 16
O enzymatic labeling Isotope-Coded Affinity Tag
(ICAT) Tandem Mass Tags (TMTs) and Isobaric Tags for Relative and Absolute Quantification
(iTRAQ) The 18
O labeling method enzymatically cleaves the peptide bond with trypsin in the
presence of 18
O-enriched H2O and introduces 4-Da mass shift in the tryptic peptides153
The
advantages of this method include 18
O-enriched water is extremely stable tryptic peptides will
be labeled with the same mass shift secondary reactions inherent to other chemical labeling can
be avoided Conversely widespread use of 18
O-labeling has been hindered due to the difficulty
of attaining complete 18
O incorporation and the lack of robustness154 155
Currently ICAT
36
TMTs and iTRAQ methods are extensively used in quantitative proteomics In ICAT cysteine
residues are specifically derivatized with a reagent containing either zero or eight deuterium
atoms as well as a biotin group for affinity purification of cysteine-containing peptides156 157
The advantage of ICAT is that the affinity purification via biotin moiety can facilitate the
detection of low-abundance cysteine-containing peptides In addition the mass difference
introduced by labeling increases mass spectral complexity with quantification from the different
precursor masses done by MS and peptide identification being achieved through tandem MS
(MSMS) This added complexity from different peptide masses was addressed by using isobaric
labeling methods such as TMTs and iTRAQ 158 159
where the same peptides in different samples
are isobaric after tagging and appear as single mz in MS scans thus enhancing the peptide limit
of detection and reducing the MS scan complexity Isobaric labeling reagents are composed of a
primary amine reactive group and an isotopic reporter group linked by an isotopic balancer group
for the normalization of the total mass of the tags The reporter group serves for quantification
purpose since it is cleaved during collision-induced dissociation (CID) to yield a characteristic
isotope-encoded fragment Moreover isobaric labeling methods allow the comparison of
multiple samples within a single experiment Recently a 6-plex version of TMTs was
reported160
and iTRAQ enables up to eight samples to be labeled and relatively quantified in a
single experiment161
8-plex iTRAQ reagents have been used for the comparison of complicated
biological samples such as CSF in the studies of neurodegenerative diseases 162
Recently our
group developed a novel N N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2)
tagging reagents with high quantitation efficacy DiLeu has the advantage of synthetic simplicity
and greatly reduced synthesis cost compared to TMTs and iTRAQ163
Xiang et al demonstrated
that DiLeu produced comparable iTRAQ ability for protein sequence coverage (~43) and
37
quantitation accuracy (lt15) for tryptically digested proteins More importantly DiLeu
reagents could promote enhanced fragmentation of labeled peptides thus allowing more
confident peptide and protein identifications
2 In Vivo Metabolic Labeling
Metabolic processes can also be employed for the incorporation of stable-isotope labels
into the proteins or organisms by enriching culture media or food with light or heavy versions of
isotope labels (2H
13C
15N) The advantage of in vivo labeling is that metabolic labeling does
not suffer from incomplete labeling which is an inherent drawback for in vitro derivatization
techniques In addition metabolic labeling occurs from the start of the experiment and proteins
with light or heavy labels are simultaneously extracted thus reducing the error and variability of
quantification introduced during sample preparation The most widely used strategy for
metabolic labeling is known as stable-isotope labeling of amino acids in cell culture (SILAC)
which was introduced by Mann and co-workers164 165
In SILAC one cell population is grown
in normal or light media while the other is grown in heavy media enriched with a heavy
isotope-encoded (typically 13
C or 15
N) amino acid such as arginine or leucine Cells from the
two populations are then combined proteins are extracted digested and analyzed by MS The
relative protein expression differences are then determined from the extracted ion
chromatograms from both the light and heavy peptide forms SILAC has been shown to be a
powerful tool for the study of intracellular signal transduction In addition this technique has
recently been applied to the quantitative analysis of phosphotyrosine (pTyr) proteomes to
characterize pTyr-dependent signaling pathways166 167
38
Labe-free quantification
Although various isotope labeling methods have provided powerful tools for quantitative
proteomics several limitations of these approaches are noted Labeling increases the cost and
complexity of sample preparation introduces potential errors during the labeling reaction It also
requires a higher sample concentration and complicates data processing and interpretation In
addition so far only TMTs and iTRAQ allow the comparison of multiple (up to eight) samples
simultaneously The comparison of more than eight samples in a single experiment cannot be
achieved by isotope labeling In order to address these concerns there has been significant
interest in the development of label-free quantitative approaches Current label-free
quantification methods for MS-based proteomics were developed based on the observation that
the chromatographic peak area of a peptide168 169
or frequency of MSMS spectra170
correlating
to the protein or peptide concentration Therefore the two most common label-free
quantification approaches are conducted by comparing (i) area under the curve (AUC) of any
given peptides171 172
or (ii) by frequency measurements of MSMS spectra assigned to a protein
commonly referred to as spectral counting173
Several recent reviews provided detailed and
comprehensive knowledge comparing label-free methods with labeling methods data processing
and commercially available software for label-free quantitative proteomics174-177
Dissociation Techniques
The vast majority of proteomic experiments have proteins or peptides being identified by
two critical pieces of data obtained from the mass spectrometer The first is the precursor ion
identified by its mz which is informative to the mass of the peptide being analyzed The second
is the use of tandem mass spectrometry to fragment or dissociate the precursor ion and record the
39
generated fragment ion pattern to discern the amino acid sequence The three most popular
dissociation or fragmentation techniques for peptides are CID electron-transfer dissociation
(ETD) and high-energy collision dissociation (HCD) A recent study on the human plasma
proteome demonstrated that combined fragmentation techniques enhance coverage by providing
complementary information for identifications CID enabled the greatest number of protein
identifications while HCD identified an additional 25 proteins and ETD contributed an
additional 13 protein identifications178
ETDECD
Electron capture dissociation (ECD) 179
preceded ETD but ECD was developed for use
in a Penning trap for Fourier transform ion cyclotron resonance (FTICR) mass spectrometers
ECD requires the ion of interest to be in contact with near-thermal electrons and for the electron
capture event to occur on the millisecond time scale but the time scale is inadequate for electron
trapping in Paul traps or quadrupoles in the majority of mass spectrometers180
ETD involves a
radical anion like fluoranthene with low electron affinity to be transferred to peptide cation
which results in more uniform cleavage along the peptide backbone The cation accepts an
electron and the newly formed odd-electron protonated peptide undergoes fragmentation by
cleavage of the N-Cα bond which results in fragmentation ions consisting of c- and zbull-type
product ions The uniformed cleavage results in reduced sequence discrimination to labile bonds
such as PTMs and also provides improved sequencing for larger peptides compared to CID181
The realization that larger peptides produced better MSMS quality spectra compared to CID led
to a decision tree analysis strategy where peptide charge states and size determined whether the
precursor peptide would be fragmented with CID or ETD182
One of the main benefits of
ETDECD is the ability to sequence peptides with labile PTMs such as phosphorylation77 183
40
sulfation184
glycosylation185
ubiquitination186
and histone modifications187
ETD also has the
benefit of providing better sequence information on larger neuropeptides when compared to
CID188
However a thorough analysis suggested that CID still yielded more peptideprotein
identifications than ETD in large scale proteoimcs189
HCD
High energy collision dissociation (HCD)190
is an emerging fragmentation technique that
offers improved detection of small reporter ions from iTRAQ-based studies191 192
HCD is
performed at a higher energy in a collision cell instead of an ion trap like CID thus HCD does
not suffer from the low-mass cutoff limitation Furthermore HCD offers enhanced
fragmentation efficiency assisting in MSMS spectra interpretation and protein identification193
A major drawback for HCD is that the spectral acquisition times are up to two-fold longer due to
increased ion requirement for Fourier transform detection in the orbitrap194
HCD has been
reported to increase phosphopeptide identifications over CID74
but in a different study CID was
reported to offer more phosphopeptide identifications over HCD194
Work has also been done to
transfer the decision tree analysis for HCD which basically switches CID with HCD claiming
better quality data determined by higher Mascot scores with more peptide identifications195
MSE
Data dependent acquisition (DDA) is the most commonly used ion selection process in
mass spectrometers for proteomic experiments An alternative process which does not have ion
selection nor switch between MS and MSMS modes is termed MSE MS
E is a data independent
mode and does not require precursor ions of a significant intensity to be selected for MSMS
analysis196
A data independent mode decouples the mass spectrometer choosing which
precursor ions to fragment and when the ions are fragmented MSE works by a low or high
41
energy scan and no ion isolation is occurring The low energy scan is where the precursor ion is
not fragmented and the high energy scan allows fragmentation The resulting mix of precursor
and fragmentation ions is then detected simultaneously197
The data will then need to be
deconvoluted using a proprietary time-aligned algorithm that is discussed elsewhere198
The
continuous data independent acquisition allows multiple MSMS spectra to be collected during
the natural analyte peak broadening observed in chromatography which provides more data
points for AUC label-free quantification In addition lower abundance peptides can be
sequenced as more MSMS spectra are collected throughout the elution of an LC peak allowing
better signal averaging for smaller analyte peak of interest during coelution and reducing
sampling bias in typical DDA experiments where only more abundant peaks can be selected for
fragmentation
A comparison of spiked internal protein standards into a complex protein digest provided
evidence that MSE was comparable to DDA analysis in LC-MS
199 MS
E has been used for label
free proteomics of immunodepleted serum in large scale proteomics samples200
In addition
MSE was performed for the characterization of human cerebellum and primary visual cortex
proteomes Hundreds of proteins were identified including many previously reported in
neurological disorders201
MSE is quickly becoming a versatile data acquisition method recently
used in such studies as cancer cells202
schizophrenia203
and pituitary proteome discovery204
The usefulness of MSE as an unbiased data acquisition method is being assimilated into multiple
proteomics studies including studies involving neurological disorders
Data Analysis
42
One of the major bottlenecks in non-targeted proteomic experiments is how to handle the
enormous amount of data obtained Database searches biostatistical analysis de novo
sequencing PTM validation all have their place and multiple available platforms are available
If the organism being studied has had its genome sequenced databases can be created
with a list of proteins in the FASTA format to be used in database searching There are
numerous database searching algorithms for sequence identification of MSMS data including
Mascot205
Sequest206
Xtandem207
OMSSA208
and PEAKS209
These searching algorithms are
performed by matching MSMS spectra and precursor mass to sequences found within proteins
How well the actual spectra match the theoretical spectra determines a score which is unique to
the searching algorithm and usually can be extrapolated to the probability of a random hit
Recently a database has been developed for PTM analysis by the use of the program SIMS210
Specifically for phosphopeptides Ascorersquos algorithm scans the MSMS data to determine the
likelihood of correct phosphosite identification from the presence of site identifying product
ions211
If the organism that is being analyzed has not had its genome sequenced and no (or very
limited) FASTA database is available a homology search can be performed using SPIDER212
available with PEAKS software Alternatively individual MSMS spectrum can be de novo
sequenced but software is available to perform automated de novo sequencing of numerous
spectra (PEAKS208
DeNovoX and PepSeq)
For large-scale protein identifications the false discovery rate (FDR) must be established
by the searching algorithm and that is accomplished by re-searching the data with a false
database created by reversing or scrambling the amino acid sequence of the original database
used for the protein search Any hits from the false database will contribute to the FDR and this
value can be adjusted usually around 1 An additional layer of confidence in the obtained data
43
can be achieved in shotgun proteomics experiments by removing all the proteins that are
identified by only one peptide
Once a set of confident proteins or peptides have been generated from database
searching bioinformatic analysis or biostatistical analysis is needed Numerous software
packages are available for different purposes FLEXIQuant is an example for absolute
quantitation of isotopically labeled protein or peptides of interest213
FDR analysis of
phosphopeptides or other specific PTMs can be adjusted with such software as Scaffold
providing data consisting only of a specific modification214
Bioinformatic tools such as
Scaffold or ProteoIQ also include gene ontology (GO) analysis which can classify identified
proteins by three categories cellular component molecular function or biological process
Custom bioinformatics programs can also be developed and are often useful in various proteomic
studies including biomarker discovery in neurological diseases215
More detailed review of
bioinformatics in peptidomics216
and proteomics217
can be found elsewhere
Validation of Biomarkers by Targeted Proteomics
The validation of putative biomarkers identified by MS-based proteomic analysis is often
required to provide orthogonal analysis to rule out a false positive by MS and providing
additional evidence for the biomarker candidate(s) from the study for future potential clinical
assays At present antibody-based assays such as Western blotting ELISA and
immunochemistry are the most widely used methods for biomarker validation Although accurate
and well established these methods rely on protein specific antibodies for the measurement of
the putative biomarker and could be difficult for large-scale validation of all or even a subset of a
long list of putative protein biomarkers typically obtained by MS-based comparative proteomic
44
analysis Large scale validation is impractical due to the cost for each antibody the labor to
develop a publishable Western blot or ELISA and the antibody availability for certain proteins
As an alternative strategy quantitative assays based on multiple-reaction monitoring (MRM) MS
using a triple quadrupole mass spectrometer have been employed in biomarker verification
MRM is the most common use of MSMS for absolute quantitation It is a hypothesis
driven experiment where the peptide of interest and its subsequent fragmentation pattern must be
known prior to the quantitative MRM experiments MRM involves selecting a specific mz (first
quadrupole) to be isolated for fragmentation (second quadrupole) followed by one or more of
the most intense fragment ions (third quadrupole) being monitored The ability to quantitate and
thus validate the proteins or peptides as potential biomarkers is achieved by performing MRM on
isotopically labeled reference peptide for targeted peptideprotein of interest The main obstacle
for quantification of peptides is interference and ion suppression effects from co-eluting
substances Since the isotopically labeled and native peptide will co-elute the same interference
and ion suppression will occur for both peptides and thus correcting these interfering effects
Peptides need to be systematically chosen for a highly sensitive and reproducible MRM
experiment to ensure proper validation of putative biomarkers Peptides require certain intrinsic
properties which include an mz within the practical mass detection range for the instrument and
high ionization efficiency If the desired peptide to be quantified is derived from a digestion
then peptides that have detectable incomplete digestion or missed cleavage site can be a major
source of variability Peptides with a methionine and to a lesser extent tryptophan are
traditionally removed from consideration from MRM quantitative experiments due to the
variable nature of the oxidation that can occur In addition if chromatographic separation is
performed the retention behavior of the peptide must be well behaved with little tailing effects
45
eluting late causing broadening of the peak and even irreversible binding to the column As an
example hydrophilic peptides being eluted off a C18 column may exhibit the previously
described concerns and a different chromatographic separation will need to be explored for
improved limits of detection quantitation and validation To determine consistent peptide
detection or usefulness of certain peptides databases such as Proteomics Database218
PRIDE219
PeptideAtlas220
have been developed to compile proteomic data repositories from initial
discovery experiments
After the peptide is selected for analysis the proper MRM transitions need to be selected
to optimize the sensitivity and selectivity of the experiment It is common for investigators to
select two or three of the most intense transitions for the proposed experiment It is imperative
that the same instrument is used for the determination of transition ions as different mass
spectrometers may have a bias towards different fragment ions
MRM experiments are still highly popular experiments for hypothesis directed
experiments221
biomarker analysis222
and validation223
Validation of putative biomarkers is
increasingly becoming a necessary step when performing large scale non-hypothesis driven
proteomics experiments The traditional validation techniques of ELISA Western blotting and
immunohistochemistry are still used but MRM experiments are becoming an attractive
alternative for validation of putative biomarkers due to its enhanced throughput and specificity
Current work is still being performed to both expand the linear dynamic range224
and
sensitivity225
of MRM A recent endeavor to increase the sensitivity for MRM experiments was
accomplished by ldquoPulsed MRMrdquo via the use of an ion funnel trap to enhance confinement and
accumulation of ions The authors claimed an increase by 5-fold for peak amplitude and a 2-3
fold reduction in chemical background225
46
Remaining Challenges and Emerging Technologies
Large sample numbers for mass spectrometry analysis
Multiple conventional studies in proteomics have been performed on a single or a few
biological samples As bio-variability can be exceedingly high the need for larger sample sizes
is currently being investigated Prentice et al used a starting point of 3200 patient samples
from the Womenrsquos Health Institute (WHI) to probe the plasma proteome using MS for
biomarkers The study did not test the 3200 patient samples by MS because even a simple one
hour one dimensional RP analysis on a mass spectrometer would take months of instrument time
for uninterrupted analysis Instead the authors pooled 100 samples together to bring the total
number of pooled samples to 32 To provide relevant plasma biomarkers the samples were then
subjected to immunodepletion 2-D protein separation (96 fractions total) and then 1-D RPLC of
tryptic peptide separation on-line interface to a mass spectrometer The large sample cohorts
help address bio-variability that can be a concern from small sample size proteomic experiments
and provide ample sample amounts to investigate the low abundance proteins226
Hemoglobin-derived neuropeptides and non-classical neuropeptides
Neuropeptides such as neuropeptide Y and enkephalin are short chains of amino acids
that are secreted from a range of neuronal cells that signal nearby cells In contrast non-classical
neuropeptides are termed as neuropeptides or ldquomicroproteinsrdquo which are derived from
intracellular protein fragments and synthesized from the cytosol227
MS was recently used to
determine that hemopressins which are hemoglobin-derived peptides are upregulated in Cpefatfat
mice brains Gelman et al designed an MS experiment to compare hemoglobin-derived
47
peptides comparing the brain blood and heart peptidome in mice The authors provided data
that specific hemoglobin peptides were produced in the brain and were not produced in the
blood Certain alpha and beta hemoglobin peptides were also up regulated in the brain for
Cpefatfat
mice and bind to CB1 cannabinoid receptors228
As discussed earlier in the review
peptidomics and specifically neuropeptidomics are popular fields of study utilizing MS and non-
classical neuropeptides is an exciting emerging area of research that could further expand the
diversity of cell-cell signaling molecules
Ultrasensitive mass spectrometry for single cell analysis
In addition to large scale analysis MS-based proteomics and peptidomics are making
progress into ultrasensitive single cell analysis The most successful MS-based techniques for
single cell analysis was performed with MALDI and studies that have been performed on
relatively large neurons are reviewed elsewhere229
The ultrasensitive MS analysis is currently
directed towards single cell analysis of smaller cells including cancer cells The first challenge
in single cell analysis is the isolation and further sample preparation to yield relevant data
Collection and isolation of a cell type can be accomplished using antibodies for fluorescence
activated cell sorting (FACS) and immune magnetic separation FACS works by flow cytometry
sorting cells by a laser that excites a fluorescent tag that is attached to an antibody Immune
magnetic separation allows separation by antibodies with magnetic properties such as
Dynabeads230
One exciting study combining FACS and MS termed mass cytometry This
technology works by infusing a droplet into an inductively coupled plasma mass spectrometer
(ICP-MS) containing a single cell bound to antibodies chelated to transition elements allowing a
quantifying response between single cells231
Clearly the future of single cell analysis for
48
biomarker analysis and proteomics is encouraging and has the potential to be an emerging field
in MS-based proteomics and peptidomics
Laserspray ionization (LSI)
Laserspray ionization (LSI) is an exciting new method to produce multiply charged mass
spectra from MALDI that is nearly identical to ESI232-234
Recently it has been reported that LSI
can be performed in lieu of matrix to produce a total solvent-free analysis234
The benefits of
being able to generate multiply charged peptides without any solvent may offer advantages
including MS analysis of insoluble membrane proteins or hydrophobic peptides avoidance of
chemical reactions while in solvents a reduction of sample loss due to liquid sample preparation
and ability to avoid diffusion effects from tissue imaging studies234
The multiply charged peptide and protein ions produced by LSI expand the mass range
for tissue imaging analysis More importantly the multiply-charged peptide ions are amenable
for electron-based fragmentation methods such as ETD or ECD which can be employed in
conjunction with tissue imaging experiments to yield in situ sequencing and identification of
peptides of interest235
Paper spray ionization
Paper spray (PS) is an ambient ionization method which was first reported using
chromatography paper allowing detection of metabolites from dried blood spots The original
method used a cut out piece of paper with a voltage clipped on the back while applying 10 microL of
methanolH2O236
Improvements have been made to this technology to enhance analysis
efficiency with a new solvent 91 dichloromethaneisopropanol (vv) and the use of silica paper
49
over chromatography paper237
Interesting applications or modifications have been made to PS
including direct analysis of biological tissue238
and leaf spray for direct analysis of plant
materials239
but both detect metabolites instead of proteins or peptides Paper spray ionization
was previously shown to enable detection of cytochrome c and bradykinin [2-9] standards in a
proof of principle study240
Clearly the utility of PS analysis in proteomics and peptidomics is
yet to be explored
niECD
New fragmentation techniques have been investigated for their utility in proteomics and
peptidomics including a recently reported negative-ion electron capture dissociation (niECD)
Acidic peptides which usually contain PTMs such as phosphorylation or sulfonation are often
difficult to be detected as multiply charged peptides in the positive ion mode As discussed
earlier multiply charged peptides are required for ECDETD fragmentation The fragmentation
of niECD is accomplished by a multiply negatively charged peptide adding an electron The
resulting fragmentation of multiply sulfated and phosphorylated peptide and protein standards
showed no sulfate loss and preserved phosphorylation site The resulting fragmentation pattern
from niECD was also improved in the peptide anions and provides a new strategy for de novo
sequencing with PTM localization241
Conclusions and Perspectives
Proteomics methodologies have produced large datasets of proteins involved in various
biological and disease progression processes Numerous mass spectrometry-based proteomics
and peptidomics tools have been developed and are continuously being improved in both
50
chromatographic or electrophoretic separation and MS hardware and software However several
important issues that remain to be addressed rely on further technical advances in proteomics
analysis When large proteomes consisting of thousands of proteins are analyzed and quantified
dynamic range is still limited with more abundant proteins being preferentially detected
Development and optimization of chemical tagging reagents that target specific protein classes
maybe necessary to help enrich important signaling proteins and assess cellular and molecular
heterogeneity of the proteome and peptidome Furthermore a significant bottleneck in
usefulness of proteomics research is the ability to validate the results and provide clear
significant biological relevance to the results The idea of P4 medicine242 243
is an attractive
concept where the four Prsquos stand for predictive preventive personalized and participatory
Proteomics is one of the critical ldquoomicsrdquo fields and has led to the development of enabling
innovative strategies to P4 medicine244
A goal of P4 medicine is to assess both early disease
detection and disease progression in a person A simplified example of how proteomics fits into
P4 medicine is that certain brain-specific proteins could be used for diagnosis with
presymptomatic prion disease244
The concept of proteomic experiments providing an individual
biomarker is becoming more obsolete with the revised vision being a biomolecular barcode that
could potentially be ldquoscannedrdquo or be a fingerprint for a specific disease or early onset to that
disease being closer to reality An excellent review on what biomarker analysis can do for true
patients is available245
Proteomics can also generate new hypothesis that can be tested by classical biochemical
approaches If a disease has an unknown pathogenesis proteomics is a good starting point to try
to assemble putative markers that can lead to further hypothesis for evaluation If a particular
protein or PTM is associated with a disease state either qualitatively or quantitatively potential
51
treatments could target that protein of interest or investigators could monitor that protein or
PTM during potential treatments of the disease Proteomics has expanded greatly over the last
few decades with the goal of providing revealing insights to some of the most complex
biological problems currently facing the scientific community
Acknowledgements
Preparation of this manuscript was supported in part by the University of Wisconsin Graduate
School Wisconsin Alzheimerrsquos Disease Research Center Pilot Grant and a Department of
Defense Pilot Award LL acknowledges an H I Romnes Faculty Fellowship
52
Figure 1 A summary of general workflows of biomarker discovery pipeline by MS-based
proteomic approaches
Biological sample (CSF blood urine saliva cell
lysate tissue homogenates secreted proteins etc)
Protein extraction Sample pretreatment
2D-GE2D-DIGE MS 1D or 2D LC-MSMS
MALDI-IMS
Identification of
differentially
expressed proteins
Protein identification
Potential biomarkers
Biomarker validation
- Antibody based immunoassays
- MRM
Quantitative analysis
- Isotope labeling
- Label free
Identification and
localization of
differentially expressed
biomolecules
Intact tissue
Sample preparation Slice frozen tissues
thaw-mounted on plate
Apply Matrix
53
Figure 2 Tissue expression and dynamic range of the human plasma proteome A pie chart
representing the tissue of origin for the high abundance proteins shows that the majority of
proteins come from the liver (A) Conversely the lower abundance plasma proteins have a much
more diverse tissue of origin (B and C) The large dynamic range of plasma proteins is presented
and the proteins can be grouped into three categories (classical plasma proteins tissue leakage
products interleukinscytokines) (D) Adapted from Zhang et al11
and Schiess et al246
with
permission
54
55
Table 1 A summary of the common strategies applied to MS-based quantitative proteomic
analysis
Gel based Stable isotope labeling Label free
2D-GE
2D-DIGE 110
In vitro derivatization
18O
16O
153
ICAT 156
TMT 159
iTRAQ 158
Formaldehyde 247
ICPL 248
In vivo metabolic labeling
14N
15N
249
SILAC 164
AUC measurement 169 172
Spectral counting 173
AUC Area Under Curve ICAT Isotope-Coded Affinity Tag TMT Tandem Mass Tags iTRAQ Isobaric Tags for
Relative and Absolute Quantification ICPL Isotope Coded Protein Labeling SILAC Stable Isotope Labeling by
Amino Acids in Cell Culture Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
56
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Purvine S O Camp D G 2nd Smith R D Evaluation of multiprotein
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K Yoo J S Ping P Pounds J Adkins J Qian X Wang R Wasinger V Wu C Y
Zhao X Zeng R Archakov A Tsugita A Beer I Pandey A Pisano M Andrews P
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(13) 3226-45
10 Liu T Qian W J Gritsenko M A Xiao W Moldawer L L Kaushal A Monroe
M E Varnum S M Moore R J Purvine S O Maier R V Davis R W Tompkins R
G Camp D G 2nd Smith R D High dynamic range characterization of the trauma patient
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Sakuma T Nakamori S Sata N Nagai H Ioka T Okusaka T Kosuge T Tsuchida A
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13 Murakoshi Y Honda K Sasazuki S Ono M Negishi A Matsubara J Sakuma
T Kuwabara H Nakamori S Sata N Nagai H Ioka T Okusaka T Kosuge T
Shimahara M Yasunami Y Ino Y Tsuchida A Aoki T Tsugane S Yamada T Plasma
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14 Addona T A Shi X Keshishian H Mani D R Burgess M Gillette M A
Clauser K R Shen D Lewis G D Farrell L A Fifer M A Sabatine M S Gerszten R
E Carr S A A pipeline that integrates the discovery and verification of plasma protein
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25
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24
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4825-35
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Proteomics 2010 73 (4) 769-77
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7757-65
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Evaluation of HCD- and CID-type fragmentation within their respective detection platforms for
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Proteomics 2009 9 (6) 1683-95
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Analysis of the human pituitary proteome by data independent label-free liquid chromatography
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(5) 958-64
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Anal Chem 2008 80 (20) 7846-54
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211 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
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Criekinge W Peptidomics coming of age a review of contributions from a bioinformatics
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Hermjakob H PRIDE new developments and new datasets Nucleic Acids Res 2008 36
(Database issue) D878-83
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Hilgendorf C Development of a highly sensitive method using liquid chromatography-multiple
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Mass Spectrometry for Quantification of Heat Shock Proteins Anal Chem 2012
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validation in blood specimens by selected reaction monitoring mass spectrometry of N-
glycosites Methods Mol Biol 2011 728 179-94
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natural isotopologue transitions Talanta 2011 87 307-10
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Smith R D Pulsed multiple reaction monitoring approach to enhancing sensitivity of a tandem
quadrupole mass spectrometer Anal Chem 2011 83 (6) 2162-71
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McIntosh M Wang P Buson Busald T Hsia J Jackson R D Rossouw J E Manson J
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227 Gelman J S Fricker L D Hemopressin and other bioactive peptides from cytosolic
proteins are these non-classical neuropeptides AAPS J 2010 12 (3) 279-89
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228 Gelman J S Sironi J Castro L M Ferro E S Fricker L D Hemopressins and
other hemoglobin-derived peptides in mouse brain comparison between brain blood and heart
peptidome and regulation in Cpefatfat mice J Neurochem 2010 113 (4) 871-80
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profiling Trends Biotechnol 2000 18 (4) 151-60
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Pavlov S Vorobiev S Dick J E Tanner S D Mass cytometry technique for real time
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spectrometry Anal Chem 2009 81 (16) 6813-22
232 Trimpin S Inutan E D Herath T N McEwen C N Laserspray ionization a new
atmospheric pressure MALDI method for producing highly charged gas-phase ions of peptides
and proteins directly from solid solutions Mol Cell Proteomics 2010 9 (2) 362-7
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charged ions Anal Chem 2010 82 (12) 4998-5001
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charged ions without solvent using laserspray ionization a total solvent-free analysis approach at
atmospheric pressure Anal Chem 2011 83 (11) 4076-84
235 Inutan E D Richards A L Wager-Miller J Mackie K McEwen C N Trimpin
S Laserspray ionization a new method for protein analysis directly from tissue at atmospheric
pressure with ultrahigh mass resolution and electron transfer dissociation Mol Cell Proteomics
2010 10 (2) M110 000760
236 Wang H Liu J Cooks R G Ouyang Z Paper spray for direct analysis of complex
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substrate for paper-spray analysis of therapeutic drugs in dried blood spots Anal Chem 84 (2)
931-8
238 Wang H Manicke N E Yang Q Zheng L Shi R Cooks R G Ouyang Z
Direct analysis of biological tissue by paper spray mass spectrometry Anal Chem 83 (4) 1197-
201
239 Liu J Wang H Cooks R G Ouyang Z Leaf spray direct chemical analysis of plant
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Am Chem Soc 2011 133 (42) 16790-3
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243 Hood L Friend S H Predictive personalized preventive participatory (P4) cancer
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(2) 111-21
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N-metabolic labelingmass
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Rapid Commun Mass Spectrom 2002 16 (14) 1389-97
73
Chapter 3
Protein changes in immunodepleted cerebrospinal fluid from transgenic
mouse models of Alexander disease detected using mass spectrometry
Adapted from ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse
models of Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P
Messing A Li L Submitted
74
ABSTRACT
Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range
spanning at least nine orders of magnitude in protein content and is in direct contact with the
brain A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the
low volumes of CSF that are obtainable from mice As a model system in which to test this
approach we utilized transgenic mice that over-express the intermediate filament glial fibrillary
acidic protein (GFAP) These mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we report the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates were performed to address animal variability as well as reproducibility in
mass spectrometric analysis Relative quantitation was performed using distributive normalized
spectral abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins
with significant changes in the CSF of GFAP transgenic mice has been identified with validation
from ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
75
INTRODUCTION
Alexander disease (AxD) is a fatal neurogenetic disorder caused by heterozygous point
mutations in the coding region of GFAP (encoding glial fibrillary acidic protein)1 The hallmark
diagnostic feature of this disease is the accumulation of astrocytic cytoplasmic inclusions known
as Rosenthal fibers containing GFAP Hsp27 αB-crystallin and other components2-5
Although
several potential treatment strategies6-8
are under investigation clinical trial design is hampered
by the absence of a standardized clinical scoring system or means to quantify lesions in MRI
that could serve to monitor severity and progression of disease One solution to this problem
would be the identification of biomarkers in readily sampled body fluids as indirect indicators of
disease
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy site for brain or spinal
cord in evaluating diseases of the central nervous system The protein composition of CSF is
well defined at least for the most abundant species of proteins and numerous studies exist that
characterize individual biomarkers or complex patterns of biomarkers in various diseases9 10
GFAP itself is present in CSF (albeit at much lower levels than in brain parenchyma) and in one
study of three Alexander disease patients its levels were markedly increased11
Whether an
increase in CSF GFAP will be a consistent finding in Alexander disease or whether other useful
biomarkers for this disease could be identified through an unbiased analysis of the CSF
proteome is not yet known
The rarity of Alexander disease makes analysis of human samples difficult However
mouse models exist that replicate key features of the disease such as formation of Rosenthal
fibers Unfortunately mouse CSF poses particular problems for proteomic studies and there is
76
an urgent need for technical improvements for dealing with this fluid For instance collection
from an adult mouse typically yields ~10 μL of CSF often with some contamination by blood12
To further complicate analysis CSF has an exceedingly low protein content (~04 μgμL) with
over 60 of the total protein content consisting of a single protein albumin13 14
A number of
techniques have been developed to remove albumin from biological samples including Cibacron
Blue15
IgG immunodepletion16
and IgY immunodepletion17-19
IgY which is avian in origin
offers reduced non-specific binding and increased avidity when compared to IgG antibodies from
rabbits goats and mice20-23
One widely used IgY cocktail is IgY-14 which contains fourteen
specific antibodies specific for albumin IgG transferrin fibrinogen α1-antitrypsin IgA IgM
α2-macroglobulin haptoglobin apolipoproteins A-I A-II and B complement C3 and α1-acid
glycoprotein Since existing protocols for IgY-14 depletion are optimized for use with large
volumes of serum new protocols must be developed to permit its use with the low volumes of a
low protein fluid represented by mouse CSF
Various improvements have also taken place in the field of proteomic analysis that could
facilitate analysis of mouse CSF Data dependent tandem mass spectrometry followed by
quantification of proteins is used in standard shotgun proteomics24-29
Several methods now exist
for introducing quantitation into mass spectrometry including stable isotope labeling30-32
isobaric tandem mass tags33 34
and spectral counting35 36
Spectral counting which is a
frequency measurement that uses MSMS counts of identified peptides as the metric to enable
protein quantitation is attractive because it is label-free and requires no additional sample
preparation Finally recent advances in spectral counting has produced a data refinement
strategy termed normalized spectral abundance factor (NSAF)37 38
and further developed into
distributive NSAF (dNSAF) to address issues with peptides shared by multiple proteins39
77
To identify potential biomarkers in AxD we report a novel scaled-down version of IgY
antibody depletion strategy to reduce the complexity and remove high abundance proteins in
mouse CSF samples The generated spectral counts data were then subjected to dNSAF natural
log data transformation and t-test analysis to determine which proteins differ in abundance when
comparing GFAP transgenics and controls with multiple biological and technical replicates
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) urea (gt99) sodium chloride (997) and ammonium
bicarbonate (certified) were purchased from Fisher Scientific (Fair Lawn NJ) Deionized water
(182 MΩcm) was prepared with a Milli-Q Millipore system (Billerica MA) Optima LCMS
grade acetonitrile and water were purchased from Fisher Scientific (Fair Lawn NJ) DL-
Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega
(Madison WI) Formic acid (ge98) was obtained from Fluka (Buchs Switzerland)
Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris ge999 ) ammonium formate
(ge99995) glycine (ge985) and IgY-14 antibodies were purchased from Sigma-Aldrich
(Saint Louis MO)
Mice
Transgenic mice over-expressing the human GFAP gene (Tg737 line) were maintained
as hemizygotes in the FVBN background and genotyped via PCR on DNA prepared from tail
samples as described previously40
The mice were housed on a 14-10 light-dark cycle with ad
libitum access to food and water All procedures were conducted using protocols approved by
the UW-Madison IACUC
78
CSF collection
CSF was collected from mice as described previously12
Briefly mice were anesthetized
with avertin (400-600 mgkg ip) A midline sagittal incision was made over the dorsal aspect
of the hindbrain and three muscle layers carefully peeled back to expose the cisterna magna The
membrane covering the cisterna magna was pierced with a 30 gauge needle and CSF was
collected immediately using a flexible plastic pipette Approximately ten microliters of CSF was
collected per animal All samples used for MS analysis showed no visible contamination of
blood
Enzyme-linked immunosorbent assay (ELISA)
A sandwich ELISA was used to quantify GFAP8 Briefly a microtiter plate was coated
with a cocktail of monoclonal antibodies (Covance SMI-26R) Plates were blocked with 5
milk before addition of sample or standards diluted in PBS with Triton-X and BSA A rabbit
polyclonal antibody (DAKO Z334) was used to detect the GFAP followed by a peroxidase
conjugated anti-rabbit IgG antibody (Sigma A6154) secondary antibody The peroxidase activity
was detected with SuperSignal ELISA Pico Chemiluminescent Substrate (PIERCE) and
quantified with a GloRunner Microplate Luminometer Values below the biological limit of
detection (16ngL) were given the value 16ngL before multiplying by the dilution factor
Immunodepletion of abundant proteins
Currently there are no commercial immunodepletion products available for use with CSF
and to address the low protein content of this fluid (~04 microgmicroL) Therefore 100 microL of
purchased IgY-14 resin was coupled with a Pierce Spin Cup using a paper filter from Thermo
Scientific (Waltham MA) CSF samples from either transgenics or controls were first pooled to
100 microL and then added to 100 microL of 20 mM Tris-HCl 300 mM pH 74 (2x dilution buffer) and
79
allowed to mix with the custom IgY-14 depletion spin column on an end-over-end rotor for 30
minutes at 4oC The sample was then centrifuged at 04 rcf for 45 seconds with an Eppendorf
Centrifuge 5415D (Hamburg Germany) The antibodies were then washed with 50 microL 1x
dilution buffer vortexed for 5 minutes centrifuged at 04 rcf for 45 seconds and the flow through
was collected for tryptic digestion The antibodies were then stripped of the bound proteins with
four 025 mL washes of 01 M glycine pH 25 neutralized with two 025 mL washes of 01 M
Tris-HCl pH 80 and washed once with 025mL of 1x dilution buffer The immunodepletion
protocol was repeated for each transgenic or control pooled 100 microL sample (N=3)
Preparation of tryptic digests
The immunodepleted pooled mouse CSF samples (200 microL total volume) were
concentrated to 10 microL using a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA)
To each sample 8 microL of 133 M urea and 1 microL of 050 M DTT were added and allowed to
incubate at 37oC for one hour After incubation 27 microL of 055 M IAA was added for
carboxymethylation and the sample was allowed to incubate for 15 minutes in the dark To
quench the IAA 1 microL of 050 M DTT was added and allowed to react for 10 minutes To
perform trypsin digestion 70 microL of 50 mM NH4HCO3 was then added along with 025 microg
trypsin which had previously been dissolved in 50 mM acetic acid at a concentration of 05
microgmicroL Digestion was performed overnight at 37oC and quenched by addition of 25 microL of 10
formic acid The tryptic peptides were then subjected to solid phase extraction using a Varian
Omix Tip C18 100 microL (Palo Alto CA) Peptides were eluted with 50 ACN in 01 formic
acid concentrated and reconstituted in 30 microL H2O in 01 formic acid
RP nanoLC separation
80
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto an Agilent
Technologies Zorbax 300 SB-C18 5 microm 5x03 mm trap cartridge (Santa Clara CA) at a flow
rate of 5 microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm
Atlantis dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B
at 250 nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
81
range of 300-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot41
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt mus musculus
(house mouse) database (version 575) False positive analyses42
were calculated using an
automatic decoy option of Mascot Results from the Mascot results were reported using
Proteinscape 21 and technical replicates were combined and reported as a protein compilation
using ProteinExtractor (Bruker Daltonics Bremen Germany)
Mascot search parameters were as follows Allowed missed cleavages 2 enzyme
trypsin variable modifications carboxymethylation (C) and oxidation (M) peptide tolerance
plusmn12 Da maximum number of 13
C 1 MSMS tolerance plusmn05 Da instrument type ESI-Trap
Reported proteins required a minimum of one peptide with a Mascot score ge300 with bold red
characterization Spectral counts were determined from the number of MSMS spectra identified
from accepted proteins A bold red peptide combines a bold peptide which represents the first
query result from a submitted MSMS spectrum with the red peptide which indicates the top
peptide for the identified protein Requiring one bold red peptide assists in removal of
homologous redundant proteins and further improves protein results In addition requiring one
82
peptide to be identified by a score gt300 removes the ability for proteins to be identified by
multiple low Mascot scoring peptides
Each immunodepleted biological replicate had technical triplicates performed and the
technical triplicates were summed together by ProteinExtractor Peptide spectral counts were
then summed for each protein and subjected to dNSAF analysis Details for this method can be
found elsewhere37 39
but briefly peptide spectral counts are summed per protein (SpC) based on
unique peptides and a weighted distribution of any shared peptides with homologous proteins
ProteinScape removed 83 homologous proteins found in the current study to bring the total
number of proteins identified to 266 but some non-unique homologous peptides which are
shared by multiple proteins are still present in the resulting 266 remaining proteins To address
these non-unique homologous peptides distributive spectral counting was performed as
described elsewhere39
The dSpC is divided by the proteinrsquos length (L) and then divided by the
summation of the dSpCL from all proteins in the experiment (N) to produce each proteinrsquos
specific dNSAF value
N
i
i
kk
LdSpC
LdSpCdNSAF
1
)(
)()(
The resulting data were then transformed by taking the natural log of the dNSAF value The
means for each protein were calculated using the ln(dNSAF) from N=3 biological replicates and
the whole data set was subjected to the Shapiro-Wilk test for normalityGaussian distribution
performed on the software PAST (Version 198 University of Oslo Norway Osla) The
Shapiro-Wilk test was repeated multiple times to determine a non-zero value for zero spectral
83
counts A non-zero value is required to alleviate the errors of dividing by zero which was
experimentally determined to be 043 The Gaussian data were then subjected to the t-test to
identify statistically significant changes in protein expression
RESULTS AND DISCUSSION
General workflow
Individual CSF samples were manually inspected and samples were only selected that
showed no visual blood contamination Preliminary experiments showed that the maximum
degree of blood contamination estimated from counts of red blood cells in the CSF that was not
visible to the eye was less than 005 by total mass Approximately 10 individual mouse CSF
samples were pooled to achieve the desired 100 μL volume for a single biological replicate The
CSF samples were IgY immunodepleted followed by digestion with trypsin The resulting
digested samples were then de-salted concentrated reconstituted in 30 μL of 01 formic acid
and 5 μL was loaded onto a reversed phase nanoflow HPLC column subjected to a 90 minute
gradient and infused into the amaZon ETD ion trap mass spectrometer The workflow for
mouse CSF analysis is shown in Figure 1 The remainder of the desalted sample was saved for
technical replicates
Immunodepletion for CSF
Currently there are no immunodepletion techniques specifically designed for CSF
Nonetheless the protein profiles between CSF and serum are similar enough to use currently
available immunodepletion techniques designed for serum as a starting point The smallest
commercially available IgY-14 immunodepletion kit is for 15 μL of serum which is equivalent in
protein content to ~15 mL of CSF Therefore for 100 μL CSF one fifteenth of the total IgY-14
84
beads would be used for the equivalent immunodepletion which is 66 μL of proprietary bead
slurry The potential for irreversible binding of abundant proteins to their respective IgY
antibody even after an extra stripping wash and low amounts of total beads made using 66 μL
of IgY-14 bead slurry unrealistic In practice almost doubling the amount of IgY-14 beads (100
μL) used was not sufficient and resulted in albumin and transferrin peptides to be observed in
high abundance (data not shown) The most important protein to immunodeplete is albumin and
it has been reported to be a greater percentage of total CSF protein content (~60) than serum
(~49) in humans14
The difference in albumin percentage supports the results that proprietary
blends of immunodepletion beads for high abundance proteins such as albumin cannot be
scaled down on a strict protein scale and further modifications to the serum immunodepletion
protocol need to be made
Since IgY-14 beads were developed for use with serum all of its protocols need to be
taken into account to modify the protocol for CSF Serum samples should be diluted fifty times
before mixing with the IgY-14 beads but CSF protein concentration is at least one hundred times
lower than serum Therefore CSF is below half the recommended diluted protein concentration
for IgY immunodepletion Consequently multiple steps have been devised to address this
limitation First the binding time between the proteins targeted for removal from the CSF and
IgY-14 beads was doubled during the end-over-end rotor depletion step from the recommended
15 minutes to 30 minutes Second to prevent non-specific protein binding to the antibodies the
CSF samples were diluted with an equal amount (100 μL) of 2X dilution buffer The dilution
buffer of NaCl and Tris-HCl was needed to assist in reducing non-specific binding of proteins to
the 14 antibodies and ensuring the sample is held at physiological pH In addition to these
modifications a doubling of the starting IgY-14 bead slurry to 200 μL provided the desired
85
results Overall this modified protocol results in effective depletion of CSF abundant proteins
using only one-fifth of the antibodies provided by the smallest commercially available platform
Data Analysis
Spectral counting technique for relative quantitation provides numerous benefits for the
study of mouse CSF due to its label-free advantage In contrast isotopic labeling method often
involves additional sample processing that could cause sample loss which is highly undesirable
for low protein content and low volume samples Labeling methods also require a mixing of two
sets of isotopically labeled samples which would effectively increase the sample complexity and
reduce the amount of sample that can be loaded onto the nanoLC column by half In addition
more than two sets of samples can be compared by label-free methods The use of label-free
spectral counting method does not lead to an increase in sample complexity or interference in
quantitation from peptides in the mz window selected for tandem MS Using spectral counting
for relative quantitation however is dependent on reproducible HPLC separation and careful
mass spectrometry operation to minimize technical variability during the study To address
concerns of analytical reliability and run to run deviations base peak chromatograms from two
transgenic IgY-14 immunodepleted biological replicates including two technical replicates of
each were shown to be highly reproducible (Figure 2)
Each biological sample was analyzed in triplicate with the same protocols on the amaZon
ETD with three control and three transgenic samples From the three technical replicates for
each biological replicate the spectral counts of the peptides for the proteins identified were
summed The results from these mouse CSF biological triplicates are shown in Figure 3A for
GFAP overexpressor and Figure 3B for control The summation of spectral counts for each
biological replicate was performed to remove the inherent bias related to data dependent analysis
86
for protein identification One concern in grouping technical replicates is a potential loss of
information regarding analytical variability Figure 4 provides a graphical representation of
variability of technical replicates illustrating the standard deviation of technical replicates with
error bars Figure 4 shows that a statistically changed protein creatine kinase M (A) and an
unchanged protein kininogen-1 (B) have similar variability within (technical replicates) and
between samples (biological replicates) for each protein In addition Figure 4B illustrates that
even with the variability of kininogen-1 the resulting mean shown by the dashed line of control
and transgenic samples were almost equal whereas Figure 4A shows significantly different
expression level of creatine kinase M Performing replicate analysis of each biological sample
(n=3) helps correct for systematic errors deriving from sample handling and pooling of samples
helps reduce random error during the CSF sample collection process
Protein Identification and Spectral Counting Analysis
The data for dNSAF analysis like any mass spectrometry proteomics experiment
requires multiple layers of verification to ensure reliable data Our initial protein identifications
were subjected to a database search using a decoy database from Mascot which resulted in an
average false positive rate below 1 for all the experimental data collected Representative
MSMS spectra between control and GFAP transgenic samples are illustrated in Figure 5
Overall 266 proteins were identified in a combination of control and transgenic samples
(Supplemental Table 1) A total of 349 proteins were initially identified but 83 proteins were
isoforms of previously identified proteins and automatically excluded by ProteinExtractor The
next level of quality control was to only include ln(dNSAF) values from proteins identified by 2
or more unique peptides having a Mascot score of ge300 and observed in two out of three
biological replicates These selection parameters resulted in 106 proteins remaining after
87
dNSAF analysis (Supplemental Table 2) The resulting spectral counts were then subjected to
dSpC in order to account and correct for the systematic error of peptides shared by multiple
proteins (Supplemental Table 3)
It is inevitable in large scale and complex proteomics experiments that some proteins will
be seen in some samples and not others In addition when controls were compared to transgenic
samples as shown in Figure 3C 127 proteins were unique to either control or GFAP transgenic
mice and 139 proteins were seen in both samples From dNSAF equation if the spectral count
is zero the numerator is zero and the value will not be normalized between runs In order to
circumvent the zero spectral counts in dNSAF analysis zero spectral counts must be replaced by
an experimentally determined non-zero value determined to be 043 The 043 spectral counts
for zero spectral counts was calculated by serial Shapiro-Wilk tests to determine the lowest value
(043) for zero spectral counts and maintain a Gaussian distribution for all data sets The 043
value for zero spectral counts in the current study was higher than the 016 reported value for
zero spectral counts in the original NSAF spectral counting study37
Our study may have a
higher zero spectral count value than the previous study because the spectral counting data were
an addition of three technical replicates and three times 016 is close to 043 The normalized
Gaussian data were then subjected to t-test analysis and P-values below 005 are considered as
statistically significant and are presented in Table 1 The proteins with significant up or down
regulation from Table 1 can be further evaluated as how close significant proteins were to a p-
value of 005 such as ganglioside GM2 activator serine protease inhibitor A3N and collagen
alpha-2(I) chain having t-test scores of 0045 0047 and 0043 respectively Proteins exhibiting
a P-value close to 005 were more likely to be highly variable proteins or have smaller fold
changes between control and transgenic samples and thus provide less biological relevancy to
88
future studies The modest change in antithrobin-III which is reduced by 14-fold in transgenic
is included due a low pooled standard deviation in spectral counts
Spectral counting has been analyzed with fold changes derived directly from the average
spectral counts from the technical replicates and then the average of the three biological
replicates We decided to perform additional analysis using fold changes to dig deeper into
proteins that statistically support the null hypothesis from the dNSAF analysis To only pull out
highly confident protein identifications we used the same strict cut-off of two unique peptides
identified per protein as in dNSAF analysis We only accepted proteins with greater than three-
fold change in total spectral counts listed in Table 2 Two proteins contactin-1 (cntn1) and
cathepsin B (CB) illustrate the potential biomarkers missed by dNSAF analysis Cntn1 had zero
spectral count in the transgenic sample and had an average spectral count of 41 in control
samples The lack of any spectral counts in one biological control for cntn1 resulted in a large
standard deviation in the ln(dNSAF) means for the control which resulted in the t-test supporting
the null hypothesis Another example is CB which was detected by numerous spectral counts in
every GFAP transgenic biological replicate with an average spectral count of 97 (Table 2) The
presence of CB in one biological control sample (23 average spectral counts) resulted in a high
standard deviation in the mean of the control samples These examples exhibit a limitation of
dNSAF analysis which could cause a loss of potentially useful information
Previously Identified Proteins with Expression Changes
Previously three proteins have been described as increased in CSF from individual(s)
suffering from AxD In one study a single patientrsquos CSF was found to have elevated levels of
αβ-crystallin and HSP2744
In a second study three patients were reported to have elevated
levels of GFAP with concentrations from 4760 ngL to 30000 ngL (compared to lt175 ngL for
89
controls)11
GFAP was detected in our current study however the other two proteins were not
detected One possibility is that αB-crystallin and Hsp-27 levels in mouse CSF are too low for
detection by MS analysis In addition while the transgenic mice display the hallmark
pathological feature of AxD in the form of Rosenthal fibers they do not have any evident
leukodystrophy and thus may not display the full range of changes in CSF as might be found in
human patients
Creatine Kinase M
Creatine kinase M (M-CK) is a 43 kDa protein whose role is to reversibly catalyze
phosphate transfer between ATP and energy storage compounds M-CK has been primarily
found in muscle tissue and for humans creatine kinase M is diagnostically analyzed in the blood
for myocardial infarction (heart attack) In addition M-CK is also found in Purkinje neurons of
the cerebellum45 46
A related protein creatine kinase B (B-CK) also exhibited an apparent 21
fold increase in transgenic CSF over control but this difference was not statistically different
B-CK concentration is known to be elevated in CSF following head trauma47
or cerebral
infarction48
but decreased in astrocytes in individuals affected by multiple sclerosis49
Cathepsin
The data showed multiple cathepsins were up regulated in the CSF of transgenic mice
when compared to control mice The up regulated cathepsins were S L1 and B isoforms which
are all cysteine proteases Cathepsin S (CS) was never observed in control samples but
observed with an average of 73 spectral counts per analysis Cathepsin L1 (CL1) was up
regulated by 94 times in transgenic mice These two cathepsins exhibited significant changes
using dNSAF and t-test statistics as shown in Table 1 and Cathepsin B (CB) showed a 42 fold
increase in transgenic CSF as shown in Table 2
90
Cathepsins regulate apoptosis in cells50
which is the major mechanism for elimination of
cells deemed by the organism to be dangerous damaged or expendable CL and CB are
redundant in the system as thiol proteases and inhibition of CB by CA-047 causes a diminished
apoptosis response in multiple cell lines51
Intriguingly increased levels of CB or CL are
correlated with poor prognosis for cancer patients and shorter disease-free intervals It is
believed that these proteases degrade the extracellular membrane which allows tumor cells to
invade adjacent tissue and metastasize52
With regards to AxD the up regulation of these
cathepsins may be indicative of the bodyrsquos natural defense and response to Rosenthal fibers
Thus stimulation of these cathepsins may provide a further protective stress response but the
positive correlation between these proteases and cancer highlights the multiple roles of these
proteins in pathological response Alternatively it has been shown that increased CB is involved
with the tumor necrosis factor α (TNFα) induced apoptosis cascade53
The activation of the
TNFα could produce oligodendrocyte toxicity54
with the expression of TNFα being elevated in
tissue samples from mouse models and AxD patients55
The potential for a positive or a negative
effect in increasing or decreasing cathepsins warrant future research with cathepsins and AxD
Contactin-1
Contactin-1 (Cntn1) is a 133 kDa cell surface protein that is highly glycosylated and
belongs to a family of immunoglobulin domain-containing cell adhesion molecues56
Table 2
shows that Cntn1 is down regulated in transgenic mice since spectral counts were only observed
in control mice Cntn1 is expressed in neurons and oligodendrocytes and higher levels were
observed during brain development57
In addition Cntn1 leads to activation of Notch1 which
mediates differentiation and maturation of oligodendrocyte precursor cells (OPC) Although the
mechanism leading to a reduction of Cntn1 in CSF is not known it may reflect a change in
91
astrocyte interactions with either neurons or oligodendrocytes to alter their expression of this
protein
Validation of putative biomarkers and MS proteomics data using ELISA and RNA
microarray data
To further validate the relative protein expression data obtained via MS-based spectral
counting techniques orthogonal immunological and molecular biological approaches have been
examined As GFAP was shown to be elevated in the CSF of transgenic animals we used a
well-defined GFAP ELISA protocol to test its CSF concentration CSF from 8 week old male
mice was collected from both transgenic and control animals Five samples of transgenic CSF
was prepared by pooling four to eight animals for the GFAP ELISA In the case of controls
each sample represents a single animal GFAP concentrations observed by both the MS and
ELISA showed significant increases of GFAP in the CSF when comparing transgenic to control
animals
Another validation of MS spectral counts is observed in a microarray analysis performed
on transgenic mouse olfactory bulb tissue 55
In this paper nine of the proteins found by MS
showed similar gene expression in the microarray (Table 3) It is not surprising that all the genes
observed in the microarray are not the same as the proteins observed by MS analysis Gene
expression and protein synthesis and expression are not always correlated but the similarities
and overlapping trends observed with these two assays are encouraging As shown in Table 3
gene expression analysis also revealed the up regulation of several cathepsin proteins GFAP
and down regulation of contactin-1 in CSF collected from transgenic mice corroborating the
MS-based proteomics results
92
CONCLUSIONS
In this study we have produced a panel of proteins with significant up or down regulation
in the CSF of transgenic GFAP overexpressor mice This mouse model for AxD was consistent
with the previous studies showing elevation of GFAP in CSF The development of a modified
IgY-14 immunodepletion technique for low amounts of CSF was presented This improved
protocol is useful for future investigations to deal with the unique challenges of mouse CSF
analysis Modified proteomics protocols were employed to profile mouse CSF with biological
and technical triplicates addressing the variability and providing quantitation with dNSAF
spectral counting Validation of the MS-based proteomics data were performed using both
ELISA and RNA microarray data to provide further confidence in the changes in the putative
protein biomarkers This study presents three classes of interesting targets for future study in
AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
93
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1127-37
54 Butt A M Jenkins H G Morphological changes in oligodendrocytes in the intact
mouse optic nerve following intravitreal injection of tumour necrosis factor J Neuroimmunol
1994 51 (1) 27-33
55 Hagemann T L Gaeta S A Smith M A Johnson D A Johnson J A Messing
A Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal
fibers reveals a stress response followed by glial activation and neuronal dysfunction Hum Mol
Genet 2005 14 (16) 2443-58
56 Falk J Bonnon C Girault J A Faivre-Sarrailh C F3contactin a neuronal cell
adhesion molecule implicated in axogenesis and myelination Biol Cell 2002 94 (6) 327-34
57 Eckerich C Zapf S Ulbricht U Muumlller S Fillbrandt R Westphal M Lamszus
K Contactin is expressed in human astrocytic gliomas and mediates repulsive effects Glia
2006 53 (1) 1-12
97
Table 1 Statistically changed proteins between transgenic and control mouse CSF using
dNSAF analysis
Accession Protein Pa SC
b Fold
Changec
Control
dSpCd
Transgenic
dSpCd
KCRM_MOUSE Creatine kinase M-type 0034 425 30 182 541
HEXB_MOUSE Βeta-hexosaminidase subunit β 0012 183 uarr 0 59
CMGA_MOUSE Chromogranin-A 000078 153 darr 44 0
ANT3_MOUSE Antithrombin-III 0017 176 -14 67 47
SAP3_MOUSE Ganglioside GM2 activator 0045 415 darr 28 0
SPA3N_MOUSE Serine protease inhibitor A3N 0047 170 42 10 42
CO1A2_MOUSE Collagen alpha-2(I) chain 0043 56 darr 19 0
BLVRB_MOUSE Flavin reductase 00054 204 uarr 0 12
CATS_MOUSE Cathepsin S 00032 232 uarr 0 73
GFAP_MOUSE Glial fibrillary acidic protein 0021 107 uarr 0 21
RET4_MOUSE Retinol-binding protein 4 0040 189 darr 13 0
CBPE_MOUSE Carboxypeptidase E 0043 139 darr 11 0
CATL1_MOUSE Cathepsin L1 0015 87 94 02 19
The statistics are performed using the t-test from the ln(dNSAF) Gaussian data
a P p-value of the t-test where the null hypothesis states that there was no change in expression between
control and transgenic GFAP overexpressor b SC percentage of sequence coverage of the listed protein from
sequenced tryptic peptides c Fold Change positive values are indicative of a fold change for transgenic CSF
negative values are fold changes for control CSF (ie down-regulated in transgenic CSF) uarr indicates the protein
was only detected in transgenic CSF and darr indicates the protein was only detected in control CSF d dSpC
distributive spectral counts which represent the average spectral counts observed per run analysis on the mass
spectrometer and corrected using distributive analysis for peptides shared by more than one protein
98
Table 2 Proteins showing greater than three-fold changes with at least two unique
peptides identified for each protein
Accession Protein SC ()a Fold
Change b
Control
dSpC c
Transgenic
dSpC c
MUG1_MOUSE Murinoglobulin-1 precursor 147 42 155 37
CO4B_MOUSE Complement C4-B 113 54 22 118
PRDX6_MOUSE Peroxiredoxin-6 576 46 14 64
CNTN1_MOUSE Contactin-1 65 darr 41 0
CATB_MOUSE Cathepsin B 263 42 23 97
CAH3_MOUSE Carbonic anhydrase 3 396 76 11 84
APOH_MOUSE Beta-2-glycoprotein 1 128 44 44 1
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
PGK1_MOUSE Phosphoglycerate kinase 1 355 36 17 61
NDKB_MOUSE Nucleoside diphosphate kinase B 408 34 13 44
FHL1_MOUSE
Four and a half LIM domains
protein 1 243 39 13 51
NELL2_MOUSE
Protein kinase C-binding protein
NELL2 45 -43 13 03
MDHM_MOUSE
Malate dehydrogenase
mitochondrial 385 41 12 49
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
a SC percentage of sequence coverage of the listed protein from sequenced tryptic peptides b Fold
Change positive values are indicative of a fold change for transgenic CSF negative values are fold changes for
control CSF and darr indicates the protein was only detected in control CSF c dSpC distributive spectral counts
which represent the average spectral counts observed per run analysis on the mass spectrometer and corrected using
distributive analysis for peptides shared by more than one protein
99
Table 3 Validation of changes in proteins revealed by MS-based spectral counting
consistent with previously published microarray data
Consistent changes in RNA and proteomic data
uarr regulated in transgenic darr regulated in transgenic
Cathepsin S Contactin-1
Cathepsin B Carboxypeptidase E
Cathepsin L1
Peroxiredoxin-6
Complement C4-B
Glial fibrillary acidic protein
Serine protease inhibitor A3N
Note Validation of putative biomarkers from the current proteomics dataset by previously
published RNA microarray data55
Both up and down regulated proteins were consistent with the
RNA microarray data
_
100
___________________________________________
SUPPLEMENTAL INFORMATION (Available upon request)
Table S1 Compilation list of proteins identified from all the control and transgenic biological
replicates
Table S2 Distributive spectral counting calculations performed for proteins observed to share
identified peptides
Table S3 Proteins with dNSAF spectral counting performed including t-test analysis for a
comparison between transgenic and control CSF
101
FIGURE LEGENDS
Figure 1 The general workflow indicating the major steps involved in sample collection sample
processing mass spectrometric data acquisition and analysis of mouse CSF samples
Figure 2 Assessment of run to run variability of the base peak chromatograms within and
between two biological and technical replicates The peak profile and intensity scale is
consistent between the four chromatograms The four panels show two biological replicates (Tg
4 and Tg5) with two technical replicates for each biological sample
Figure 3 A Venn-diagram of the biological triplicates (1 2 and 3) of transgenic mouse
CSF showing 78 proteins identified in all three experiments B Venn-diagram of the biological
triplicates (1 2 and 3) of control mouse CSF having 61 proteins identified by all three
replicates C The overlap between control and transgenic CSF proteomic analysis showing 139
proteins identified by both groups and 73 and 54 uniquely identified by respective groups
Figure 4 Assessment of technical replicate variability between biological replicates The error
bars in both A and B are the standard deviation derived from the technical triplicates for each
biological replicate Panel A shows creatine kinase M having more or equal variability in the
biological triplicates than each technical triplicate The means of the biological triplicates are
illustrated by the dashed line Panel B represents kininogen-1 which is unchanged between
control (Ctl) and transgenic (Tg) samples The biological variability coupled with the technical
replicates provides a barely noticeable difference in the pooled mean between control and
102
transgenic spectral counts The difference in means is contrasted with the three fold change
observed from creatine kinase M (A)
Figure 5 MSMS profile of the tryptic peptide LSVEALNSLTGEFK from creatine kinase M
(A) The top MSMS is from a control sample with an elution at 463 minutes (B) The bottom
MSMS is from the GFAP transgenic sample with an elution at 46 minutes These tandem MS
spectra show instrument reliability and consistent fragmentation patterns which are necessary for
spectral counting analysis
Figure 6 Validation of MS spectral counts using a GFAP ELISA GFAP content (ngL)
measured within mouse CSF from both transgenic and control animals The data represents the
average with standard deviation (n=5 each sample represents pooling of 1 to 8 animals) The
statistics are performed using a student t-test plt00001
103
Figure 1
104
Figure 2
105
Figure3
106
Figure 4
107
Figure 5
108
Figure 6
Ctl Tg
100
1000
10000
100000
Mouse CSF Sample
GF
AP
(n
gL
)
109
Table of Contents Summary
Cerebrospinal fluid (CSF) was obtained from transgenic mouse models of Alexander disease as
well as healthy controls We immunodepleted pooled CSF from mice by a modified IgY-14
protocol Shotgun proteomics utilizing trypsin digestion 1-D nanoRP separation CID tandem
mass spectrometry analysis Mascot database searching and relative quantitation via distributive
normalized spectral abundance factor resulted in the identification of 266 proteins and 27
putative biomarkers
110
Chapter 4
Genomic and proteomic profiling of rat adapted scrapie
Adapted from ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A
Cunningham R Wang J Wellner D Ma D Li L Aiken J In preparation
111
Abstract
A novel model of prion disease using rats called Rat Adapted Scrapie (RAS) was
developed with the genomics from brain and proteomics from cerebrospinal fluid (CSF) profiled
The CSF proteome from control and RAS (biological N=5 technical replicates N=3) were
digested and separated using one dimensional reversed-phase nanoLC coupled to data-
dependent tandem MS acquisition on an ion trap mass spectrometer In total 512 proteins 167
non-redundant protein groups and 1032 unique peptides were identified with a 1 false
discovery rate (FDR) Of the proteins identified 21 were statistically up-regulated (plt005) and
7 statistically down-regulated in the RAS CSF We also identified 1048 genes which were
differentially regulated in rat prion disease and upon mapping these changes to mouse gene
expression however only 22 of these genes were in common with mRNAs responding to
prion infection in mice suggesting that the molecular pathology observed in mice may not be
applicable to other species The proteins are compared to the differentially regulated genes as
well as to previously published proteins showing changes consistent with other prion animal
models
112
Introduction
Prion diseases are an unusual class of fatal transmissible neurodegenerative disorders
that affect the mammalian central nervous system They are caused by the accumulation of an
abnormal conformation of the normal host encoded cellular prion protein PrPC This
conformational rearrangement of PrPC is brought about by template directed misfolding wherein
seed molecules of the abnormal isoform PrPScrapie
PrPSc
convert PrPC into new PrP
Sc molecules
Scrapie in sheep and goats is the prototypic prion disease and is so named because clinically
affected animals scrape their wool off on pen and stall walls Laboratory investigation of prion
diseases typically relies upon rodents which can be infected with natural isolates of scrapie1
albeit with some difficulty as ovid scrapie isolates need time to adapt to rodents This adaptation
is characteristic of prion disease interspecies transmissions and properly reflects the molecular
adaptation that must occur to allow interaction between exogenous foreign PrPSc
and host PrPC
molecules selecting for conformers which exhibit template directed misfolding In some cases
no conformational solution is found reflecting a species barrier to disease transmission
In recent years advances in genomics and proteomics technologies have allowed
unprecedented examination of the biomolecules that are altered upon exposure to prion agents
These studies2 3
have relied upon analysis of gene and protein expression changes in response to
prion infection with the aim of trying to identify pathways that might underlie the mechanism of
prion-induced neurotoxicity A second important aim has been to identify signature molecules
that might act as surrogate biomarkers for these diseases as there are significant analytical
challenges associated with sensitively detecting and specifically distinguishing disease-induced
conformational changes (PrPSc
) of the prion protein from normal host conformations (PrPC)
113
Mass spectrometry (MS)-based proteomics has become a promising tool for biomarker
discovery from biological fluids such as CSF blood and urine4-6
Two-dimensional gel
electrophoresis MS (2D-GE MS) and two-dimensional differential gel electrophoresis (2D-DIGE
MS) are the most widely used methods for proteomic profiling of prion infected CSF so far due
to the advantage of ready separation and quantification of proteins in complex biological samples
Studies using 2D-GE MS or 2D-DIGE MS to profile proteins in CSF have led to the
identifications of currently accepted biomarker for sCJD 14-3-3 protein and other potential
biomarkers for prion diseases7-9
However the application of this method in biomarker
discovery is limited by insufficient sensitivity and potential bias against certain classes of
proteins as gel-based separation does not work well for the low abundance proteins very basic
or acidic proteins very small or large proteins and hydrophobic proteins 10 11
In contrast to 2D-
GE or 2D-DIGE shotgun proteomics employs enzymatic digestion of the protein samples
followed by chromatographic separation prior to introduction into a mass spectrometer for
tandem MS analyses Shotgun proteomics has become increasingly popular in proteomic
research because these methods are reproducible highly automated and have a greater
likelihood of detecting low abundance proteins12 13
Due to the sample complexity in CSF and
because albumin comprises over half of the protein content in CSF removal of high-abundance
proteins including albumin is necessary to improve proteomic coverage and identify low-
abundance proteins One method is IgY immunodepletion14 15
which is performed prior to LC-
MSMS and uses species specific antibodies from chicken yolk to remove abundant proteins in
biological samples such as CSF In the present work CSF from control and rat adapted scrapie
animal models (biological N=5 technical N=3 replicates) were analyzed by LC-MSMS and we
114
indentified 512 proteins and 167 non-redundant protein isoforms (referred to as protein groups)
with 21 proteins being statistically up-regulated (p lt 005) and 7 statistically down-regulated
By and large this work has been performed using laboratory mice for the gene
expression studies and human cerebrospinal fluid for proteomics mice do not provide sufficient
volumes of CSF for proteomic studies Both approaches are exceedingly valuable as the mouse
model allows cross-sectional time course experiments to be performed including the important
pre-clinical phase of disease Critically however the relevance and generalizability of mouse
prion responses to other prion diseases especially human disease is unknown Human proteomic
studies on CSF guarantee relevant protein identifications but are limited to the clinical phase of
the disease when apparent markers may reflect gross neurodegeneration covering up subtle but
more specific responses To address these issues we have adapted mouse RML prions into rats
with the aim of expanding the knowledge of prion disease responses addressing the limitations
of mice and opening up the possibility of preclinical proteomic profiling in a laboratory rodent
In the present work CSF samples from control and rat adapted scrapie were analyzed by system
biology approaches Here we show that Rat Adapted Scrapie (RAS) is a powerful tool for an -
omics based approach to decipher the molecular impact of prion disease in vivo with
applicability to the molecular mechanisms of disease and biomarker discovery We identified
1048 genes which were differentially regulated in rat prion disease Astonishingly on the whole
mouse orthologs of these genes did not change in mouse adapted scrapie and vice versa
questioning the universality of previous mouse gene expression profiles These RAS gene
expression changes were identified in the CSF proteome where we detected 512 proteins and 167
protein groups with 21 proteins being statistically up-regulated (plt005) and 7 statistically down-
115
regulated in the CSF of prion diseased rats Many of the proteins detected have previously been
observed in human CSF from CJD patients
Materials and Methods
Ethics Statement
This study was carried out in accordance with the recommendations in the NIH Guide for Care
and Use of Laboratory Animals and the guidelines of the Canadian Council on Animal Care The
protocols used were approved by the Institutional Animal Care and Use Committees at the
University of Wisconsin and University of Alberta
Chemicals
Acetonitrile (HPLC grade) and ammonium bicarbonate (certified) were purchased from
Fisher Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) Optima LCMS grade acetonitrile and water were purchased
from Fisher Scientific (Fair Lawn NJ) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris
ge999 ) ammonium formate (ge99995) glycine (ge985) and IgY-7 antibodies were
purchased from Sigma-Aldrich (Saint Louis MO)
Rat Transmission and Adaptation
Prion agents used were the rocky mountain lab RML strain of mouse adapted scrapie
Stetsonville transmissible mink encephalopathy16
(TME) Hyper (Hy) strain of Hamster TME 17
1st passage Skunk adapted TME prepared as described and C from genetically defined
transmissions18
116
Brains from animals clinically affected with prion disease were aseptically removed and
prepared as 10 wv homogenates in sterile water 50 microL of 10 brain homogenate was
inoculated into weanling wistar rats intracranially After one year of incubation preclinical rats
from RML infections were euthanized by CO2 inhalation and the brain excised homogenized
and re-inoculated into naive animals Subsequent serial passages were from rats clinically
affected with rat adapted scrapie
Brains from rat passages were aseptically removed and bisected sagittally Brain halves
were reserved for immunohistochemistry (IHC) in formalin or frozen for immunoblotting RNA
isolation or subsequent passage For IHC tissues were first fixed in 10 buffered formalin
followed by antigen retrieval at 121 oC 21 bar in 10mM citrate buffer for 2 min After cooling
to room temperature sections were treated with 88 formic acid for 30 min and 4M guanidine
thiocyanate for 2 h Endogenous peroxidases were inactivated by 003 hydrogen peroxide and
tissue was blocked with 1 normal mouse serum Mouse anti-PrP mAB SAF83 (Cayman
Chemical) was biotinylated and applied at a 1250 dilution in blocking buffer overnight at 4 oC
Washes were performed in 10mM PBS with 005 tween-20 Streptavidin-peroxidase
(Invitrogen) was added for Diaminobenzidine (DAB) color reaction Anti-GFAP
immunohistochemistry was performed as above except that formic acid and guanidine treatment
steps were excluded Mouse anti-GFAP mab G-A-5 (Sigma) was used at a 1400 dilution
Brain samples for immunoblot were treated with or without Proteinase K (Roche) at a
ratio of 35 microg PK 100 ug protein at 50 microg mL for 30 minutes at 37 oC Phosphotungstenic acid
enrichments were performed as described14 19
Bis-Tris SDS-PAGE was performed on 12
polyacrylamide gels (Invitrogen) and transferred to PVDF Immunoblotting was performed using
117
mABs SAF83 (Cayman Chemical) 6H4 (Prionics) or 3F10 (a kind gift from Yong-Sun Kim) all
at a 120000 dilution
Gene Expression Profiling
RNA was extracted from frozen brain halves from clinically affected and control animals with
the QIAshredder and RNeasy mini kit (Qiagen Valencia CA) in accordance with the
manufacturerrsquos recommendations Due to the relatively large mass of rat brain an initial
homogenization was performed with a needle and syringe in 5mL of buffer RLT before further
diluting an aliquot in accordance with the manufacturers protocol Total RNA was amplified and
labeled in preparation for chemical fragmentation and hybridization with the MessageAmp
Premier RNA amplification and labeling kit (Life Technologies Grand Island NY) Amplified
and labeled cRNAs were hybridized on Affymetrix (Santa Clara CA) rat genome 230 20 high
density oligonucleotide arrays in accordance with the manufacturers recommendations
Differentially Expressed Genes were identified using Arraystar 50 (DNAStar Madison WI)
Robust multi-array normalization using the quantile approach was used to normalize all
microarray data A moderated T-test with a multiple comparison adjustment20
was used to reduce
the false discovery rate yet preserve a meaningful number of genes for pathway analysis
Pathway analysis was performed using the DAVID Bioinformatics database21
Comparative
analysis of genes induced by prions in mouse22
and rat disease was performed on genes
exhibiting at least a 2-fold change in expression Orthologs of rat and mouse genes were
identified using ENSEMBLE biomart release 6823
CSF Proteomic Profiling
118
CSF was collected from rats anaesthetized with isoflorane via puncture of the cisterna
magna A volume of 100-200 microL was routinely obtained CSF was touch spun for 30s at 2000xg
on a benchtop nano centrifuge to identify any blood contamination by the presence of a red
pellet CSF with blood contamination was not used for proteomic analysis CSF was prepared
for profiling by first depleting abundant proteins with an antibody based immunopartitioning
column IgY-R7 (Sigma-Aldrich Saint Louis MO) The manufacturers recommendations were
followed with a few deviations 40 microg of protein (~100 microL CSF) was bound to 100 microL of IgY
bead slurry in total volume of 600 microL formulated in 150mM ammonium bicarbonate The flow
through was collected and denatured by the addition of 10 microL of 8 M guanidine thiocyanate and
lyophilized in a speed-vac apparatus The sample was then reconstituted in 10 microL of water and 1
microL of 050 M DTT were added and allowed to incubate at 37oC for 30 minutes After incubation
27 microL of 055 M IAA was added for carboxymethylation and the sample was allowed to
incubate in the dark for 15 minutes After that 1 microL of 050 M DTT was added and allowed to
sit at room temperature for 10 minutes To perform trypsin digestion 70 microL of 50 mM
NH4HCO3 was then added along with 025 microg trypsin Digestion was performed overnight at
37oC and quenched by addition of 5 microL of 10 formic acid The tryptic peptides were then
subjected to solid phase extraction using an Agilent OMIX Tip C18 100 microL (Santa Clara CA)
Peptides were eluted with 50 ACN in 01 formic acid concentrated and reconstituted in 30
microL H2O with 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of a nanoAcquity (Milford MA) with a 10 microL injection
loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B consisted of
ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto a Waters 5 microm
119
Symmetry C18 180 microm x 20 mm trap cartridge (Milford MA) at a flow rate of 75 microLmin for 5
minutes at 95 A 5 B Separation was performed on a Waters 17 microm BEH130 C18 75 microm x
100 mm (Milford MA) with a gradient of 5 to 15 B over 10 minutes followed by 15 to
40 B over 80 minutes at room temperature
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with a Bruker CaptiveSpray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Waters Acquity console software to perform MS acquisitions for all experiments Smart
parameter setting (SPS) was set to 700 mz compound stability and trap drive level was set at
100 Optimization of the CaptiveSpray source resulted in dry gas temperature of 160oC dry
gas flow of 30 Lmin capillary voltage of -1325 V end plate offset of -500 V MSMS
fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
range of 350-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
120
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot24
(Version 24 Matrix
Science London UK) Database searching was performed against a forward and reversed
concatenated SwissProt Rattus rattus Mascot search parameters were as follows Allowed
missed cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) peptide tolerance plusmn12 Da maximum number of 13
C 1 MSMS
tolerance plusmn05 Da instrument type ESI-Trap Data was then transformed into Dat file formats
and transferred to ProteoIQ (NuSep Bogart GA) False positive analyses were calculated using
ProteoIQ and set at 1
Results
Development of Rat Adapted Scrapie
To develop a rat adapted strain of prion disease we introduced 6 different prion agents RML
TME Hy Skunk Adapted TME and Chronic Wasting Disease (CWD) from wild type 96G and
96S deer16-18
into 6 rats (Fig 1) Of these primary transmissions only RML induced the
accumulation of Proteinase K resistant PrP after one year of incubation as determined by western
blotting on 10 brain homogenates and PrPSc
enriched phoshotungstenic acid precipitated brain
homogenates Second passage of rat adapted RML scrapie resulted in clinically affected rats at
565 days post inoculation Serial passages of TME or CWD agents were not performed Clinical
symptoms consisted of ataxia lethargy wasting kyphosis and myoclonus Prion affected rats
121
also showed low level porphyrin staining around their head Subsequent serial passage decreased
incubation time to 215 days
Proteinase K resistant prion protein was observed from all clinically affected animals both by
immunoblot (Fig 2) and by immunohistochemistry (Fig 3) Di- and mono-glycosylated bands
were the most abundant isoforms of PrPSc
PrPSc
was extensively deposited in the cerebral cortex
hippocampus thalamus inferior colliculus and granular layer of the cerebellum GFAP
expressing activated astrocytes were found throughout the brain particularly in the white matter
of the hippocampus thalamus and cerebellum Spongiform change was a dominant feature of
clinical rat
Gene expression Profiling
In total 1048 genes were differentially regulated within a 95 confidence interval
(Supplementary Table 1) and 305 genes were differentially expressed by greater than 2-fold (Fig
4) The 1048 genes that were statistically significant were used for pathway analysis using
DAVID Pathway analysis suggested that the gene expression profile was consistent with
immune activation and maturation as well as inflammation (Supplementary Table 2) a likely
interpretation given the observable astrocytosis (Fig 3) and gliosis associated with prion disease
Other pathways highlighted by the analysis included increases in transcription of genes involved
in lysosomes and endosomes
To further probe the gene expression data we compared genes which were differentially
expressed in rat adapted scrapie against their orthologs expression in mouse scrapie and vice
versa (Figs 5 and 6) We did not observe a high degree of correlation between expression fold
changes For example GFAP a gene whose up-regulation in prion disease is well known was
122
increased 25 fold in rat adapted scrapie but up-regulated 93 fold in mouse scrapie A
qualitative analysis of expression of orthologs in prion disease suggests that many genes
deregulated at least 2-fold in mouse or rat do not have orthologs that are differentially expressed
For example the gene RNAseT2 was up-regulated greater than threefold in rat adapted scrapie
but was not significantly up-regulated in mouse Similarly three genes important in metals
homeostatsis metallothionein 1a and 2a and ceruloplasmin were up-regulated in rat 46 43 and
3 fold respectively but were not differentially expressed in mouse prion disease
CSF Proteomics
Each immunodepleted biological replicate (N=5 for each control and RAS) had technical
triplicates performed The triplicates were summed together using ProteoIQ Peptide spectral
counts were then summed for each protein and subjected to dNSAF analysis using ProteoIQ
internal algorithms Details for this method can be found elsewhere25 26
but briefly peptide
spectral counts are summed per protein (SpC) based on unique peptides and a weighted
distribution of any shared peptides with homologous proteins T-tests were used to identify
significant changes in protein expression 1032 unique peptides which identify 512 proteins and
167 protein groups were found Of these 512 proteins 437 were identified in both RAS and
control CSF samples (Fig 7) The complete list of all 512 proteins can be viewed in
Supplemental Table 3 Using the ProteoIQ the most likely isoform was selected such as 14-3-3
protein gamma
From Table 1 we observe five proteins that agree with the genomic data for up
regulations in RAS granulin macrophage colony-stimulating factor 1 receptor cathepsin D
complement factor H and ribonuclease T2 In addition serine protease inhibitor A3N was not
123
detected as up regulated in the RAS genomic data but was found to be up-regulated in previous
genomic profiling of the mouse prion model22
One interesting trend from the data in Table 1 is
that the majority of proteins found to be up-regulated in the RAS model were not detected in the
control samples The absence of the detection of those proteins such as ribonuclease T2 in the
control CSF does not necessarily suggest the absence of the protein nonetheless it is below the
detection limits for this current proteomics protocol and instrumentation
Discussion
Mice have been the preferred laboratory rodent for prion diseases research because they
can be inexpensively housed and are amenable to transgenesis which allows for short incubation
periods as well as hypothesis testing upon targeted pathways Pursuant to the determination of
the mouse genome and the development of high density transcriptional arrays for measurements
of gene expression profiling mice have been used extensively to examine the molecular
pathology of prion disease probing the impact of disease and animal strain In order to expand
upon this foundation we adapted mouse prions to infect rats We reasoned that by taking a
comparative approach to the molecular pathology of prion disease inferences could be obtained
into the variability of the molecular response to prion diseases and that understanding this
variability might suggest whether human prion disease responses are more or less similar to
mouse responses A second rationale is the desire to identify surrogate markers of prion disease
While this approach has been taken before using gene expression profiling a more direct
approach has been to use proteomic approaches on cerebral spinal fluid (CSF) identifying
proteins that are increase in abundance with disease A rat prion disease is valuable for this
because the rat proteome is established and rats allow for the collection of relatively large
volumes of CSF (~100 microL per animal) which allows for robust analytical separations enhancing
124
detection of biomarkers Finally rats unlike humans can be used in a time course study of prion
disease This allows for the identification of early transcriptional and proteomic responses to
prion infection responses which are particularly valuable for the identification of surrogate
disease biomarkers
To initiate the development of a rat prion disease we attempted to adapt six different
prion disease agents PrPres
molecules to rat via intracranial inoculation of weanling animals
(Figure 1) Of these six agents only mouse RML prions were able to surmount the species
barrier to prion disease transmission Mouse PrP differs from rat by eight amino acid changes
six in the mature protein and four in the N-terminal octa-peptide repeat region (Supplementary
Fig 1) As rat shares the most prion protein sequence homology with mouse it is perhaps not
surprising that it transmitted whereas the other did not confirming that the primary prion protein
sequence is the most important determinant for interspecies transmission We conclude that there
is a large molecular species barrier preventing conversion of rat PrPc into PrP
res
The transmission of mouse RML into rats was characterized by a shortening of the
incubation period following each passage This is indicative of agent adaption to the new host
and increases in the titer present in end-stage brain Overall our adaptation of mouse prion
disease into rats resulted in a similar agent to that observed by Kimberlin27
The differences in
incubation period at second passage are largely due to our collecting the animals at 365 days post
inoculation upon first passage whereas Kimberlin and Walker allowed the first passage animals
to reach end-stage clinical rats
Rat adapted scrapie is in many ways similar to other prion diseases The clinical period of
disease is marked by a progressive neurological dysfunction with prominent ataxia kyphosis and
125
wasting The protease resistant PrP observed is typical as is the widespread deposition of PrPSc
in
the brain Spongiosis and reactive astrogliosis are as expected of a prion disease
Gene expression profiles from rats clinically affected with prion disease revealed a strong
neuronal inflammation associated with proliferated and activated astrocytes This is perhaps best
observed through the up-regulation of GFAP GFAP positive astrocytes were omnipresent
throughout the brain and GFAP mRNA was up-regulated 25 fold The up-regulation of GFAP is
a hallmark of the molecular response to prion infection and has been routinely observed Our
comparative analysis of gene expression changes in mouse RML versus rat adapted scrapie
suggest substantial differences in gene expression in response to prion disease despite the fact
that the overall response is neuro-inflammatory This suggests that the potential overlap between
mouse expression profiles and a putative human CJD expression profile could be quite different
at the level of individual transcripts that might be expected to be changed
CSF Proteomics
CSF proteomics can be exceedingly challenging due to the small sample available large
dynamic range in proteins and abundant proteins limiting sample loading onto nanoscale
columns Dynamic range reduction in the CSF sample was achieved using a custom amount of
IgY-7 antibodies to abundant proteins such as albumin After immunodepletion the total
protein content was reduced by ~90 limiting the proteomics analysis to one dimensional
separation Label free quantitation spectral counting was performed because it requires less
protein and does not increase sample complexity The proteins identified from the affected and
control N=5 are shown in Supplemental Table 2 Consistent detection of proteins in CSF from
both control and infected rats was observed (Fig 7C) Only two proteins were identified in
126
controls that were not observed in RAS and only 10 proteins were only observed in RAS Some
of these proteins that were only identified in RAS are significantly changed (Supplemental Table
3) One concern in proteomics data is the variability from run to run and the possibility that
certain proteins are identified from different unique peptides Figure 7A shows that the vast
majority of the unique peptides detected (783) were identified with a 1 FDR in both RAS and
control CSF samples highlighting the analytical reproducibility of our methodology
Proteomic analysis of the infected rat CSF provides a reasonable approach to cross
validate biomarkers detected by gene expression profiling For example RNAseT2 a secreted
ribonuclease was up-regulated 3-fold at the mRNA level and was also enriched in CSF from
infected rats (Table 1) Similarly coordinated increases in detection of colony stimulating factor
1 receptor complement factor H granulin and cathepsin D were also observed Conversely
proteomic analysis of CSF also allows for the observation of post-transcriptional responses to
prion disease that might be absent in gene expression profiling The 14-3-3 proteins and neuron
specific enolase both known markers for CJD are only detected by proteomic analysis Thus
gene expression profiling and proteomic detection serve to increase confidence in the
observation of up-regulation enhancing the likelihood that proteins detected by both
methodologies are specific and perhaps may be more sensitive at earlier time points
Comparison to human CSF prion disease proteome
In our comparative analysis 21 proteins were found to be up-regulated while 7 proteins
down-regulated in prion infected rats Included in the up-regulated proteins were the 14-3-3
proteins epsilon and gamma 14-3-3 zetadelta was also up-regulated but not statistically
significant Enolase 2 or neuron specific enolase (NSE) was also up-regulated in CSF of infected
127
rats These proteins are all in agreement with results from previous proteomic profiling of human
CSF from patients with CJD8 9
The detection of 14-3-3 protein is included in the diagnostic
criteria approved by World Health Organization for the pre-mortem diagnosis of clinically
suspected cases of sCJD28
although its application in large-scale screening of CJD is still
debated due to high false positive rate where elevated 14-3-3 protein levels were also reported in
other conditions associated with acute neuronal damage29 30
It was suggested that other brain-
derived proteins that are also specific to CJD can be used in conjunction with 14-3-3 protein to
increase diagnosis accuracy and specificity31
NSE is present in high concentration in neurons
and in central and peripheral neuroendocrine cells NSE serves as another valuable biomarker in
diagnosis of CJD as the elevated level of NSE in CSF was found in early and middle stages of
CJD 32
Other proteins detected in CSF included cystatin C and serpina3N although both of
these were not statistically changed These proteins were both previously identified as being
putative biomarkers for CJD33 34
Utility for biomarker discovery with emphasis on onset of biomarker appearance in CSF
The investigation of the protein changes in CSF from RAS compared to control rats
provides a solid foundation when investigating potential biomarkers with prion disease onset
The cross-validation of the genomic and proteomics data further emphasizes the targets for
consideration during disease onset Biomarker discovery provides the potential to determine if
animals such as cows in bovine spongiform encephalopathy (BSE) are affected instead of
having to be euthanized when BSE is expected In addition CSF collection in mice or hamsters
Prion models is extremely difficult and limited alternatively with the advent of the RAS model
CSF can be collected in sufficient amounts for CSF proteomics CSF collection for mice or
hamsters can be ~10 microL and could be contaminated with blood further complicating proteomic
128
analysis unlike rats which over 10 times more CSF can be collected per animal35
Due to the
amount of CSF available time course studies analyzing CSF proteomics is possible in RAS due
to animal numbers that are manageable and reasonable The RAS model further allows
investigators to bypass working with highly infections CJD CSF samples to investigate the CSF
proteome changes
Conclusion
In this study we have described the gene and protein expression changes in brain and
spinal fluid from a transmission of mouse prions into rats We find that while the overall gene
expression profile in rats is similar to that in mice the specific genes that make up that profile
are different suggesting that genes that change in response to prion disease in different species
may not be as conserved Analysis of CSF from rat adapted scrapie identified similar protein
changes as known in human CJD The rat will be a useful model to identify surrogate markers
that appear prior to the onset of clinical disease and thus may be of higher specificity and
sensitivity
Supplemental Information Available Upon Request
1 Chandler R L Encephalopathy in mice produced by inoculation with scrapie brain material Lancet 1961 1 (7191) 1378-9 2 Silva C J Onisko B C Dynin I Erickson M L Requena J R Carter J M Utility of Mass Spectrometry in the Diagnosis of Prion Diseases Anal Chem 2011 3 Wei X Herbst A Ma D Aiken J Li L A quantitative proteomic approach to prion disease biomarker research delving into the glycoproteome J Proteome Res 2011 10 (6) 2687-702 4 Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C Mann M Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7 (1) 386-99 5 Sjodin M O Bergquist J Wetterhall M Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers J Chromatogr B Analyt Technol Biomed Life Sci 2003 878 (22) 2003-12 6 Cunningham R Ma D Li L Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery Frontiers in Biology 2012 7 (4) 313-335
129
7 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66 8 Harrington M G Merril C R Asher D M Gajdusek D C Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease N Engl J Med 1986 315 (5) 279-83 9 Piubelli C Fiorini M Zanusso G Milli A Fasoli E Monaco S Righetti P G Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping Proteomics 2006 6 Suppl 1 S256-61 10 Lilley K S Razzaq A Dupree P Two-dimensional gel electrophoresis recent advances in sample preparation detection and quantitation Curr Opin Chem Biol 2002 6 (1) 46-50 11 Oh-Ishi M Maeda T Separation techniques for high-molecular-mass proteins J Chromatogr B Analyt Technol Biomed Life Sci 2002 771 (1-2) 49-66 12 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset J Proteome Res 2008 7 (2) 698-707 13 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422 (6928) 198-207 14 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample preparation and analysis Proteomics 2005 5 (13) 3314-28 15 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep Biochem Biotechnol 2009 39 (3) 221-47 16 Marsh R F Bessen R A Lehmann S Hartsough G R Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy J Gen Virol 1991 72 ( Pt 3) 589-94 17 Bessen R A Marsh R F Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent J Virol 1992 66 (4) 2096-101 18 Johnson C J Herbst A Duque-Velasquez C Vanderloo J P Bochsler P Chappell R McKenzie D Prion protein polymorphisms affect chronic wasting disease progression PLoS One 2011 6 (3) e17450 19 Safar J Wille H Itri V Groth D Serban H Torchia M Cohen F E Prusiner S B Eight prion strains have PrP(Sc) molecules with different conformations Nat Med 1998 4 (10) 1157-65 20 Benjamini Y Hochberg Y Controlling the False Discovery Rate A Practical and Powerful Approach to Multiple Testing Journal of the Royal Statistical Society Series B (Methodological) 1995 57 (1) 289-300 21 Huang da W Sherman B T Lempicki R A Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat Protoc 2009 4 (1) 44-57 22 Moody L R Herbst A J Aiken J M Upregulation of interferon-gamma-induced genes during prion infection J Toxicol Environ Health A 2009 74 (2-4) 146-53 23 Flicek P Amode M R Barrell D Beal K Brent S Carvalho-Silva D Clapham P Coates G Fairley S Fitzgerald S Gil L Gordon L Hendrix M Hourlier T Johnson N Kahari A K Keefe D Keenan S Kinsella R Komorowska M Koscielny G Kulesha E Larsson P Longden I McLaren W Muffato M Overduin B Pignatelli M Pritchard B Riat H S Ritchie G R Ruffier M Schuster M Sobral D Tang Y A Taylor K Trevanion S Vandrovcova J White S Wilson M Wilder S P Aken B L Birney E Cunningham F Dunham I Durbin R Fernandez-Suarez X M Harrow J Herrero J
130
Hubbard T J Parker A Proctor G Spudich G Vogel J Yates A Zadissa A Searle S M Ensembl 2012 Nucleic Acids Res 2012 40 (Database issue) D84-90 24 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 1999 20 (18) 3551-67 25 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J Proteome Res 2006 5 (9) 2339-47 26 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome quantitation how to deal with peptides shared by multiple proteins Anal Chem 2010 82 (6) 2272-81 27 Kimberlin R H Cole S Walker C A Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters J Gen Virol 1987 68 ( Pt 7) 1875-81 28 World Health Organization Global surveillance diagnosis and therapy of human transmissible spongiform encephalopathies report from a WHO consultation 1998 World Health Organization Geneva Switzerland 9-11 February 1998 1-26 29 Bartosik-Psujek H Archelos J J Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG J Neurol 2004 251 (4) 414-20 30 Saiz A Graus F Dalmau J Pifarre A Marin C Tolosa E Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders Ann Neurol 1999 46 (5) 774-7 31 Sanchez-Juan P Green A Ladogana A Cuadrado-Corrales N Saanchez-Valle R Mitrovaa E Stoeck K Sklaviadis T Kulczycki J Hess K Bodemer M Slivarichova D Saiz A Calero M Ingrosso L Knight R Janssens A C van Duijn C M Zerr I CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease Neurology 2006 67 (4) 637-43 32 Zerr I Bodemer M Racker S Grosche S Poser S Kretzschmar H A Weber T Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease Lancet 1995 345 (8965) 1609-10 33 Sanchez J C Guillaume E Lescuyer P Allard L Carrette O Scherl A Burgess J Corthals G L Burkhard P R Hochstrasser D F Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease Proteomics 2004 4 (8) 2229-33 34 Miele G Seeger H Marino D Eberhard R Heikenwalder M Stoeck K Basagni M Knight R Green A Chianini F Wuthrich R P Hock C Zerr I Aguzzi A Urinary alpha1-antichymotrypsin a biomarker of prion infection PLoS One 2008 3 (12) e3870 35 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
131
Figure 1 Interspecies transmission of prion disease to rats End-stage clinical brain homogenates
were used to passage prion disease After one year of incubation animals were euthanized to
determine the extent of PrPres
accumulation Protease resistance PrP was only observed in those
animals infected with RML scrapie prions This material was serially passaged for two more
incubations before becoming rat-adapted as indicated by the shortening of the incubation period
132
Table 1 A list of selected proteins and mRNAs up-regulated in RAS CSF and brain tissue If
the protein is detected in the RAS CSF but not the control CSF the CSF expression is reported
with a infin If there is no change or data on certain genes related to an up regulated protein nd is
noted The mouse genomic data presented here was previously published22
Gene Protein Symbol Accession CSF
Expression
Rat
GEX
Mouse
GEX
14-3-3 protein zetadelta Ywhaz NP_037143 infin nd nd
14-3-3 protein epsilon Ywhae NP_113791 infin nd nd
14-3-3 protein gamma Ywhag NP_062249 infin nd nd
serine protease inhibitor A3N Serpin A3N NP_113719 54 nd 975
enolase 2 (gamma neuronal) Eno2 NP_647541 infin nd nd
granulin GRN NP_058809 62 364 184
macrophage colony-stimulating
factor 1 receptor
Csf1r NP_001025072 infin 293 205
cathepsin D CTSD NP_599161 infin 255 299
complement factor H Cfh NP_569093 376 234 nd
ribonuclease T2 RNAset2 NP_001099680 infin 302 nd
133
Figure 2 Accumulation of PrPSc
in rat adapted scrapie First second and third passage brain
homogenates were assayed for the presence of protease resistant PrP by immunoblot PrPSc
was
observed following phosphotungstenic acid enrichment at first passage By clinical disease in 2nd
and 3rd
passage rats PrPSc
had substantially accumulated
134
Figure 3 Histological appearance of rat adapted scrapie in the hippocampus at clinical disease
Infected animals showed intense immuno-staining for deposits of PrPSc
and GFAP expressing
astrocytes Spongiform change is an abundant feature in rat adapted scrapie
135
Figure 4 Scatter plot of gene expression profiling of rat adapted scrapie The expression of
individual genes from uninfected and infected animals were plotted to display up and down
regulation The dashed green line is no change Solid green lines are 2-fold changes in gene
expression
136
Figure 5 Quantitative relationship between orthologous pairs of two-fold up-regulated genes in
mouse and rat scrapie Genes up-regulated more than two fold were mapped to their orthologs
and the fold change was plotted Expression is log2 transformed
137
Figure 6 Comparison of genes upregulated in rat and mouse prion diseases Genes up-regulated
two fold in rodent scrapie were identified and the expression of their orthologs was determined
138
Figure 7 Venn diagram comparison of the CSF proteomics data between rat adapted scrapie
(RAS) model and control rats (A) Unique peptides from tryptic peptides produced for the
proteins identified (B) The total proteins identified including all isoforms within the protein
groups (C) The protein groups comparing only the top protein hit of the protein isoforms
showing very consistent protein identifications between RAS and control
139
Chapter 5
Investigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiae
Adapted from ldquoInvestigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M
Heideman W Li L In preparation
140
Abstract
This work explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Kinases such as protein
kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose response
Thus performing a large scale phosphoproteomic MS study can be highly beneficial to map the
signaling networks and cascades involved in glucose-dependent stimulated yeast growth Yeast
cell extract was digested and phosphopeptides were enriched by immobilized metal affinity
chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase (RP)-RP
separation The low pH separation was infused directly into an ion trap mass spectrometer with
neutral loss electron-transfer dissociation (ETD)-triggered fragmentation to improve
phosphopeptide identifications In total 477 unique phosphopeptides are identified with 06
false discovery rate (FDR) and 669 unique phosphopeptides identified with 54 FDR This
study includes comparative phosphoproteins for one specific motif of PKA xxxKRKRxSxxxx
which is presented and differences between starved vs glucose fed are highlighted Phosphosite
validation is performed using a localization algorithm Ascore to provide more confident and
site-specific characterization of phosphopeptides
141
Introduction
Microorganisms such as Saccharomyces cerevisiae (yeast) cycle between growth when
nutrients are available and quiescence when nutrients are scarce When glucose is limited yeast
go into growth arrest state but when glucose is added growth quickly resumes Kinases such as
protein kinase A (PKA) and target of rapamycin (TOR) regulate growth in response to nutrient
conditions and have been well studied through transcriptional control1-4
Yeast execute large
transcriptome alterations in response to changing environmental growth conditions5 6
Gene
regulation by glucose introduction in yeast has been studied including genes used for growth on
alternative carbon sources and activation of genes coding for glucose transport and protein
synthesis7-10
Response to nutrients for survival is not limited to yeast biology and indeed all
living creatures both prokaryotes and multicellular eukaryotes like mammals require nutrient
responsiveness and coordinating cellular functions to survive
With regulation of certain genes well studied by glucose introduction the mechanism and
global protein modulation caused by glucose introduction remain unknown6 Large-scale
phosphorylation analysis has been previously performed in yeast using mass spectrometry11-14
Mapping the phosphoproteome in yeast transitioning from quiescence to growth conditions to
better understand the roles of phosphorylation in orchestrating growth is needed The
phosphorylation state during growth or starvation could be used to engineer cells to drive ectopic
activity (or inhibition) to promote growth and ethanol production on non-native sugars like
xylose
It has been reported that the phosphorylation state can be affected by the introduction of
glucose to carbon-starved yeast15
and phosphorylation plays a significant role in the cell cycle
and signal transduction16
Specifically O-Phosphorylation can function as a molecular switch by
142
changing the structure of a protein via alteration of the chemical nature of an amino acid for
serine threonine or tyrosine It has been reported that up to 30 of the proteome can undergo
phophorylation17
Mass spectrometry has evolved as a powerful tool to accomplish phosphosite
mapping using shotgun proteomics With available technology tens of thousands of
phosphorylation sites can be mapped to amino acids on thousands of proteins using shotgun
proteomics18-20
Mass spectrometry can offer sensitive automated non-targeted global analysis of
phosphorylation events in proteomic samples but in any large scale phosphoproteomic
investigation enrichment of phosphoproteinspeptides is required First phosphorylation is
usually a sub-stoichiometric process where only a percentage of all protein copies are
phosphorylated21
Various enrichment methods have been used for phosphopeptide enrichment
including metal oxide affinity chromatography (MOAC)22
such as TiO223
immobilized metal
affinity chromatography (IMAC)12 24 25
electrostatic repulsion-hydrophilic interaction
chromatography (ERLIC)26
and immunoaffinity of tyrosine phosphorylation27 28
After
enrichment MS analyses of phosphorylated peptides are still challenging due to ion suppression
from non-phosphorylated peptides
Even after phosphopeptide enrichment further sample preparation is needed for large
scale proteomic experiments Additional fractionation can increase protein coverage of a
sample by over ten fold such as MudPIT29
(multidimensional protein identification technology)
In online MudPIT a sample is injected onto a strong cation exchange (SCX) column coupled to
a RP column Successive salt bumps followed by low pH gradients provide the separation of
peptides in two dimensions Since the phosphate groups on phosphopeptides have a low pKa
value due to being more acidic then their unmodified counterparts they tend to elute earlier and
143
disproportionally in ion exchange chromatography like SCX Alternatively reversed-phase
reversed-phase (RP-RP) separation has been proposed as an alternative to MudPIT for offline
two dimensional (2D) separation30
One of the caveats of 2D separation is the potential for
wasted mass spectrometry time from early and late fractions having very few peptides present
all while having too much sample for middle fractions One simple method to reduce these
ldquowasted fractionsrdquo is to combine early or late fractions with the middle fractions to reduce MS
runs with little peptide content to analyze thus shortening the overall analysis time31
In addition the labile phosphorylation group has a large propensity to undergo cleavage
during collision induced dissociation (CID) producing a neutral loss The neutral loss can
produce insufficient backbone fragment ions for MSMS identification32
A solution to neutral
loss fragmentation in phosphopeptides is MS3 using CID to produce improved backbone
fragmentation13 14 33
An alternative fragmentation method to CID for fast sampling ion traps is
electron transfer dissociation (ETD)34-36
ETD produces a more uniform back-bone cleavage
where the cation peptide receives an electron from a low affinity radical anion37
The transferred
electron induces a dissociation at the N-Cα bond to produce c- and zbull-type product ions while
retaining the labile post-translational modifications (PTM) such as phosphorylation bound to the
product ions38
The Bruker amaZon ETD ion trap mass spectrometer has the potential to trigger
ETD fragmentation after a labile PTM produces a neutral loss from CID fragmentation This
method is termed neutral loss-triggered ETD fragmentation and provides a complementary
fragmentation pathway to labile poor fragmenting phosphorylated peptides
In this work we provide a qualitative comparative list of yeast phosphopeptides observed
in glucose fed vs glucose starved conditions
144
Experimental
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) ammonium hydroxide (ACS plus certified) urea (gt99)
sodium chloride (997) ammonium bicarbonate (certified) Optima LCMS grade acetonitrile
Optima LCMS grade water and EDTA disodium salt (99) were purchased from Fisher
Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) ammonium formate (ge99995) Trizma
hydrochloride (Tris HCl ge990) Triton X-100 (laboratory grade) and iron (III) chloride
hexahydrate (97) were purchased from Sigma-Aldrich (Saint Louis MO) Sodium dodecyl
sulfate (SDS) (998) was purchased from US Biological (Marblehead MA) Nickel
nitrilotriacetic acid (Ni-NTA) magnetic agarose beads were purchased from Qiagen (Valencia
CA) Autoclaved yeast peptone dextrose (YPD) media was prepared in 1 L of deionized water
using 10 g yeast extract 20 g Peptone from Becton Dickinson and Company (Sparks MD) and
20 g D-(+)-Glucose (ge995) purchased from Sigma-Aldrich (Saint Louis MO)
Modified Mary Miller Yeast Protein Isolation
The yeast culture was prepared and protein extraction was performed using a modified
Mary Miller protocol39
Briefly yeast strain s288c was inoculated with YPD media and shook
for 72 hours to allow the yeast to be glucose starved After the 72 hours the yeast culture was
partitioned into two flasks To one flask glucose was added at 2 of the final concentration and
allowed to incubate and shake for 10 minutes Then 100 optical density units (ODU) of the yeast
145
culture were collected from each flask Each yeast sample was spun down in a Beckman-Coulter
J6B centrifuge (Indianapolis IN) at 2500xg for three minutes The media was aspirated and the
tubes were flash frozen in liquid nitrogen and stored at -80oC The yeast pellets were thawed on
ice and resuspended in 1 mL of lysis buffer (50 mM Tris HCl 01 triton x-100 05 SDS
pH=80) and 1X protease and phosphatase inhibitor cocktail from Thermo Scientific (Rockford
IL) and transferred to an Eppendorf tube To the yeast 200 microL of glass beads were added and
amalgamated for 25 minutes at 4oC The sample was inverted and the Eppendorf tube was
pierced with a hot 23 gauge needle through the bottom and the tube was placed atop a 5 mL
culture tube and centrifuge in the Beckman-Coulter J6B at 2500xg for three minutes at 4oC to
collect the liquid containing the yeast cells while the glass beads remain trapped in the
Eppendorf tubes The protein lysate was then centrifuged at 3000xg for five minutes at 4oC and
the supernatant was collected and stored at -80oC
Preparation of tryptic digests
The Saccharomyces cerevisiae protein cell extract for fed or starved were analyzed with a
BCA protein assay kit (Thermo Scientific Rockford IL) and 2 mg of protein was added to four
parts -20oC acetone and allowed to precipitate the protein for 1 hour at -20
oC The samples were
then centrifuged at 14000xg for 5 minutes and the supernatant was removed and the protein
pellet was reconstituted in 360 microl of 8 M urea To each sample 20 microL of 050 M DTT was
added and allowed to incubate at 37oC for 45 minutes After incubation 54 microL of 055 M IAA
was added for carbamidomethyl of cysteine and the sample was allowed to incubate for 15
minutes in the dark To quench the IAA 20 microL of 050 M DTT was added and allowed to react
for 10 minutes To perform trypsin digestion 1400 microL of 50 mM NH4HCO3 was then added
along with 20 microg trypsin to each sample Digestion was performed overnight at 37oC and
146
quenched by addition of 25 microL of 10 formic acid For each sample the tryptic peptides were
then subjected to solid phase extraction using a Hypersep C18 100 mg solid phase extraction
(SPE) column (Thermo Scientific Bellefonte PA) Peptides were eluted with 50 ACN in
01 formic acid concentrated and reconstituted in 500 microL of 80 ACN and 01 formic acid
Phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC)
One ml of Ni-NTA was washed three times with 800 microL of H2O and then the Ni was
removed with 800 microL of 100 mM EDTA in 50 mM ammonium bicarbonate and vortexed for 30
minutes The supernatant was removed and the NTA was washed with 800 microL of H2O three
times followed by addition of 800 microL 10 mM FeCl3 hexahydrate and vortexed for 30 minutes
The Fe-NTA was then washed three times with 800 microL of H2O and once with 80 ACN in 01
formic acid before being combined with the cell extract for phosphopeptide enrichment and
vortexed for 40 minutes The sample was washed three times with 200 microL of 80 ACN in 01
formic acid before elution with two aliquots of 200 microL of 5 ammonium hydroxide in 5050
ACNH2O with 15 minutes of vortexing The enriched phosphopeptides were then dried down
with a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA) and reconstituted in 55 microL
25mM ammonium formate pH=75
First dimension neutral pH separation
Individually 50 microL of each yeast phosphopeptide enriched sample was injected onto a
Phenomenex Gemini-NX 3microm C18 110 Aring 150 x 2 mm column (Torrance CA) The Gemini
column was coupled to a Phenomenex SecurityGuard Gemini C18 2 mm guard cartridge
(Torrance CA) Mobile phase A consisted of 25 mM ammonium formate at pH=75 and mobile
phase B consisted of 25 mM ammonium formateACN with a 19 ratio respectively at pH=75
The column was operated at 50oC at a flow rate of 05 mLmin The flow profile was 2-30 B
147
over 65 minutes and then 5 min at 100 B A total of 22 fractions were collected every 3
minutes and the fractions were combined as follows 1 with 12 2 with 13 etc to 11 with 22
The combined fractions were dried down and reconstituted in 25 microL of 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL from each fraction onto an Agilent Technologies
Zorbax 300 SB-C18 5 microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5
microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm Atlantis
dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B at 250
nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) for MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
148
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions An additional mode of MSMS fragmentation electron transfer dissociation
(ETD) was triggered on the precursor ion when a neutral loss was observed in CID
fragmentation from phosphoric acid H3PO4 (Δmz of 49 and 327 for 2+ and 3+ charge states
respectively) or for metaphosphoric acid (HPO3) (Δmz of 40 and 267 for 2+ and 3+ charge
states respectively) For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms rolling average 2 acquisition range of 300-1500 mz
and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target
was set to 300000 maximum accumulation time 5000 ms two spectral averages acquisition
range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required to prevent artificial data
reduction Identification of peptides were performed using Mascot40
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt Saccharomyces
cerevisiae database (version 575) Mascot search parameters were as follows Allowed missed
cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) and phosphorylation (STY) peptide tolerance plusmn12 Da maximum
number of 13
C 1 MSMS tolerance peptide decoy (Mascot) checked plusmn05 Da instrument type
149
ESI-Trap Results from the Mascot search was exported as DAT files for analysis by Scaffold 3
and Scaffold PTM
Scaffold and Ascore data processing
Scaffold 3 (Proteome Software Portland OR)was used for MSMS proteomics data
comparison and false discovery rate analysis The DAT files were loaded into Scaffold 3 and
the fractions for the two dimensional fractionation were combined The resulting biological
triplicates for starved and glucose fed yeast were filtered with a 1 false discovery rate (FDR)
on the protein level (Supplemental Table 1) Due to the inherent poor fragmentation of
phosphopeptides a lenient FDR level may still yield biologically relevant data and thus a 54
FDR was generated for phosphopeptides (Supplemental Table 2) For a more conservative list of
phosphopeptides with a 06 FDR was generated from Scaffold 3 (Supplemental Table 3) FDR
analysis is sufficient at preventing poor data from being reported but does not prevent false
phosphosite identification in phosphopeptides To provide confidence in site identification
Scaffold PTM was used to perform Ascore41
analysis Ascore uses an algorithm to score the
probability of the phosphosite from a phosphopeptide identified by a database searching
algorithm such as Mascot Ascore can be freely accessed at httpascoremedharvardedu
Cell collection RNA isolation and microarray data analysis
All experiments were performed in biological duplicates Cell samples (10 ODU) were
taken at starved and fed states spun at 5000xg for 2 minutes at 30 oC the supernatant was
removed and the pellets snap frozen using liquid nitrogen RNA was isolated using the Epicentre
MasterPure Yeast Purification Kit (Epicentre Technologies) and quality was checked using gel
electrophoresis cRNA synthesis was carried out using the GeneChipreg Expression 3
Amplification One-Cycle Target Labeling and Control Reagents kit (Affymetrix) All
150
experiments followed the manufactures instructions cRNA samples were hybridized to
GeneChip Yeast Genome 20 Arrays for 16 hours Arrays were washed stained and scanned
according the manufactures recommendations Affymetrix CEL files were RMA normalized
with R and the Bioconductor Suite Data analysis was performed within TIGR Multiexperiment
Viewer v451 in-house Perl scripting R and Bioconductor
Results
Sample preparation for shotgun proteomics
As discussed in the introduction the purpose of this study is to provide an exploratory list
of qualitative differences in the phosphoproteome between starved vs glucose fed yeast After
yeast cell lysate production a substantial amount of sample preparation is performed to enhance
the depth of the phosphoproteome coverage A workflow of the steps following cell lysis is
outlined in Figure 1A The yeast proteins are isolated via acetone precipitation followed by
digestion with trypsin to produce peptides Phosphopeptides are intermixed with the entire
tryptic peptide sample and therefore Fe-NTA IMAC is used to enrich the phosphopeptides To
improve upon the number of phosphopeptides we then performed two dimensional separation
with high pH RP followed by low pH RP C18 and infused the eluent directly onto an ion trap
mass spectrometer Figure 1B show an improved technique for the first dimension of separation
to combine the early eluting and late eluting fractions from the first phase of separation to reduce
overall MS analysis time The combination of fractions 1 and 12 2 and 13 etc essentially
improves the duty cycle of the mass spectrometer by ensuring sufficient peptide content is
injected onto a low pH nanoLC RP C18 column
ETD-triggered mass spectrometry
151
In the present study labile phosphorylation can lead to non-informative neutral loss
During MS scanning mode the instrument will choose the 6 most abundant peaks with correct
isotopic distribution and select them for MSMS CID fragmentation During CID fragmentation
it is common for labile PTMs such as phosphorylation to undergo neutral loss and thus limited
informative b and y-type ions are formed Alternatively ETD fragmentation can be used on
specific MSMS scans which produce a neutral loss indicative of phosphorylation (98Daz or
80Daz) The subsequent ETD fragmentation on these putative phosphopeptides will lead to
uniform backbone cleavage resulting in confident identification of phosphopeptides with site-
specific localization during MSMS It is important to note that CID fragmentation still produces
very informative fragmentation for phosphorylation but ETD provides an orthogonal
fragmentation pathway to further increase the phosphoproteome coverage Additionally the
duty cycle for ETD fragmentation is roughly half that of CID therefore about half as many
potential peptides would be fragmented and sequenced if the instrument was using ETD
fragmentation exclusively
Protein Data
Even with phosphopeptide enrichment it is inevitable that non-phosphopeptides will also
be identified All data were searched with Mascot and in total over 1000 proteins were identified
with a 1 FDR rate and are listed in Supplemental Table 1 The proteins listed in Supplemental
Table 1 include both phosphoproteins and non-phosphorylated proteins In addition to the
proteins identified in the fed and starved states the unique peptides and spectral counts are also
listed for reference The phosphopeptides are specifically listed with a 54 FDR rate in
Supplemental Table 2 and comparisons between starved and fed with Ascore analysis are listed
for every phosphopeptide identified A higher confidence of phosphopeptide identification is
152
sometimes required before investing in time consuming biological experiments so a list of
phosphopeptides identified in starved and fed was taken from a setting directing Scaffold to
produce a 05 FDR which resulted in the generated FDR of 06 FDR and listed in
Supplemental Table 3
A summary of specific phosphorylation sites are listed in Table 1 with a 06 FDR and
Table 2 with a 54 FDR Each table gives totals for unique phosphopeptides identified having
an Ascore localization score ge80 without Ascore and phosphorylation events on each unique
peptides As expected the majority of phosphorylation events (over 50) occurred on serine
whereas fewer phosphorylation events (~10) occurred on tyrosine In addition the vast
majority of phosphorylation events were single phosphorylation (ge65) with very few
identifications having more than two phosphosites per peptide For specific phosphopeptide
identification and subsequent phosphoprotein identification refer to Supplemental Table 2 and 3
Discussion
Transcriptional response to glucose feeding
Yeast responds to the repletion of glucose after glucose-depletion by broad
transcriptional changes to induce growth Roughly one-third of the yeast genome is altered by at
least two fold when glucose is introduced as shown in Figure 2 Figure 2 is a heat map from a
microarray analysis showing 1601 genes up regulated and 1457 genes down regulated after
addition of glucose compared to the starved state The arbitrary cut-offs for these values were as
follows two fold induction after nutrient repletion and a false discovery rate (q-value) of 001
Red color in the heat map indicates a particular gene is up-regulated in the fed state compared to
the starved state Alternatively genes coded in green are less expressed in the fed state
compared to the starved condition The intensity of the green or red colors is indicative of the
153
intensity of the fold change in gene expression These large transcriptional changes after glucose
repletion drive and complement the current phosphoproteomic study
PKA motif analysis
One benefit of a large scale phosphoproteomics experiment is to examine the different
phosphopeptides from known motifs for specific kinases PKA and TOR are responsible for the
majority of the transcriptional response and thus PKA is a good target for motif analysis Figure
3 shows the PKA motif sequence xxxRKRKxSxxxxxx with the phosphorylation occurring on
the serine Other than F16P (fructose-16-bisphosphatase) all the proteins listed are unique to the
starved or fed samples A motif sequence will inevitably show up by random chance in any
analysis For this study the control for motif analysis uses the swissprot protein list for the
entire yeast proteome for the background Compared to background this specific PKA kinase
from Figure 3 is up-regulated by 264 fold when compared to the background One interesting
protein emerged from this motif analysis in the fed sample but not the starved sample is
Ssd1which is implicated in the control of the cell cycle in G1 phase42
Ssd1 also is
phosphorylated by Cbk1 and Cbk1 has been proposed to inhibit Ssd143
and provides an
intriguing target for future studies on starved vs glucose fed yeast growth
Localization of the phosphorylation sites
When a phosphopeptide contains any number of serine threonine or tyrosine amino
acids the localization of the phosphosite can sometimes be ambiguous Database searches used
to identify peptides like Mascot do not provide any probability for localization of correct
phosphorylation sites Ascore is an algorithm that uses the same MSMS spectra as Mascot but
instead of comparing theoretical peptide MSMS to experimental spectra Ascore searches for
informative a b or y-type peptide fragments giving evidence for phosphosite The Scaffold
154
program adds a localization probability to the Ascore values and the values are listed in
Supplemental Table 2 and 3 Figure 4 has a representative Ascore mass spectra assigning the
peaks identified and providing evidence that the phosphorylation site occurs at the threonine
instead of the serine Incorporating Ascore into this study provides a layer of validation for
putative phosphosite identification
Plasma Membrane 2-ATPase
A previous study identified and localized phosphorylation sites on plasma membrane 1-
ATPase after glucose was introduced to starved yeast15
In the current study PMA2 (plasma
membrane ATPase 2) was identified in glucose fed and not starved samples The doubly
threonine phosphorylated peptide identified in fed yeast is PMA2 with amino acid sequence
IKMLTGDAVGIAKETCR (583-599) whereas the homologous peptide in PMA1 has the exact
same amino acid sequence except for the first isoleucine substituted for valine
VKMLTGDAVGIAKETCR (554-570 not observed in data) PMA2 was observed in the 06
FDR list of phosphopeptides and has an Ascore localization probably of 100 A plant study
showed that PMA2 phosphorylation level was higher in early growth phase than when in
stationary phase44
In addition PMA2 expression in yeast permits the growth of yeast and
threonine phosphorylation has been reported on Thr-95545
The identification of PMA2 in the
fed glucose cell extract provides an interesting target for future study on the molecular
mechanisms involved in regulation growth in starved vs glucose fed yeast
Conclusion
In conclusion this work provides a qualitative comparison in the phosphoproteome
between starved and glucose fed yeast A large scale IMAC enrichment of yeast cell lysate
followed by 2-D separation provided a rich source of phosphopeptides for neutral loss triggered
155
ETD analysis using mass spectrometry A specific motif for PKA is highlighted to show the
differences in proteins identified between starved vs fed conditions In total 477 unique
phosphopeptides are identified with 06 FDR and 669 unique phosphopeptides identified with
54 FDR Phosphosite validation is performed using a localization algorithm Ascore to
provide further confidence on the site-specific characterization of these phosphopeptides The
proteins Ssd1 and PMA2 emerged as potential intriguing targets for more in-depth studies on
protein phosphorylation involved in glucose response
Supplemental Tables 1 2 and 3 are available upon request
References
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Rodriguez A L Aragon A D Quinones G A Allen C Werner-Washburne M Genomic
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identification of novel essential genes Mol Biol Cell 2004 15 (12) 5295-305
2 Radonjic M Andrau J C Lijnzaad P Kemmeren P Kockelkorn T T van Leenen
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Cell 2005 18 (2) 171-83
3 Slattery M G Heideman W Coordinated regulation of growth genes in
Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
4 Wang Y Pierce M Schneper L GAtildefrac14ldal C G k e Zhang X Tavazoie S
Broach J R Ras and Gpa2 Mediate One Branch of a Redundant Glucose Signaling Pathway in
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5 Newcomb L L Hall D D Heideman W AZF1 is a glucose-dependent positive
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14
6 Newcomb L L Diderich J A Slattery M G Heideman W Glucose regulation of
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7 Carlson M Glucose repression in yeast Curr Opin Microbiol 1999 2 (2) 202-7
8 Gancedo J M Yeast carbon catabolite repression Microbiol Mol Biol Rev 1998 62
(2) 334-61
9 Johnston M Feasting fasting and fermenting Glucose sensing in yeast and other cells
Trends Genet 1999 15 (1) 29-33
156
10 Warner J R The economics of ribosome biosynthesis in yeast Trends Biochem Sci
1999 24 (11) 437-40
11 Li X Gerber S A Rudner A D Beausoleil S A Haas W Villen J Elias J E
Gygi S P Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces
cerevisiae J Proteome Res 2007 6 (3) 1190-7
12 Ficarro S B McCleland M L Stukenberg P T Burke D J Ross M M
Shabanowitz J Hunt D F White F M Phosphoproteome analysis by mass spectrometry and
its application to Saccharomyces cerevisiae Nat Biotechnol 2002 20 (3) 301-5
13 Gruhler A Olsen J V Mohammed S Mortensen P Faergeman N J Mann M
Jensen O N Quantitative phosphoproteomics applied to the yeast pheromone signaling
pathway Mol Cell Proteomics 2005 4 (3) 310-27
14 Peng J Schwartz D Elias J E Thoreen C C Cheng D Marsischky G Roelofs
J Finley D Gygi S P A proteomics approach to understanding protein ubiquitination Nat
Biotechnol 2003 21 (8) 921-6
15 Lecchi S Nelson C J Allen K E Swaney D L Thompson K L Coon J J
Sussman M R Slayman C W Tandem phosphorylation of Ser-911 and Thr-912 at the C
terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation J Biol
Chem 2007 282 (49) 35471-81
16 Cohen P The regulation of protein function by multisite phosphorylation--a 25 year
update Trends Biochem Sci 2000 25 (12) 596-601
17 Kalume D E Molina H Pandey A Tackling the phosphoproteome tools and
strategies Curr Opin Chem Biol 2003 7 (1) 64-9
18 Nagaraj N DSouza R C Cox J Olsen J V Mann M Feasibility of large-scale
phosphoproteomics with higher energy collisional dissociation fragmentation J Proteome Res
2010 9 (12) 6786-94
19 Olsen J V Vermeulen M Santamaria A Kumar C Miller M L Jensen L J
Gnad F Cox J Jensen T S Nigg E A Brunak S Mann M Quantitative
phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis Sci
Signal 2010 3 (104) ra3
20 Breitkopf S B Asara J M Determining In Vivo Phosphorylation Sites Using Mass
Spectrometry In Current Protocols in Molecular Biology John Wiley amp Sons Inc 2012
21 Steen H Jebanathirajah J A Rush J Morrice N Kirschner M W Phosphorylation
analysis by mass spectrometry myths facts and the consequences for qualitative and
quantitative measurements Mol Cell Proteomics 2006 5 (1) 172-81
22 Kweon H K Hakansson K Metal oxide-based enrichment combined with gas-phase
ion-electron reactions for improved mass spectrometric characterization of protein
phosphorylation J Proteome Res 2008 7 (2) 749-55
23 Larsen M R Thingholm T E Jensen O N Roepstorff P Jorgensen T J Highly
selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide
microcolumns Mol Cell Proteomics 2005 4 (7) 873-86
24 Kokubu M Ishihama Y Sato T Nagasu T Oda Y Specificity of immobilized
metal affinity-based IMACC18 tip enrichment of phosphopeptides for protein phosphorylation
analysis Anal Chem 2005 77 (16) 5144-54
25 Swaney D L Wenger C D Thomson J A Coon J J Human embryonic stem cell
phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry Proc
Natl Acad Sci U S A 2009 106 (4) 995-1000
157
26 Hao P Guo T Sze S K Simultaneous analysis of proteome phospho- and
glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction
chromatography PLoS One 2011 6 (2) e16884
27 Rush J Moritz A Lee K A Guo A Goss V L Spek E J Zhang H Zha X
M Polakiewicz R D Comb M J Immunoaffinity profiling of tyrosine phosphorylation in
cancer cells Nat Biotechnol 2005 23 (1) 94-101
28 Ficarro S Chertihin O Westbrook V A White F Jayes F Kalab P Marto J A
Shabanowitz J Herr J C Hunt D F Visconti P E Phosphoproteome analysis of
capacitated human sperm Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3
and valosin-containing proteinp97 during capacitation J Biol Chem 2003 278 (13) 11579-89
29 Washburn M P Wolters D Yates J R 3rd Large-scale analysis of the yeast
proteome by multidimensional protein identification technology Nat Biotechnol 2001 19 (3)
242-7
30 Dowell J A Frost D C Zhang J Li L Comparison of two-dimensional
fractionation techniques for shotgun proteomics Anal Chem 2008 80 (17) 6715-23
31 Song C Ye M Han G Jiang X Wang F Yu Z Chen R Zou H Reversed-
phase-reversed-phase liquid chromatography approach with high orthogonality for
multidimensional separation of phosphopeptides Anal Chem 2010 82 (1) 53-6
32 Palumbo A M Smith S A Kalcic C L Dantus M Stemmer P M Reid G E
Tandem mass spectrometry strategies for phosphoproteome analysis Mass Spectrom Rev 2011
30 (4) 600-25
33 Beausoleil S A Jedrychowski M Schwartz D Elias J E Villen J Li J Cohn M
A Cantley L C Gygi S P Large-scale characterization of HeLa cell nuclear
phosphoproteins Proc Natl Acad Sci U S A 2004 101 (33) 12130-5
34 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and
protein sequence analysis by electron transfer dissociation mass spectrometry Proc Natl Acad
Sci U S A 2004 101 (26) 9528-33
35 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion
dependence in the partitioning between proton and electron transfer in ionion reactions
International Journal of Mass Spectrometry 2004 236 (1acirceuroldquo3) 33-42
36 Hui L Cunningham R Zhang Z Cao W Jia C Li L Discovery and
characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP)
and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using
multiple tandem mass spectrometry techniques J Proteome Res 2011 10 (9) 4219-29
37 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-
site identity and position on electron-transfer dissociation of polypeptide cations J Am Chem Soc
2007 129 (40) 12232-43
38 Coon J J Collisions or electrons Protein sequence analysis in the 21st century Anal
Chem 2009 81 (9) 3208-15
39 Miller M E Cross F R Distinct subcellular localization patterns contribute to
functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae Mol Cell Biol
2000 20 (2) 542-55
40 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
158
41 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
approach for high-throughput protein phosphorylation analysis and site localization Nat
Biotechnol 2006 24 (10) 1285-92
42 Sutton A Immanuel D Arndt K T The SIT4 protein phosphatase functions in late
G1 for progression into S phase Mol Cell Biol 1991 11 (4) 2133-48
43 Jansen J M Wanless A G Seidel C W Weiss E L Cbk1 regulation of the RNA-
binding protein Ssd1 integrates cell fate with translational control Curr Biol 2009 19 (24)
2114-20
44 Kanczewska J Marco S Vandermeeren C Maudoux O Rigaud J L Boutry M
Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3
proteins converts a dimer into a hexamer Proc Natl Acad Sci U S A 2005 102 (33) 11675-80
45 Maudoux O Batoko H Oecking C Gevaert K Vandekerckhove J Boutry M
Morsomme P A plant plasma membrane H+-ATPase expressed in yeast is activated by
phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the
absence of fusicoccin J Biol Chem 2000 275 (23) 17762-70
159
Figure 1 The general workflow indicating the major steps involved in sample collection
sample processing mass spectrometric data acquisition and analysis of comparative
phosphoproteomics for starved vs glucose fed yeast (A) The high pH RP separation
procedure for combining fractions to reduce low peptide containing fractions from the
UV-VIS trace is also shown (B)
160
Figure 2 Heat map for the global transcriptional response to glucose fed vs starved samples
S288C cells starved for glucose until growth was arrested and subsequently glucose was added
161
Samples for microarray analysis were taken at the starved state and 60 minutes after glucose was
added The heat map shows the fed log2 fold change for each gene relative to the starved state
across the genome performed in biological replicate (A) Black indicates no change in
expression level while red indicates higher expression for the fed relative to the starved state
Green indicates higher expression for the starved state compared to the fed state (Adapted from
Dr Michael Conways Thesis)
162
Figure 3 The motif for protein kinase A (PKA) that was observed for fed vs starved which is
xxxRKRKxSxxxxxx with the phosphorylation occurring on the serine The motif occurred at a
rate 264 fold higher than the yeast proteome used for background In addition one protein was
observed in both starved and fed with accession identification of F16P (Fructose-16-
bisphosphatase)
163
06 FDR phosphopeptide analysis
Table 1 The number of phosphopeptides identified with a 06 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
Starved Fed All
Ascore ge80 score
unique
STY 164 153 317
S 87 (530) 82 (536) 169 (533)
T 60 (366) 55 (359) 115 (363)
Y 17 (104) 16 (105) 33 (104)
Unique no Ascore
STY 242 235 477
S 131 (541) 133 (566) 264 (553)
T 86 (355) 78 (332) 164 (344)
Y 25 (103) 24 (102) 49 (103)
Phosphorylation events
on each unique peptide
1 102 113 187
2 36 40 68
3 12 11 22
4 or more 8 3 11
164
54 FDR phosphopeptide analysis
Starved Fed All
Ascore ge80 score
unique
STY 217 217 434
S 115 (530) 113 (521) 228 (525)
T 78 (359) 78 (359) 156 (359)
Y 24 (111) 26 (120) 50 (115)
Unique no Ascore
STY 337 332 669
S 193 (573) 180 (542) 373 (558)
T 111 (329) 116 (349) 227 (339)
Y
Phosphorylation events
on each unique peptide
1
2
3
4 or more
33 (98)
135
56
16
11
36 (108)
169
55
14
3
69 (103)
280
100
27
13
Table 2 The number of phosphopeptides identified with a 54 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
165
Figure 4 Ascore validation of the phosphopeptide LSLAtKK from the protein SGM1 (slow
growth on galactose and mannose protein 1) with 100 localization probability observed
in only fed but not starved yeast samples Ascorersquos algorithm identifies a b and y-type
ions and looks to identify peaks that provide evidence for a specific phosphorylation site
For this example Ascore assigns this phosphosite to threonine (T5) instead of the serine
(S2) The intense peaks at 4024 mz and 5250 mz are not identified by any a b or y-
type ions From the ladder sequence of the peptide shown numerous ions indicate the
threonine is phosphorylated while the serine is not Among these ions used for
localization are b2 y2 y5+H2O y3 y4 and y5
166
Chapter 6
Use of electron transfer dissociation for neuropeptide sequencing and
identification
Adapted from ldquoDiscovery and Characterization of the Crustacean Hyperglycemic Hormone
Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue
Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniquesrdquo Hui L
Cunningham R Zhang Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
167
Abstract
The crustacean Callinectes sapidus sinus gland (SG) is a well-defined neuroendocrine site that
produces numerous hemolymph-borne agents including the most complex class of endocrine
signaling moleculesmdashneuropeptides One such peptide is the crustacean hyperglycemic hormone
(CHH) precursor-related peptide (CPRPs) which was not previously sequenced Electron
transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and
high charge state In addition ETD was used to sequence the sulfated peptide Cholecystokinin
CCK-like Homarus americanus using a salt adduct Collectively these two examples
demonstrated the utility of ETD in sequencing neuropeptides with large molecular weight or
with labile modifications
168
Introduction
Neuropeptides are the largest and most diverse group of endocrine signaling molecules in
the nervous system They are necessary and critical for initiation and regulation of numerous
physiological processes such as feeding reproduction and development1 2
Mass spectrometry
(MS) with unique advantages such as high sensitivity high throughput chemical specificity and
the capability of de novo sequencing with limited genomic information is well suited for the
detection and sequencing of neuropeptides in endocrine glands and simultaneously provides the
potential for information on post-translational modifications such as sulfonation3-6
The sinus glands (SG) are located between the medulla interna and medulla externa of the
eyestalk in Callinectes sapidus It is a well-known neuroendocrine site that synthesizes and
secretes peptide hormones regulating various physiological activities such as molting
hemolymph glucose levels integument color changes eye pigment movements and
hydro-mineral balance7 Our labrsquos previous neuropeptidomic studies of SG in several
crustacean species including Cancer borealis8-11
Carcinus maenas12
and Homarus americanus13
14 used multiple MS strategies combining MALDI-based high resolution accurate mass profiling
biochemical derivatization and nanoscale separation coupled to tandem MS for de novo
sequencing In the current study we explore the neuropeptidome of the SG in the blue crab
Callinectes sapidus a vital species of economic importance on the seafood market worldwide In
total 70 neuropeptides are identified including 27 novel neuropeptides and among them the
crustacean hyperglycemic hormone precursor-related peptides (CPRPs) and orcokinins represent
major neuropeptide families known in the SG
The crustacean hyperglycemic hormone (CHH) and its precursor-related peptide (CPRP) are
produced concurrently during the cleavage of CHH from the CHH preprohormone protein15
The
169
CPRP peptide is located between the signal peptide and the CHH sequence and is separated from
the CHH by a dibasic cleavage site The functions of CPRPs are currently unknown16
However
the complete characterization of CPRPs provides a foundation for future experiments elucidating
their functional roles in crustaceans The cDNA of CHH preprohormone in SG of Callinectes
sapidus has been characterized17
but the direct detection of CPRP has not been reported due to
its relatively large size and possible post-translational modifications While small fragments of
CPRPs can be identified using nanoLC-ESI-Q-TOF tandem mass spectrometry the intact
peptide is difficult to detect due to the large molecular weight of CPRPs
Peptidomics traditionally uses collision induced dissociation (CID) for tandem MS
experiments for de novo sequencing Recently an alternative peptide fragmentation method has
been developed using the transfer of an electron termed electron transfer dissociation (ETD)18 19
ETD involves a radical anion with low electron affinity to be transferred to peptide cation which
results in reduced sequence discrimination and thus provides improved sequencing for larger
peptides compared to CID20
Specifically for an ion trap ETD the fluoranthene radical anion is
allowed to react with peptide cations in the three dimensional trap The resulting dissociation
cleaves at the N-Cα bond to produce c and zbull type product ions The peptidome of model
organisms like Callinectes sapidus can be further explored and expanded utilizing ETD as a
complementary fragmentation technique to CID Previous peptidomic analysis has been
completed using ETD as an additional fragmentation method21
It was observed that
enzymatically produced peptides with a higher mz produced improved sequence coverage using
ETD This observation termed decision tree analysis determined that a charge state of ge6 all
peptides endogenous or enzymatic should be fragmented via ETD22
In the present study the
highly charged peptide CPRP with an intact molecular weight of 3837 readily produces plus six
170
charge states The higher charge state of CPRP is highly amenable to fragmentation by ETD
which produces remarkably improved fragmentation and thus increased sequence coverage when
compared to CID
Sulfonation of tyrosine is a post-translational modification (PTM) that is thought to occur on
trans-membrane spanning and secreted proteins23
Cholecystokinin-8 (CCK-8) is a sulfated
peptide which has been linked to satiety24-26
and a CCK-like peptide has been observed in
lobster27
Sulfonation is an extremely labile modification and is often lost during soft
ionization such as ESI or MALDI but can be avoided by optimizing the ionization process28
One potential strategy to identify sulfopeptides is to scan for a neutral loss of 80 Da after CID
but this method does not allow for identification of site of sulfonation and has the risk to be
mistaken for phosphorylation The sulfonation PTM is a negatively charged modification on
the peptide which allows for negative ion scanning in the mass spectrometer but provides
minimal MSMS sequence coverage during CID due to preferential loss of the sulfate group
Alternatively electron-based dissociation technique enables more rapid radical driven
fragmentation where the cleavage pattern is more uniform along the peptide backbone without
initially cleaving the labile sulfated group thus preserving the site of modification These types
of dissociation techniques only work for multiply-charged ions thus a method to install a
multiply-charged cation on the target peptide is desirable It has been shown that the electron
capture dissociation (ECD) can be used to sequence sulfated peptides when a multi-charged
cation is added to the solution29
We use a similar multi-charge cation solution technique to
sequence a sulfated peptide from Homarus americanus via ETD on an ion trap mass
spectrometer Here we presented the use of the ETD fragmentation technique for the analysis
of large peptides and peptides containing labile post-translational modification
171
Experimental Section
Chemical and materials
Acetonitrile methanol magnesium chloride (ACS grade) potassium chloride (ACS grade) and
calcium chloride (ACS grade) were purchased from Fisher Scientific (Pittsburgh PA) Formic
acid was from Sigma-Aldrich (St Louis MO) Human sulfated CCK-8 and CCK-like peptide
(E(pyro)FDEY(Sulfo)GHMRFamide) were purchased from American Peptide (Sunnyvale CA)
Fused-silica capillary with 75 μm id and 360 μm od was purchased from Polymicro
Technologies (Phoenix AZ) Millipore C18 Ziptip column was used for sample cleaning and all
water used in this study was deionized water (182 MΩcm) prepared from a Milli-Q Millipore
system (Billerica MA) The physiological saline consisted of 440 mM NaCl 11mM KCl 26
mM MgCl2 13 mM CaCl2 11 mM Trizma base and 5 mM maleic acid in pH 745
Animals and dissection
Callinectes sapidus (blue crab) were obtained from commercial food market and maintained
without food in artificial sea water at 10-12 ordmC Animals were cold-anesthetized by packing on
ice for 15-30min before dissection The optic ganglia with the SG attached were dissected in
chilled (~4ordmC) physiological saline and individual SGs were isolated from the optic ganglia by
micro-dissection and immediately placed in acidified methanol (90 methanol 9 glacial acetic
acid and 1 water) and stored at -80ordmC until tissue extraction
Tissue homogenization
Acidified methanol was used during the homogenization of SGs The homogenized SGs were
immediately centrifuged at 16100 g using an Eppendorf 5415D tabletop centrifuge (Eppendorf
172
AG) The pellet was washed using acidified methanol and combined with the supernatant and
further dried in a Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation) The
resulting dried sample was re-suspended with a minimum amount (20 microL) of 01 formic acid
Fractionation of homogenates using reversed phase (RP)-HPLC
The re-suspended extracts were then vortexed and briefly centrifuged The resulting supernatants
were subsequently fractionated via high performance liquid chromatography (HPLC) HPLC
separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax
UV-D II absorbance detector (Rainin Instrument Inc Woburn MA) The mobile phases included
Solution A (deionized water containing 01 formic acid) and Solution B (acetonitrile containing
01 formic acid) About 20 μl of extract was injected onto a Macrosphere C18 column (21 mm
id x 250 mm length 5 microm particle size Alltech Assoc Inc Deerfield IL) The separation
consisted of a 120 minute gradient of 5-95 Solution B Fractions were automatically collected
every two minutes using a Rainin Dynamax FC-4 fraction collector (Rainin Instrument Inc
Woburn MA) The fractions were then concentrated using in Savant SC 110 SpeedVac
concentrator (Thermo Electron Corporation) and re-suspended with minimum amount of 01
formic acid
Nano-LC-ESI-Q-TOF MSMS
Nanoscale LC-ESI-Q-TOF MSMS was performed using a Waters nanoAcquity UPLC system
coupled to a Q-TOF Micro mass spectrometer (Waters Corp Milford MA)
Chromatographic separations were performed on a homemade C18 reversed phase capillary
column (75 microm internal diameter x100 mm length 3 microm particle size 100 Aring) The mobile phases
173
used were 01 formic acid in deionized water (A) 01 formic acid in acetonitrile (B) An
aliquot of 50 microl of a tissue extract or HPLC fraction was injected and loaded onto the trap
column (Zorbax 300SB-C18 Nano trapping column Agilent Technologies Santa Clara CA)
using 95 mobile phase A and 5 mobile phase Bat a flow rate of 10 microlmin for 10 minutes
Following this the stream select module was switched to a position at which the trap column
came in line with the analytical capillary column and a linear gradient of mobile phases A and B
was initiated For neuropeptides the linear gradient was from 5 buffer B to 45 buffer B over
90 min The nanoflow ESI source conditions were set as follows capillary voltage 3200 V
sample cone voltage 35 V extraction cone voltage 1 V source temperature 100ordmC A data
dependent acquisition was employed for the MS survey scan and the selection of three precursor
ions and subsequent MSMS of the selected parent ions The MS scan range was from mz
400-1800 and the MSMS scan was from mz 50-1800
Peptide Prediction De Novo Sequencing and Database Searching
De novo sequencing was performed using a combination of MassLynxTM
41 PepSeq software
(Waters) and manual sequencing Tandem mass spectra acquired from the Q-TOF were first
deconvoluted using MaxEnt 3 software (Waters) to convert multiply charged ions into their
singly charged forms The resulting spectra were pasted into the PepSeq window for sequencing
analysis The candidate sequences generated by the PepSeq software were compared and
evaluated for homology with previous known peptides The online program blastp (National
Center for Biotechnology Information Bethesda MD httpwwwncbinlmnihgovBLAST)
was used to search the existing NCBI crustacean protein database using the candidate peptide
sequences as queries For all searches the blastp database was set to non-redundant protein
174
sequences (ie nr) and restricted to crustacean sequences (ie taxid 6657) For each of the
proteins putatively identified via blastp the BLAST score and BLAST-generated E-value for
significant alignment are provided in the appropriate subsection of the results Peptides with
partial sequence homology were selected for further examination by comparing theoretical
MSMS fragmentation spectra generated by PepSeq with the raw MSMS spectra If the
fragmentation patterns did not match well manual sequencing was performed
NanoLC Coupled to MSMS by CID and ETD
Setup for RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN (Fisher Scientific Optima LCMS grade Fair Lawn NJ) Injections
consisted of 5 microL of prepared tissue extract onto an Agilent Technologies Zorbax 300 SB-C18 5
microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5 microLmin for 5 minutes at 95
A5 B Separation was performed on a Waters HPLC column with 3 microm Atlantis dC18 75 microm
x 150 mm (Milford MA) on a gradient from 5 to 45 mobile phase B at 250 nLmin over 90
minutes at room temperature Emitter tips were pulled from 75 microm inner diameter 360 microm
outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using a commercial
laser puller model P-2000 (Sutter Instrument Co Novato CA)
Mass Spectrometry Data Acquisitions for Collision Induced Dissociation (CID)
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany) equipped
with an on-line nanospray source was used for mass spectrometry data acquisition Hystar
(Version 32 Bruker Daltonics Bremen Germany) was used to couple and control Eksigent
175
nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for all
experiments Smart parameter setting (SPS) was set to mz 700 compound stability and trap
drive level were set at 100 Optimization of the nanospray source resulted in dry gas
temperature 125oC dry gas 40 Lmin capillary voltage -1300 V end plate offset -500 V
MSMS fragmentation amplitude 10V and SmartFragmentation set at 30-300
Data was generated in data dependent mode with strict active exclusion set after two spectra and
released after one minute MSMS was obtained via CID fragmentation for the six most
abundant MS peaks with a preference for doubly charged ions and excluded singly charged ions
For MS generation the ion charge control (ICC) target was set to 200000 maximum
accumulation time 5000 ms four spectral average acquisition range of mz 300-1500 and scan
speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target was set to
200000 maximum accumulation time 5000 ms three spectral averages acquisition range of
mz 100-2000 and scan speed (Ultrascan) of 32000 mz s-1
Mass Spectrometry Data Acquisitions for Electron Transfer Dissociation (ETD)
The amaZon ETD ion-trap mass spectrometer was operated in electron transfer dissociation for
MSMS fragmentation with the same optimized settings as reported for CID unless otherwise
stated Smart parameter setting (SPS) was set to 500 mz compound stability and trap drive
level were set at 100 MSMS was obtained via ETD fragmentation for the four most
abundant MS peaks with no preference for specifically charged ions except to exclude singly
charged ions The ETD reagent parameters were set to 400000 target ICC for the fluoranthene
radical anion The fluoranthene radical anion has an mz of 210 and therefore the 210 mz value
was removed from the resulting MSMS spectra and 160 mz was set for the MSMS low mz
cut-off
176
Direct Infusion of Sulfated Peptide Standards into the Mass Spectrometer using ETD and
CID Fragmentation
The sulfated peptide standards were infused into the mass spectrometer at a flow rate of 300
nlmin using a fused-silica capillary with 75 μm id and 360 μm od coupled to a custom pulled
tip The CCK-8 human peptide (20 microM) was infused in 5050 ACNH2O and detected in
negative ionization mode with an ICC of 70000 and fragmented with CID using the same
settings as the previous CID experiments Alternatively the CCK-like lobster sulfated peptide
(2 microM) was mixed with 30 microM of different salts (MgCl2 KCl or CaCl2) and 01 formic acid in
5050 ACNH2O The plus three charge of the CCK-like lobster peptide was selected for ETD
fragmentation in positive mode with the same setting as the previous ETD experiments The
data were then de novo sequenced for coverage and localization of the sulfation site
Data Analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen Germany)
Deviations in parameters from the default Protein Analysis in DataAnalysis were as follows
intensity threshold 1000 maximum number of compounds 1E7 and retention time window 05
minutes These parameter changes assisted in providing better quality spectra for sequencing
Identification of peptides was performed using Mascot (Version 23 Matrix Science London
UK) Searches were performed against a custom crustacean database none enzyme allow up to
1 missed cleavage amidated C-terminal as variable modifications peptide tolerance mass error
12 Da MSMS mass error tolerance is 06 Da
Results and Discussion
177
Identification and Characterization of Intact CPRPs Using ETD
Using ESI-Q-TOF 9 truncated CPRPs fragments were sequenced to cover the amino acid
sequence of the intact CPRP peptide RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE
A representative CID MSMS spectrum of ASLKSDTVTPLR is shown in Figure 1 (a) CID
using ESI-Q-TOF mainly produces fragmentation for doubly and triply charge molecules which
is adequate to sequence peptides with relatively small molecular weight (less than 2000 Da)
However CID fragmentation is insufficient to sequence the larger intact CPRP in a complex
sample whose molecular mass is 3837 Da and therefore it is extremely difficult to directly
sequence by traditional CID ESI-Q-TOF ETD is an attractive fragmentation alternative to
sequence the highly charged CPRP peptide In ETD a protonated peptide reacts with an anion
(fluoranthene radical) where the anion transfers an electron to the peptide cation and the resulting
fragmentation occurs along the N-Cα bond by free-radical-driven-cleavage The large size of
CPRP offers multiple basic amino acid sites to sequester charge and reduce the sequence
coverage from collision induced dissociate by preventing random backbone cleavage whereas
ETD does not cleave the weakest bond and provides the alternative fragmentation pathway to
obtain superior sequence coverage to larger peptides As shown in Figure 1 (cd) the
fragmentation of ETD is highly amenable to ETD compared to the CID fragmentation in Figure
1 (b) As evident from comparison ETD offers more extensive fragmentation than CID thus
providing enhanced coverage for large intact peptides such as CPRPs Specifically we observe
125 of the b- and y- cleavages for CID as compared to an average of 535 of c- and zbull-
fragments More than a four-fold increase in fragments using ETD suggests the utility of this
relatively new tandem MS fragmentation method as complementary techniques for de novo
sequencing of large intact endogenous peptides and novel neuropeptide discovery endeavors
178
Negative Mode Sulfated Peptide Identification
An accepted method for identification and quantification for sulfated peptides is negative
ionization30
CCK-8 sulfated peptide standards show intense signal in negative ionization mode
without needing to have additives added such as salts Figure 2 shows the CID MSMS
spectrum from the negative ionization and produces an intense b6 ion at 430 mz The transition
from the doubly negatively charged MS ion of 570 mz to the b6 ion provides a selected reaction
monitoring transition for quantification studies but the sequence information is limited and the
presence of the methionine produces variable oxidation In addition Figure 2 shows the
MSMS product ions with the loss of the sulfate group thus making site-specific location of
sulfation more difficult
Investigation of Cation Salt Adducts to Produce Multiply Charged Sulfated Peptides
Sulfated peptides can be isolated in positive scanning mode but usually only in a plus one
state with low signal intensity If ETD is performed on the singly charged peptide cation a
neutral is formed and is lost in the mass spectrometer and thus no sequence information can be
obtained In order to remedy this situation a technique that adding metal salts to peptides to
increase charge state for ECD used in Fourier transform ion cyclotron resonance mass
spectrometry (FTICR-MS)29
inspired the investigation of increasing charge state of targeted
peptide via metal salt adduction followed by ETD analysis in a Bruker amaZon ion trap
Various salts were investigated including MgCl2 CaCl2 and KCl each used at a concentration of
30 microM and mixed with a sulfated peptide standard at 2 microM The addition of KCl produced
mostly doubly charged ion of the lobster CCK-like peptide standard K adduct which produced
insufficient sequence information from ETD fragmentation (data not shown) In comparison
the MgCl2 and CaCl2 produced intense triply charged peptide ions but CaCl2 produced lower
179
signal intensity compared to MgCl2 (data not shown)
Cation Assisted ETD Fragmentation of CCK-like Lobster Sulfated Peptide and Future
Directions
The addition of MgCl2 to the lobster CCK-like sulfated peptide is shown in Figure 3
Except for z1 the complete z-series is obtained including the z7 ion with and without the
sulfation PTM The spectrum is complicated by the additional Mg adducts but several peaks
are shown without any Mg adduct such as z2 z3 z4 etc Clearly the method of cation
assisted ETD fragmentation can be useful using ion trap instruments and can properly sequence
sulfated peptides that are prone to neutral loss from the labile PTM One concern about future
direction in sulfopeptide isolation in non-genome sequenced species is isolation of sulfopeptides
Currently antibodies to specific sulfopeptides is the only commonly used enrichment technique
for sulfopeptides Also since metal cations are needed for this method direct infusion into an
ESI mass spectrometer is preferred to prevent running large amounts of adduct forming salts
through the LC system With direct infusion the lack of separation confounds the problem in
sulfopeptide detection because any co-eluting peptides could have ionization efficiency and thus
reduce the signal of the sulfopeptide Once discovered and sequenced a selected reaction
monitoring (SRM) method could be developed using LC retention coupled with negative
ionization mode followed by CID MSMS or possibly MSMSMS to perform quantitative
studies for sulfopeptides
Conclusions
In this study ETD was performed to improve the sequence coverage of large endogenous
neuropeptides with higher charge and bigger size The intact CPRP from Callinectes sapidus was
identified and characterized with 68 sequence coverage by MS for the first time Cation
180
assisted ETD fragmentation was also shown to be a powerful tool in de novo sequencing of
sulfated neuropeptides These endeavors into using ETD for certain neuropeptides will assist in
future analysis of large neuropeptides and PTM containing neuropeptides
181
Reference
1 Schwartz M W Woods S C Porte D Jr Seeley R J Baskin D G Central nervous system control of
food intake Nature 2000 404 (6778) 661-71
2 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R
Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide
family of aplysia J Neurosci 2002 22 (17) 7797-808
3 Baggerman G Cerstiaens A De Loof A Schoofs L Peptidomics of the larval Drosophila melanogaster
central nervous system Journal of Biological Chemistry 2002 277 (43) 40368-40374
4 Desiderio D M Mass spectrometric analysis of neuropeptidergic systems in the human pituitary and
cerebrospinal fluid Journal of Chromatography B 1999 731 (1) 3-22
5 Li L J Kelley W P Billimoria C P Christie A E Pulver S R Sweedler J V Marder E Mass
spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab Cancer
borealis Journal of Neurochemistry 2003 87 (3) 642-656
6 Li L J Moroz T P Garden R W Floyd P D Weiss K R Sweedler J V Mass spectrometric survey of
interganglionically transported peptides in Aplysia Peptides 1998 19 (8) 1425-1433
7 Strand F L Neuropeptides regulators of physiological processes 1st ed ed MIT Press Cambridge Mass
1999 p 658 p
8 Fu Q Goy M F Li L J Identification of neuropeptides from the decapod crustacean sinus glands using
nanoscale liquid chromatography tandem mass spectrometry Biochemical and Biophysical Research
Communications 2005 337 (3) 765-778
9 Fu Q Christie A E Li L J Mass spectrometric characterization of crustacean hyperglycemic hormone
precursor-related peptides (CPRPs) from the sinus gland of the crab Cancer productus Peptides 2005 26 (11)
2137-2150
10 Ma M M Chen R B Ge Y He H Marshall A G Li L J Combining Bottom-Up and Top-Down Mass
Spectrometric Strategies for De Novo Sequencing of the Crustacean Hyperglycemic Hormone from Cancer borealis
Analytical Chemistry 2009 81 (1) 240-247
11 Ma M M Sturm R M Kutz-Naber K K Fu Q Li L J Immunoaffinity-based mass spectrometric
characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis Biochemical
and Biophysical Research Communications 2009 390 (2) 325-330
12 Ma M M Bors E K Dickinson E S Kwiatkowski M A Sousa G L Henry R P Smith C M Towle
D W Christie A E Li L J Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and
functional genomics General and Comparative Endocrinology 2009 161 (3) 320-334
13 Chen R B Jiang X Y Conaway M C P Mohtashemi I Hui L M Viner R Li L J Mass Spectral
Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus
Journal of Proteome Research 2010 9 (2) 818-832
14 Ma M M Chen R B Sousa G L Bors E K Kwiatkowski M A Goiney C C Goy M F Christie A
E Li L J Mass spectral characterization of peptide transmittershormones in the nervous system and
neuroendocrine organs of the American lobster Homarus americanus General and Comparative Endocrinology
2008 156 (2) 395-409
15 Ollivaux C Gallois D Amiche M Boscameric M Soyez D Molecular and cellular specificity of
post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family FEBS J 2009 276
(17) 4790-802
16 Wilcockson D C Chung J S Webster S G Is crustacean hyperglycaemic hormone precursor-related
peptide a circulating neurohormone in crabs Cell and Tissue Research 2002 307 (1) 129-138
17 Choi C Y Zheng J Watson R D Molecular cloning of a cDNA encoding a crustacean hyperglycemic
hormone from eyestalk ganglia of the blue crab Callinectes sapidus General and Comparative Endocrinology 2006
148 (3) 383-387
18 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and protein sequence analysis
by electron transfer dissociation mass spectrometry Proc Natl Acad Sci U S A 2004 101 (26) 9528-33
19 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion dependence in the partitioning
between proton and electron transfer in ionion reactions International Journal of Mass Spectrometry 2004 236
(1-3) 33-42
20 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-site identity and
position on electron-transfer dissociation of polypeptide cations J Am Chem Soc 2007 129 (40) 12232-43
182
21 Altelaar A F Mohammed S Brans M A Adan R A Heck A J Improved identification of endogenous
peptides from murine nervous tissue by multiplexed peptide extraction methods and multiplexed mass spectrometric
analysis J Proteome Res 2009 8 (2) 870-6
22 Swaney D L McAlister G C Coon J J Decision tree-driven tandem mass spectrometry for shotgun
proteomics Nat Methods 2008 5 (11) 959-64
23 Monigatti F Hekking B Steen H Protein sulfation analysis--A primer Biochim Biophys Acta 2006 1764
(12) 1904-13
24 Chandler P C Wauford P K Oswald K D Maldonado C R Hagan M M Change in CCK-8 response
after diet-induced obesity and MC34-receptor blockade Peptides 2004 25 (2) 299-306
25 Little T J Feltrin K L Horowitz M Meyer J H Wishart J Chapman I M Feinle-Bisset C A
high-fat diet raises fasting plasma CCK but does not affect upper gut motility PYY and ghrelin or energy intake
during CCK-8 infusion in lean men Am J Physiol Regul Integr Comp Physiol 2008 294 (1) R45-51
26 Blevins J E Morton G J Williams D L Caldwell D W Bastian L S Wisse B E Schwartz M W
Baskin D G Forebrain melanocortin signaling enhances the hindbrain satiety response to CCK-8 Am J Physiol
Regul Integr Comp Physiol 2009 296 (3) R476-84
27 Turrigiano G G Selverston A I A cholecystokinin-like hormone activates a feeding-related neural circuit in
lobster Nature 1990 344 (6269) 866-8
28 Wolfender J L Chu F Ball H Wolfender F Fainzilber M Baldwin M A Burlingame A L
Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB further investigation
of labile sulfo- and phosphopeptides by electrospray matrix-assisted laser desorptionionization (MALDI) and
atmospheric pressure MALDI mass spectrometry J Mass Spectrom 1999 34 (4) 447-54
29 Liu H Hakansson K Electron capture dissociation of tyrosine O-sulfated peptides complexed with divalent
metal cations Anal Chem 2006 78 (21) 7570-6
30 Young S A Julka S Bartley G Gilbert J R Wendelburg B M Hung S C Anderson W H
Yokoyama W H Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using
immunoprecipitation liquid chromatography-mass spectrometrymass spectrometry Anal Chem 2009 81 (21)
9120-8
183
Figure 1 Comparison of tandem MS spectra of truncated CPRP (ASLKSDTVTPL mz 128766)
by ESI-Q-TOF (a) and intact CPRP (MW 3837) by the amaZon ETD for both CID and ETD
fragmentation (a) MSn of precursor ion with mz 640 with charge state +6 by CID represent
loss of NH3 ordm represent loss of H2O (b) MS+6
of precursor ion with mz 640 with charge state +6
by ETD at z represent z+1 z represent z+2 (c) MS+5
of precursor ion with mz 768 with charge
state +5 by ETD z represent z+1 z represent z+2 For all labels in Figure 1 charge state +1 is
not specified
184
185
Figure 2 Negative CID spectrum of CCK-8 peptide standard (20 microM) via direct infusion show
the doubly charged b6 ion provides the most intense MSMS transition
186
Figure 3 ETD spectrum of E(pyro)FDEY(Sulfo)GHMRF-amide (2 microM) obtained on the
amaZon ETD via direct infusion with 30 microM MgCl2 The sulfated peptide can be identified
with a visible z-series of z2 to z9 and identified sulfate loss
187
Chapter 7
Investigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysis
Adapted from ldquoInvestigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J
Wellner D Li L Journal of Mass Spectrometry In Press
188
ABSTRACT
This work investigates the introduction of methanol and a salt modifier to molecular
weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide
quantities Using a neuropeptide standard bradykinin sample loss was reduced over two orders
of magnitude with and without undigested protein present Additionally a bovine serum
albumin (BSA) tryptic digestion was investigated Twenty-seven tryptic peptides were identified
from MALDI mass spectra after enriching with methanol while only two tryptic peptides were
identified after the standard MWCO protocol The strategy presented here enhances recovery
from MWCO separation for sub-microg peptide samples
INTRODUCTION
Molecular weight cut-off membrane-based centrifugal filter devices (MWCO) are
commonly used to desalt and concentrate large molecular weight proteins [1] Greeing and
Simpson recently investigated various MWCO membranes for large amounts of starting material
(~6 mg) focusing on optimal conditions for the sub 25 kDa protein fraction [2] The authors
recovered 200 μg to 29 mg of protein from multiple MWCO experiments and demonstrated that
a 1090 acetonitrile (ACN)H2O elution solvent produced optimal results [3] In addition Manza
et al provided an alternative approach to isolate proteins with a 5 kDa MWCO by using
NH4HCO3 and recovering the retained proteins [4] Alternatively the elution from MWCOs can
be collected to recover only low molecular weight peptides Multiple peptidomic studies have
utilized MWCOs for peptide isolation during the first few steps of sample preparation [5 6]
When sample amount is limited or peptide content is below 1 microg sample loss is a significant
concern when using MWCOs to isolate endogenous peptides Optimized protocols have been
189
investigated using ACN [3 7] salt [4 8] SDS [5] or native sample [6 9 10] but these
experiments primarily focused on large sample amounts rather than sub-microgram peptide
quantities
MWCOs separate large molecules from small molecules The small molecule fraction
may be rich with signaling peptides (SP) cytokines and other small molecules involved in cell-
cell signaling Signaling peptides perform various functions in the body including cell growth
cell survival and hormonal signaling between organs [11] Individual SP contribute to different
aspects of behavior such as pain (enkephalins) [12] feeding (neuropeptide Y) [13] and blood
pressure (bradykinin) [14] MWCO separations can be used to enrich biologically important SP
and explore the peptide content from biological fluids with relatively low peptide content like
blood or cerebrospinal fluid (CSF) In a recent investigation the detection of neuropeptides and
standards in crustacean hemolymph was improved when methanol and protease inhibitors were
present before performing MWCO neuropeptide isolation The impact of methanol on MWCO
sample loss was not investigated in the study [15] In another study a large-scale mass
fingerprinting protocol of endogenous peptides from CSF used a combination of salts before
MWCO fractionation but the impact of adding salts was not discussed [16] The most
commonly used brand of MWCO in the publications and in peptidomic studies is Millipore
Therefore Millipore MWCOs (using regenerated cellulose as the membrane) were used in the
present study The purpose of this work is to provide an optimized sample preparation technique
for MWCO filtering to reduce sample loss and allow sub-microg detection of peptides using MALDI
mass spectrometry
MATERIALS AND METHODS
190
Materials and Chemicals
Water acetonitrile methanol (optima LCMS grade) and sodium chloride (995) were
purchased from Fisher Scientific (Fair Lawn NJ) The α-cyano-4-hydroxy-cinnamic acid (99)
formic acid (FA) (ge98) and bovine serum albumin (ge96) were purchased from Sigma-
Aldrich (St Louis MO) Amicon Ultra 05 mL 10000 MWCO centrifugal filters and ZipTips
packed with C18 reversed-phase resin were purchased from Millipore (Billerica MA) Trypsin-
digested bovine serum albumin (BSA) was purchased from Waters (Milford MA) Bradykinin
was purchased from American Peptide Company (Sunnyvale CA)
MALDI MS Instrumentation
An AutoFLEX III MALDI TOFTOF mass spectrometer (Bruker Daltonics Billerica
MA) was operated in positive ion reflectron mode The MALDI MS instrument is equipped with
a proprietary smart beam (Bruker Daltonics Billerica MA) laser operating at 200 Hz The
instrument was internally calibrated over the mass range of mz 500minus2500 using a standard
peptide mix Two thousand laser shots were collected per sample spot at an accelerating voltage
of 19 kV and a constant laser power using random shot selection The acquired data were
analyzed using FlexAnalysis software (Bruker Daltonics Billerica MA) Mass spectrometry
data acquisition was obtained by averaging 2000 laser shots
Molecular weight cut off separation procedure
The MWCO separations were performed using Amicon Ultra 05 mL 10000 MWCO
centrifugal filters (Billerica MA) Before MWCO separation three washing steps were
performed to remove contaminants from the filter The three washes were 500 μL of 5050
H2OMeOH 500 μL of H2O and 400 μL of the solution used for MWCO separation For the
191
100 H2O solution 1 microg of BSA or bradykinin was used for separation All the other MWCO
separation experiments used 500 ng of BSA or 100 ng or less of bradykinin The MWCO filter
was then centrifuged at 14000 rcf for 5 min at room temperature in an Eppendorf 5415 D
microcentrifuge (Brinkmann Instruments Inc Westbury NY) The filtrate was concentrated in a
Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation West Palm Beach FL)
and acidified The resulting sample was desalted according to the manufacturer using C18
ZipTips from Millipore (Billerica MA) by washing the ZipTip with three times 100 ACN
three aqueous washes of 01 FA binding the peptides from the solution and one aqueous wash
of 01 FA Peptides were then eluted from the ZipTips using 15 microL of 50 ACN in 01 FA
Matrix deposition
Equal volumes of 05 μL from the 15 μL sample solution (including standards not subject
to MWCO filtering) and α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution in 50
ACN50 H2O were mixed using a dried-droplet method and spotted on a MALDI target The
resulting droplets were allowed to air dry prior to mass spectrometry acquisition
RESULTS AND DISCUSSION
Analysis of two orders of magnitude increase for bradykinin standard
Bradykinin was selected to assess the potential peptide loss in the flow-through after
performing MWCO separation As shown in Figure 1 1 microg of bradykinin standard does not
produce a detectable signal by MALDI mass spectrometry analysis after a 10 kDa MWCO
separation in water (performed in triplicate) For comparison 1 ng of bradykinin standard
diluted to 15 microL produced an intense signal on the MALDI mass spectrometer suggesting
192
significant sample loss occurs when the target analyte is low in quantity (data not shown
performed in triplicate) Figure 1 shows that the addition of a salt in this case NaCl improves
the limits of detection and decreases sample loss when 7030 watermethanol was compared to
7030 aqueous 1 M NaClmethanol The reproducible results gave a relative standard deviation
(RSD) of 6 for peak intensity Figure 1 shows that even when starting with 1 μg of bradykinin
too much sample is lost during the MWCO separation in water to detect the remainder
However an intense signal is observed for 10 ng of bradykinin after the MWCO separation when
7030 aqueous 1 M NaClmethanol is used as an elution solvent Sample loss from zip-tipping
was estimated in triplicate by calculating the decreases in peak intensity When 10 ng of
bradykinin was used sample loss was ~41 In comparison the calculated yield for 10 ng of
bradykinin after the 7030 aqueous 1 M NaClmethanol MWCO separation and zip-tip desalting
showed an estimated sample loss of 63 meaning more loss can be attributed to sample clean-
up than MWCO filtration
A series of experiments were performed to determine if 7030 aqueous 1 M
NaClmethanol is an optimal solution to recover peptides during a MWCO separation (data not
shown) A 5050 aqueous 1 M NaClmethanol and a 5050 watermethanol elution were
performed in duplicate but signal intensity of the resulting bradykinin was poor and numerous
polymer peaks were detected in the flow through A lower salt concentration 01 M NaCl was
used but also produced greatly reduced signal when compared to aqueous 1 M NaCl To assess
the optimized methodrsquos compatibility with lower sample amounts the 7030 aqueous 1 M
NaClmethanol solution was added to 1 ng of bradykinin for MWCO separation but no signal
was obtained (data not shown) Using a neuropeptide standard the addition of methanol and
NaCl salt significantly improved the sample recovery in sub-microg amounts
193
BSA tryptic peptide mixture analysis
After demonstrating the importance of using an optimized solution for MWCO
separations with an individual peptide the new method was applied to 500 ng of BSA tryptic
digest to investigate its utility with more complex peptide mixtures Table 1 lists the BSA
tryptic peptides identified in the MALDI MS analysis from different solution conditions
processed by MWCO separation As shown in Table 1 a directly spotted BSA tryptic peptide
standard in the absence of any MWCO filtration enabled identification of 39 tryptic peptides by
accurate peptide mass measurements Once again when using 100 H2O for MWCO
separations the starting amount was doubled to 1 microg (also done with 500 ng data not shown)
However many tryptic peptides were not detected due to low signal intensities and non-optimal
elution conditions Instead of H2O a 1 M NaCl solution was used for the MWCO elution but
only two tryptic peptides were identified (Table 1) The addition of 30 methanol into the
sample before MWCO filtration produced the first increase in identified BSA tryptic peptides
The remaining data from Table 1 shows improved BSA tryptic peptide identifications as the
sample (elution) conditions were further optimized Figure 2 shows the actual mass spectra
associated with the three most promising elution solutions along with 100 H2O
The BSA tryptic peptide intensities are shown in Figure 2A and the most intense tryptic
peptide YLYEIAR mz 92749 was observed in the four different solutions shown in Figure 2B
but not in the 100 H2O or 100 1 M NaCl solutions (data not shown) In Figure 2A all mass
spectra are normalized to an intensity of 7 x 104 to illustrate two points First the MWCO
filtering step still produced sample loss regardless of the solvent conditions chosen Second
there is a noticeable increase in peptide peak intensity using the optimized solvent 6040
194
aqueous 1 M NaClmethanol (Figure 2A) Figure 2C displays a zoomed-in view of a BSA
tryptic peptide signal LKECC
DKPLLEK mz 153266 (
carbamidomethyl) observed only in
the optimized solvent The detection of the mz 153266 peptide in Figure 2C highlights the
potential gain in sample and detectable peptides by using an optimized saltMeOH combination
A non-optimized saltMeOH combination will still reduce sample loss but further minimizing
sample loss during sample preparation will always be desirable in any analytical protocol
MWCO composition
The purpose of this application note is to provide evidence of sub-microg sample loss during
MWCO separations of peptide samples and a solution to overcome this limitation The
explanation of why adding MeOH and NaCl to the sample solution provides a significant
reduction in sample loss is beyond the scope of this application note Regardless Supplemental
Table S1 is an expanded version of Table 1 showing the amino acid sequence hydrophobicity
calculated using GRAVY scores and pI of the identified peptides in this study No discernible
trend was obtained from the data The membrane of commonly used MWCO in peptidomics and
for this study is comprised of chemically treated (regenerated) cellulose which is a
polysaccharide containing β (1rarr4) linked D-glucose Glucose has numerous free hydroxyl
groups which could non-specifically adsorb peptides flowing through the MWCO The addition
of MeOH has the most significant effect on signal which could be due to disrupting the
interaction between peptides and hydroxyl groups from glucose NaCl has a less significant
effect on sample recovery compared to MeOH but a detectable reduction in sample loss is noted
This improvement in sample recovery could be analogous to the use of NaCl in
195
immunodepletion protocols to reduce non-specific binding which is accomplished by adding
150 mM NaCl [17]
Analysis of bradykinin in the presence of undigested BSA
When using MWCO for peptide isolation proteins are typically present in the samples
usually in larger amounts Figure 3 shows the effect that adding BSA protein to a 10 ng
bradykinin solution before MWCO fractionation has on the resulting recovery of bradykinin
Adding 10 μg of BSA to the optimized 7030 aqueous 1 M NaClmethanol solution slightly
decreased bradykininrsquos signal with a RSD of 10 More severe signal reduction occurred after
adding 100 microg BSA with a RSD of 2 (N=2) It is not unexpected that more signal reduction
due to sample loss would occur especially in the 100 microg BSA sample because the BSA protein
has a molar ratio of 160 BSA protein molecules to one bradykinin peptide Figure 3 shows the
usefulness of the MWCO method with samples containing large amounts of proteins
RecommendationConclusion
The present work provides solutions to reduce sample loss from the use of MWCO for
sub-microg peptide isolation with or without non-digested proteins in the sample Despite its
widespread utility significant sample loss often occurs during the MWCO fractionation step
which is particularly problematic when analyzing low-abundance peptides from limited starting
material This application note aims to reduce sample loss during MWCO separations
specifically for sub-microg peptide isolation If complex samples are processed with MWCO
separation the authors recommend eluting the sample with 6040 aqueous 1 M NaClmethanol
solution as a starting point to minimize sample loss This application note provides a viable
196
alternative for sub-microg peptide MWCO separation circumventing the need to increase the starting
material by minimizing sample loss from using a MWCO membrane-based centrifugal filter
device
References
[1] HM Georgiou GE Rice MS Baker Proteomic analysis of human plasma failure of
centrifugal ultrafiltration to remove albumin and other high molecular weight proteins
Proteomics 2001 1 1503
[2] DW Greening RJ Simpson Low-molecular weight plasma proteome analysis using
centrifugal ultrafiltration Methods Mol Biol 2011 278 109
[3] DW Greening RJ Simpson A centrifugal ultrafiltration strategy for isolating the low-
molecular weight (ltor=25K) component of human plasma proteome J Proteomics 2010 73
637
[4] LL Manza SL Stamer AJ Ham SG Codreanu DC Liebler Sample preparation and
digestion for proteomic analyses using spin filters Proteomics 2005 5 1742
[5] D Theodorescu D Fliser S Wittke H Mischak R Krebs M Walden M Ross E Eltze O
Bettendorf C Wulfing A Semjonow Pilot study of capillary electrophoresis coupled to mass
spectrometry as a tool to define potential prostate cancer biomarkers in urine Electrophoresis
2005 26 2797
[6] K Antwi G Hostetter MJ Demeure BA Katchman GA Decker Y Ruiz TD Sielaff LJ
Koep DF Lake Analysis of the plasma peptidome from pancreas cancer patients connects a
peptide in plasma to overexpression of the parent protein in tumors J Proteome Res 2009 8
4722
[7] LP Aristoteli MP Molloy MS Baker Evaluation of endogenous plasma peptide extraction
methods for mass spectrometric biomarker discovery J Proteome Res 2007 6 571
[8] A Zougman B Pilch A Podtelejnikov M Kiehntopf C Schnabel C Kumar M Mann
Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7
386
[9] X Yuan DM Desiderio Human cerebrospinal fluid peptidomics J Mass Spectrom 2005 40
176
[10] X Zheng H Baker WS Hancock Analysis of the low molecular weight serum peptidome
using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer J Chromatogr A
2006 1120 173
[11] L Li JV Sweedler Peptides in the brain mass spectrometry-based measurement approaches
and challenges Annu Rev Anal Chem 2008 1 451
[12] GB Stefano G Fricchione Y Goumon T Esch Pain immunity opiate and opioid
compounds and health Med Sci Monit 2005 11 MS47
[13] J Jensen Regulatory peptides and control of food intake in non-mammalian vertebrates Comp
Biochem Physiol A Mol Integr Physiol 2001 128 471
197
[14] A Kuoppala KA Lindstedt J Saarinen PT Kovanen JO Kokkonen Inactivation of
bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma Am
J Physiol Heart Circ Physiol 2000 278 H1069
[15] R Chen M Ma L Hui J Zhang L Li Measurement of neuropeptides in crustacean
hemolymph via MALDI mass spectrometry J Am Soc Mass Spectrom 2009 20 708
[16] H Jahn S Wittke P Zurbig TJ Raedler S Arlt M Kellmann W Mullen M Eichenlaub H
Mischak K Wiedemann Peptide fingerprinting of Alzheimers disease in cerebrospinal fluid
identification and prospective evaluation of new synaptic biomarkers PLoS One 2011 6
e26540
[17] NA Cellar AS Karnoup DR Albers ML Langhorst SA Young Immunodepletion of high
abundance proteins coupled on-line with reversed-phase liquid chromatography a two-
dimensional LC sample enrichment and fractionation technique for mammalian proteomics J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877 79
198
Table 1 Identified BSA tryptic peptides from various MWCO separation conditions
BSA tryptic
peptide (MH+)
100
H2O 1microg
100
1 M NaCl
70
H2O
80
1 M NaCl
70
1 M NaCl
60
H2O
60
1 M NaCl
5083
5453
6894
7124
8985
9275
10345
10725
11385
11636
12496
12837
13057
13997
14157
14197
14398
14636
14798
15026
15118
15328
15547
15677
15768
16399
16678
16738
17248
17408
17477
17497
18809
18890
19019
19079
20450
21139
22479
Total 39 2 2 6 8 15 15 27
199
Figure 1 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard showing improvement over two orders of magnitude in detection limits Each MWCO
separation was performed at minimum in triplicate with representative spectrum selected for
each with a calculated RSD from the peak heights Three different amounts of bradykinin were
tested to assess the magnitude of sample loss under different MWCO solvent conditions The
top panel shows 1 microg of bradykinin standard after MWCO separation with 100 H2O elution
produced no signal The addition of 40 or 30 MeOH produced very low bradykinin signals
for both 100 ng (RSD of 28) and 10 ng (SNlt3 no RSD calculated) respectively In the
bottom two spectra each showed very large intensity but the 7030 aqueous 1 M NaClmethanol
10 ng bradykinin was processed with a 10kDa MWCO and zip-tipped and was reproducible with
200
a RSD of 6 The last spectrum was from 10 ng bradykinin (RSD of 3) which was diluted to
an equivalent volume as all the other experiments and directly spotted onto the MALDI plate
201
Figure 2 Representative MALDI mass spectra from MWCO separation of a BSA tryptic
peptide standard showing sample loss Stacked mass spectra from mz range 875-2150
normalized to 7 x 104 intensity representing the detection difference from a BSA tryptic peptide
standard from different MWCO separation conditions (A) It should be noted that when the
solvent for MWCO elution was 100 H2O 1 microg of BSA tryptic peptides was processed instead
of 500 ng A zoomed in view of the most abundant BSA tryptic peptide detected YLYEIAR
mz 92749 (B) Various percentages of MeOH produced significant signal but addition of a salt
(1 M NaCl) increases the signal which is closest to a directly spotted BSA tryptic peptide
standard A zoomed in view of a representative low intensity BSA tryptic peptide detected
LKECC
DKPLLEK mz 153266 (C) The optimized solution to be used for MWCO filtration
202
6040 aqueous 1 M NaClmethanol was the only procedure that enabled the detection of the
tryptic peptide in Figure 2C which was also detected in the directly spotted BSA tryptic peptide
standard All experiments were performed a minimum of two times with nearly identical results
) Carbamidomethyl amino acid modification
ordm) Tryptic peptide identified in three of the spectra in Figure 2A
dagger) Tryptic peptide identified in two of the spectra in Figure 2A
) Tryptic peptide identified in a single spectrum in Figure 2A
203
Figure 3 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard with a BSA protein present showing optimized solvent conditions minimized samples
losses Each experiment was performed in duplicate Two different amounts of BSA protein
were tested to assess the magnitude of sample loss caused by the presence of a protein The top
panel shows 10 microg of BSA protein and the second panel shows 100 microg of BSA protein added
while only 10 ng of bradykinin was added Detectable sample loss was observed when the BSA
protein was added but panel two shows that the amount of BSA protein was 1 x 104 greater
(equivalent to 160 fold molar excess) than bradykinin The last two spectra were controls using
a MWCO with the optimized solution in panel 3 and panel 4 using 10 ng bradykinin which was
diluted to an equivalent volume as all the other experiments and directly spotted onto the
MALDI plate
204
Supplemental Table 1 Expanded Table 1 including grand average of hydropathicity (GRAVY)
score theoretical pI and the sequence from the underlying amino acid sequence for the peptides
identified in the BSA digest The GRAVY and pI scores were obtained from ExPASy
Bioinformatics and modifications were not taken into consideration
(httpwebexpasyorgprotparam) Peptide assignments to the recorded peaks were done by
BSA
tryptic
peptide
(MH+)
GRAVY
score
Theoretical
pI
Sequence 100
H2O
1microg
100
1 M
NaCl
70
H2O
80
1 M
NaCl
70
1 M
NaCl
60
H2O
60
1 M
NaCl
5083 NA NA FGER
5453 0900 972 VASLR
6894 0267 979 AWSVAR
7124 -0950 647 SEIAHR
8985 0529 674 LcVLHEK
9275 -0071 600 YLYEIAR
10345 -0725 674 NEcFLSHK
10725 -0211 538 SHcIAEVEK
11385 0 599 ccTESLVNR
11636 0130 453 LVNELTEFAK
12496 -1250 545 FKDLGEEHFK
12837 0264 675 HPEYAVSVLLR
13057 -0582 532 HLVDEPQNLIK
13997 0567 437 TVMENFVAFVDK
14157 0567 437 TVmENFVAFVDK
14197 0058 530 SLHTLFGDELcK
14398 -0133 875 RHPEYAVSVLLR
14636 -0515 465 TcVADESHAGcEK
14798 0292 600 LGEYGFQNALIVR
15026 -0625 409 EYEATLEEccAK
15118 0207 597 VPQVSTPTLVEVSR
15328 -0617 617 LKEccDKPLLEK
15547 -0823 441 DDPHAcYSTVFDK
15677 -0085 437 DAFLGSFLYEYSR
15768 -0985 456 LKPDPNTLcDEFK
16399 -0067 875 KVPQVSTPTLVEVSR
16678 0064 437 MPCTEDYLSLILNR
16738 -1723 550 QEPERNEcFLSHK
17248 0064 437 MPcTEDYLSLILNR
17408 0064 437 mPcTEDYLSLILNR
17477 -0914 414 YNGVFQEccQAEDK
17497 -0621 410 EccHGDLLEcADDR
18809 -0537 606 RPcFSALTPDETYVPK
18890 -0567 674 HPYFYAPELLYYANK
19019 -1275 466 NEcFLSHKDDSPDLPK
19079 0044 454 LFTFHADIcTLPDTEK
20450 -0812 839 RHPYFYAPELLYYANK
21139 -0682 480 VHKEccHGDLLEcADDR
22479 -0458 423 EccHGDLLEcADDRADLAK
Total 39 2 2 6 8 15 15 27
205
mass matching with no tandem mass spectrometry performed Lower case amino acids indicate
a modification present in the peptide of carbamidomethyl (c) or oxidation (m)
206
Chapter 8
Conclusions and Future Directions
207
Summary
Comparative shotgun proteomics investigating numerous biological changes in various
species and sample media and peptidomic method development have been reported The
developed comparative shotgun proteomics based on label-free spectral counting with nanoLC
MSMS platform have been employed in mouse CSF rat CSF yeast culture and other biological
specimens Mass spectrometry (MS)-based peptidomic enhancements using ETD and improved
sample preparation methods for molecular weight cut-offs have been reported Together these
studies add to the Li Labs capabilities in comparative shotgun proteomics and expand available
methods for peptidomic research
Immunodepletion of CSF for comparative proteomics
Chapters 3 and 4 used similar methods to generate a list of differentially expressed
proteins providing insight into how Alexander disease (AxD) perturbs the proteome and how the
new prion model rat adapted scrapie (RAS) proteome varies from control rats In the GFAP
overexpressor mouse CSF study in Chapter 3 we have produced a panel of proteins with
significant up or down regulation in the CSF of transgenic GFAP overexpressor mice MS-based
proteomic study of this mouse model for AxD was consistent with the previous studies showing
elevation of GFAP in CSF The development of a modified IgY-14 immunodepletion technique
for low amounts of CSF with recommendations for future antibody depletion techniques to deal
with the unique challenges of mouse CSF was presented Modified proteomics protocols were
employed to profile mouse CSF with biological and technical triplicates addressing the
variability and providing quantitation with dNSAF spectral counting Validation of the data was
performed using both ELISA and RNA microarray data to provide corroboration with the
208
changes in the putative biomarkers This work presents numerous interesting targets for future
study in AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
Using the protocol developed in Chapter 3 we performed a similar study in RAS CSF
compared to control rat CSF Two differences in sample preparation for the rat CSF compared
to the mouse CSF are noted (1) rat IgY-7 immunodepletion was performed and (2) each rat
CSF sample was collected from one animal due to sufficient volume instead of pooling from
multiple animals for the mouse CSF sample After immunodepletion the CSF samples from
control and RAS (biological N=5 technical replicates N=3) were digested and separated using
one dimensional RP nanoLC separation We were able to identify 167 proteins with redundant
isoforms removed which was derived from total 512 proteins with a 1 FDR from all the CSF
samples Comparative analysis was performed using dNSAF spectral counting and 21 proteins
were significantly changed Our data were consistent with previous prion CSF studies showing
14-3-3 protein increased in CSF of prion infected animals Genomic analysis was also
performed and was used to cross-validate our proteomic data and numerous proteins were found
to be consistent including a novel marker related to prion disease RNAset2 (ribonuclease T2)
In summary this work provides a foundation for investigation of the perturbed proteome of a
new prion model RAS
Yeast Phosphoproteomic Comparison between Starved and Glucose Fed Conditions
This work presented a qualitative comparison of the phosphoproteome between starved
and glucose fed Saccharomyces cerevisiae (bakers yeast) A large scale IMAC enrichment of
yeast cell lysate followed by 2-D separations provided a rich source of phosphopeptides for CID
MSMS and neutral loss triggered ETD analysis using mass spectrometry A specific motif for
PKA was highlighted to show the differences in proteins identified between starved and glucose
209
fed conditions In total 477 unique phosphopeptides were identified with 06 FDR and 669
unique phosphopeptides identified with 54 FDR Phosphosite validation was performed using
a localization algorithm Ascore to provide further confidence on the site-specific
characterization of these phosphopeptides Two proteins Ssd1 and PMA2 emerged as potential
intriguing targets for more in-depth studies on protein phosphorylation involved in glucose
response
Methods for Peptide Sample Preparation and Sequencing
In this study ETD was performed to improve the sequence coverage of endogenous large
neuropeptides and labile tyrosine sulfation CCK-like neuropeptides found in the blue crab
Callinectes sapidus The intact CPRP from Callinectes sapidus was identified and characterized
with 68 sequence coverage by MS for the first time Cation assisted ETD fragmentation using
MgCl2 was also shown to be a powerful tool in de novo sequencing of sulfated neuropeptides
These endeavors into using ETD for certain neuropeptides will assist in future analysis of large
neuropeptides and PTM containing neuropeptides
In addition to ETD sequencing I presented a protocol on improving recovery of minute
quantities of peptides by adding methanol and a salt modifier (NaCl) to molecular weight cut-off
membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities
Despite its widespread utility significant sample loss often occurs during the MWCO
fractionation step which is particularly problematic when analyzing low-abundance peptides
from limited starting material This work presented a method to reduce sample loss during
MWCO separations specifically for sub-microg peptide isolation Using a neuropeptide standard
bradykinin sample loss was reduced by over two orders of magnitude with and without
210
undigested protein present The presence of bovine serum albumin (BSA) undigested protein
and the bradykinin standard lends evidence that sample loss occurs because of the MWCO and
not the presence of the protein Additionally a BSA tryptic digestion is presented where twenty-
seven tryptic peptides are identified from MALDI mass spectra after enriching with methanol
while only two tryptic peptides are identified after the standard MWCO protocol
Ongoing Projects and Future Directions
CSF Projects
Rat Adapted Scrapie and Time Course Study of Rat CSF
In ongoing experiments from the work described in Chapter 4 related to rat adapted
scrapie (RAS) we are working towards more complete proteome coverage of CSF and a time
course study of RAS After the promising results of the 1-D proteomic comparison between
RAS and control CSF we want to perform IgY immunodepletion on 1 mL of rat CSF followed
by 2-D high pH-low pH RP-RP LC-MSMS IgY immunodepletion is still required and
afterwards approximately 40 microg of low abundance protein would remain Following traditional
urea tryptic digestion (see Appendix 1) and solid phase extraction (SPE) clean-up the sample
would be dried down and reconstituted in mobile phase A (25 mM ammonium formate basic
pH) for high pH RP separation Fractions would be dried and subjected to MS analysis similar to
the work described in Chapter 4 The purpose of this work would be to increase the proteome
coverage from a few hundred to a thousand CSF proteins identified A time course study of RAS
is also desirable to gain insight into disease progression Rats at different stages will be
sacrificed and CSF will be collected The goal is to perform isobaric labeling between the time
courses with spectral counting being an alternative for relative protein expression We will use
the targets identified in Chapter 4 to track certain proteins for time course analysis Overall
211
these future projects will dig deeper into the proteome and how this novel prion model RAS
perturbs the proteins expressed in rats over time
Glycoproteomics and Peptidomic Analysis of CSF Samples from Individuals with
Alzheimerrsquos Disease
Alzheimerrsquos disease is an incurable progressive neurodegenerative disorder which results
in cognitive impairment Our aim is to provide additional biomarkers andor targets for drug
treatment Currently we used 500 microL of human CSF for non-membrane glycoprotein
enrichment (see Appendix 1) followed by 2-D high-pH-low-pH RP-RP amaZon ion trap LC-
MSMS analysis The initial work was a failure due to low amount of signal and significant
sample loss (data not shown) Through a comparison with Dr Xin Weirsquos PhD thesis we
estimated roughly 20 microg of proteins was obtained after glycoprotein enrichment and 1-D analysis
produced over 100 protein identifications (data not shown) but the additional off-line 2-D
separation and sample clean up resulted in low number of protein identifications for each fraction
analyzed by MS (data not shown) We have recently obtained 1 mL of human CSF samples
from healthy controls and individuals suffering from Alzheimerrsquos disease and plan to perform
the same experiments with double the starting amount and reduce the fractions collected from 2-
D separation from 11 to 6 In addition to the glycoproteomics anything not enriched will be
subjected to MWCO separation using the method presented in Chapter 7 The obtained peptide
sample will be subjected to Q-TOF nanoLC MSE data acquisition followed by de novo
sequencing using various programs including PEAKS and Mascot Collectively we feel this
project has great potential to lead to interesting targets and further expand the proteomic
knowledge of Alzheimerrsquos disease
GFAP Knock-in Mouse CSF
212
In a direct follow-up to Chapter 3 we aim to perform comparative proteomics on control
vs glial fibrillary acidic protein (GFAP) knock-in mouse CSF samples The sample preparation
protocol as presented in Chapter 3 will be used For quantitative proteomics we plan on
performing isobaric labeling followed by performing high energy collision induced dissociation
(HCD) on the Orbitrap Elite The MS acquisition will also perform a top ten scan where the top
ten MS peaks will be selected for tandem MS analysis In addition targeted quantitation of
specific proteins using multiple reaction monitoring (MRM) can be performed on potential
biomarkers and identified in this follow-up study and the work presented in Chapter 3 Also any
CSF samples with noticeable blood content cannot be used for the exploratory proteomics
experiments but can potentially be used for the MRM analysis and should be kept for such
experiments in the future
Large Scale Proteomics
Proteomics of Mouse Amniotic Fluid for Lung Maturation
The overall goal of this project is to determine what proteins are present in amniotic fluid
when the embryo is 175 weeks compared to amniotic fluid at 155 weeks The rationale behind
why these two time points matter was investigated through a lung explant culture where amniotic
fluid was added at 155 weeks and 175 weeks Visual maturation of lungs occurred with the
175 week amniotic fluid and lung maturation genes were up regulated in the mRNA in the lung
explant culture when compared to the 155 week amniotic fluid The compound which is
causing the maturation of the lungs is unknown and search for a secreted protein might provide a
clue to this process In order to test this hypothesis we carried out discovery proteomics
experiments The workflow involves tryptic digestion SPE and 1-D low pH nanoLC separation
coupled to an amaZon ETD ion trap for tandem MS analysis The resulting mass spectrometric
213
acquisition identified less than 100 proteins with a 1 FDR (data not published) To address the
lack of depth in the proteome coverage we purchased an IgY immunodepletion column to
remove the most abundant proteins in amniotic fluid similarly to Chapter 3 and 4 but on a larger
scale One abundant protein alpha-fetoprotein is present in amniotic fluid but absent or present
in very low concentration in CSF serum or plasma Alpha-fetoprotein is similar to albumin and
thus we ran amniotic fluid on an IgY immunodepletion column and observed significant
reduction in alpha-fetoprotein amount (data not published) After immunodepletion 2-D high
pH-low pH RP-RP will be performed followed by nanoLCMSMS on the amaZon ETD ion trap
We will perform mass spectrometry experiments using the 155 week amniotic fluid and 175
week amniotic fluid which have average protein contents ~2 mgmL We anticipate a minimum
of tenfold increase in the proteome coverage of amniotic fluid and provide meaningful
hypothesis driven biological experiments from this work
Phosphoproteomics of JNK Activation
c-Jun N-terminal kinase (JNK) is an important cellular mediator of stress activated
signaling Under conditions of oxidative stress JNK is activated resulting in the downstream
phosphorylation of a large number of proteins including c-Jun However the cellular response
to JNK activation is extremely complex and JNK activation can result in extremely different
physiological outcomes For example JNK is at the crossroads of cellular death and survival
and exhibits both pro-death and pro-survival activities These highly disparate outcomes of JNK
activation are highly contextual and depend on the type of stressor and duration of stress In the
brain JNK has been shown to be activated in models of Alzheimerrsquos disease Parkinsonrsquos
disease amyotrophic lateral sclerosis and stroke It is thought that JNK is activated in these
diseases as a result of oxidative stress and it is unknown whether this activation is pro-survival or
214
pro-death To elucidate JNK signaling in the brain we chose to analyze primary cortical
astrocytes under conditions of oxidative stress Since hydrogen peroxide is a physiologically
relevant oxidant in the brain and known to activate JNK we chose to use this to treat astrocytes
and then analyze changes to the phosphoproteome by mass spectrometry By doing this we
hope to elucidate important JNK-dependent targets involved in stress signaling in the brain and
that identifying these targets could lead to the identification of novel disease mechanisms and
potentially new therapeutic targets for neurodegeneration Specifically we plan on performing
stable isotope labeling by amino acids (SILAC) of astrocytes for control hydrogen peroxide
treated and hydrogen peroxide treated with JNK inhibitor The SILAC labeled astrocyte cell
lysate will be digested enriched using Fe-NTA IMAC and separated using 2-D high pH-low pH
RP-RP and infused into the amaZon ETD ion trap The protocol is similar to the yeast
comparative phosphoproteomics from Chapter 5 After analysis we plan on processing the data
using ProteoIQ to identify phosphoproteins with significant changes
Immunoprecipitation Followed by Mass Spectrometry
Stb3 Mass Spectrometry Analysis
Stb3 (Sin3-binding protein) has previously been shown to change location depending on
the presence or absence of glucose (Heideman Lab Dr Michael Conways Thesis) An
immunoprecipitation of Stb3 was performed in parallel to the work described in Chapter 5 Two
separate experiments were performed one with wild type Stb3 and another tagged with myc for
improved protein immunoprecipitation Myc tagging is a polypeptide tag which can be
recognized by an antibody and provide a better enrichment compared to using a Stb3 antibody
alone The myc tagging was done because of the low abundance of Stb3 and the limited amount
of protein that was pulled down by the antibody for Stb3 Immunopreciptation experiments were
215
performed for both starved and glucose fed samples All samples were tryptically digested
followed by low pH RP nanoLCMSMS Both CID and ETD were used for fragmentation
analysis of each sample (n=2) The Stb3 wild type had two peptide hits in one MS run which is
not surprising due to low Stb3 antibody affinity In contrast a significant amount of Stb3 was
pulled down from Myc tagged starved and glucose fed samples For the glucose starved
samples 22 unique peptides from Stb3 were identified whereas glucose fed samples yielded 21
unique Stb3 peptides identified (unpublished data) The identified Stb3 in the myc samples
allowed us to investigate what other proteins were pulled down that are not present in the wild
type samples
From previous work by our collaborator Dr Heideman it had been suggested that Stb3
forms a complex with Sin3 and Rpd3 Our proteomic data shows multiple significant peptide
hits for Sin3 and Rpd3 in myc tagged but not in wild type where Stb3 is only observed once
with a low Mascot score When looking at the unique proteins identified in myc tagged glucose
fed sample but not included in the wild type samples the myc fed sample contained eight unique
ribosomal proteins with numerous unique peptides for each The ribosomal proteins identified in
myc fed sample supports the novel hypothesis that in the glucose fed state the complex of Stb3
Rpd3 and Sin3 interact with ribosomal proteins Other proteins unique to myc tagged glucose
starved samples are glyceraldehyde-3-phosphate dehydrogenase 2 and transcriptional regulatory
protein UME6 Also three proteins were observed in both myc fed and starved but not in the
wild type samples including transposon Ty1-DR1 Gag-Pol polyprotein(YD11B) SWIRM
domain-containing protein (FUN19) and RNA polymerase II degradation factor 1 (DEF1) Our
proteomics data for the Stb3 immunoprecipitation experiments with starved vs glucose fed
216
samples provide exciting evidence to support previous observation made by the Heideman group
and highlight the utility of MS-based approach to deciphering protein-protein interactions
Conclusions
The majority of the work described in this dissertation revolves around sample
preparation for proteomics and peptidomics with a focus on generating biologically testable
hypotheses In the area of neuropeptides CPRP was fully sequenced analytical methods were
transformed for use in an ETD ion trap and sub-microg peptides can now be analyzed by mass
spectrometry after MWCO separation In the field of comparative proteomics comparisons
between mouse CSF in GFAP overexpressors and control or rat adapted scrapie model and
control CSF yeast fed vs glucose starved cell extract have all been presented Collectively this
thesis has developed new techniques for neuropeptide sample preparation and presented
numerous comparative proteomic analyses of various biological samples and how the proteomes
are dynamically perturbed by various treatments and disease conditions
217
Appendix 1
Protocols for sample preparation for mass spectrometry based
proteomics and peptidomics
218
Small Scale Urea Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 8 μL of urea solution
(400mg05mL) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Allow to digest overnight in 37degC water bath
10 Acidify with 10μL 10 formic acid
11 Perform solid phase extraction using tips dependent of sample amount
a Sub-5μg amounts ndash Millipore Ziptips
b 5-75μg amounts ndash Bond-Elut tips from Agilent (OMIX tips)
12 Dry down in Speedvac as needed for experiment
219
Small Scale ProteaseMAX Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 2 μL of 1 of
ProtesaeMAX (Promega) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Add 1 μL ProteaseMAX and let sit for 3-4 hours
10 Acidify with 2μL 10 formic acid
11 Dry down in Speedvac as needed for experiment
220
Large Scale Urea Tryptic Digestion (mg of proteins)
1 All solutions are in a 50 mM NH4HCO3 buffer (395 mgmL)
2 Add 20μL of 05M Dithiothreitol (DTT) (77mg100μL) and 160μL of urea solution
(400mg05mL) to sample
3 Allow sample to denature 45-60 minutes in a 37degC water bath
4 Add 54μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
5 Quench reaction with 20μL of DTT solution
6 Dilute with 14mL of NH4HCO3 solution
7 Add 100μg of trypsin
8 Allow to digest overnight in 37degC water bath
9 Acidify sample with 100μL of 10 formic acid
10 Perform solid phase extraction with Hypersep C18 Cartridges with 100 mg of C18
bead volume (Thermo)
11 Dry down with the Speedvac as needed for experiment
221
Fe-NTA Preparation from Ni-NTA Beads
1 Centrifuge (5 min at 140 rcf) and use a magnet to keep beads in place as supernatant
is removed
2 Wash 3 times with 800μL of H2O (using same technique of centrifuging and using
magnet to keep beads in places as supernatant is removed)
3 Add 800μL of 100mM EDTA (372mgmL) in a 50mM NH4HCO3 (395mgmL)
buffered solution (pH around 8) and vortex for 30 minutes to chelate the Ni
centrifuge and remove supernatant
4 Wash 3 times with 800μL of H2O
5 Add 800μL of 10mM FeCl3 hexahydrate (27mgmL) and vortex for 30 minutes to
bind Fe to beads centrifuge and remove supernatant
6 Wash 3 times with 800μL H2O
7 Resuspend in a storage solution of 111 ACNMeOH001 Acetic Acid (1mL total)
222
Fe-NTA IMAC Phospho-enrichment
1 Use wash solution (80ACN 01Formic Acid) to rinse beads vortex 1 minute
centrifuge and remove supernatant
2 Add sample (around 500μL in 80ACN 01Formic Acid) and vortex 40 minutes to
allow sample to bind dispose of supernatant after centrifuging
3 Wash 3 times with 200μL of wash solution discard supernatant
4 Add 200μL of elution solution (5050 ACN5Ammonium Hydroxide) vortex 15
minutes and save supernatant
5 Add 200μL of elution solution vortex 10 minutes and save supernatant
6 Wash 2 time with wash solution (collect supernatant of first wash)
7 If reusing store in 1mL solution of 111 ACNMeOH001 Acetic Acid
223
High pH Off-line Separation
1) In general a minimum of 20 microg of peptides are needed to gain any benefit
from off-line 2D fractionation It is better to inject 100 microg of peptides on
column
2) Use a Gemini column or a similar column that can handle pH=10 and for this
protocol a 21 mm x 150 mm column was used
3) Prepare ldquobuffer Ardquo 25 mM NH4 formate pH=10 Also prepare ldquobuffer Brdquo
4) Dry down desired sample and reconstitute in buffer A
5) Inject based off sample loop (current Alliance HPLC has a 50 microL sample
loop)
6) Run gradient at bottom of the page collecting fractions every 3 minutes except
for the 1st minute which is the void volume
7) Optional If you want to reduce instrument time you can combine fractions 1
with 12 2 with 13 etc until 11 with 22
Time Mobile phase A Mobile phase B Flow Rate
05mlmin
0 98 2 05 mLmin
65rsquo 70 30 05 mLmin
65rsquo1rdquo 5 95 05 mLmin
70 5 95 05 mLmin
224
Non Membrane Glycoprotein Enrichment
1 For protocols involving membrane glycoprotein enrichment see Dr Xin Weirsquos
thesis
2 Add 75 microL of Wheat germ agglutinin (WGA) bound to agarose beads and 150 microL
of Concanavalin A (ConA) to a Handee spin cup (Thermo) Wash 2 times with
lectin affinity chromatography (LAC) binding buffer (015 M NaCl 002 M Tris-
HCl 1 mM CaCl2 pH=74) centrifuging at 400 g for 30 seconds
3 Place stopper on bottom of spin cup and add 200 microg of sample (500 microL for CSF)
Bring up to 300 microL using lectin LAC binding buffer
4 Incubate for 1 hour with continuous mixing at room temperature
5 Centrifuge at 400 g for 30 seconds
6 Perform two more 300 microL LAC binding washes followed by centrifugation
7 Add 300 microL LAC eluting buffer (0075 M NaCl 001 M Tris-HCl 02 M alpha-
methyl mannoside 02 M alpha-methyl glucoside and 05 M acetyl-D-
glucosamine) vortex for 10 minutes (have stopper in place while vortexing)
centrifuge and collect
7 Add another 300 microL LAC eluting buffer centrifuge and collect
225
MWCO separation for Sub-microg peptide concentrations
1 Wash molecular weight cut-off (MWCO) with 5050 waterMeOH centrifuge at
14000 g for 5 minutes for 10 kDa filter (time will vary Millipore Amicon Ultra
filters)
2 Wash with 100 water centrifuge at 14000 g for 5 minutes
3 Add methanol to the sample to get the concentration to 30 methanol and add
salt to achieve a roughly 05 M NaCl before adding the sample to the MWCO
4 Centrifuge at 14000 for 10 minutes collect flow through
226
Immunoprecipitation
Modified from Thermo Fisher Scientific Classic IP Kit
1 10 microL Polyclonal antibody added to 1 microg protein standard in a Handee spin cup
(Thermo) diluted with 1x TBS buffer to 400 microL and incubated overnight at on
shakerend-over-end rotator
2 Wash Protein GA beads with 2x 200microL TBS buffer and added to the
antibodysample for 15 hours at 4oC
3 Centrifuge at 400 g for 30 seconds and discard flow through
4 Wash 3x with 100microL TBS buffer centrifuge at 400 g for 30 seconds and discard
flow through
5 Wash with 1x conditioning solution (neutral pH buffer) centrifuge at 400 g for 30
seconds and discard flow through
6 Eluted with elution buffer (2x100microL) centrifuge at 400 g for 30 seconds and
collect flow through
227
C18 Solid Phase Extraction (SPE)
1 Determine amount of peptides for SPE ge5 microg use Ziptips from Millipore If
between 5-75 microg use OMIX from Agilent 100 microL version and if ge75 microg use SPE
cartridges such as 100 mg Hypersep from Thermo
2 Ensure no detergents are in the sample and it is acidified
3 The three SPE procedures all use the same sets of solutions only volumes vary
4 3x Wetting solution (100 ACN) for 60 microL OMIX (20 microL ziptip and 15 mL for
100 mg cartridge)
5 3x Wash solution (01 formic acid in 100 H2O) same volumes as 4
6 Draw up the sample about 10 times minimum (generally 30-40 is preferred)
without letting the bead volume dry out
7 1X Wash solution same volumes as 4
8 Back load the eluting solution 01 formic acid in 5050 H2OACN into the
Ziptips (15 microL) or OMIX (50 microL) tips For the SPE cartridge add 700 microL of
eluting solution
9 Dry down and prepare for next step in sample preparation
228
Laser Puller Programs and Protocols
1) Obtain about two feet of Fused-silica capillary with 75 μm id and 360 μm od
2) Wash with methanol and then air dry using the bomb
3) Cut into one foot or desired length
4) Use a lighter to burn the middle portion (about an inch in length) of the tubing
5) Remove the ash by rinsing with methanol and rubbing with a Kimwipe
6) Make sure the laser puller has been on for at least 30 minutes before use to allow
for the instrument to warm up
7) Place capillary in instrument with the burnedexposed portion in the center
making sure that the length of the capillary is pulled taut
8) Enter desired program (next page) and press pull
229
Laser Puller Programs
Program 99 (default lab program)
Heat Filament Velocity Delay Pull
250 0 25 150 15
240 0 25 150 15
235 0 25 150 15
245 0 25 150 15
Program 97 (developed for larger inner diameter tips)
Heat Filament Velocity Delay Pull
230 - 25 150 -
220 - 25 150 -
215 - 25 150 8
230
On column Immunodepletion (serum plasma CSF amniotic fluid)
1) Prepare buffer A ldquoDilution Bufferrdquo 10 mM Tris-HCl pH=74 150 mM NaCl
2) Prepare buffer B ldquoStripping Bufferrdquo 01 M Glycine-HCl pH=25
3) Prepare buffer C ldquoNeutralization Bufferrdquo 100 mM Tris-HCl pH=80
4) Determine loading amount (LC1=~1000 ug of serum or plasma less for CSF due
to the increased amount of albumin percentage in CSF)
5) Add Dilution buffer to sample before injection and ensure the sample is proper
pH (~7)
6) Use gradient below
Time A B C Flow Rate
(mLmin)
0rsquo 100 0 0 02
4rsquo59rdquo 100 0 0 02
5rsquo 100 0 0 05
8rsquo59rdquo 100 0 0 05
9rsquo 0 100 0 05
22rsquo 0 100 0 05
22rsquo1rdquo 0 0 100 05
39rsquo 0 0 100 05
7) Store the column in 1x dilution buffer until next use
231
Small Scale Immunodepletion (100 microL of CSF)
1) Use 75 microg (per 100 microL CSF) of Sigma Seppro IgY-14 or IgY-7 bead slurry
2) Add an equal volume of 2x dilution buffer (20 mM Tris-HCl pH=74 300 mM
NaCl) to the starting amount of CSF
3) Add to a spin cup with a filter and allow to mix for 30 minutes
4) Centrifuge at 400 g for 30 seconds and collect the flow through
5) Add 50 microL of 1x dilution buffer mix for 5 minutes centrifuge at 400 g for 30
seconds and collect the flow through
6) Use 1x stripping washes (01 M Glycine-HCl pH=25) centrifuge as before and
discard Repeat four times
7) Use 1x neutralization buffer (100 mM Tris-HCl pH=80) centrifuge as before
and discard Repeat two times
8) Store the beads in the spin column in 1x dilution buffer until next use
232
Alliance Maintenance Protocol
Seal Wash
10 methanol no acetonitrile This wash cleans behind the pump-head seals to
ensure proper lubrication Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime SealWsh gt Start
2 Press Stop after 5 minutes
Prime Injector
10 methanol for maintenance high organic solvent for dirty runs (eg 95
acetonitrile) Done before injecting any real samples to ensure no bubbles are
present in the injector Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime NdlWsh gt Start
2 After completion press Close
Purge Injector
Solvent is dependent on run Run this protocol at beginning of experiments each day
Minimum once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Navigate Direct Function gt 4 Purge Injector gt OK
3 Set Sample loop volumes 60 leave Compression Check unchecked gt OK
Prime Solvent Pumps
Solvent is dependent on run If solvents are changed run this protocol Minimum
once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys choose composition A type 100 Enter x4
3 Navigate Direct Function gt 3 Wet Prime gt OK
4 Set Flow Rate 7000 mLmin Time 100 min gt OK
5 Repeat for all changedactive solvent pumps
Condition Column
Dependent on user Use starting conditions for run
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys type starting solvent compositions for run
3 Navigate Direct Function gt 6 Condition Column gt OK
4 Set Time as desired
233
Appendix 2
List of Publications and Presentations
234
PUBLICATIONS
ldquoDiscovery and characterization of the crustacean hyperglycemic hormone precursor related
peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes
sapidus using multiple tandem mass spectrometry techniquesrdquo Hui L Cunningham R Zhang
Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
ldquoInvestigation and reduction of sub-microgram peptide loss using molecular weight cut-off
fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J Wellner D Li L
Journal of Mass Spectrometry In Press
ldquoMass spectrometry-based proteomics and peptidomics for systems biology and biomarker
discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse models of
Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P Messing A Li
L Journal of Proteome Research Submitted
ldquoInvestigation of the differences in the phosphoproteome between starved vs glucose fed
Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M Heideman
W Li L In preparation
ldquoIdentification of astrocytic JNK targets by phosphoproteomics using mass spectrometryrdquo
Cunningham R Dowell J Wang J Wellner D Li L Milhelm M In preparation
ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A Cunningham R Wellner
D Wang J Ma D Li L Aiken J In preparation
235
INVITED SEMINARS AND CONFERENCE PRESENTATIONS
Robert Cunningham Davenne Mayour and Shoshanna Coon ldquoMorphology and Thermal
Stability of Monolayers on Porous Siliconrdquo The 231th
ACS National Meeting 2006 Atlanta
GA
Robert Cunningham Xin Wei Paige Jany Albee Messing and Lingjun Li ldquoMass
Spectrometry-Based Analysis of Cerebrospinal Fluid Peptidome and Proteome for Biomarker
Discovery in Alexander Diseaserdquo The 57th
ASMS Conference 2009 Philadelphia PA
Robert Cunningham ldquoWhat to Expect in a PhD Graduate Schoolrdquo Invited seminar University
of Northern Iowa 2010 Cedar Falls IA
Robert Cunningham Dustin Frost Albee Messing and Lingjun Li ldquoInvestigation of an
Optimized ProteaseMAX Assisted Trypsin Digestion of Human CSF for Pseudo-SRM
Quantification of GFAP and Identification of Biomarkersrdquo The 58th
ASMS Conference 2010
Salt Lake City UT
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo Oral presentation at Pittcon Conference and Expo 2011 2011 Atlanta
GA
Robert Cunningham Michael Conway Daniel Wellner Douglas Grunwald Warren
Heideman and Lingjun Li ldquoDevelopment of optimized phosphopeptide enrichment methods for
comparison of starved and glucose fed yeast Saccharomyces cerevisiaerdquo The 59th
ASMS
Conference 2011 Denver CO
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo 2011 Wisconsin Human Proteomics Symposium 2011 Madison WI
ii Greer Chris Lietz Chenxi Jia Dustin Frost Di Ma Hui (Vivian) Ye Nicole Woodards
and Claire Schmerberg for their collaboration in many challenging research projects and
fruitful discussions on various research areas There are too many people to thank each
one individually but every member of the Li lab has in some way contributed to my
learning experience Beyond research work their friendship also made my life here in
Madison much more enjoyable
I would also like to thank our collaborators Dr Albee Messing Dr Warren
Heideman Dr Xin Sun and Dr James Dowell It is my great pleasure to have the
opportunities to work with these amazing people and gain precious experience I have
learned so much from them and their achievements in the field have inspired me to strive
to do the best I could
Furthermore I would like to thank Gary Girdaukas and Dr Cameron Scarlett at
School of Pharmacy for the access of the MALDI-FTMS and Bruker amaZon ion trap
instruments
In particular I wish to thank my family my Mom and Step-Dad for raising me
and my Dad for always being there for me They all supported me in my decision to
pursue science and specifically a career in chemistry I would like to thank my Sister
who grew up with me and always led by example in academics Most importantly I
would like to thank my wife Na Liu for her constant support She has inspired and
helped me finish my PhD and always encouraged me to be the best I could be To them
I dedicate this thesis
iii
Table of Contents
Page
________________________________________________________________________
Acknowledgements i
Table of Contents iii
Abstract iv
Chapter 1 Introduction brief background and research summary 1
Chapter 2 Mass spectrometry-based proteomics and peptidomics for
biomarker discovery and the current state of the field 15
Chapter 3 Protein changes in immunodepleted cerebrospinal fluid from
transgenic mouse models of Alexander disease detected
using mass spectrometry 73
Chapter 4 Genomic and proteomic profiling of rat adapted scrapie 110
Chapter 5 Investigation of the differences in the phosphoproteome
between starved vs glucose fed Saccharomyces cerevisiae 139
Chapter 6 Use of electron transfer dissociation for neuropeptide
sequencing and identification 166
Chapter 7 Investigation and reduction of sub-microgram peptide loss
using molecular weight cut-off fractionation prior to
mass spectrometric analysis 187
Chapter 8 Conclusions and future directions 206
Appendix 1 Protocols for sample preparation for mass spectrometry
based proteomics and peptidomics 217
Appendix 2 Publications and presentations 233
_______________________________________________________________________
iv
Mass Spectrometry Applications for Comparative Proteomics and
Peptidomic Discovery
Robert Stewart Cunningham
Under the supervision of Professor Lingjun Li
At the University of Wisconsin-Madison
Abstract
In this thesis multiple biological samples from various diseases models or
treatments are investigated using shotgun proteomics and improved methods are
developed to enable extended characterization and detection of neuropeptides In general
this thesis aims to expand upon the rapidly evolving field of mass spectrometry (MS)-
based proteomics and peptidomics by primarily enhancing small scale sample analysis
A review of the current status and progress in the field of biomarker discovery in
peptidomics and proteomics is presented To this rapidly expanding body of literature
our critical review offers new insights into MS-based biomarker studies investigating
numerous biological samples methods for post-translational modifications quantitative
proteomics and biomarker validation Methods are developed and presented including
immunodepletion for small volume cerebrospinal fluid (CSF) samples for comparison of
the CSF proteomes between an Alexander disease transgenic mouse model with
overexpression of the glial fibrillary acidic protein and a control animal This thesis also
covers the application of the small scale immunodepletion of CSF for comparative
proteomic analysis of a novel rat adapted scrapie (RAS) model for prion disease and
v
compares the RAS CSF proteome to control rat CSF using MS Large scale
phosphoproteomics of starved vs glucose fed yeast is presented to better understand the
phosphoproteome changes that occur during glucose feeding Method development for
neuropeptide analysis is expanded upon using electron transfer dissociation (ETD)
fragmentation to successfully sequence for the first time the crustacean hyperglycemic
hormone precursor-related peptide (CPRP) from the blue crab Callinectes sapidus In
addition a method for ETD sequencing of sulfonated neuropeptides using a magnesium
salt adduct in an ion trap mass spectrometer is reported This thesis also reports on a
method for sub-microg peptide isolation when using a molecular weight cut-off filtration
device to improve sample recovery by over 2 orders of magnitude All the protocols used
throughout the work are provided in an easy to use step-by-step format in the Appendix
Collectively this body of work extends the capabilities of mass spectrometry as a
bioanalytical tool for shotgun proteomics and expands upon methods for neuropeptide
discovery and analysis
1
Chapter 1
Introduction Brief Background and Research Summary
2
Abstract
Mass spectrometry based comparative proteomics and improved methods for
neuropeptide discovery have been reported in this thesis A very brief introduction of crustacean
neuropeptidomics is covered in this chapter followed by Chapter 2 which covers in great detail
comparative proteomics using mass spectrometry with an emphasis on biomarker discovery
Chapter 3 reports a novel immunodepletion technique used for comparative proteomics between
glial fibrillary acidic protein (GFAP) overexpressor and control mouse cerebrospinal fluid (CSF)
Chapter 4 involves rat CSF comparative proteomics between rat adapted scrapie and control
animals Chapter 5 details comparisons of phosphoproteomes in Saccharomyces cerevisiae
(Bakerrsquos yeast) between starved vs glucose fed conditions Chapter 6 investigates the use of
electron transfer dissociation (ETD) for kDa sized neuropeptides and neuropeptides with tyrosine
sulfonation Chapter 7 presents a molecular weight cut-off (MWCO) protocol to allow sub-microg
peptides to be recovered Chapter 8 provides a conclusion to this thesis and outlines future
directions for certain projects
3
Background
Mass spectrometry (MS) requires gas phase ions for experimental measurement and
intact large nonvolatile biomolecules proved difficult to be analyzed by electron ionization or
chemical ionization until the invention of two soft ionization techniques matrix-assisted laser
desorptionionization (MALDI)1 and electrospray ionization (ESI)
2 ESI and MALDI are the
two most common soft ionization techniques for mass spectrometry Once ionized molecules
such as peptides or proteins can be separated by their mass to charge ratios (mz) using various
mass analyzers manipulating electric andor magnetic fields ESI and MALDI mass
spectrometric techniques have become central analytical methods in biological sciences because
they are suitable for nonvolatile thermally labile analytes in femtomole quantities3 ESI allows
the coupling of high pressure liquid chromatography and the constant flow of solvent is
electrically charged at the outlet to produce a Taylor cone4 During desolvation the Raleigh
limit is reached and a coulombic explosion occurs commonly producing multiply charged ions
A modification of ESI is nanoLC-ESI which operates at nLmin flow rates to analyze low sample
amounts such as proteins or peptides in biological samples5 NanoLC-ESI uses less solvent as
the source because of the reduced flow rate of standard LCMS with an ESI source and nanoLC-
ESI is the predominate inlet for shotgun proteomic ESI MS experiments6 Conversely MALDI
can use sub-microL volume of sample and be co-crystallized with an ultraviolet absorbing organic
matrix that is obliterated using a laser at appropriate wavelength to generate gas phase ions
Alternatively MALDI has the unique capability to work with tissue samples and ionize in the
solid state instead of liquid like ESI
4
Mass analyzers require an operating pressure between 10-4
-10-10
Torr to allow proper ion
transfer and separationisolation of the ion based upon its mz Numerous mass analyzers are
currently available and each have their own strengths and weaknesses as shown in Figure 1 The
biomolecules are separated by the mass analyzers and detected without fragmentation which is
termed MS analysis Following MS detection tandem mass spectrometry (MSMS) of the
original precursor ion can be performed to provide additional structural information such as a
ladder sequence of amino acids for peptides Numerous fragmentation techniques are available
for proteomics such as collision induced dissociation (CID) electron transfer dissociation (ETD)
or high energy collision induced dissociation (HCD) Each of these fragmentation techniques
have their own benefits in proteomics experiments and are discussed in depth in Chapter 2 The
background and current status for comparative proteomics with specific emphasis on biomarker
analysis are covered in Chapter 2
Neuropeptidomic Method Development in the Crustacean Model System
Utilizing Mass Spectrometry
Chapter 6 and 7 focus on sample preparation and ETD fragmentation techniques to
characterize neuropeptides in the neuroendocrine organs in the crustacean nervous system
Neuropeptides are short chains of amino acids which are a diverse class of cell-cell signaling
molecules in the nervous system Neuropeptides have been investigated for being involved in
numerous physiological processes such as memory7 learning
8 depression
9 pain
10 reward
11
reproduction12
sleep-wake cycles13
homeostasis14
and feeding15-17
Figure 2 depicts how
neuropeptides are synthesized as pre-prohormones in the rough endoplasmic reticulum and
5
packaged in the Golgi apparatus After being packaged these pre-prohormones are processed
into bioactive peptides within the vesicle which is occurring during vesicular transport down an
axon and released into the synaptic cleft Secreted neuropeptides stimulate the post synaptic
neurons by interacting with G-protein coupled receptors at the chemical synapse
The crustacean model nervous system is well-defined neural network which has been
used in neurobiology and neuroendocrine (neurosecretory) research and thus lends itself for
studying neuromodulation18-22
Figure 3 shows the locations of several neuroendocrine organs in
the crustacean Callinectes sapidus including the sinus gland (SG) which is studied in Chapter 6
The Lingjun Li lab has focused on neuropeptide discovery and function in crustacean
neuroendocrine organs using mass spectrometry23-25
The work presented in Chapters 6 and 7
expand on sample preparation and analytical tools to further investigate the neuropeptidome
Research Overview
Comparative Proteomics of Biological Samples
Chapter 2 provides a thorough review of comparative proteomics and biomarker analysis
using mass spectrometry The scientific community has shown great interest in the field of mass
spectrometry-based proteomics and peptidomics for its applications in biology Proteomics
technologies have evolved to generate large datasets of proteins or peptides involved in various
biological and disease progression processes producing testable hypotheses for complex
biological questions This chapter provides an introduction and insight into relevant topics in
proteomics and peptidomics including biological material selection sample preparation
separation techniques peptide fragmentation post-translational modifications quantification
6
bioinformatics and biomarker discovery and validation In addition current literature and
remaining challenges and emerging technologies for proteomics and peptidomics are discussed
Chapter 3 investigates comparative proteomics between a GFAP overexpressor mouse
model and a control mouses cerebrospinal fluid (CSF) CSF is a low protein content biological
fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in
direct contact with the brain but consist of very abundant proteins similar to serum which require
removal A modified IgY-14 immunodepletion treatment is presented to remove abundant
proteins such as albumin to enhance analysis of the low volumes of CSF that are obtainable
from mice These GFAP overexpressor mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we present the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates are performed to address animal variability as well as reproducibility in mass
spectrometric analysis Relative quantitation is performed using distributive normalized spectral
abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins with
significant changes in the CSF of GFAP transgenic mice are identified with validation from
ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
Chapter 4 explores the CSF proteomics of a novel prion model called rat adapted scrapie
(RAS) A similar IgY immunodepletion technique is presented as in Chapter 3 and is similarly
used to reduce the abundant proteins in CSF CSF from control and RAS (biological N=5
technical replicates N=3) were digested and separated using one dimensional reversed-phase
nanoLC separation In total 512 proteins 167 non-redundant protein groups and 1032 unique
peptides are identified with a 1 FDR Comparative analysis was done using dNSAF spectral
7
counting and 21 proteins were significantly up or down-regulated The proteins are compared to
the 1048 differentially regulated genes and additionally compared to previously published
proteins showing changes consistent with other prion animal models Of particular interest is
RAS specific proteins like RNAseT2 which is not up-regulated in mouse prion disease but is
designated as upregulated in both the genomic and proteomics data for RAS
Chapter 5 explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Previous work by the
Heideman lab investigated the transcriptional response to fresh glucose in yeast26
Kinases such
as protein kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose
response so we described a large scale phosphoproteomic MS based study in this chapter
Yeast cell extract was digested and phosphopeptides were enriched by immobilized metal
affinity chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase
(RP)-RP separation The low pH separation was infused directly into an ion trap mass
spectrometer with neutral loss triggered ETDfragmentation Specifically the CID fragmentation
can cause a neutral loss of 98z from the H3PO4 and can produce an incomplete fragmentation
pattern and lead to ambiguous identification so when the neutral loss is detected ETD MSMS
fragmentation is performed The neutral loss triggered ETD fragmentation is included in this
study to improve phosphopeptide identifications In total 477 phosphopeptides are identified
with 06 FDR and 669 phosphopeptides identified with 54 FDR Motif analysis of PKA and
phosphosite validation are performed as well
8
The future of comparative proteomics investigating small sample amounts or PTMs is
promising Further advances in enrichment separations science mass spectrometry
analyzersdetectors and bioinformatics will continue to create more powerful tools that enable
digging deeper in proteomics with both small (mouse CSF) and large (cell extracts) sample
amounts
Methods for Neuropeptide Analysis Using ETD fragmentation and Sample
Preparation
Chapter 6 investigates the utility of ETD fragmentation to sequence endogenous large
neuropeptides and labile tyrosine sulfation in the crustacean Callinectes sapidus The sinus
gland (SG) of the crustacean is a well-defined neuroendocrine site that produces numerous
hemolymph-borne agents including the most complex class of endocrine signaling moleculesmdash
neuropeptides One such peptide is the crustacean hyperglycemic hormone (CHH) precursor-
related peptide (CPRPs) which was not previously sequenced Electron transfer dissociation
(ETD) is used for sequencing of intact CPRPs due to their large size and high charge state In
addition ETD is used to sequence a sulfated peptide cholecystokinin (CCK)-like peptide in the
lobster Homarus americanus using a salt adduct Collectively this chapter presents two
examples of the utility of ETD in sequencing neuropeptides with large molecular weight or with
labile modifications
Chapter 7 reports a protocol on improving recovery of minute quantities of peptides by
adding methanol and a salt modifier (NaCl) to molecular weight cut-off membrane-based
centrifugal filters (MWCO) to enrich sub-microgram peptide quantities In some biological
9
fluids such as CSF the endogenous peptide content is very low and using pure water to perform
the MWCO separation produces too much sample loss Using a neuropeptide standard
bradykinin sample loss is reduced over two orders of magnitude with and without undigested
protein present The presence of bovine serum albumin (BSA) undigested protein and the
bradykinin standard lends evidence that sample loss occurs because of the MWCO and not the
presence of the protein Additionally a BSA tryptic digestion is presented where twenty-seven
tryptic peptides are identified from MALDI mass spectra after enriching with methanol while
only two tryptic peptides are identified after the standard MWCO protocol The strategy
presented in Chapter 7 greatly enhances recovery from MWCO separation for sub-microg peptide
samples
10
References
1 Tanaka K Waki H Ido Y Akita S Yoshida Y Yoshida T Matsuo T Protein and polymer analyses up to mz 100 000 by laser ionization time-of-flight mass spectrometry Rapid Communications in Mass Spectrometry 1988 2 (8) 151-153
2 Fenn J B Mann M Meng C K Wong S F Whitehouse C M Electrospray ionization for mass spectrometry of large biomolecules Science 1989 246 (4926) 64-71
3 Domon B Aebersold R Mass spectrometry and protein analysis Science 2006 312 (5771) 212-7
4 Whitehouse C M Dreyer R N Yamashita M Fenn J B Electrospray interface for liquid chromatographs and mass spectrometers Anal Chem 1985 57 (3) 675-9
5 Wilm M Mann M Analytical properties of the nanoelectrospray ion source Anal Chem 1996 68 (1) 1-8
6 Karas M Bahr U Dulcks T Nano-electrospray ionization mass spectrometry addressing analytical problems beyond routine Fresenius J Anal Chem 2000 366 (6-7) 669-76
7 Gulpinar M Yegen B The physiology of learning and memory Role of peptides and stress Curr Protein Pept Sci 2004 5 (6) 457-473
8 Ogren S O Kuteeva E Elvander-Tottie E Hokfelt T Neuropeptides in learning and memory processes with focus on galanin In Eur J Pharmacol 2010 Vol 626 pp 9-17
9 Strand F Neuropeptides general characteristics and neuropharmaceutical potential in treating CNS disorders Prog Drug Res 2003 61 1-37
10 Li W Chang M Peng Y-L Gao Y-H Zhang J-N Han R-W Wang R Neuropeptide S produces antinociceptive effects at the supraspinal level in mice Regul Pept 2009 156 (1-3) 90-95
11 Wu C-H Tao P-L Huang E Y-K Distribution of neuropeptide FF (NPFF) receptors in correlation with morphine-induced reward in the rat brain Peptides 2010 31 (7) 1374-1382
12 Tsutsui K Bentley G E Kriegsfeld L J Osugi T Seong J Y Vaudry H Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin new key neuropeptides controlling reproduction J Neuroendocrinol 2010 22 (7) 716-727
13 Piper D C Upton N Smith M I Hunter A J The novel brain neuropeptide orexin-A modulates the sleep-wake cycle of rats Eur J Neurosci 2000 12 (2) 726-730
14 Tsuneki H Wada T Sasaoka T Role of orexin in the regulation of glucose homeostasis - Tsuneki - 2009 - Acta Physiologica - Wiley Online Library Acta Physiol 2010
15 Kageyama H Takenoya F Shiba K Shioda S Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding Neuropeptides 2010 44 (2) 133-138
16 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide family of aplysia J Neurosci 2002 22 (17) 7797-7808
11
17 Jensen J Regulatory peptides and control of food intake in non-mammalian vertebrates Comp Biochem Physiol Part A Mol Integr Physiol 2001 128 (3) 469-477
18 Billimoria C P Li L Marder E Profiling of neuropeptides released at the stomatogastric ganglion of the crab Cancer borealis with mass spectrometry J Neurochem 2005 95 (1) 191-199
19 Cape S S Rehm K J Ma M Marder E Li L Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster Homarus americanus J Neurochem 2008 105 (3) 690-702
20 Cruz Bermuacutedez N D Fu Q Kutz Naber K K Christie A E Li L Marder E Mass
spectrometric characterization and physiological actions of GAHKNYLRFamide a novel FMRFamide‐like peptide from crabs of the genus Cancer J Neurochem 2006 97 (3) 784-799
21 Grashow R Brookings T Marder E Reliable neuromodulation from circuits with variable underlying structure Proc Natl Acad Sci USA 2009 106 (28) 11742-11746
22 Ma M Szabo T M Jia C Marder E Li L Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins Peptides 2009 30 (9) 1660-1668
23 Chen R Hui L Cape S S Wang J Li L Comparative Neuropeptidomic Analysis of Food Intake via a Multi-faceted Mass Spectrometric Approach ACS Chem Neurosci 2010 1 (3) 204-214
24 Li L Sweedler J V Peptides in the brain mass spectrometry-based measurement approaches and challenges Annu Rev Anal Chem 2008 1 451-483
25 Ma M Wang J Chen R Li L Expanding the crustacean neuropeptidome using a multifaceted mass spectrometric approach J Proteome Res 2009 8 (5) 2426-2437
26 Slattery M G Heideman W Coordinated regulation of growth genes in Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
12
Figure 1 Capabilities and performances of certain mass analyzers Check marks indicate
availability check marks in parentheses indicate optional + ++ and +++ indicate possible or
moderate goodhigh and excellentvery high respectively Adapted with permission from
reference 3
13
Figure 2 Synthesis processing and release of neuropeptides (a) Cartoon showing two
interacting neurons (b) The synthesis of neuropeptides in the neuronal organelles and their
transport down the axon to the presynaptic terminal is depicted (c) Shows neuropeptide release
and interaction with the dendrite of the post-synaptic cell (Adapted from PhD thesis by Dr
Stephanie Cape)
14
Figure 3 Schematic showing location of the neuronal tissues being analyzed in the MS studies
of neuropeptides in Callinectes sapidus The SGs (sinus glands located in the eyestalks of the
crab) and the POs (pericardial organs located in the chamber surrounding the heart) release
neurohormones into the hemolymph (crab circulatory fluid) The brain is near the STNS
(stomatogastric nervous system neural network that controls the motion of the gut and foregut)
which has direct connections to the STG (stomatogastric ganglion) The STG is located in an
artery so it is continually in contact with hemolymph (Adapted from PhD thesis by Dr Robert
Sturm)
15
Chapter 2
Mass Spectrometry-based Proteomics and Peptidomics for Biomarker
Discovery and the Current State of the Field
Adapted from ldquoMass spectrometry-based proteomics and peptidomics for systems biology and
biomarker discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
16
Abstract
The scientific community has shown great interest in the field of mass spectrometry-based
proteomics and peptidomics for its applications in biology Proteomics technologies have
evolved to produce large datasets of proteins or peptides involved in various biological and
disease progression processes producing testable hypothesis for complex biological questions
This review provides an introduction and insight to relevant topics in proteomics and
peptidomics including biological material selection sample preparation separation techniques
peptide fragmentation post-translation modifications quantification bioinformatics and
biomarker discovery and validation In addition current literature and remaining challenges and
emerging technologies for proteomics and peptidomics are presented
17
Introduction
The field of proteomics has seen a huge expansion in the last two decades Multiple factors have
contributed to the rapid expansion of this field including the ever evolving mass spectrometry
instrumentation new sample preparation methods genomic sequencing of numerous model
organisms allowing database searching of proteomes improved quantitation capabilities and
availability of bioinformatic tools The ability to investigate the proteomes of numerous
biological samples and the ability to generate future hypothesis driven experiments makes
proteomics and biomarker studies exceedingly popular in biological studies today In addition
the advances in post-translational modification (PTM) analysis and quantification ability further
enhance the utility of mass spectrometry (MS)-based proteomics A subset of proteomics
research is devoted to profiling and quantifying neurologically related proteins and endogenous
peptides which has progressed rapidly in the past decade This review provides a general
overview as outlined in Figure 1 of proteomics technology including methodological and
conceptual improvements with a focus on recent studies and neurological biomarker studies
Biological Material Selection
The choice of biological matrix is an important first step in any proteomics analysis The
ease of sample collection (eg urine plasma or saliva) versus usefulness or localization of
sample (eg specific tissue or proximity fluid) needs to be evaluated early on in a study design
Plasma derived by centrifugation of blood to remove whole cells is a very popular
choice in proteomics due to the high protein content (~65 mg ml1) and the ubiquitous nature of
blood in the body and the ability to obtain large sample amounts or various time points without
the need to sacrifice the animal or to perform invasive techniques Plasma is centrifuged
18
immediately after sample collection unlike serum where coagulation needs to occur first To
obtain plasma blood is collected in a tube with an anticoagulant added (ETDA heparin or
citrate) and centrifuged but previous reports have shown variable results when heparin has been
used as an anticoagulant2 Human Proteome Organization (HUPO) specifically recommends the
anticoagulants EDTA or citrate to treat plasma3 4
One of the primary concerns with plasma is
degradation of the protein content via endogenous proteases found in the sample5 One way to
address this problem is the use of protease inhibitors In addition freezethaw cycles need to be
minimized to prevent protein degradation and variability6 7
Plasma proteomics has seen
extensive coordinated efforts to start assessing the diagnostic needs using plasma8 HUPO also
has established a public human database for plasma and serum proteomics from 35 collaborating
labratories9 Large dynamic range studies have been performed on plasma with a starting sample
amount of 2625 microl (1575 mg) resulting in 3654 proteins identified with a sub 5 false
discovery rate10
The large dynamic range spanning across eleven orders of magnitude as visualized in
Figure 2 is one of the biggest obstacles in plasma proteomics Figure 2 also shows that as lower
abundance proteins are investigated the origins of those identified proteins are more diverse than
the most abundant proteins Recent mining of the plasma proteome showed an ability to search
for disease biomarker applications across seven orders of magnitude In addition the tissue of
origin for the identified plasma proteins were identified and its origin was more diverse as the
protein concentration decreased11
Plasma has been used as a source for biomarker studies such
as colorectal cancer12 13
cardiovascular disease14
and abdominal aortic aneurysm15
Even
though the blood brain barrier prevents direct blood to brain interaction neurological disorders
such as Alzheimerrsquos disease (AD) have had their proteomes studied using plasma16
19
An alternative sample derived from blood is serum which is plasma allowed to coagulate
instead of adding anti-coagulates The time for coagulation is usually 30 minutes and during that
time significant and random degradation from endogenous proteases can occur The additional
variability caused from the coagulation process can change the concentration of multiple
potentially valuable biomarkers As biodiversity between samples or organisms is a challenging
endeavor additional sample variability due to serum generation may be undesirable but serum is
still currently being used for biomarker disease studies17
Serum has been used to compare the
proteome differences in neurological diseases such as AD Parkinsonrsquos disease and amyotrophic
lateral sclerosis and a review can be found elsewhere discussing the subject18
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy of brain or spinal cord
in evaluating diseases of the central nervous system and has been used for studies in neurological
disorders due to being a rich source of neuro-related proteins and peptides19
The protein
composition of the most abundant proteins in CSF is well defined and numerous studies exist to
broaden the proteins identified20-22
CSF has an exceedingly low protein content (~04 μgμL)
which is ~100 times lower than serum or plasma and over 60 of the total protein content in
CSF consists of a single protein albumin23-25
In addition the variable concentrations of proteins
span up to twelve orders of magnitude further complicating analysis and masking biologically
relevant proteins to any given study26
One of the highest number of identified proteins is from
Schutzer et al with 2630 non-redundant proteins from 14 mL of pooled human CSF This study
involved the removal of highly abundant proteins by performing IgY-14 immunodepletion
followed by two dimensional (2D) liquid chromatography (LC) separation27
Studies have also
been performed to characterize individual biomarkers or complex patterns of biomarkers in
various diseases in the CSF28 29
One potential pitfall of CSF proteomic analysis is
20
contamination from blood which can be identified by counting red blood cells present or
examining surrogate markers from blood contamination other than hemoglobin such as
peroxiredoxin catalase and carbonic anhydrase30
A proof of principle CSF peptidomics study
identified numerous endogenous peptides associated with the central nervous system which can
be used as a bank for neurological disorder studies31
Numerous recent reports highlighted the
utility of CSF analysis for biomarker studies in AD32 33
medulloblastoma34
both post-mortem
and ante-mortem35
Cellular lysates offer the distinct advantage to work with a cell line yeast or bacteria
with large amounts of proteins available for analysis36 37
with Saccharomyces cerevisiae being
the most common cell lysate38 39
Other cell lines are also used including HeLa40
and E coli41
The ability to obtain milligrams of proteins easily to scale up experiments without animal
sacrifice offers a clear advantage in biological sample selection Current literature supports
cellular lysate as a valued and sought after source of proteins for large scale proteomics
experiments because of the ability to assess treatments conditions and testable hypotheses42-44
Cellular lysate from rat B104 neuroblastoma cell line was used as an in vitro model for cerebral
ischemia and showed abundance changes in multiple proteins involved in various neurological
disorders45
Other Sources of Biological Samples
Urine
The urine proteome appears to be another attractive reservoir for biomarker discovery
due to the relatively low complexity compared with the plasma proteome and the noninvasive
collection of urine Urine is often considered as an ideal source to identify biomarkers for renal
21
diseases due to the fact that in healthy adults approximately 70 of the urine proteome originate
from the kidney and the urinary tract 46
thus the use of urine to identify neurological disorders is
neglected However strong evidence have shown that proteins that are associated with
neurodegenerative diseases can be excreted in the urine47-49
indicating the application of urine
proteomics could be a useful approach to the discovery of biomarkers and development of
diagnostic assays for neurodegenerative diseases However the current view of urine proteome
is still limited by factors such as sample preparation techniques and sensitivity of the mass
spectrometers There has been a tremendous drive to increase the coverage of urine proteome
In a recent study Court et al compared and evaluated several different sample preparation
methods with the objective of developing a standardized robust and scalable protocol that could
be used in biomarkers development by shotgun proteomics50
In another study Marimuthu et al
reported the largest catalog of proteins in urine identified in a single study to date The
proteomic analysis of urine samples pooled from healthy individuals was conducted by using
high-resolution Fourier transform mass spectrometry A total of 1823 proteins were identified
of which 671 proteins have not been previously reported in urine 51
Saliva
For diagnosis purposes saliva collection has the advantage of being an easy and non-
invasive technique The recent studies on saliva proteins that are critically involved in AD and
Parkinsonrsquos diseases suggested that saliva could be a potentially important sample source to
identify biomarkers for neurodegenerative diseases Bermejo-Pareja et al reported the level of
salivary Aβ42 in patients with mild AD was noticeably increased compared to a group of
controls 52
In another study Devic et al identified two of the most important Parkinsons
22
disease related proteinsmdash α-synuclein (α-Syn) and DJ-1 in human saliva 53
They observed that
salivary α-Syn levels tended to decrease while DJ-1 levels tended to increase in Parkinsons
disease The published results from this study also suggest that α-Syn might correlate with the
severity of motor symptoms in Parkinsons disease Due in part to recent advancements in MS-
based proteomics has provided promising results in utilizing saliva to explore biomarkers for
both local and systemic diseases 54 55
the further profiling of saliva proteome will provide
valuable biomarker discovery source for neurodegenerative diseases
Tissue
Compared to body fluids such as plasma serum and urine where the proteomic analysis
is complicated by the wide dynamic range of protein concentration the analysis of tissue
homogenates using the well-established and conventional proteomic analysis techniques has the
advantage of reduced dynamic range However the homogenization and extraction process may
suffer from the caveat that spatial information is lost which would be inadequate for the
detection of biomarkers whose localization and distribution play important roles in disease
development and progression Matrix-assisted laser desorptionionization (MALDI) imaging
mass spectrometry (IMS) is a method that allows the investigation of a wide range of molecules
including proteins peptides lipids drugs and metabolites directly in thin slices of tissue 56-59
Because this technology allows for identification and simultaneous localization of biomolecules
of interests in tissue sections linking the spatial expression of molecules to histopathology
MALDI-IMS has been utilized as a powerful tool for the discovery of new cancer biomarker
candidates as well as other clinical applications60 61
The utilization of MALDI-IMS for human
or animal brain tissue to identify or map the distribution of molecules related to
neurodegenerative diseases were also recently reported62 63
23
Secretome
There has been an increasing interest in the study of proteins secreted by various cells
(the secretomes) from tissue-proximal fluids or conditioned media as a potential source of
biomarkers Cell secretomes mainly comprise proteins that are secreted or are shed from the cell
surface and these proteins can play important role in both physiological processes (eg cell
signaling communication and migration) and pathological processes including tumor
angiogenesis differentiation invasion and metastasis In particular the study of cancer cell
secretomes by MS based proteomics has offered new opportunities for cancer biomarker
discovery as tumor proteins may be secreted or shed into the bloodstream and could be used as
noninvasive biomarkers The latest advances and challenges of sample preparation sample
concentration and separation techniques used specifically for secretome analysis and its clinical
applications in the discovery of disease specific biomarkers have been comprehensively
reviewed64 65
Here we only highlight the proteomic profiling of neural cells secretome that has
been applied to neurosciences for a better understanding of the roles secreted proteins play in
response to brain injury and neurological diseases The LC-MS shotgun identification of
proteins released by astrocytes has been recently reported66-68
In these studies the changes
observed in the astrocyte secretomes induced by inflammatory cytokines or cholinergic
stimulation were investigated6667
Alternatively our group performed 2D-LC separation and
included cytoplasmic protein extract from astrocytes as a control to identify cytoplasmic protein
contaminants which are not actively secreted from cells68
Sample Preparation
24
Proteomic analysis and biomarker discovery research in biological samples such as body
fluids tissues and cells are often hampered by the vast complexity and large dynamic range of
the proteins Because disease identifying biomarkers are more likely to be low-abundance
proteins it is imperative to remove the high-abundance proteins or apply enrichment techniques
to allow detection and better coverage of the low-abundance proteins for MS analysis Several
strategies including depletion and protein equalizer approach have been used during sample
preparation to reduce sample complexity69 70
and the latest advances of these methods have been
reviewed by Selvaraju et al 71
Alternatively the complexity of biological samples can be
reduced by capturing a specific subproteome that may have the biological information of interest
The latter strategy is especially useful in the biomarker discovery where the changes in the
proteome are not solely reflected through the concentration level of specific proteins but also
through changes in the post-translational modifications (PTMs) Here we will mainly discuss
the enrichment of phosphoproteinpeptides glycoproteinpeptides and sample preparation for
peptidomics and membrane proteins
Phosphoproteomics
Phosphorylation can act as a molecular switch on a protein by turning it on or off within
the cell It is thought that up to 30 of the proteins can be phosphorylated72
and it plays
significant roles in such biological processes as the cell cycle and signal transduction73
Currently tens of thousands of phosphorylation sites can be proposed using analytical methods
available today74 75
The amino acids that are targeted for phosphorylation studies are serine
threonine and tyrosine with the abundance of detection decreasing typically in that order Other
25
amino acids have been reported to be phosphorylated but traditional phosphoproteomics
experiments ignore these rare events76
In a typical large-scale phosphoproteomics experiment the sample size is usually in
milligram amounts to account for the low stoichiometry of phosphorylated proteins The large
amount of protein is then digested typically with trypsin but alternatively experiments have
been performed with Lys-C digestion to produce large enzymatic peptides The larger peptides
produced from Lys-C render higher charged peptides during electrospray ionization (ESI) and
allow improved electron-based fragmentation to determine specific sites of phosphorylation77
From the pool of peptides phosphopeptides must be enriched otherwise they will be masked by
the vast number and higher ionization efficiency of non-phosphorylated peptides The two most
common enrichment techniques are immobilized metal ion affinity chromatography (IMAC) and
metal oxide affinity chromatography (MOAC) TiO2 being the most common oxide used for this
purpose A recent study reported that phosphorylation of neuronal intermediate filament proteins
in neurofibrillary tangles are involved in Alzheimerrsquos disease78
Glycoproteomics
Protein glycosylation is one of the most common and complicated forms of PTM Types
of protein glycosylation in eukaryotes are categorized as either N-linked where glycans are
attached to asparagine residues in a consensus sequence N-X-ST (X can be any amino acid
except proline) via an N-acetylglucosamine (N-GlcNAc) residue or the O-glycosylation where
the glycans are attached to serine or threonine Glycosylation plays a fundamental role in
numerous biological processes and aberrant alterations in protein glycosylation are associated
with neurodegenerative disease states such as Creutzfeld-Jakob Disease (CJD) and AD79 80
26
Due to the low abundance of glycosylated forms of proteins compared to non-glycosylated
proteins it is essential to enrich glycoproteins or glycopeptides in complex biological samples
prior to MS analysis Two of the most common enrichment methods used in glycoproteomics are
lectin affinity chromatography (LAC) and hydrazide chemistry The detailed methodologies of
LAC hydrazide chemistry and other enrichment methods in glycoproteomics have been
extensively reviewed in the past81 82
In particular LAC is of great interest in studies of
glycosylation alterations as markers of AD and other neurodegenerative diseases due to its recent
applications in brain glycoproteomics83
Our group has utilized multi-lectin affinity
chromatography containing concanavalin A (ConA) and wheat germ agglutinin (WGA) to enrich
N-linked glycoproteins in control and prion-infected mouse plasma84
This method enabled us to
identify a low-abundance glycoprotein serum amyloid P-component (SAP) PNGase F digestion
and Western blotting validation confirmed that the glycosylated form of SAP was significantly
elevated in mice with early prion infection and it could be potentially used as a diagnostic
biomarker for prion diseases
Membrane proteins
Membrane proteins play an indispensable role in maintaining cellular integrity of their
structure and perform many important functions including signaling transduction intercellular
communication vesicle trafficking ion transport and protein translocationintegration85
However due to being relatively insoluble in water and low abundance it is challenging to
analyze membrane proteins by traditional MS-based proteomics approaches Numerous efforts
have been made to improve the solubility and enrichment of membrane proteins during sample
preparation Several comprehensive studies recently covered the commonly used technologies in
27
membrane proteomics and different strategies that circumvent technical issues specific to the
membrane 86-90
Recently Sun et al reported using 1-butyl-3-methyl imidazolium
tetrafluoroborate (BMIM BF4) an ionic liquid (IL) as a sample preparation buffer for the
analysis of integral membrane proteins (IMPs) by microcolumn reversed phase liquid
chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MSMS)
The authors compared BMIM BF4 to the other commonly used solvents such as sodium dodecyl
sulfate methanol Rapigest and urea but they found that the number of identified IMPs from rat
brain extracted by ILs was significantly increased The improved identifications could be due to
the fact that BMIM BF4 has higher thermal stability and thus offered higher solubilizing ability
for IMPs which provided better compatibility for tryptic digestion than traditionally used solvent
systems38
In addition to characterization of membrane proteome the investigation of PTMs on
membrane proteins is equally important for characterization of disease markers and drug
treatment targets Phosphorylations and glycosylations are the two most important PTMs for
membrane proteins In many membrane protein receptors the cytoplasmic domains can be
phosphorylated reversibly and function as signal transducers whereas the receptor activities of
the extracellular domains are mediated via N-linked glycosylation Wiśniewski et al provides an
informative summary on recent advances in proteomic technology for the identification and
characterization of these modifications91
Our group has pioneered the development of detergent
assisted lectin affinity chromatography (DALAC) for the enrichment of hydrophobic
glycoproteins using mouse brain extract92
We compared the binding efficiency of lectin affinity
chromatography in the presence of four commonly used detergents and determined that under
certain concentrations detergents can minimize the nonspecific bindings and facilitate the
elution of hydrophobic glycoproteins In summary NP-40 was suggested as the most suitable
28
detergent for DALAC due to the higher membrane protein recovery glycoprotein recovery and
membranous glycoprotein identifications compared to other detergents tested In a different
study on mouse brain membrane proteome Zhang et al reported an optimized protocol using
electrostatic repulsion hydrophilic interaction chromatography (ERLIC) for the simultaneous
enrichment of glyco- and phosphopeptides from mouse brain membrane protein preparation93
Using this protocol they successfully identified 544 unique glycoproteins and 922 glycosylation
sites which were significantly higher than those using the hydrazide chemistry method
Additionally a total of 383 phosphoproteins and 915 phosphorylation sites were identified
suggesting that the ERLIC separation has the potential for simultaneous analysis of both glyco-
and phosphoproteomes
Peptidomics
Peptidomics can be loosely defined as the study of the low molecular weight fraction of
proteins encompassing biologically active endogenous peptides protein fragments from
endogenous protein degradation products or other small proteins such as cytokines and signaling
peptides Studies can involve endogenous peptides94
peptidomic profiling33
and de novo
sequencing of peptides95 96
Neuropeptidomics focuses on biologically active short segments of
peptides and have been investigated in numerous species including Rattus97 98
Mus musculus99
100 Bovine taurus
101 Japanese quail diencephalon
102 and invertebrates
103-106 The isolation of
peptides is typically performed through molecular weight cut-offs from either biofluids such as
CSF plasma or tissue extracts If the protein and peptide content is high such as for tissue or cell
lysates protein precipitation can be done via high organic solvents and the resulting supernatant
can be analyzed for extracted peptides where extraction solvent and conditions could have a
29
significant effect on what endogenous peptides are extracted from tissue107
A comparative
peptidomic study of human cell lines highlights the utility of finding peptide signatures as
potential biomarkers108
A thorough review of endogenous peptides and neuropeptides is beyond
the scope of this review and an excellent review on this topic is available elsewhere109
Fractionation and Separation
The mass spectrometer has a limited duty cycle and data dependent analysis can only
scan a limited number of mz peaks at any given time In addition significant ion suppression
can occur if there is a difference in concentration between co-eluting peptides or if too many
peptides co-elute Therefore one of the biggest challenges in biomarker discovery is the
complexity of the sample and the presence of high-abundance proteins in body fluids such as
CSF serum and plasma In addition to the removal of the most abundant proteins by
immunodepletion the reduction of the complexity of the sample by further fractionation is
indispensable to facilitate the characterization of unidentified biomarkers from the low
abundance proteins Traditionally used techniques for complex protein analysis include gel
based fractionation methods such as two-dimensional gel electrophoresis (2D-GE) and its
variation two-dimensional differential gel electrophoresis (2D-DIGE) or non-gel based such as
one- or multidimensional liquid chromatography (LC) and microscale separation techniques
such as capillary electrophoresis (CE)
2D-GE MS has been widely used as a powerful tool to separate proteins and identify
differentially expressed proteins ever since 2D gels were coupled to mass spectrometry In 2D-
GE MS thousands of proteins can be separated on a single gel according to pI and molecular
weight Individual protein spots that show differences in abundance between different samples
30
can then be excised from the gel digested into peptides and analyzed by MALDI MS or by
liquid chromatography tandem mass spectrometry (LC-MSMS) for protein identification The
introduction of 2D-DIGE adds a quantitative strategy to gel electrophoresis by enabling multiple
protein extracts to be separated on the same 2D gel thus providing comparative analysis of
proteomes in complex samples In 2D-DIGE protein extracts from two different conditions and
an internal standard can be labeled with fluorescent dyes for example Cy3 Cy5 and Cy2
respectively prior to two-dimensional gel electrophoresis Compared to traditional 2D-GE 2D-
DIGE provides the clear advantage of overcoming the inter-gel variation problem 110
Proteomic
profiling of CSF by 2D-GE and 2D-DIGE has led to the identification of putative biomarkers in
multiple neurological disorders For example Brechlin et al reported an optimized 2-DIGE
protocol profiled CSF from 36 CJD patients The applicability of their approach was proven by
the detection of known CJD biomarkers such as 14-3-3 protein neuron-specific enolase lactate
dehydrogenase and other proteins that are potentially relevant to CJD 111
In another study to
identify novel CSF biomarkers for multiple sclerosis CSF from 112 multiple sclerosis patients
and control individuals were analyzed by 2D-GE MS for comparative proteomics Ten potential
multiple sclerosis biomarkers were selected for validation by immunoassay 112
These
methodologies sample preparation techniques and applications of 2D-DIGE in
neuroproteomics were reviewed by Diez et al113
Although 2D gel provides excellent resolving
power and capability to visualize abundance changes there are some limitations to the method
For example gel based separation is not suitable for low abundance proteins extremely basic or
acidic proteins very small or large proteins and hydrophobic proteins114 115
Complementary to gel-based approaches shotgun proteomics coupled to LC have
become increasingly popular in proteomic research because they are reproducible highly
31
automated and capable of detecting low abundance proteins Furthermore another advantage of
LC-MS shotgun proteomics is the suitability for isotope labeling for protein quantification which
is reviewed in a later section In shotgun proteomics a protein mixture is digested and resulting
peptides are separated by LC prior to tandem MS fragmentation to identify different proteins by
peptide sequencing The most common separation for shotgun proteomics peptidomics or top-
down proteomics experiments use low-pH reversed phase (RP) C18 C8 or C4 columns RPLC
is well established which provides high resolution desalts the sample which can interfere with
ionization and the mobile phase is compatible with ESI Nanoscale C18 columns allow for
separation and introduction of sub microgram samples If larger amounts of sample are
available two dimensional separations are usually preferred to greatly enhance the coverage of
the investigated proteome which will be discussed in depth later It is preferable to have an
orthogonal separation method and since RP separates via hydrophobicity strong cation exchange
(SCX) was the original choice due to its separation by charge MudPIT (multidimensional
protein identification technology) usually refers to the use of SCX as the first phase of separation
and is a well-established platform116
SCX has the advantage over RP separation technologies to
effectively remove interfering detergents from the sample SCX separation is not based solely
off charge and hydrophobicity contributes to elution therefore a small amount of organic
modifier usually 10-15 is added to lessen the hydrophobicity effects117
The addition of
organic modifiers needs to be minimized otherwise binding to the C18 trap cartridge or C18
column will be reduced if performed on-line SCX can be used for PTMs and offers specific
applications for proteomic studies and an excellent current review is offered on this subject
elsewhere118
An alternative MudPIT separation scheme employing high pH RPLC as the first
phase of separation and low pH RPLC in the second dimension (RP-RP) has been successfully
32
applied to the proteomic analysis of complex biological samples119 120
The advantage of using
RP as the first dimension is the higher resolution for separation and better compatibility with
down-stream MS detection by eliminating salt Song et al reported a phosphoproteome analysis
based on this 2D RP-RP coupling scheme121
Hydrophilic interaction chromatography (HILIC) employs distinct separation modality
where the retention of peptides is increased with increasing polarity122
The loading of sample is
done by high organic and eluted by increasing the percentage of the aqueous phase or polarity of
the mobile phase opposite from RPLC thus establishing orthogonality of the two separation
modes123
HILIC has quickly become a very useful method and is actively used for proteomic
experiments124
for increased sensitivity125
phosphoproteomics126
glycoproteins127
and
quantification studies128
An alternative and modification to HILIC is ERLIC which adds an
additional mode of separation by electrostatic attraction An earlier study using ERLIC
demonstrated the ability to separate phosphopeptides from non-phosphorylated peptides at
pH=2129
A recent study looking into changes in the phosphoproteome of Marekrsquos Disease
applied ERLIC to chicken embryonic fibroblast lysate identifying only 13 phosphopeptides
out of all the identified peptides Due to the lack of isolation of phosphopeptides from ERLIC
the investigators performed immobilized metal affinity chromatography (IMAC) enrichment on
the fractions increasing identification of phosphopeptides over 50 fold130
A comparative study
of ERLIC to HILIC and SCX following TiO2 phospho-enrichment reported that
SCXgtERLICgtHILIC for phosphopeptide identifications126
Recent developments in instrumentation to combine LC with ion mobility spectrometry
(IMS) and MS (LC-IMS-MS) offered more advantages than conventional LC due to the rapid
high-resolution separations of analytes based on their charge mass and shape as reflected by
33
mobility in a given buffer gas The mobility of an ion in a buffer gas is determined by the ionrsquos
charge and its collision cross-section with the buffer gas The methodologies of IMS separations
and the application of LC-IMS-MS for the proteomics analysis of complex systems including
human plasma have been reviewed by Clemmerrsquos group131-133
They proposed a method that
employs intrinsic amino acid size parameters to obtain ion mobility predictions which can be
used to rank candidate peptide ion assignments and significantly improve peptide identification
134
Although 2D gel and LC are routinely used as separation techniques in MS-based
proteomics capillary electrophoresis (CE) has received increasing attention as a promising
alternative due to the fast and high-resolution separation it offers CE has a wide variety of
operation modes among which capillary zone electrophoresis (CZE) and capillary isoelectric
focusing (CIEF) have the greatest potential applications in MS-based proteomics thus will be
highlighted here CZE separates analytes by their charge-to-size ratios in buffers under a high
electrical field and is often used as the final dimension prior to MS analysis while the separation
feature of CIEF is based on isoelectric point and this technique is more suitable to be used as the
first dimension separation Detailed description of different CEndashMS interfaces sample
preconcentration and capillary coating to minimize analyte adsorption could be found in several
reviews135-141
CE technique is complementary to conventional LC in that it is suitable for the
analysis of polar and chargeable compounds Dovichirsquos group conducted proteomic analysis of
the secreted protein fraction of Mycobacterium marinum which has intermediate protein
complexity142
The tryptic digests were either analyzed by UPLC-ESI-MSMS in triplicates or
prefactionated by RPLC followed by CZE-ESI-MSMS It was demonstrated that the two
methods identified similar numbers of peptides and proteins within similar analysis times
34
However CZE-ESI-MSMS analysis of the prefractionated sample tended to identify more
peptides that are basic and have lower mz values than those identified by UPLC-ESI-MSMS
This analysis also presented the largest number of protein identifications by using CE-MSMS
suggesting the effectiveness of prefractionation of complex samples by LC method prior to CZE-
ESI-MSMS The use of CIEF as the first dimension of separation provides both sample
concentration and excellent resolving power The combination of CIEF and RPLC separation
has been applied to the proteomic analyses where the amount of protein sample is limited and
cannot meet the requirement of minimal load amount for 2D LC-MSMS143 144
So far CE-MS
has been widely applied to the proteomic analysis of various biological samples such as urine145
146 CSF
147 blood
148 frozen tissues
149 and the formalin-fixed and paraffin-embedded (FFPE)
tissue samples150
The recent CEndashMS applications to clinical proteomics have been summarized
in several reviews135 151 152
Protein Quantification
In 2D gel electrophoresis the quantitative analysis of protein mixtures is performed on
the gel by comparing the intensity of the protein stain The development of 2D-DIGE eliminated
the gel-to-gel variation and greatly improved the quantitative capability and reliability of 2D gel
methodology110
However the accuracy of 2D gel based protein quantification suffers from the
limitations that a seemingly single gel spot often contains multiple proteins and the difficulty of
detecting proteins with extreme molecular weights and pI values as well as highly hydrophobic
proteins such as membrane proteins Therefore non-gel based shotgun proteomics technology is
more suitable for accurate and large-scale protein identification and quantification in complex
samples Briefly the quantification in non-gel based shotgun proteomics can be categorized into
35
two major approaches stable isotope labeling-based and label-free methods The common
strategies for quantitative proteomic analysis are reviewed and summarized in Table 1
Isotope labeling methods
Because stable isotope-labeled peptides have the same chemical properties as their
unlabeled counterparts the two peptides within a mixture should exhibit identical behaviors in
MS ionization The mass difference introduced by isotope labeling enables the detection of a
pair of two distinct peptide masses by MS within the mixture and allowing for the measurement
of the relative abundance differences between two peptides Depending on how isotopes are
incorporated into the protein or peptide these labeling methods can be divided into two groups
In vitro chemical derivatization techniques which incorporate a label or tag into the peptide or
protein during sample preparation metabolic labeling techniques which introduce the isotope
label directly into the organism via isotope-enriched nutrients from food or media
1 In vitro derivatization techniques
There are multiple methods to introduce heavy isotopes into proteins or peptides in vitro
The commonly used strategies include 18
O 16
O enzymatic labeling Isotope-Coded Affinity Tag
(ICAT) Tandem Mass Tags (TMTs) and Isobaric Tags for Relative and Absolute Quantification
(iTRAQ) The 18
O labeling method enzymatically cleaves the peptide bond with trypsin in the
presence of 18
O-enriched H2O and introduces 4-Da mass shift in the tryptic peptides153
The
advantages of this method include 18
O-enriched water is extremely stable tryptic peptides will
be labeled with the same mass shift secondary reactions inherent to other chemical labeling can
be avoided Conversely widespread use of 18
O-labeling has been hindered due to the difficulty
of attaining complete 18
O incorporation and the lack of robustness154 155
Currently ICAT
36
TMTs and iTRAQ methods are extensively used in quantitative proteomics In ICAT cysteine
residues are specifically derivatized with a reagent containing either zero or eight deuterium
atoms as well as a biotin group for affinity purification of cysteine-containing peptides156 157
The advantage of ICAT is that the affinity purification via biotin moiety can facilitate the
detection of low-abundance cysteine-containing peptides In addition the mass difference
introduced by labeling increases mass spectral complexity with quantification from the different
precursor masses done by MS and peptide identification being achieved through tandem MS
(MSMS) This added complexity from different peptide masses was addressed by using isobaric
labeling methods such as TMTs and iTRAQ 158 159
where the same peptides in different samples
are isobaric after tagging and appear as single mz in MS scans thus enhancing the peptide limit
of detection and reducing the MS scan complexity Isobaric labeling reagents are composed of a
primary amine reactive group and an isotopic reporter group linked by an isotopic balancer group
for the normalization of the total mass of the tags The reporter group serves for quantification
purpose since it is cleaved during collision-induced dissociation (CID) to yield a characteristic
isotope-encoded fragment Moreover isobaric labeling methods allow the comparison of
multiple samples within a single experiment Recently a 6-plex version of TMTs was
reported160
and iTRAQ enables up to eight samples to be labeled and relatively quantified in a
single experiment161
8-plex iTRAQ reagents have been used for the comparison of complicated
biological samples such as CSF in the studies of neurodegenerative diseases 162
Recently our
group developed a novel N N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2)
tagging reagents with high quantitation efficacy DiLeu has the advantage of synthetic simplicity
and greatly reduced synthesis cost compared to TMTs and iTRAQ163
Xiang et al demonstrated
that DiLeu produced comparable iTRAQ ability for protein sequence coverage (~43) and
37
quantitation accuracy (lt15) for tryptically digested proteins More importantly DiLeu
reagents could promote enhanced fragmentation of labeled peptides thus allowing more
confident peptide and protein identifications
2 In Vivo Metabolic Labeling
Metabolic processes can also be employed for the incorporation of stable-isotope labels
into the proteins or organisms by enriching culture media or food with light or heavy versions of
isotope labels (2H
13C
15N) The advantage of in vivo labeling is that metabolic labeling does
not suffer from incomplete labeling which is an inherent drawback for in vitro derivatization
techniques In addition metabolic labeling occurs from the start of the experiment and proteins
with light or heavy labels are simultaneously extracted thus reducing the error and variability of
quantification introduced during sample preparation The most widely used strategy for
metabolic labeling is known as stable-isotope labeling of amino acids in cell culture (SILAC)
which was introduced by Mann and co-workers164 165
In SILAC one cell population is grown
in normal or light media while the other is grown in heavy media enriched with a heavy
isotope-encoded (typically 13
C or 15
N) amino acid such as arginine or leucine Cells from the
two populations are then combined proteins are extracted digested and analyzed by MS The
relative protein expression differences are then determined from the extracted ion
chromatograms from both the light and heavy peptide forms SILAC has been shown to be a
powerful tool for the study of intracellular signal transduction In addition this technique has
recently been applied to the quantitative analysis of phosphotyrosine (pTyr) proteomes to
characterize pTyr-dependent signaling pathways166 167
38
Labe-free quantification
Although various isotope labeling methods have provided powerful tools for quantitative
proteomics several limitations of these approaches are noted Labeling increases the cost and
complexity of sample preparation introduces potential errors during the labeling reaction It also
requires a higher sample concentration and complicates data processing and interpretation In
addition so far only TMTs and iTRAQ allow the comparison of multiple (up to eight) samples
simultaneously The comparison of more than eight samples in a single experiment cannot be
achieved by isotope labeling In order to address these concerns there has been significant
interest in the development of label-free quantitative approaches Current label-free
quantification methods for MS-based proteomics were developed based on the observation that
the chromatographic peak area of a peptide168 169
or frequency of MSMS spectra170
correlating
to the protein or peptide concentration Therefore the two most common label-free
quantification approaches are conducted by comparing (i) area under the curve (AUC) of any
given peptides171 172
or (ii) by frequency measurements of MSMS spectra assigned to a protein
commonly referred to as spectral counting173
Several recent reviews provided detailed and
comprehensive knowledge comparing label-free methods with labeling methods data processing
and commercially available software for label-free quantitative proteomics174-177
Dissociation Techniques
The vast majority of proteomic experiments have proteins or peptides being identified by
two critical pieces of data obtained from the mass spectrometer The first is the precursor ion
identified by its mz which is informative to the mass of the peptide being analyzed The second
is the use of tandem mass spectrometry to fragment or dissociate the precursor ion and record the
39
generated fragment ion pattern to discern the amino acid sequence The three most popular
dissociation or fragmentation techniques for peptides are CID electron-transfer dissociation
(ETD) and high-energy collision dissociation (HCD) A recent study on the human plasma
proteome demonstrated that combined fragmentation techniques enhance coverage by providing
complementary information for identifications CID enabled the greatest number of protein
identifications while HCD identified an additional 25 proteins and ETD contributed an
additional 13 protein identifications178
ETDECD
Electron capture dissociation (ECD) 179
preceded ETD but ECD was developed for use
in a Penning trap for Fourier transform ion cyclotron resonance (FTICR) mass spectrometers
ECD requires the ion of interest to be in contact with near-thermal electrons and for the electron
capture event to occur on the millisecond time scale but the time scale is inadequate for electron
trapping in Paul traps or quadrupoles in the majority of mass spectrometers180
ETD involves a
radical anion like fluoranthene with low electron affinity to be transferred to peptide cation
which results in more uniform cleavage along the peptide backbone The cation accepts an
electron and the newly formed odd-electron protonated peptide undergoes fragmentation by
cleavage of the N-Cα bond which results in fragmentation ions consisting of c- and zbull-type
product ions The uniformed cleavage results in reduced sequence discrimination to labile bonds
such as PTMs and also provides improved sequencing for larger peptides compared to CID181
The realization that larger peptides produced better MSMS quality spectra compared to CID led
to a decision tree analysis strategy where peptide charge states and size determined whether the
precursor peptide would be fragmented with CID or ETD182
One of the main benefits of
ETDECD is the ability to sequence peptides with labile PTMs such as phosphorylation77 183
40
sulfation184
glycosylation185
ubiquitination186
and histone modifications187
ETD also has the
benefit of providing better sequence information on larger neuropeptides when compared to
CID188
However a thorough analysis suggested that CID still yielded more peptideprotein
identifications than ETD in large scale proteoimcs189
HCD
High energy collision dissociation (HCD)190
is an emerging fragmentation technique that
offers improved detection of small reporter ions from iTRAQ-based studies191 192
HCD is
performed at a higher energy in a collision cell instead of an ion trap like CID thus HCD does
not suffer from the low-mass cutoff limitation Furthermore HCD offers enhanced
fragmentation efficiency assisting in MSMS spectra interpretation and protein identification193
A major drawback for HCD is that the spectral acquisition times are up to two-fold longer due to
increased ion requirement for Fourier transform detection in the orbitrap194
HCD has been
reported to increase phosphopeptide identifications over CID74
but in a different study CID was
reported to offer more phosphopeptide identifications over HCD194
Work has also been done to
transfer the decision tree analysis for HCD which basically switches CID with HCD claiming
better quality data determined by higher Mascot scores with more peptide identifications195
MSE
Data dependent acquisition (DDA) is the most commonly used ion selection process in
mass spectrometers for proteomic experiments An alternative process which does not have ion
selection nor switch between MS and MSMS modes is termed MSE MS
E is a data independent
mode and does not require precursor ions of a significant intensity to be selected for MSMS
analysis196
A data independent mode decouples the mass spectrometer choosing which
precursor ions to fragment and when the ions are fragmented MSE works by a low or high
41
energy scan and no ion isolation is occurring The low energy scan is where the precursor ion is
not fragmented and the high energy scan allows fragmentation The resulting mix of precursor
and fragmentation ions is then detected simultaneously197
The data will then need to be
deconvoluted using a proprietary time-aligned algorithm that is discussed elsewhere198
The
continuous data independent acquisition allows multiple MSMS spectra to be collected during
the natural analyte peak broadening observed in chromatography which provides more data
points for AUC label-free quantification In addition lower abundance peptides can be
sequenced as more MSMS spectra are collected throughout the elution of an LC peak allowing
better signal averaging for smaller analyte peak of interest during coelution and reducing
sampling bias in typical DDA experiments where only more abundant peaks can be selected for
fragmentation
A comparison of spiked internal protein standards into a complex protein digest provided
evidence that MSE was comparable to DDA analysis in LC-MS
199 MS
E has been used for label
free proteomics of immunodepleted serum in large scale proteomics samples200
In addition
MSE was performed for the characterization of human cerebellum and primary visual cortex
proteomes Hundreds of proteins were identified including many previously reported in
neurological disorders201
MSE is quickly becoming a versatile data acquisition method recently
used in such studies as cancer cells202
schizophrenia203
and pituitary proteome discovery204
The usefulness of MSE as an unbiased data acquisition method is being assimilated into multiple
proteomics studies including studies involving neurological disorders
Data Analysis
42
One of the major bottlenecks in non-targeted proteomic experiments is how to handle the
enormous amount of data obtained Database searches biostatistical analysis de novo
sequencing PTM validation all have their place and multiple available platforms are available
If the organism being studied has had its genome sequenced databases can be created
with a list of proteins in the FASTA format to be used in database searching There are
numerous database searching algorithms for sequence identification of MSMS data including
Mascot205
Sequest206
Xtandem207
OMSSA208
and PEAKS209
These searching algorithms are
performed by matching MSMS spectra and precursor mass to sequences found within proteins
How well the actual spectra match the theoretical spectra determines a score which is unique to
the searching algorithm and usually can be extrapolated to the probability of a random hit
Recently a database has been developed for PTM analysis by the use of the program SIMS210
Specifically for phosphopeptides Ascorersquos algorithm scans the MSMS data to determine the
likelihood of correct phosphosite identification from the presence of site identifying product
ions211
If the organism that is being analyzed has not had its genome sequenced and no (or very
limited) FASTA database is available a homology search can be performed using SPIDER212
available with PEAKS software Alternatively individual MSMS spectrum can be de novo
sequenced but software is available to perform automated de novo sequencing of numerous
spectra (PEAKS208
DeNovoX and PepSeq)
For large-scale protein identifications the false discovery rate (FDR) must be established
by the searching algorithm and that is accomplished by re-searching the data with a false
database created by reversing or scrambling the amino acid sequence of the original database
used for the protein search Any hits from the false database will contribute to the FDR and this
value can be adjusted usually around 1 An additional layer of confidence in the obtained data
43
can be achieved in shotgun proteomics experiments by removing all the proteins that are
identified by only one peptide
Once a set of confident proteins or peptides have been generated from database
searching bioinformatic analysis or biostatistical analysis is needed Numerous software
packages are available for different purposes FLEXIQuant is an example for absolute
quantitation of isotopically labeled protein or peptides of interest213
FDR analysis of
phosphopeptides or other specific PTMs can be adjusted with such software as Scaffold
providing data consisting only of a specific modification214
Bioinformatic tools such as
Scaffold or ProteoIQ also include gene ontology (GO) analysis which can classify identified
proteins by three categories cellular component molecular function or biological process
Custom bioinformatics programs can also be developed and are often useful in various proteomic
studies including biomarker discovery in neurological diseases215
More detailed review of
bioinformatics in peptidomics216
and proteomics217
can be found elsewhere
Validation of Biomarkers by Targeted Proteomics
The validation of putative biomarkers identified by MS-based proteomic analysis is often
required to provide orthogonal analysis to rule out a false positive by MS and providing
additional evidence for the biomarker candidate(s) from the study for future potential clinical
assays At present antibody-based assays such as Western blotting ELISA and
immunochemistry are the most widely used methods for biomarker validation Although accurate
and well established these methods rely on protein specific antibodies for the measurement of
the putative biomarker and could be difficult for large-scale validation of all or even a subset of a
long list of putative protein biomarkers typically obtained by MS-based comparative proteomic
44
analysis Large scale validation is impractical due to the cost for each antibody the labor to
develop a publishable Western blot or ELISA and the antibody availability for certain proteins
As an alternative strategy quantitative assays based on multiple-reaction monitoring (MRM) MS
using a triple quadrupole mass spectrometer have been employed in biomarker verification
MRM is the most common use of MSMS for absolute quantitation It is a hypothesis
driven experiment where the peptide of interest and its subsequent fragmentation pattern must be
known prior to the quantitative MRM experiments MRM involves selecting a specific mz (first
quadrupole) to be isolated for fragmentation (second quadrupole) followed by one or more of
the most intense fragment ions (third quadrupole) being monitored The ability to quantitate and
thus validate the proteins or peptides as potential biomarkers is achieved by performing MRM on
isotopically labeled reference peptide for targeted peptideprotein of interest The main obstacle
for quantification of peptides is interference and ion suppression effects from co-eluting
substances Since the isotopically labeled and native peptide will co-elute the same interference
and ion suppression will occur for both peptides and thus correcting these interfering effects
Peptides need to be systematically chosen for a highly sensitive and reproducible MRM
experiment to ensure proper validation of putative biomarkers Peptides require certain intrinsic
properties which include an mz within the practical mass detection range for the instrument and
high ionization efficiency If the desired peptide to be quantified is derived from a digestion
then peptides that have detectable incomplete digestion or missed cleavage site can be a major
source of variability Peptides with a methionine and to a lesser extent tryptophan are
traditionally removed from consideration from MRM quantitative experiments due to the
variable nature of the oxidation that can occur In addition if chromatographic separation is
performed the retention behavior of the peptide must be well behaved with little tailing effects
45
eluting late causing broadening of the peak and even irreversible binding to the column As an
example hydrophilic peptides being eluted off a C18 column may exhibit the previously
described concerns and a different chromatographic separation will need to be explored for
improved limits of detection quantitation and validation To determine consistent peptide
detection or usefulness of certain peptides databases such as Proteomics Database218
PRIDE219
PeptideAtlas220
have been developed to compile proteomic data repositories from initial
discovery experiments
After the peptide is selected for analysis the proper MRM transitions need to be selected
to optimize the sensitivity and selectivity of the experiment It is common for investigators to
select two or three of the most intense transitions for the proposed experiment It is imperative
that the same instrument is used for the determination of transition ions as different mass
spectrometers may have a bias towards different fragment ions
MRM experiments are still highly popular experiments for hypothesis directed
experiments221
biomarker analysis222
and validation223
Validation of putative biomarkers is
increasingly becoming a necessary step when performing large scale non-hypothesis driven
proteomics experiments The traditional validation techniques of ELISA Western blotting and
immunohistochemistry are still used but MRM experiments are becoming an attractive
alternative for validation of putative biomarkers due to its enhanced throughput and specificity
Current work is still being performed to both expand the linear dynamic range224
and
sensitivity225
of MRM A recent endeavor to increase the sensitivity for MRM experiments was
accomplished by ldquoPulsed MRMrdquo via the use of an ion funnel trap to enhance confinement and
accumulation of ions The authors claimed an increase by 5-fold for peak amplitude and a 2-3
fold reduction in chemical background225
46
Remaining Challenges and Emerging Technologies
Large sample numbers for mass spectrometry analysis
Multiple conventional studies in proteomics have been performed on a single or a few
biological samples As bio-variability can be exceedingly high the need for larger sample sizes
is currently being investigated Prentice et al used a starting point of 3200 patient samples
from the Womenrsquos Health Institute (WHI) to probe the plasma proteome using MS for
biomarkers The study did not test the 3200 patient samples by MS because even a simple one
hour one dimensional RP analysis on a mass spectrometer would take months of instrument time
for uninterrupted analysis Instead the authors pooled 100 samples together to bring the total
number of pooled samples to 32 To provide relevant plasma biomarkers the samples were then
subjected to immunodepletion 2-D protein separation (96 fractions total) and then 1-D RPLC of
tryptic peptide separation on-line interface to a mass spectrometer The large sample cohorts
help address bio-variability that can be a concern from small sample size proteomic experiments
and provide ample sample amounts to investigate the low abundance proteins226
Hemoglobin-derived neuropeptides and non-classical neuropeptides
Neuropeptides such as neuropeptide Y and enkephalin are short chains of amino acids
that are secreted from a range of neuronal cells that signal nearby cells In contrast non-classical
neuropeptides are termed as neuropeptides or ldquomicroproteinsrdquo which are derived from
intracellular protein fragments and synthesized from the cytosol227
MS was recently used to
determine that hemopressins which are hemoglobin-derived peptides are upregulated in Cpefatfat
mice brains Gelman et al designed an MS experiment to compare hemoglobin-derived
47
peptides comparing the brain blood and heart peptidome in mice The authors provided data
that specific hemoglobin peptides were produced in the brain and were not produced in the
blood Certain alpha and beta hemoglobin peptides were also up regulated in the brain for
Cpefatfat
mice and bind to CB1 cannabinoid receptors228
As discussed earlier in the review
peptidomics and specifically neuropeptidomics are popular fields of study utilizing MS and non-
classical neuropeptides is an exciting emerging area of research that could further expand the
diversity of cell-cell signaling molecules
Ultrasensitive mass spectrometry for single cell analysis
In addition to large scale analysis MS-based proteomics and peptidomics are making
progress into ultrasensitive single cell analysis The most successful MS-based techniques for
single cell analysis was performed with MALDI and studies that have been performed on
relatively large neurons are reviewed elsewhere229
The ultrasensitive MS analysis is currently
directed towards single cell analysis of smaller cells including cancer cells The first challenge
in single cell analysis is the isolation and further sample preparation to yield relevant data
Collection and isolation of a cell type can be accomplished using antibodies for fluorescence
activated cell sorting (FACS) and immune magnetic separation FACS works by flow cytometry
sorting cells by a laser that excites a fluorescent tag that is attached to an antibody Immune
magnetic separation allows separation by antibodies with magnetic properties such as
Dynabeads230
One exciting study combining FACS and MS termed mass cytometry This
technology works by infusing a droplet into an inductively coupled plasma mass spectrometer
(ICP-MS) containing a single cell bound to antibodies chelated to transition elements allowing a
quantifying response between single cells231
Clearly the future of single cell analysis for
48
biomarker analysis and proteomics is encouraging and has the potential to be an emerging field
in MS-based proteomics and peptidomics
Laserspray ionization (LSI)
Laserspray ionization (LSI) is an exciting new method to produce multiply charged mass
spectra from MALDI that is nearly identical to ESI232-234
Recently it has been reported that LSI
can be performed in lieu of matrix to produce a total solvent-free analysis234
The benefits of
being able to generate multiply charged peptides without any solvent may offer advantages
including MS analysis of insoluble membrane proteins or hydrophobic peptides avoidance of
chemical reactions while in solvents a reduction of sample loss due to liquid sample preparation
and ability to avoid diffusion effects from tissue imaging studies234
The multiply charged peptide and protein ions produced by LSI expand the mass range
for tissue imaging analysis More importantly the multiply-charged peptide ions are amenable
for electron-based fragmentation methods such as ETD or ECD which can be employed in
conjunction with tissue imaging experiments to yield in situ sequencing and identification of
peptides of interest235
Paper spray ionization
Paper spray (PS) is an ambient ionization method which was first reported using
chromatography paper allowing detection of metabolites from dried blood spots The original
method used a cut out piece of paper with a voltage clipped on the back while applying 10 microL of
methanolH2O236
Improvements have been made to this technology to enhance analysis
efficiency with a new solvent 91 dichloromethaneisopropanol (vv) and the use of silica paper
49
over chromatography paper237
Interesting applications or modifications have been made to PS
including direct analysis of biological tissue238
and leaf spray for direct analysis of plant
materials239
but both detect metabolites instead of proteins or peptides Paper spray ionization
was previously shown to enable detection of cytochrome c and bradykinin [2-9] standards in a
proof of principle study240
Clearly the utility of PS analysis in proteomics and peptidomics is
yet to be explored
niECD
New fragmentation techniques have been investigated for their utility in proteomics and
peptidomics including a recently reported negative-ion electron capture dissociation (niECD)
Acidic peptides which usually contain PTMs such as phosphorylation or sulfonation are often
difficult to be detected as multiply charged peptides in the positive ion mode As discussed
earlier multiply charged peptides are required for ECDETD fragmentation The fragmentation
of niECD is accomplished by a multiply negatively charged peptide adding an electron The
resulting fragmentation of multiply sulfated and phosphorylated peptide and protein standards
showed no sulfate loss and preserved phosphorylation site The resulting fragmentation pattern
from niECD was also improved in the peptide anions and provides a new strategy for de novo
sequencing with PTM localization241
Conclusions and Perspectives
Proteomics methodologies have produced large datasets of proteins involved in various
biological and disease progression processes Numerous mass spectrometry-based proteomics
and peptidomics tools have been developed and are continuously being improved in both
50
chromatographic or electrophoretic separation and MS hardware and software However several
important issues that remain to be addressed rely on further technical advances in proteomics
analysis When large proteomes consisting of thousands of proteins are analyzed and quantified
dynamic range is still limited with more abundant proteins being preferentially detected
Development and optimization of chemical tagging reagents that target specific protein classes
maybe necessary to help enrich important signaling proteins and assess cellular and molecular
heterogeneity of the proteome and peptidome Furthermore a significant bottleneck in
usefulness of proteomics research is the ability to validate the results and provide clear
significant biological relevance to the results The idea of P4 medicine242 243
is an attractive
concept where the four Prsquos stand for predictive preventive personalized and participatory
Proteomics is one of the critical ldquoomicsrdquo fields and has led to the development of enabling
innovative strategies to P4 medicine244
A goal of P4 medicine is to assess both early disease
detection and disease progression in a person A simplified example of how proteomics fits into
P4 medicine is that certain brain-specific proteins could be used for diagnosis with
presymptomatic prion disease244
The concept of proteomic experiments providing an individual
biomarker is becoming more obsolete with the revised vision being a biomolecular barcode that
could potentially be ldquoscannedrdquo or be a fingerprint for a specific disease or early onset to that
disease being closer to reality An excellent review on what biomarker analysis can do for true
patients is available245
Proteomics can also generate new hypothesis that can be tested by classical biochemical
approaches If a disease has an unknown pathogenesis proteomics is a good starting point to try
to assemble putative markers that can lead to further hypothesis for evaluation If a particular
protein or PTM is associated with a disease state either qualitatively or quantitatively potential
51
treatments could target that protein of interest or investigators could monitor that protein or
PTM during potential treatments of the disease Proteomics has expanded greatly over the last
few decades with the goal of providing revealing insights to some of the most complex
biological problems currently facing the scientific community
Acknowledgements
Preparation of this manuscript was supported in part by the University of Wisconsin Graduate
School Wisconsin Alzheimerrsquos Disease Research Center Pilot Grant and a Department of
Defense Pilot Award LL acknowledges an H I Romnes Faculty Fellowship
52
Figure 1 A summary of general workflows of biomarker discovery pipeline by MS-based
proteomic approaches
Biological sample (CSF blood urine saliva cell
lysate tissue homogenates secreted proteins etc)
Protein extraction Sample pretreatment
2D-GE2D-DIGE MS 1D or 2D LC-MSMS
MALDI-IMS
Identification of
differentially
expressed proteins
Protein identification
Potential biomarkers
Biomarker validation
- Antibody based immunoassays
- MRM
Quantitative analysis
- Isotope labeling
- Label free
Identification and
localization of
differentially expressed
biomolecules
Intact tissue
Sample preparation Slice frozen tissues
thaw-mounted on plate
Apply Matrix
53
Figure 2 Tissue expression and dynamic range of the human plasma proteome A pie chart
representing the tissue of origin for the high abundance proteins shows that the majority of
proteins come from the liver (A) Conversely the lower abundance plasma proteins have a much
more diverse tissue of origin (B and C) The large dynamic range of plasma proteins is presented
and the proteins can be grouped into three categories (classical plasma proteins tissue leakage
products interleukinscytokines) (D) Adapted from Zhang et al11
and Schiess et al246
with
permission
54
55
Table 1 A summary of the common strategies applied to MS-based quantitative proteomic
analysis
Gel based Stable isotope labeling Label free
2D-GE
2D-DIGE 110
In vitro derivatization
18O
16O
153
ICAT 156
TMT 159
iTRAQ 158
Formaldehyde 247
ICPL 248
In vivo metabolic labeling
14N
15N
249
SILAC 164
AUC measurement 169 172
Spectral counting 173
AUC Area Under Curve ICAT Isotope-Coded Affinity Tag TMT Tandem Mass Tags iTRAQ Isobaric Tags for
Relative and Absolute Quantification ICPL Isotope Coded Protein Labeling SILAC Stable Isotope Labeling by
Amino Acids in Cell Culture Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
56
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1 Liu T Qian W J Mottaz H M Gritsenko M A Norbeck A D Moore R J
Purvine S O Camp D G 2nd Smith R D Evaluation of multiprotein
immunoaffinity subtraction for plasma proteomics and candidate biomarker discovery
using mass spectrometry Mol Cell Proteomics 2006 5 (11) 2167-74
2 Holten-Andersen M N Murphy G Nielsen H J Pedersen A N Christensen I J
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Proteomics 2010 73 (4) 769-77
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(5) 958-64
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Anal Chem 2008 80 (20) 7846-54
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211 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
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storing protein identification data J Proteome Res 2004 3 (6) 1234-42
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Hermjakob H PRIDE new developments and new datasets Nucleic Acids Res 2008 36
(Database issue) D878-83
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Hilgendorf C Development of a highly sensitive method using liquid chromatography-multiple
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Mass Spectrometry for Quantification of Heat Shock Proteins Anal Chem 2012
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glycosites Methods Mol Biol 2011 728 179-94
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natural isotopologue transitions Talanta 2011 87 307-10
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Smith R D Pulsed multiple reaction monitoring approach to enhancing sensitivity of a tandem
quadrupole mass spectrometer Anal Chem 2011 83 (6) 2162-71
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McIntosh M Wang P Buson Busald T Hsia J Jackson R D Rossouw J E Manson J
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227 Gelman J S Fricker L D Hemopressin and other bioactive peptides from cytosolic
proteins are these non-classical neuropeptides AAPS J 2010 12 (3) 279-89
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228 Gelman J S Sironi J Castro L M Ferro E S Fricker L D Hemopressins and
other hemoglobin-derived peptides in mouse brain comparison between brain blood and heart
peptidome and regulation in Cpefatfat mice J Neurochem 2010 113 (4) 871-80
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profiling Trends Biotechnol 2000 18 (4) 151-60
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Pavlov S Vorobiev S Dick J E Tanner S D Mass cytometry technique for real time
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spectrometry Anal Chem 2009 81 (16) 6813-22
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atmospheric pressure MALDI method for producing highly charged gas-phase ions of peptides
and proteins directly from solid solutions Mol Cell Proteomics 2010 9 (2) 362-7
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charged ions Anal Chem 2010 82 (12) 4998-5001
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charged ions without solvent using laserspray ionization a total solvent-free analysis approach at
atmospheric pressure Anal Chem 2011 83 (11) 4076-84
235 Inutan E D Richards A L Wager-Miller J Mackie K McEwen C N Trimpin
S Laserspray ionization a new method for protein analysis directly from tissue at atmospheric
pressure with ultrahigh mass resolution and electron transfer dissociation Mol Cell Proteomics
2010 10 (2) M110 000760
236 Wang H Liu J Cooks R G Ouyang Z Paper spray for direct analysis of complex
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substrate for paper-spray analysis of therapeutic drugs in dried blood spots Anal Chem 84 (2)
931-8
238 Wang H Manicke N E Yang Q Zheng L Shi R Cooks R G Ouyang Z
Direct analysis of biological tissue by paper spray mass spectrometry Anal Chem 83 (4) 1197-
201
239 Liu J Wang H Cooks R G Ouyang Z Leaf spray direct chemical analysis of plant
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Am Chem Soc 2011 133 (42) 16790-3
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243 Hood L Friend S H Predictive personalized preventive participatory (P4) cancer
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(2) 111-21
72
245 Belda-Iniesta C de Castro J Perona R Translational proteomics what can you do for
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N-metabolic labelingmass
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Rapid Commun Mass Spectrom 2002 16 (14) 1389-97
73
Chapter 3
Protein changes in immunodepleted cerebrospinal fluid from transgenic
mouse models of Alexander disease detected using mass spectrometry
Adapted from ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse
models of Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P
Messing A Li L Submitted
74
ABSTRACT
Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range
spanning at least nine orders of magnitude in protein content and is in direct contact with the
brain A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the
low volumes of CSF that are obtainable from mice As a model system in which to test this
approach we utilized transgenic mice that over-express the intermediate filament glial fibrillary
acidic protein (GFAP) These mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we report the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates were performed to address animal variability as well as reproducibility in
mass spectrometric analysis Relative quantitation was performed using distributive normalized
spectral abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins
with significant changes in the CSF of GFAP transgenic mice has been identified with validation
from ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
75
INTRODUCTION
Alexander disease (AxD) is a fatal neurogenetic disorder caused by heterozygous point
mutations in the coding region of GFAP (encoding glial fibrillary acidic protein)1 The hallmark
diagnostic feature of this disease is the accumulation of astrocytic cytoplasmic inclusions known
as Rosenthal fibers containing GFAP Hsp27 αB-crystallin and other components2-5
Although
several potential treatment strategies6-8
are under investigation clinical trial design is hampered
by the absence of a standardized clinical scoring system or means to quantify lesions in MRI
that could serve to monitor severity and progression of disease One solution to this problem
would be the identification of biomarkers in readily sampled body fluids as indirect indicators of
disease
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy site for brain or spinal
cord in evaluating diseases of the central nervous system The protein composition of CSF is
well defined at least for the most abundant species of proteins and numerous studies exist that
characterize individual biomarkers or complex patterns of biomarkers in various diseases9 10
GFAP itself is present in CSF (albeit at much lower levels than in brain parenchyma) and in one
study of three Alexander disease patients its levels were markedly increased11
Whether an
increase in CSF GFAP will be a consistent finding in Alexander disease or whether other useful
biomarkers for this disease could be identified through an unbiased analysis of the CSF
proteome is not yet known
The rarity of Alexander disease makes analysis of human samples difficult However
mouse models exist that replicate key features of the disease such as formation of Rosenthal
fibers Unfortunately mouse CSF poses particular problems for proteomic studies and there is
76
an urgent need for technical improvements for dealing with this fluid For instance collection
from an adult mouse typically yields ~10 μL of CSF often with some contamination by blood12
To further complicate analysis CSF has an exceedingly low protein content (~04 μgμL) with
over 60 of the total protein content consisting of a single protein albumin13 14
A number of
techniques have been developed to remove albumin from biological samples including Cibacron
Blue15
IgG immunodepletion16
and IgY immunodepletion17-19
IgY which is avian in origin
offers reduced non-specific binding and increased avidity when compared to IgG antibodies from
rabbits goats and mice20-23
One widely used IgY cocktail is IgY-14 which contains fourteen
specific antibodies specific for albumin IgG transferrin fibrinogen α1-antitrypsin IgA IgM
α2-macroglobulin haptoglobin apolipoproteins A-I A-II and B complement C3 and α1-acid
glycoprotein Since existing protocols for IgY-14 depletion are optimized for use with large
volumes of serum new protocols must be developed to permit its use with the low volumes of a
low protein fluid represented by mouse CSF
Various improvements have also taken place in the field of proteomic analysis that could
facilitate analysis of mouse CSF Data dependent tandem mass spectrometry followed by
quantification of proteins is used in standard shotgun proteomics24-29
Several methods now exist
for introducing quantitation into mass spectrometry including stable isotope labeling30-32
isobaric tandem mass tags33 34
and spectral counting35 36
Spectral counting which is a
frequency measurement that uses MSMS counts of identified peptides as the metric to enable
protein quantitation is attractive because it is label-free and requires no additional sample
preparation Finally recent advances in spectral counting has produced a data refinement
strategy termed normalized spectral abundance factor (NSAF)37 38
and further developed into
distributive NSAF (dNSAF) to address issues with peptides shared by multiple proteins39
77
To identify potential biomarkers in AxD we report a novel scaled-down version of IgY
antibody depletion strategy to reduce the complexity and remove high abundance proteins in
mouse CSF samples The generated spectral counts data were then subjected to dNSAF natural
log data transformation and t-test analysis to determine which proteins differ in abundance when
comparing GFAP transgenics and controls with multiple biological and technical replicates
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) urea (gt99) sodium chloride (997) and ammonium
bicarbonate (certified) were purchased from Fisher Scientific (Fair Lawn NJ) Deionized water
(182 MΩcm) was prepared with a Milli-Q Millipore system (Billerica MA) Optima LCMS
grade acetonitrile and water were purchased from Fisher Scientific (Fair Lawn NJ) DL-
Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega
(Madison WI) Formic acid (ge98) was obtained from Fluka (Buchs Switzerland)
Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris ge999 ) ammonium formate
(ge99995) glycine (ge985) and IgY-14 antibodies were purchased from Sigma-Aldrich
(Saint Louis MO)
Mice
Transgenic mice over-expressing the human GFAP gene (Tg737 line) were maintained
as hemizygotes in the FVBN background and genotyped via PCR on DNA prepared from tail
samples as described previously40
The mice were housed on a 14-10 light-dark cycle with ad
libitum access to food and water All procedures were conducted using protocols approved by
the UW-Madison IACUC
78
CSF collection
CSF was collected from mice as described previously12
Briefly mice were anesthetized
with avertin (400-600 mgkg ip) A midline sagittal incision was made over the dorsal aspect
of the hindbrain and three muscle layers carefully peeled back to expose the cisterna magna The
membrane covering the cisterna magna was pierced with a 30 gauge needle and CSF was
collected immediately using a flexible plastic pipette Approximately ten microliters of CSF was
collected per animal All samples used for MS analysis showed no visible contamination of
blood
Enzyme-linked immunosorbent assay (ELISA)
A sandwich ELISA was used to quantify GFAP8 Briefly a microtiter plate was coated
with a cocktail of monoclonal antibodies (Covance SMI-26R) Plates were blocked with 5
milk before addition of sample or standards diluted in PBS with Triton-X and BSA A rabbit
polyclonal antibody (DAKO Z334) was used to detect the GFAP followed by a peroxidase
conjugated anti-rabbit IgG antibody (Sigma A6154) secondary antibody The peroxidase activity
was detected with SuperSignal ELISA Pico Chemiluminescent Substrate (PIERCE) and
quantified with a GloRunner Microplate Luminometer Values below the biological limit of
detection (16ngL) were given the value 16ngL before multiplying by the dilution factor
Immunodepletion of abundant proteins
Currently there are no commercial immunodepletion products available for use with CSF
and to address the low protein content of this fluid (~04 microgmicroL) Therefore 100 microL of
purchased IgY-14 resin was coupled with a Pierce Spin Cup using a paper filter from Thermo
Scientific (Waltham MA) CSF samples from either transgenics or controls were first pooled to
100 microL and then added to 100 microL of 20 mM Tris-HCl 300 mM pH 74 (2x dilution buffer) and
79
allowed to mix with the custom IgY-14 depletion spin column on an end-over-end rotor for 30
minutes at 4oC The sample was then centrifuged at 04 rcf for 45 seconds with an Eppendorf
Centrifuge 5415D (Hamburg Germany) The antibodies were then washed with 50 microL 1x
dilution buffer vortexed for 5 minutes centrifuged at 04 rcf for 45 seconds and the flow through
was collected for tryptic digestion The antibodies were then stripped of the bound proteins with
four 025 mL washes of 01 M glycine pH 25 neutralized with two 025 mL washes of 01 M
Tris-HCl pH 80 and washed once with 025mL of 1x dilution buffer The immunodepletion
protocol was repeated for each transgenic or control pooled 100 microL sample (N=3)
Preparation of tryptic digests
The immunodepleted pooled mouse CSF samples (200 microL total volume) were
concentrated to 10 microL using a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA)
To each sample 8 microL of 133 M urea and 1 microL of 050 M DTT were added and allowed to
incubate at 37oC for one hour After incubation 27 microL of 055 M IAA was added for
carboxymethylation and the sample was allowed to incubate for 15 minutes in the dark To
quench the IAA 1 microL of 050 M DTT was added and allowed to react for 10 minutes To
perform trypsin digestion 70 microL of 50 mM NH4HCO3 was then added along with 025 microg
trypsin which had previously been dissolved in 50 mM acetic acid at a concentration of 05
microgmicroL Digestion was performed overnight at 37oC and quenched by addition of 25 microL of 10
formic acid The tryptic peptides were then subjected to solid phase extraction using a Varian
Omix Tip C18 100 microL (Palo Alto CA) Peptides were eluted with 50 ACN in 01 formic
acid concentrated and reconstituted in 30 microL H2O in 01 formic acid
RP nanoLC separation
80
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto an Agilent
Technologies Zorbax 300 SB-C18 5 microm 5x03 mm trap cartridge (Santa Clara CA) at a flow
rate of 5 microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm
Atlantis dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B
at 250 nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
81
range of 300-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot41
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt mus musculus
(house mouse) database (version 575) False positive analyses42
were calculated using an
automatic decoy option of Mascot Results from the Mascot results were reported using
Proteinscape 21 and technical replicates were combined and reported as a protein compilation
using ProteinExtractor (Bruker Daltonics Bremen Germany)
Mascot search parameters were as follows Allowed missed cleavages 2 enzyme
trypsin variable modifications carboxymethylation (C) and oxidation (M) peptide tolerance
plusmn12 Da maximum number of 13
C 1 MSMS tolerance plusmn05 Da instrument type ESI-Trap
Reported proteins required a minimum of one peptide with a Mascot score ge300 with bold red
characterization Spectral counts were determined from the number of MSMS spectra identified
from accepted proteins A bold red peptide combines a bold peptide which represents the first
query result from a submitted MSMS spectrum with the red peptide which indicates the top
peptide for the identified protein Requiring one bold red peptide assists in removal of
homologous redundant proteins and further improves protein results In addition requiring one
82
peptide to be identified by a score gt300 removes the ability for proteins to be identified by
multiple low Mascot scoring peptides
Each immunodepleted biological replicate had technical triplicates performed and the
technical triplicates were summed together by ProteinExtractor Peptide spectral counts were
then summed for each protein and subjected to dNSAF analysis Details for this method can be
found elsewhere37 39
but briefly peptide spectral counts are summed per protein (SpC) based on
unique peptides and a weighted distribution of any shared peptides with homologous proteins
ProteinScape removed 83 homologous proteins found in the current study to bring the total
number of proteins identified to 266 but some non-unique homologous peptides which are
shared by multiple proteins are still present in the resulting 266 remaining proteins To address
these non-unique homologous peptides distributive spectral counting was performed as
described elsewhere39
The dSpC is divided by the proteinrsquos length (L) and then divided by the
summation of the dSpCL from all proteins in the experiment (N) to produce each proteinrsquos
specific dNSAF value
N
i
i
kk
LdSpC
LdSpCdNSAF
1
)(
)()(
The resulting data were then transformed by taking the natural log of the dNSAF value The
means for each protein were calculated using the ln(dNSAF) from N=3 biological replicates and
the whole data set was subjected to the Shapiro-Wilk test for normalityGaussian distribution
performed on the software PAST (Version 198 University of Oslo Norway Osla) The
Shapiro-Wilk test was repeated multiple times to determine a non-zero value for zero spectral
83
counts A non-zero value is required to alleviate the errors of dividing by zero which was
experimentally determined to be 043 The Gaussian data were then subjected to the t-test to
identify statistically significant changes in protein expression
RESULTS AND DISCUSSION
General workflow
Individual CSF samples were manually inspected and samples were only selected that
showed no visual blood contamination Preliminary experiments showed that the maximum
degree of blood contamination estimated from counts of red blood cells in the CSF that was not
visible to the eye was less than 005 by total mass Approximately 10 individual mouse CSF
samples were pooled to achieve the desired 100 μL volume for a single biological replicate The
CSF samples were IgY immunodepleted followed by digestion with trypsin The resulting
digested samples were then de-salted concentrated reconstituted in 30 μL of 01 formic acid
and 5 μL was loaded onto a reversed phase nanoflow HPLC column subjected to a 90 minute
gradient and infused into the amaZon ETD ion trap mass spectrometer The workflow for
mouse CSF analysis is shown in Figure 1 The remainder of the desalted sample was saved for
technical replicates
Immunodepletion for CSF
Currently there are no immunodepletion techniques specifically designed for CSF
Nonetheless the protein profiles between CSF and serum are similar enough to use currently
available immunodepletion techniques designed for serum as a starting point The smallest
commercially available IgY-14 immunodepletion kit is for 15 μL of serum which is equivalent in
protein content to ~15 mL of CSF Therefore for 100 μL CSF one fifteenth of the total IgY-14
84
beads would be used for the equivalent immunodepletion which is 66 μL of proprietary bead
slurry The potential for irreversible binding of abundant proteins to their respective IgY
antibody even after an extra stripping wash and low amounts of total beads made using 66 μL
of IgY-14 bead slurry unrealistic In practice almost doubling the amount of IgY-14 beads (100
μL) used was not sufficient and resulted in albumin and transferrin peptides to be observed in
high abundance (data not shown) The most important protein to immunodeplete is albumin and
it has been reported to be a greater percentage of total CSF protein content (~60) than serum
(~49) in humans14
The difference in albumin percentage supports the results that proprietary
blends of immunodepletion beads for high abundance proteins such as albumin cannot be
scaled down on a strict protein scale and further modifications to the serum immunodepletion
protocol need to be made
Since IgY-14 beads were developed for use with serum all of its protocols need to be
taken into account to modify the protocol for CSF Serum samples should be diluted fifty times
before mixing with the IgY-14 beads but CSF protein concentration is at least one hundred times
lower than serum Therefore CSF is below half the recommended diluted protein concentration
for IgY immunodepletion Consequently multiple steps have been devised to address this
limitation First the binding time between the proteins targeted for removal from the CSF and
IgY-14 beads was doubled during the end-over-end rotor depletion step from the recommended
15 minutes to 30 minutes Second to prevent non-specific protein binding to the antibodies the
CSF samples were diluted with an equal amount (100 μL) of 2X dilution buffer The dilution
buffer of NaCl and Tris-HCl was needed to assist in reducing non-specific binding of proteins to
the 14 antibodies and ensuring the sample is held at physiological pH In addition to these
modifications a doubling of the starting IgY-14 bead slurry to 200 μL provided the desired
85
results Overall this modified protocol results in effective depletion of CSF abundant proteins
using only one-fifth of the antibodies provided by the smallest commercially available platform
Data Analysis
Spectral counting technique for relative quantitation provides numerous benefits for the
study of mouse CSF due to its label-free advantage In contrast isotopic labeling method often
involves additional sample processing that could cause sample loss which is highly undesirable
for low protein content and low volume samples Labeling methods also require a mixing of two
sets of isotopically labeled samples which would effectively increase the sample complexity and
reduce the amount of sample that can be loaded onto the nanoLC column by half In addition
more than two sets of samples can be compared by label-free methods The use of label-free
spectral counting method does not lead to an increase in sample complexity or interference in
quantitation from peptides in the mz window selected for tandem MS Using spectral counting
for relative quantitation however is dependent on reproducible HPLC separation and careful
mass spectrometry operation to minimize technical variability during the study To address
concerns of analytical reliability and run to run deviations base peak chromatograms from two
transgenic IgY-14 immunodepleted biological replicates including two technical replicates of
each were shown to be highly reproducible (Figure 2)
Each biological sample was analyzed in triplicate with the same protocols on the amaZon
ETD with three control and three transgenic samples From the three technical replicates for
each biological replicate the spectral counts of the peptides for the proteins identified were
summed The results from these mouse CSF biological triplicates are shown in Figure 3A for
GFAP overexpressor and Figure 3B for control The summation of spectral counts for each
biological replicate was performed to remove the inherent bias related to data dependent analysis
86
for protein identification One concern in grouping technical replicates is a potential loss of
information regarding analytical variability Figure 4 provides a graphical representation of
variability of technical replicates illustrating the standard deviation of technical replicates with
error bars Figure 4 shows that a statistically changed protein creatine kinase M (A) and an
unchanged protein kininogen-1 (B) have similar variability within (technical replicates) and
between samples (biological replicates) for each protein In addition Figure 4B illustrates that
even with the variability of kininogen-1 the resulting mean shown by the dashed line of control
and transgenic samples were almost equal whereas Figure 4A shows significantly different
expression level of creatine kinase M Performing replicate analysis of each biological sample
(n=3) helps correct for systematic errors deriving from sample handling and pooling of samples
helps reduce random error during the CSF sample collection process
Protein Identification and Spectral Counting Analysis
The data for dNSAF analysis like any mass spectrometry proteomics experiment
requires multiple layers of verification to ensure reliable data Our initial protein identifications
were subjected to a database search using a decoy database from Mascot which resulted in an
average false positive rate below 1 for all the experimental data collected Representative
MSMS spectra between control and GFAP transgenic samples are illustrated in Figure 5
Overall 266 proteins were identified in a combination of control and transgenic samples
(Supplemental Table 1) A total of 349 proteins were initially identified but 83 proteins were
isoforms of previously identified proteins and automatically excluded by ProteinExtractor The
next level of quality control was to only include ln(dNSAF) values from proteins identified by 2
or more unique peptides having a Mascot score of ge300 and observed in two out of three
biological replicates These selection parameters resulted in 106 proteins remaining after
87
dNSAF analysis (Supplemental Table 2) The resulting spectral counts were then subjected to
dSpC in order to account and correct for the systematic error of peptides shared by multiple
proteins (Supplemental Table 3)
It is inevitable in large scale and complex proteomics experiments that some proteins will
be seen in some samples and not others In addition when controls were compared to transgenic
samples as shown in Figure 3C 127 proteins were unique to either control or GFAP transgenic
mice and 139 proteins were seen in both samples From dNSAF equation if the spectral count
is zero the numerator is zero and the value will not be normalized between runs In order to
circumvent the zero spectral counts in dNSAF analysis zero spectral counts must be replaced by
an experimentally determined non-zero value determined to be 043 The 043 spectral counts
for zero spectral counts was calculated by serial Shapiro-Wilk tests to determine the lowest value
(043) for zero spectral counts and maintain a Gaussian distribution for all data sets The 043
value for zero spectral counts in the current study was higher than the 016 reported value for
zero spectral counts in the original NSAF spectral counting study37
Our study may have a
higher zero spectral count value than the previous study because the spectral counting data were
an addition of three technical replicates and three times 016 is close to 043 The normalized
Gaussian data were then subjected to t-test analysis and P-values below 005 are considered as
statistically significant and are presented in Table 1 The proteins with significant up or down
regulation from Table 1 can be further evaluated as how close significant proteins were to a p-
value of 005 such as ganglioside GM2 activator serine protease inhibitor A3N and collagen
alpha-2(I) chain having t-test scores of 0045 0047 and 0043 respectively Proteins exhibiting
a P-value close to 005 were more likely to be highly variable proteins or have smaller fold
changes between control and transgenic samples and thus provide less biological relevancy to
88
future studies The modest change in antithrobin-III which is reduced by 14-fold in transgenic
is included due a low pooled standard deviation in spectral counts
Spectral counting has been analyzed with fold changes derived directly from the average
spectral counts from the technical replicates and then the average of the three biological
replicates We decided to perform additional analysis using fold changes to dig deeper into
proteins that statistically support the null hypothesis from the dNSAF analysis To only pull out
highly confident protein identifications we used the same strict cut-off of two unique peptides
identified per protein as in dNSAF analysis We only accepted proteins with greater than three-
fold change in total spectral counts listed in Table 2 Two proteins contactin-1 (cntn1) and
cathepsin B (CB) illustrate the potential biomarkers missed by dNSAF analysis Cntn1 had zero
spectral count in the transgenic sample and had an average spectral count of 41 in control
samples The lack of any spectral counts in one biological control for cntn1 resulted in a large
standard deviation in the ln(dNSAF) means for the control which resulted in the t-test supporting
the null hypothesis Another example is CB which was detected by numerous spectral counts in
every GFAP transgenic biological replicate with an average spectral count of 97 (Table 2) The
presence of CB in one biological control sample (23 average spectral counts) resulted in a high
standard deviation in the mean of the control samples These examples exhibit a limitation of
dNSAF analysis which could cause a loss of potentially useful information
Previously Identified Proteins with Expression Changes
Previously three proteins have been described as increased in CSF from individual(s)
suffering from AxD In one study a single patientrsquos CSF was found to have elevated levels of
αβ-crystallin and HSP2744
In a second study three patients were reported to have elevated
levels of GFAP with concentrations from 4760 ngL to 30000 ngL (compared to lt175 ngL for
89
controls)11
GFAP was detected in our current study however the other two proteins were not
detected One possibility is that αB-crystallin and Hsp-27 levels in mouse CSF are too low for
detection by MS analysis In addition while the transgenic mice display the hallmark
pathological feature of AxD in the form of Rosenthal fibers they do not have any evident
leukodystrophy and thus may not display the full range of changes in CSF as might be found in
human patients
Creatine Kinase M
Creatine kinase M (M-CK) is a 43 kDa protein whose role is to reversibly catalyze
phosphate transfer between ATP and energy storage compounds M-CK has been primarily
found in muscle tissue and for humans creatine kinase M is diagnostically analyzed in the blood
for myocardial infarction (heart attack) In addition M-CK is also found in Purkinje neurons of
the cerebellum45 46
A related protein creatine kinase B (B-CK) also exhibited an apparent 21
fold increase in transgenic CSF over control but this difference was not statistically different
B-CK concentration is known to be elevated in CSF following head trauma47
or cerebral
infarction48
but decreased in astrocytes in individuals affected by multiple sclerosis49
Cathepsin
The data showed multiple cathepsins were up regulated in the CSF of transgenic mice
when compared to control mice The up regulated cathepsins were S L1 and B isoforms which
are all cysteine proteases Cathepsin S (CS) was never observed in control samples but
observed with an average of 73 spectral counts per analysis Cathepsin L1 (CL1) was up
regulated by 94 times in transgenic mice These two cathepsins exhibited significant changes
using dNSAF and t-test statistics as shown in Table 1 and Cathepsin B (CB) showed a 42 fold
increase in transgenic CSF as shown in Table 2
90
Cathepsins regulate apoptosis in cells50
which is the major mechanism for elimination of
cells deemed by the organism to be dangerous damaged or expendable CL and CB are
redundant in the system as thiol proteases and inhibition of CB by CA-047 causes a diminished
apoptosis response in multiple cell lines51
Intriguingly increased levels of CB or CL are
correlated with poor prognosis for cancer patients and shorter disease-free intervals It is
believed that these proteases degrade the extracellular membrane which allows tumor cells to
invade adjacent tissue and metastasize52
With regards to AxD the up regulation of these
cathepsins may be indicative of the bodyrsquos natural defense and response to Rosenthal fibers
Thus stimulation of these cathepsins may provide a further protective stress response but the
positive correlation between these proteases and cancer highlights the multiple roles of these
proteins in pathological response Alternatively it has been shown that increased CB is involved
with the tumor necrosis factor α (TNFα) induced apoptosis cascade53
The activation of the
TNFα could produce oligodendrocyte toxicity54
with the expression of TNFα being elevated in
tissue samples from mouse models and AxD patients55
The potential for a positive or a negative
effect in increasing or decreasing cathepsins warrant future research with cathepsins and AxD
Contactin-1
Contactin-1 (Cntn1) is a 133 kDa cell surface protein that is highly glycosylated and
belongs to a family of immunoglobulin domain-containing cell adhesion molecues56
Table 2
shows that Cntn1 is down regulated in transgenic mice since spectral counts were only observed
in control mice Cntn1 is expressed in neurons and oligodendrocytes and higher levels were
observed during brain development57
In addition Cntn1 leads to activation of Notch1 which
mediates differentiation and maturation of oligodendrocyte precursor cells (OPC) Although the
mechanism leading to a reduction of Cntn1 in CSF is not known it may reflect a change in
91
astrocyte interactions with either neurons or oligodendrocytes to alter their expression of this
protein
Validation of putative biomarkers and MS proteomics data using ELISA and RNA
microarray data
To further validate the relative protein expression data obtained via MS-based spectral
counting techniques orthogonal immunological and molecular biological approaches have been
examined As GFAP was shown to be elevated in the CSF of transgenic animals we used a
well-defined GFAP ELISA protocol to test its CSF concentration CSF from 8 week old male
mice was collected from both transgenic and control animals Five samples of transgenic CSF
was prepared by pooling four to eight animals for the GFAP ELISA In the case of controls
each sample represents a single animal GFAP concentrations observed by both the MS and
ELISA showed significant increases of GFAP in the CSF when comparing transgenic to control
animals
Another validation of MS spectral counts is observed in a microarray analysis performed
on transgenic mouse olfactory bulb tissue 55
In this paper nine of the proteins found by MS
showed similar gene expression in the microarray (Table 3) It is not surprising that all the genes
observed in the microarray are not the same as the proteins observed by MS analysis Gene
expression and protein synthesis and expression are not always correlated but the similarities
and overlapping trends observed with these two assays are encouraging As shown in Table 3
gene expression analysis also revealed the up regulation of several cathepsin proteins GFAP
and down regulation of contactin-1 in CSF collected from transgenic mice corroborating the
MS-based proteomics results
92
CONCLUSIONS
In this study we have produced a panel of proteins with significant up or down regulation
in the CSF of transgenic GFAP overexpressor mice This mouse model for AxD was consistent
with the previous studies showing elevation of GFAP in CSF The development of a modified
IgY-14 immunodepletion technique for low amounts of CSF was presented This improved
protocol is useful for future investigations to deal with the unique challenges of mouse CSF
analysis Modified proteomics protocols were employed to profile mouse CSF with biological
and technical triplicates addressing the variability and providing quantitation with dNSAF
spectral counting Validation of the MS-based proteomics data were performed using both
ELISA and RNA microarray data to provide further confidence in the changes in the putative
protein biomarkers This study presents three classes of interesting targets for future study in
AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
93
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33 Xiang F Ye H Chen R Fu Q Li L NN-dimethyl leucines as novel isobaric
tandem mass tags for quantitative proteomics and peptidomics Anal Chem 82 (7) 2817-25
34 Ross P L Huang Y N Marchese J N Williamson B Parker K Hattan S
Khainovski N Pillai S Dey S Daniels S Purkayastha S Juhasz P Martin S Bartlet-
Jones M He F Jacobson A Pappin D J Multiplexed protein quantitation in
Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol Cell Proteomics
2004 3 (12) 1154-69
35 Zybailov B Coleman M K Florens L Washburn M P Correlation of relative
abundance ratios derived from peptide ion chromatograms and spectrum counting for
quantitative proteomic analysis using stable isotope labeling Anal Chem 2005 77 (19) 6218-
24
36 Old W M Meyer-Arendt K Aveline-Wolf L Pierce K G Mendoza A Sevinsky
J R Resing K A Ahn N G Comparison of label-free methods for quantifying human
proteins by shotgun proteomics Mol Cell Proteomics 2005 4 (10) 1487-502
37 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M
P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J
Proteome Res 2006 5 (9) 2339-47
38 Mosley A L Florens L Wen Z Washburn M P A label free quantitative
proteomic analysis of the Saccharomyces cerevisiae nucleus J Proteomics 2009 72 (1) 110-20
39 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome
quantitation how to deal with peptides shared by multiple proteins Anal Chem 82 (6) 2272-81
40 Messing A Head M W Galles K Galbreath E J Goldman J E Brenner M
Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice Am J Pathol 1998
152 (2) 391-8
41 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
42 Elias J E Gygi S P Target-decoy search strategy for increased confidence in large-
scale protein identifications by mass spectrometry Nat Methods 2007 4 (3) 207-14
43 You J S Gelfanova V Knierman M D Witzmann F A Wang M Hale J E The
impact of blood contamination on the proteome of cerebrospinal fluid Proteomics 2005 5 (1)
290-6
44 Takanashi J Sugita K Tanabe Y Niimi H Adolescent case of Alexander disease
MR imaging and MR spectroscopy Pediatr Neurol 1998 18 (1) 67-70
45 Hemmer W Wallimann T Functional Aspects of Creatine Kinase in Brain
Developmental Neuroscience 1993 15 (3-5) 249-260
46 Hemmer W Zanolla E Furter-Graves E M Eppenberger H M Wallimann T
Creatine kinase isoenzymes in chicken cerebellum specific localization of brain-type creatine
96
kinase in Bergmann glial cells and muscle-type creatine kinase in Purkinje neurons Eur J
Neurosci 1994 6 (4) 538-49
47 Nordby H K Tveit B Ruud I Creatine kinase and lactate dehydrogenase in the
cerebrospinal fluid in patients with head injuries Acta Neurochirurgica 1975 32 (3) 209-217
48 Bell R D Alexander G M Nguyen T Albin M S Quantification of cerebral
infarct size by creatine kinase BB isoenzyme Stroke 1986 17 (2) 254-60
49 Steen C Wilczak N Hoogduin J M Koch M De Keyser J Reduced Creatine
Kinase B Activity in Multiple Sclerosis Normal Appearing White Matter PLoS ONE 5 (5)
e10811
50 Chwieralski C Welte T Buumlhling F Cathepsin-regulated apoptosis Apoptosis 2006
11 (2) 143-149
51 Droga-Mazovec G Bojic L Petelin A Ivanova S Romih R Repnik U Salvesen
G S Stoka V Turk V Turk B Cysteine cathepsins trigger caspase-dependent cell death
through cleavage of bid and antiapoptotic Bcl-2 homologues J Biol Chem 2008 283 (27)
19140-50
52 Duffy M J Proteases as prognostic markers in cancer Clin Cancer Res 1996 2 (4)
613-8
53 Guicciardi M E Deussing J Miyoshi H Bronk S F Svingen P A Peters C
Kaufmann S H Gores G J Cathepsin B contributes to TNF-alpha-mediated hepatocyte
apoptosis by promoting mitochondrial release of cytochrome c J Clin Invest 2000 106 (9)
1127-37
54 Butt A M Jenkins H G Morphological changes in oligodendrocytes in the intact
mouse optic nerve following intravitreal injection of tumour necrosis factor J Neuroimmunol
1994 51 (1) 27-33
55 Hagemann T L Gaeta S A Smith M A Johnson D A Johnson J A Messing
A Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal
fibers reveals a stress response followed by glial activation and neuronal dysfunction Hum Mol
Genet 2005 14 (16) 2443-58
56 Falk J Bonnon C Girault J A Faivre-Sarrailh C F3contactin a neuronal cell
adhesion molecule implicated in axogenesis and myelination Biol Cell 2002 94 (6) 327-34
57 Eckerich C Zapf S Ulbricht U Muumlller S Fillbrandt R Westphal M Lamszus
K Contactin is expressed in human astrocytic gliomas and mediates repulsive effects Glia
2006 53 (1) 1-12
97
Table 1 Statistically changed proteins between transgenic and control mouse CSF using
dNSAF analysis
Accession Protein Pa SC
b Fold
Changec
Control
dSpCd
Transgenic
dSpCd
KCRM_MOUSE Creatine kinase M-type 0034 425 30 182 541
HEXB_MOUSE Βeta-hexosaminidase subunit β 0012 183 uarr 0 59
CMGA_MOUSE Chromogranin-A 000078 153 darr 44 0
ANT3_MOUSE Antithrombin-III 0017 176 -14 67 47
SAP3_MOUSE Ganglioside GM2 activator 0045 415 darr 28 0
SPA3N_MOUSE Serine protease inhibitor A3N 0047 170 42 10 42
CO1A2_MOUSE Collagen alpha-2(I) chain 0043 56 darr 19 0
BLVRB_MOUSE Flavin reductase 00054 204 uarr 0 12
CATS_MOUSE Cathepsin S 00032 232 uarr 0 73
GFAP_MOUSE Glial fibrillary acidic protein 0021 107 uarr 0 21
RET4_MOUSE Retinol-binding protein 4 0040 189 darr 13 0
CBPE_MOUSE Carboxypeptidase E 0043 139 darr 11 0
CATL1_MOUSE Cathepsin L1 0015 87 94 02 19
The statistics are performed using the t-test from the ln(dNSAF) Gaussian data
a P p-value of the t-test where the null hypothesis states that there was no change in expression between
control and transgenic GFAP overexpressor b SC percentage of sequence coverage of the listed protein from
sequenced tryptic peptides c Fold Change positive values are indicative of a fold change for transgenic CSF
negative values are fold changes for control CSF (ie down-regulated in transgenic CSF) uarr indicates the protein
was only detected in transgenic CSF and darr indicates the protein was only detected in control CSF d dSpC
distributive spectral counts which represent the average spectral counts observed per run analysis on the mass
spectrometer and corrected using distributive analysis for peptides shared by more than one protein
98
Table 2 Proteins showing greater than three-fold changes with at least two unique
peptides identified for each protein
Accession Protein SC ()a Fold
Change b
Control
dSpC c
Transgenic
dSpC c
MUG1_MOUSE Murinoglobulin-1 precursor 147 42 155 37
CO4B_MOUSE Complement C4-B 113 54 22 118
PRDX6_MOUSE Peroxiredoxin-6 576 46 14 64
CNTN1_MOUSE Contactin-1 65 darr 41 0
CATB_MOUSE Cathepsin B 263 42 23 97
CAH3_MOUSE Carbonic anhydrase 3 396 76 11 84
APOH_MOUSE Beta-2-glycoprotein 1 128 44 44 1
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
PGK1_MOUSE Phosphoglycerate kinase 1 355 36 17 61
NDKB_MOUSE Nucleoside diphosphate kinase B 408 34 13 44
FHL1_MOUSE
Four and a half LIM domains
protein 1 243 39 13 51
NELL2_MOUSE
Protein kinase C-binding protein
NELL2 45 -43 13 03
MDHM_MOUSE
Malate dehydrogenase
mitochondrial 385 41 12 49
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
a SC percentage of sequence coverage of the listed protein from sequenced tryptic peptides b Fold
Change positive values are indicative of a fold change for transgenic CSF negative values are fold changes for
control CSF and darr indicates the protein was only detected in control CSF c dSpC distributive spectral counts
which represent the average spectral counts observed per run analysis on the mass spectrometer and corrected using
distributive analysis for peptides shared by more than one protein
99
Table 3 Validation of changes in proteins revealed by MS-based spectral counting
consistent with previously published microarray data
Consistent changes in RNA and proteomic data
uarr regulated in transgenic darr regulated in transgenic
Cathepsin S Contactin-1
Cathepsin B Carboxypeptidase E
Cathepsin L1
Peroxiredoxin-6
Complement C4-B
Glial fibrillary acidic protein
Serine protease inhibitor A3N
Note Validation of putative biomarkers from the current proteomics dataset by previously
published RNA microarray data55
Both up and down regulated proteins were consistent with the
RNA microarray data
_
100
___________________________________________
SUPPLEMENTAL INFORMATION (Available upon request)
Table S1 Compilation list of proteins identified from all the control and transgenic biological
replicates
Table S2 Distributive spectral counting calculations performed for proteins observed to share
identified peptides
Table S3 Proteins with dNSAF spectral counting performed including t-test analysis for a
comparison between transgenic and control CSF
101
FIGURE LEGENDS
Figure 1 The general workflow indicating the major steps involved in sample collection sample
processing mass spectrometric data acquisition and analysis of mouse CSF samples
Figure 2 Assessment of run to run variability of the base peak chromatograms within and
between two biological and technical replicates The peak profile and intensity scale is
consistent between the four chromatograms The four panels show two biological replicates (Tg
4 and Tg5) with two technical replicates for each biological sample
Figure 3 A Venn-diagram of the biological triplicates (1 2 and 3) of transgenic mouse
CSF showing 78 proteins identified in all three experiments B Venn-diagram of the biological
triplicates (1 2 and 3) of control mouse CSF having 61 proteins identified by all three
replicates C The overlap between control and transgenic CSF proteomic analysis showing 139
proteins identified by both groups and 73 and 54 uniquely identified by respective groups
Figure 4 Assessment of technical replicate variability between biological replicates The error
bars in both A and B are the standard deviation derived from the technical triplicates for each
biological replicate Panel A shows creatine kinase M having more or equal variability in the
biological triplicates than each technical triplicate The means of the biological triplicates are
illustrated by the dashed line Panel B represents kininogen-1 which is unchanged between
control (Ctl) and transgenic (Tg) samples The biological variability coupled with the technical
replicates provides a barely noticeable difference in the pooled mean between control and
102
transgenic spectral counts The difference in means is contrasted with the three fold change
observed from creatine kinase M (A)
Figure 5 MSMS profile of the tryptic peptide LSVEALNSLTGEFK from creatine kinase M
(A) The top MSMS is from a control sample with an elution at 463 minutes (B) The bottom
MSMS is from the GFAP transgenic sample with an elution at 46 minutes These tandem MS
spectra show instrument reliability and consistent fragmentation patterns which are necessary for
spectral counting analysis
Figure 6 Validation of MS spectral counts using a GFAP ELISA GFAP content (ngL)
measured within mouse CSF from both transgenic and control animals The data represents the
average with standard deviation (n=5 each sample represents pooling of 1 to 8 animals) The
statistics are performed using a student t-test plt00001
103
Figure 1
104
Figure 2
105
Figure3
106
Figure 4
107
Figure 5
108
Figure 6
Ctl Tg
100
1000
10000
100000
Mouse CSF Sample
GF
AP
(n
gL
)
109
Table of Contents Summary
Cerebrospinal fluid (CSF) was obtained from transgenic mouse models of Alexander disease as
well as healthy controls We immunodepleted pooled CSF from mice by a modified IgY-14
protocol Shotgun proteomics utilizing trypsin digestion 1-D nanoRP separation CID tandem
mass spectrometry analysis Mascot database searching and relative quantitation via distributive
normalized spectral abundance factor resulted in the identification of 266 proteins and 27
putative biomarkers
110
Chapter 4
Genomic and proteomic profiling of rat adapted scrapie
Adapted from ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A
Cunningham R Wang J Wellner D Ma D Li L Aiken J In preparation
111
Abstract
A novel model of prion disease using rats called Rat Adapted Scrapie (RAS) was
developed with the genomics from brain and proteomics from cerebrospinal fluid (CSF) profiled
The CSF proteome from control and RAS (biological N=5 technical replicates N=3) were
digested and separated using one dimensional reversed-phase nanoLC coupled to data-
dependent tandem MS acquisition on an ion trap mass spectrometer In total 512 proteins 167
non-redundant protein groups and 1032 unique peptides were identified with a 1 false
discovery rate (FDR) Of the proteins identified 21 were statistically up-regulated (plt005) and
7 statistically down-regulated in the RAS CSF We also identified 1048 genes which were
differentially regulated in rat prion disease and upon mapping these changes to mouse gene
expression however only 22 of these genes were in common with mRNAs responding to
prion infection in mice suggesting that the molecular pathology observed in mice may not be
applicable to other species The proteins are compared to the differentially regulated genes as
well as to previously published proteins showing changes consistent with other prion animal
models
112
Introduction
Prion diseases are an unusual class of fatal transmissible neurodegenerative disorders
that affect the mammalian central nervous system They are caused by the accumulation of an
abnormal conformation of the normal host encoded cellular prion protein PrPC This
conformational rearrangement of PrPC is brought about by template directed misfolding wherein
seed molecules of the abnormal isoform PrPScrapie
PrPSc
convert PrPC into new PrP
Sc molecules
Scrapie in sheep and goats is the prototypic prion disease and is so named because clinically
affected animals scrape their wool off on pen and stall walls Laboratory investigation of prion
diseases typically relies upon rodents which can be infected with natural isolates of scrapie1
albeit with some difficulty as ovid scrapie isolates need time to adapt to rodents This adaptation
is characteristic of prion disease interspecies transmissions and properly reflects the molecular
adaptation that must occur to allow interaction between exogenous foreign PrPSc
and host PrPC
molecules selecting for conformers which exhibit template directed misfolding In some cases
no conformational solution is found reflecting a species barrier to disease transmission
In recent years advances in genomics and proteomics technologies have allowed
unprecedented examination of the biomolecules that are altered upon exposure to prion agents
These studies2 3
have relied upon analysis of gene and protein expression changes in response to
prion infection with the aim of trying to identify pathways that might underlie the mechanism of
prion-induced neurotoxicity A second important aim has been to identify signature molecules
that might act as surrogate biomarkers for these diseases as there are significant analytical
challenges associated with sensitively detecting and specifically distinguishing disease-induced
conformational changes (PrPSc
) of the prion protein from normal host conformations (PrPC)
113
Mass spectrometry (MS)-based proteomics has become a promising tool for biomarker
discovery from biological fluids such as CSF blood and urine4-6
Two-dimensional gel
electrophoresis MS (2D-GE MS) and two-dimensional differential gel electrophoresis (2D-DIGE
MS) are the most widely used methods for proteomic profiling of prion infected CSF so far due
to the advantage of ready separation and quantification of proteins in complex biological samples
Studies using 2D-GE MS or 2D-DIGE MS to profile proteins in CSF have led to the
identifications of currently accepted biomarker for sCJD 14-3-3 protein and other potential
biomarkers for prion diseases7-9
However the application of this method in biomarker
discovery is limited by insufficient sensitivity and potential bias against certain classes of
proteins as gel-based separation does not work well for the low abundance proteins very basic
or acidic proteins very small or large proteins and hydrophobic proteins 10 11
In contrast to 2D-
GE or 2D-DIGE shotgun proteomics employs enzymatic digestion of the protein samples
followed by chromatographic separation prior to introduction into a mass spectrometer for
tandem MS analyses Shotgun proteomics has become increasingly popular in proteomic
research because these methods are reproducible highly automated and have a greater
likelihood of detecting low abundance proteins12 13
Due to the sample complexity in CSF and
because albumin comprises over half of the protein content in CSF removal of high-abundance
proteins including albumin is necessary to improve proteomic coverage and identify low-
abundance proteins One method is IgY immunodepletion14 15
which is performed prior to LC-
MSMS and uses species specific antibodies from chicken yolk to remove abundant proteins in
biological samples such as CSF In the present work CSF from control and rat adapted scrapie
animal models (biological N=5 technical N=3 replicates) were analyzed by LC-MSMS and we
114
indentified 512 proteins and 167 non-redundant protein isoforms (referred to as protein groups)
with 21 proteins being statistically up-regulated (p lt 005) and 7 statistically down-regulated
By and large this work has been performed using laboratory mice for the gene
expression studies and human cerebrospinal fluid for proteomics mice do not provide sufficient
volumes of CSF for proteomic studies Both approaches are exceedingly valuable as the mouse
model allows cross-sectional time course experiments to be performed including the important
pre-clinical phase of disease Critically however the relevance and generalizability of mouse
prion responses to other prion diseases especially human disease is unknown Human proteomic
studies on CSF guarantee relevant protein identifications but are limited to the clinical phase of
the disease when apparent markers may reflect gross neurodegeneration covering up subtle but
more specific responses To address these issues we have adapted mouse RML prions into rats
with the aim of expanding the knowledge of prion disease responses addressing the limitations
of mice and opening up the possibility of preclinical proteomic profiling in a laboratory rodent
In the present work CSF samples from control and rat adapted scrapie were analyzed by system
biology approaches Here we show that Rat Adapted Scrapie (RAS) is a powerful tool for an -
omics based approach to decipher the molecular impact of prion disease in vivo with
applicability to the molecular mechanisms of disease and biomarker discovery We identified
1048 genes which were differentially regulated in rat prion disease Astonishingly on the whole
mouse orthologs of these genes did not change in mouse adapted scrapie and vice versa
questioning the universality of previous mouse gene expression profiles These RAS gene
expression changes were identified in the CSF proteome where we detected 512 proteins and 167
protein groups with 21 proteins being statistically up-regulated (plt005) and 7 statistically down-
115
regulated in the CSF of prion diseased rats Many of the proteins detected have previously been
observed in human CSF from CJD patients
Materials and Methods
Ethics Statement
This study was carried out in accordance with the recommendations in the NIH Guide for Care
and Use of Laboratory Animals and the guidelines of the Canadian Council on Animal Care The
protocols used were approved by the Institutional Animal Care and Use Committees at the
University of Wisconsin and University of Alberta
Chemicals
Acetonitrile (HPLC grade) and ammonium bicarbonate (certified) were purchased from
Fisher Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) Optima LCMS grade acetonitrile and water were purchased
from Fisher Scientific (Fair Lawn NJ) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris
ge999 ) ammonium formate (ge99995) glycine (ge985) and IgY-7 antibodies were
purchased from Sigma-Aldrich (Saint Louis MO)
Rat Transmission and Adaptation
Prion agents used were the rocky mountain lab RML strain of mouse adapted scrapie
Stetsonville transmissible mink encephalopathy16
(TME) Hyper (Hy) strain of Hamster TME 17
1st passage Skunk adapted TME prepared as described and C from genetically defined
transmissions18
116
Brains from animals clinically affected with prion disease were aseptically removed and
prepared as 10 wv homogenates in sterile water 50 microL of 10 brain homogenate was
inoculated into weanling wistar rats intracranially After one year of incubation preclinical rats
from RML infections were euthanized by CO2 inhalation and the brain excised homogenized
and re-inoculated into naive animals Subsequent serial passages were from rats clinically
affected with rat adapted scrapie
Brains from rat passages were aseptically removed and bisected sagittally Brain halves
were reserved for immunohistochemistry (IHC) in formalin or frozen for immunoblotting RNA
isolation or subsequent passage For IHC tissues were first fixed in 10 buffered formalin
followed by antigen retrieval at 121 oC 21 bar in 10mM citrate buffer for 2 min After cooling
to room temperature sections were treated with 88 formic acid for 30 min and 4M guanidine
thiocyanate for 2 h Endogenous peroxidases were inactivated by 003 hydrogen peroxide and
tissue was blocked with 1 normal mouse serum Mouse anti-PrP mAB SAF83 (Cayman
Chemical) was biotinylated and applied at a 1250 dilution in blocking buffer overnight at 4 oC
Washes were performed in 10mM PBS with 005 tween-20 Streptavidin-peroxidase
(Invitrogen) was added for Diaminobenzidine (DAB) color reaction Anti-GFAP
immunohistochemistry was performed as above except that formic acid and guanidine treatment
steps were excluded Mouse anti-GFAP mab G-A-5 (Sigma) was used at a 1400 dilution
Brain samples for immunoblot were treated with or without Proteinase K (Roche) at a
ratio of 35 microg PK 100 ug protein at 50 microg mL for 30 minutes at 37 oC Phosphotungstenic acid
enrichments were performed as described14 19
Bis-Tris SDS-PAGE was performed on 12
polyacrylamide gels (Invitrogen) and transferred to PVDF Immunoblotting was performed using
117
mABs SAF83 (Cayman Chemical) 6H4 (Prionics) or 3F10 (a kind gift from Yong-Sun Kim) all
at a 120000 dilution
Gene Expression Profiling
RNA was extracted from frozen brain halves from clinically affected and control animals with
the QIAshredder and RNeasy mini kit (Qiagen Valencia CA) in accordance with the
manufacturerrsquos recommendations Due to the relatively large mass of rat brain an initial
homogenization was performed with a needle and syringe in 5mL of buffer RLT before further
diluting an aliquot in accordance with the manufacturers protocol Total RNA was amplified and
labeled in preparation for chemical fragmentation and hybridization with the MessageAmp
Premier RNA amplification and labeling kit (Life Technologies Grand Island NY) Amplified
and labeled cRNAs were hybridized on Affymetrix (Santa Clara CA) rat genome 230 20 high
density oligonucleotide arrays in accordance with the manufacturers recommendations
Differentially Expressed Genes were identified using Arraystar 50 (DNAStar Madison WI)
Robust multi-array normalization using the quantile approach was used to normalize all
microarray data A moderated T-test with a multiple comparison adjustment20
was used to reduce
the false discovery rate yet preserve a meaningful number of genes for pathway analysis
Pathway analysis was performed using the DAVID Bioinformatics database21
Comparative
analysis of genes induced by prions in mouse22
and rat disease was performed on genes
exhibiting at least a 2-fold change in expression Orthologs of rat and mouse genes were
identified using ENSEMBLE biomart release 6823
CSF Proteomic Profiling
118
CSF was collected from rats anaesthetized with isoflorane via puncture of the cisterna
magna A volume of 100-200 microL was routinely obtained CSF was touch spun for 30s at 2000xg
on a benchtop nano centrifuge to identify any blood contamination by the presence of a red
pellet CSF with blood contamination was not used for proteomic analysis CSF was prepared
for profiling by first depleting abundant proteins with an antibody based immunopartitioning
column IgY-R7 (Sigma-Aldrich Saint Louis MO) The manufacturers recommendations were
followed with a few deviations 40 microg of protein (~100 microL CSF) was bound to 100 microL of IgY
bead slurry in total volume of 600 microL formulated in 150mM ammonium bicarbonate The flow
through was collected and denatured by the addition of 10 microL of 8 M guanidine thiocyanate and
lyophilized in a speed-vac apparatus The sample was then reconstituted in 10 microL of water and 1
microL of 050 M DTT were added and allowed to incubate at 37oC for 30 minutes After incubation
27 microL of 055 M IAA was added for carboxymethylation and the sample was allowed to
incubate in the dark for 15 minutes After that 1 microL of 050 M DTT was added and allowed to
sit at room temperature for 10 minutes To perform trypsin digestion 70 microL of 50 mM
NH4HCO3 was then added along with 025 microg trypsin Digestion was performed overnight at
37oC and quenched by addition of 5 microL of 10 formic acid The tryptic peptides were then
subjected to solid phase extraction using an Agilent OMIX Tip C18 100 microL (Santa Clara CA)
Peptides were eluted with 50 ACN in 01 formic acid concentrated and reconstituted in 30
microL H2O with 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of a nanoAcquity (Milford MA) with a 10 microL injection
loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B consisted of
ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto a Waters 5 microm
119
Symmetry C18 180 microm x 20 mm trap cartridge (Milford MA) at a flow rate of 75 microLmin for 5
minutes at 95 A 5 B Separation was performed on a Waters 17 microm BEH130 C18 75 microm x
100 mm (Milford MA) with a gradient of 5 to 15 B over 10 minutes followed by 15 to
40 B over 80 minutes at room temperature
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with a Bruker CaptiveSpray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Waters Acquity console software to perform MS acquisitions for all experiments Smart
parameter setting (SPS) was set to 700 mz compound stability and trap drive level was set at
100 Optimization of the CaptiveSpray source resulted in dry gas temperature of 160oC dry
gas flow of 30 Lmin capillary voltage of -1325 V end plate offset of -500 V MSMS
fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
range of 350-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
120
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot24
(Version 24 Matrix
Science London UK) Database searching was performed against a forward and reversed
concatenated SwissProt Rattus rattus Mascot search parameters were as follows Allowed
missed cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) peptide tolerance plusmn12 Da maximum number of 13
C 1 MSMS
tolerance plusmn05 Da instrument type ESI-Trap Data was then transformed into Dat file formats
and transferred to ProteoIQ (NuSep Bogart GA) False positive analyses were calculated using
ProteoIQ and set at 1
Results
Development of Rat Adapted Scrapie
To develop a rat adapted strain of prion disease we introduced 6 different prion agents RML
TME Hy Skunk Adapted TME and Chronic Wasting Disease (CWD) from wild type 96G and
96S deer16-18
into 6 rats (Fig 1) Of these primary transmissions only RML induced the
accumulation of Proteinase K resistant PrP after one year of incubation as determined by western
blotting on 10 brain homogenates and PrPSc
enriched phoshotungstenic acid precipitated brain
homogenates Second passage of rat adapted RML scrapie resulted in clinically affected rats at
565 days post inoculation Serial passages of TME or CWD agents were not performed Clinical
symptoms consisted of ataxia lethargy wasting kyphosis and myoclonus Prion affected rats
121
also showed low level porphyrin staining around their head Subsequent serial passage decreased
incubation time to 215 days
Proteinase K resistant prion protein was observed from all clinically affected animals both by
immunoblot (Fig 2) and by immunohistochemistry (Fig 3) Di- and mono-glycosylated bands
were the most abundant isoforms of PrPSc
PrPSc
was extensively deposited in the cerebral cortex
hippocampus thalamus inferior colliculus and granular layer of the cerebellum GFAP
expressing activated astrocytes were found throughout the brain particularly in the white matter
of the hippocampus thalamus and cerebellum Spongiform change was a dominant feature of
clinical rat
Gene expression Profiling
In total 1048 genes were differentially regulated within a 95 confidence interval
(Supplementary Table 1) and 305 genes were differentially expressed by greater than 2-fold (Fig
4) The 1048 genes that were statistically significant were used for pathway analysis using
DAVID Pathway analysis suggested that the gene expression profile was consistent with
immune activation and maturation as well as inflammation (Supplementary Table 2) a likely
interpretation given the observable astrocytosis (Fig 3) and gliosis associated with prion disease
Other pathways highlighted by the analysis included increases in transcription of genes involved
in lysosomes and endosomes
To further probe the gene expression data we compared genes which were differentially
expressed in rat adapted scrapie against their orthologs expression in mouse scrapie and vice
versa (Figs 5 and 6) We did not observe a high degree of correlation between expression fold
changes For example GFAP a gene whose up-regulation in prion disease is well known was
122
increased 25 fold in rat adapted scrapie but up-regulated 93 fold in mouse scrapie A
qualitative analysis of expression of orthologs in prion disease suggests that many genes
deregulated at least 2-fold in mouse or rat do not have orthologs that are differentially expressed
For example the gene RNAseT2 was up-regulated greater than threefold in rat adapted scrapie
but was not significantly up-regulated in mouse Similarly three genes important in metals
homeostatsis metallothionein 1a and 2a and ceruloplasmin were up-regulated in rat 46 43 and
3 fold respectively but were not differentially expressed in mouse prion disease
CSF Proteomics
Each immunodepleted biological replicate (N=5 for each control and RAS) had technical
triplicates performed The triplicates were summed together using ProteoIQ Peptide spectral
counts were then summed for each protein and subjected to dNSAF analysis using ProteoIQ
internal algorithms Details for this method can be found elsewhere25 26
but briefly peptide
spectral counts are summed per protein (SpC) based on unique peptides and a weighted
distribution of any shared peptides with homologous proteins T-tests were used to identify
significant changes in protein expression 1032 unique peptides which identify 512 proteins and
167 protein groups were found Of these 512 proteins 437 were identified in both RAS and
control CSF samples (Fig 7) The complete list of all 512 proteins can be viewed in
Supplemental Table 3 Using the ProteoIQ the most likely isoform was selected such as 14-3-3
protein gamma
From Table 1 we observe five proteins that agree with the genomic data for up
regulations in RAS granulin macrophage colony-stimulating factor 1 receptor cathepsin D
complement factor H and ribonuclease T2 In addition serine protease inhibitor A3N was not
123
detected as up regulated in the RAS genomic data but was found to be up-regulated in previous
genomic profiling of the mouse prion model22
One interesting trend from the data in Table 1 is
that the majority of proteins found to be up-regulated in the RAS model were not detected in the
control samples The absence of the detection of those proteins such as ribonuclease T2 in the
control CSF does not necessarily suggest the absence of the protein nonetheless it is below the
detection limits for this current proteomics protocol and instrumentation
Discussion
Mice have been the preferred laboratory rodent for prion diseases research because they
can be inexpensively housed and are amenable to transgenesis which allows for short incubation
periods as well as hypothesis testing upon targeted pathways Pursuant to the determination of
the mouse genome and the development of high density transcriptional arrays for measurements
of gene expression profiling mice have been used extensively to examine the molecular
pathology of prion disease probing the impact of disease and animal strain In order to expand
upon this foundation we adapted mouse prions to infect rats We reasoned that by taking a
comparative approach to the molecular pathology of prion disease inferences could be obtained
into the variability of the molecular response to prion diseases and that understanding this
variability might suggest whether human prion disease responses are more or less similar to
mouse responses A second rationale is the desire to identify surrogate markers of prion disease
While this approach has been taken before using gene expression profiling a more direct
approach has been to use proteomic approaches on cerebral spinal fluid (CSF) identifying
proteins that are increase in abundance with disease A rat prion disease is valuable for this
because the rat proteome is established and rats allow for the collection of relatively large
volumes of CSF (~100 microL per animal) which allows for robust analytical separations enhancing
124
detection of biomarkers Finally rats unlike humans can be used in a time course study of prion
disease This allows for the identification of early transcriptional and proteomic responses to
prion infection responses which are particularly valuable for the identification of surrogate
disease biomarkers
To initiate the development of a rat prion disease we attempted to adapt six different
prion disease agents PrPres
molecules to rat via intracranial inoculation of weanling animals
(Figure 1) Of these six agents only mouse RML prions were able to surmount the species
barrier to prion disease transmission Mouse PrP differs from rat by eight amino acid changes
six in the mature protein and four in the N-terminal octa-peptide repeat region (Supplementary
Fig 1) As rat shares the most prion protein sequence homology with mouse it is perhaps not
surprising that it transmitted whereas the other did not confirming that the primary prion protein
sequence is the most important determinant for interspecies transmission We conclude that there
is a large molecular species barrier preventing conversion of rat PrPc into PrP
res
The transmission of mouse RML into rats was characterized by a shortening of the
incubation period following each passage This is indicative of agent adaption to the new host
and increases in the titer present in end-stage brain Overall our adaptation of mouse prion
disease into rats resulted in a similar agent to that observed by Kimberlin27
The differences in
incubation period at second passage are largely due to our collecting the animals at 365 days post
inoculation upon first passage whereas Kimberlin and Walker allowed the first passage animals
to reach end-stage clinical rats
Rat adapted scrapie is in many ways similar to other prion diseases The clinical period of
disease is marked by a progressive neurological dysfunction with prominent ataxia kyphosis and
125
wasting The protease resistant PrP observed is typical as is the widespread deposition of PrPSc
in
the brain Spongiosis and reactive astrogliosis are as expected of a prion disease
Gene expression profiles from rats clinically affected with prion disease revealed a strong
neuronal inflammation associated with proliferated and activated astrocytes This is perhaps best
observed through the up-regulation of GFAP GFAP positive astrocytes were omnipresent
throughout the brain and GFAP mRNA was up-regulated 25 fold The up-regulation of GFAP is
a hallmark of the molecular response to prion infection and has been routinely observed Our
comparative analysis of gene expression changes in mouse RML versus rat adapted scrapie
suggest substantial differences in gene expression in response to prion disease despite the fact
that the overall response is neuro-inflammatory This suggests that the potential overlap between
mouse expression profiles and a putative human CJD expression profile could be quite different
at the level of individual transcripts that might be expected to be changed
CSF Proteomics
CSF proteomics can be exceedingly challenging due to the small sample available large
dynamic range in proteins and abundant proteins limiting sample loading onto nanoscale
columns Dynamic range reduction in the CSF sample was achieved using a custom amount of
IgY-7 antibodies to abundant proteins such as albumin After immunodepletion the total
protein content was reduced by ~90 limiting the proteomics analysis to one dimensional
separation Label free quantitation spectral counting was performed because it requires less
protein and does not increase sample complexity The proteins identified from the affected and
control N=5 are shown in Supplemental Table 2 Consistent detection of proteins in CSF from
both control and infected rats was observed (Fig 7C) Only two proteins were identified in
126
controls that were not observed in RAS and only 10 proteins were only observed in RAS Some
of these proteins that were only identified in RAS are significantly changed (Supplemental Table
3) One concern in proteomics data is the variability from run to run and the possibility that
certain proteins are identified from different unique peptides Figure 7A shows that the vast
majority of the unique peptides detected (783) were identified with a 1 FDR in both RAS and
control CSF samples highlighting the analytical reproducibility of our methodology
Proteomic analysis of the infected rat CSF provides a reasonable approach to cross
validate biomarkers detected by gene expression profiling For example RNAseT2 a secreted
ribonuclease was up-regulated 3-fold at the mRNA level and was also enriched in CSF from
infected rats (Table 1) Similarly coordinated increases in detection of colony stimulating factor
1 receptor complement factor H granulin and cathepsin D were also observed Conversely
proteomic analysis of CSF also allows for the observation of post-transcriptional responses to
prion disease that might be absent in gene expression profiling The 14-3-3 proteins and neuron
specific enolase both known markers for CJD are only detected by proteomic analysis Thus
gene expression profiling and proteomic detection serve to increase confidence in the
observation of up-regulation enhancing the likelihood that proteins detected by both
methodologies are specific and perhaps may be more sensitive at earlier time points
Comparison to human CSF prion disease proteome
In our comparative analysis 21 proteins were found to be up-regulated while 7 proteins
down-regulated in prion infected rats Included in the up-regulated proteins were the 14-3-3
proteins epsilon and gamma 14-3-3 zetadelta was also up-regulated but not statistically
significant Enolase 2 or neuron specific enolase (NSE) was also up-regulated in CSF of infected
127
rats These proteins are all in agreement with results from previous proteomic profiling of human
CSF from patients with CJD8 9
The detection of 14-3-3 protein is included in the diagnostic
criteria approved by World Health Organization for the pre-mortem diagnosis of clinically
suspected cases of sCJD28
although its application in large-scale screening of CJD is still
debated due to high false positive rate where elevated 14-3-3 protein levels were also reported in
other conditions associated with acute neuronal damage29 30
It was suggested that other brain-
derived proteins that are also specific to CJD can be used in conjunction with 14-3-3 protein to
increase diagnosis accuracy and specificity31
NSE is present in high concentration in neurons
and in central and peripheral neuroendocrine cells NSE serves as another valuable biomarker in
diagnosis of CJD as the elevated level of NSE in CSF was found in early and middle stages of
CJD 32
Other proteins detected in CSF included cystatin C and serpina3N although both of
these were not statistically changed These proteins were both previously identified as being
putative biomarkers for CJD33 34
Utility for biomarker discovery with emphasis on onset of biomarker appearance in CSF
The investigation of the protein changes in CSF from RAS compared to control rats
provides a solid foundation when investigating potential biomarkers with prion disease onset
The cross-validation of the genomic and proteomics data further emphasizes the targets for
consideration during disease onset Biomarker discovery provides the potential to determine if
animals such as cows in bovine spongiform encephalopathy (BSE) are affected instead of
having to be euthanized when BSE is expected In addition CSF collection in mice or hamsters
Prion models is extremely difficult and limited alternatively with the advent of the RAS model
CSF can be collected in sufficient amounts for CSF proteomics CSF collection for mice or
hamsters can be ~10 microL and could be contaminated with blood further complicating proteomic
128
analysis unlike rats which over 10 times more CSF can be collected per animal35
Due to the
amount of CSF available time course studies analyzing CSF proteomics is possible in RAS due
to animal numbers that are manageable and reasonable The RAS model further allows
investigators to bypass working with highly infections CJD CSF samples to investigate the CSF
proteome changes
Conclusion
In this study we have described the gene and protein expression changes in brain and
spinal fluid from a transmission of mouse prions into rats We find that while the overall gene
expression profile in rats is similar to that in mice the specific genes that make up that profile
are different suggesting that genes that change in response to prion disease in different species
may not be as conserved Analysis of CSF from rat adapted scrapie identified similar protein
changes as known in human CJD The rat will be a useful model to identify surrogate markers
that appear prior to the onset of clinical disease and thus may be of higher specificity and
sensitivity
Supplemental Information Available Upon Request
1 Chandler R L Encephalopathy in mice produced by inoculation with scrapie brain material Lancet 1961 1 (7191) 1378-9 2 Silva C J Onisko B C Dynin I Erickson M L Requena J R Carter J M Utility of Mass Spectrometry in the Diagnosis of Prion Diseases Anal Chem 2011 3 Wei X Herbst A Ma D Aiken J Li L A quantitative proteomic approach to prion disease biomarker research delving into the glycoproteome J Proteome Res 2011 10 (6) 2687-702 4 Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C Mann M Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7 (1) 386-99 5 Sjodin M O Bergquist J Wetterhall M Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers J Chromatogr B Analyt Technol Biomed Life Sci 2003 878 (22) 2003-12 6 Cunningham R Ma D Li L Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery Frontiers in Biology 2012 7 (4) 313-335
129
7 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66 8 Harrington M G Merril C R Asher D M Gajdusek D C Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease N Engl J Med 1986 315 (5) 279-83 9 Piubelli C Fiorini M Zanusso G Milli A Fasoli E Monaco S Righetti P G Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping Proteomics 2006 6 Suppl 1 S256-61 10 Lilley K S Razzaq A Dupree P Two-dimensional gel electrophoresis recent advances in sample preparation detection and quantitation Curr Opin Chem Biol 2002 6 (1) 46-50 11 Oh-Ishi M Maeda T Separation techniques for high-molecular-mass proteins J Chromatogr B Analyt Technol Biomed Life Sci 2002 771 (1-2) 49-66 12 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset J Proteome Res 2008 7 (2) 698-707 13 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422 (6928) 198-207 14 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample preparation and analysis Proteomics 2005 5 (13) 3314-28 15 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep Biochem Biotechnol 2009 39 (3) 221-47 16 Marsh R F Bessen R A Lehmann S Hartsough G R Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy J Gen Virol 1991 72 ( Pt 3) 589-94 17 Bessen R A Marsh R F Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent J Virol 1992 66 (4) 2096-101 18 Johnson C J Herbst A Duque-Velasquez C Vanderloo J P Bochsler P Chappell R McKenzie D Prion protein polymorphisms affect chronic wasting disease progression PLoS One 2011 6 (3) e17450 19 Safar J Wille H Itri V Groth D Serban H Torchia M Cohen F E Prusiner S B Eight prion strains have PrP(Sc) molecules with different conformations Nat Med 1998 4 (10) 1157-65 20 Benjamini Y Hochberg Y Controlling the False Discovery Rate A Practical and Powerful Approach to Multiple Testing Journal of the Royal Statistical Society Series B (Methodological) 1995 57 (1) 289-300 21 Huang da W Sherman B T Lempicki R A Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat Protoc 2009 4 (1) 44-57 22 Moody L R Herbst A J Aiken J M Upregulation of interferon-gamma-induced genes during prion infection J Toxicol Environ Health A 2009 74 (2-4) 146-53 23 Flicek P Amode M R Barrell D Beal K Brent S Carvalho-Silva D Clapham P Coates G Fairley S Fitzgerald S Gil L Gordon L Hendrix M Hourlier T Johnson N Kahari A K Keefe D Keenan S Kinsella R Komorowska M Koscielny G Kulesha E Larsson P Longden I McLaren W Muffato M Overduin B Pignatelli M Pritchard B Riat H S Ritchie G R Ruffier M Schuster M Sobral D Tang Y A Taylor K Trevanion S Vandrovcova J White S Wilson M Wilder S P Aken B L Birney E Cunningham F Dunham I Durbin R Fernandez-Suarez X M Harrow J Herrero J
130
Hubbard T J Parker A Proctor G Spudich G Vogel J Yates A Zadissa A Searle S M Ensembl 2012 Nucleic Acids Res 2012 40 (Database issue) D84-90 24 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 1999 20 (18) 3551-67 25 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J Proteome Res 2006 5 (9) 2339-47 26 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome quantitation how to deal with peptides shared by multiple proteins Anal Chem 2010 82 (6) 2272-81 27 Kimberlin R H Cole S Walker C A Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters J Gen Virol 1987 68 ( Pt 7) 1875-81 28 World Health Organization Global surveillance diagnosis and therapy of human transmissible spongiform encephalopathies report from a WHO consultation 1998 World Health Organization Geneva Switzerland 9-11 February 1998 1-26 29 Bartosik-Psujek H Archelos J J Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG J Neurol 2004 251 (4) 414-20 30 Saiz A Graus F Dalmau J Pifarre A Marin C Tolosa E Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders Ann Neurol 1999 46 (5) 774-7 31 Sanchez-Juan P Green A Ladogana A Cuadrado-Corrales N Saanchez-Valle R Mitrovaa E Stoeck K Sklaviadis T Kulczycki J Hess K Bodemer M Slivarichova D Saiz A Calero M Ingrosso L Knight R Janssens A C van Duijn C M Zerr I CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease Neurology 2006 67 (4) 637-43 32 Zerr I Bodemer M Racker S Grosche S Poser S Kretzschmar H A Weber T Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease Lancet 1995 345 (8965) 1609-10 33 Sanchez J C Guillaume E Lescuyer P Allard L Carrette O Scherl A Burgess J Corthals G L Burkhard P R Hochstrasser D F Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease Proteomics 2004 4 (8) 2229-33 34 Miele G Seeger H Marino D Eberhard R Heikenwalder M Stoeck K Basagni M Knight R Green A Chianini F Wuthrich R P Hock C Zerr I Aguzzi A Urinary alpha1-antichymotrypsin a biomarker of prion infection PLoS One 2008 3 (12) e3870 35 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
131
Figure 1 Interspecies transmission of prion disease to rats End-stage clinical brain homogenates
were used to passage prion disease After one year of incubation animals were euthanized to
determine the extent of PrPres
accumulation Protease resistance PrP was only observed in those
animals infected with RML scrapie prions This material was serially passaged for two more
incubations before becoming rat-adapted as indicated by the shortening of the incubation period
132
Table 1 A list of selected proteins and mRNAs up-regulated in RAS CSF and brain tissue If
the protein is detected in the RAS CSF but not the control CSF the CSF expression is reported
with a infin If there is no change or data on certain genes related to an up regulated protein nd is
noted The mouse genomic data presented here was previously published22
Gene Protein Symbol Accession CSF
Expression
Rat
GEX
Mouse
GEX
14-3-3 protein zetadelta Ywhaz NP_037143 infin nd nd
14-3-3 protein epsilon Ywhae NP_113791 infin nd nd
14-3-3 protein gamma Ywhag NP_062249 infin nd nd
serine protease inhibitor A3N Serpin A3N NP_113719 54 nd 975
enolase 2 (gamma neuronal) Eno2 NP_647541 infin nd nd
granulin GRN NP_058809 62 364 184
macrophage colony-stimulating
factor 1 receptor
Csf1r NP_001025072 infin 293 205
cathepsin D CTSD NP_599161 infin 255 299
complement factor H Cfh NP_569093 376 234 nd
ribonuclease T2 RNAset2 NP_001099680 infin 302 nd
133
Figure 2 Accumulation of PrPSc
in rat adapted scrapie First second and third passage brain
homogenates were assayed for the presence of protease resistant PrP by immunoblot PrPSc
was
observed following phosphotungstenic acid enrichment at first passage By clinical disease in 2nd
and 3rd
passage rats PrPSc
had substantially accumulated
134
Figure 3 Histological appearance of rat adapted scrapie in the hippocampus at clinical disease
Infected animals showed intense immuno-staining for deposits of PrPSc
and GFAP expressing
astrocytes Spongiform change is an abundant feature in rat adapted scrapie
135
Figure 4 Scatter plot of gene expression profiling of rat adapted scrapie The expression of
individual genes from uninfected and infected animals were plotted to display up and down
regulation The dashed green line is no change Solid green lines are 2-fold changes in gene
expression
136
Figure 5 Quantitative relationship between orthologous pairs of two-fold up-regulated genes in
mouse and rat scrapie Genes up-regulated more than two fold were mapped to their orthologs
and the fold change was plotted Expression is log2 transformed
137
Figure 6 Comparison of genes upregulated in rat and mouse prion diseases Genes up-regulated
two fold in rodent scrapie were identified and the expression of their orthologs was determined
138
Figure 7 Venn diagram comparison of the CSF proteomics data between rat adapted scrapie
(RAS) model and control rats (A) Unique peptides from tryptic peptides produced for the
proteins identified (B) The total proteins identified including all isoforms within the protein
groups (C) The protein groups comparing only the top protein hit of the protein isoforms
showing very consistent protein identifications between RAS and control
139
Chapter 5
Investigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiae
Adapted from ldquoInvestigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M
Heideman W Li L In preparation
140
Abstract
This work explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Kinases such as protein
kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose response
Thus performing a large scale phosphoproteomic MS study can be highly beneficial to map the
signaling networks and cascades involved in glucose-dependent stimulated yeast growth Yeast
cell extract was digested and phosphopeptides were enriched by immobilized metal affinity
chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase (RP)-RP
separation The low pH separation was infused directly into an ion trap mass spectrometer with
neutral loss electron-transfer dissociation (ETD)-triggered fragmentation to improve
phosphopeptide identifications In total 477 unique phosphopeptides are identified with 06
false discovery rate (FDR) and 669 unique phosphopeptides identified with 54 FDR This
study includes comparative phosphoproteins for one specific motif of PKA xxxKRKRxSxxxx
which is presented and differences between starved vs glucose fed are highlighted Phosphosite
validation is performed using a localization algorithm Ascore to provide more confident and
site-specific characterization of phosphopeptides
141
Introduction
Microorganisms such as Saccharomyces cerevisiae (yeast) cycle between growth when
nutrients are available and quiescence when nutrients are scarce When glucose is limited yeast
go into growth arrest state but when glucose is added growth quickly resumes Kinases such as
protein kinase A (PKA) and target of rapamycin (TOR) regulate growth in response to nutrient
conditions and have been well studied through transcriptional control1-4
Yeast execute large
transcriptome alterations in response to changing environmental growth conditions5 6
Gene
regulation by glucose introduction in yeast has been studied including genes used for growth on
alternative carbon sources and activation of genes coding for glucose transport and protein
synthesis7-10
Response to nutrients for survival is not limited to yeast biology and indeed all
living creatures both prokaryotes and multicellular eukaryotes like mammals require nutrient
responsiveness and coordinating cellular functions to survive
With regulation of certain genes well studied by glucose introduction the mechanism and
global protein modulation caused by glucose introduction remain unknown6 Large-scale
phosphorylation analysis has been previously performed in yeast using mass spectrometry11-14
Mapping the phosphoproteome in yeast transitioning from quiescence to growth conditions to
better understand the roles of phosphorylation in orchestrating growth is needed The
phosphorylation state during growth or starvation could be used to engineer cells to drive ectopic
activity (or inhibition) to promote growth and ethanol production on non-native sugars like
xylose
It has been reported that the phosphorylation state can be affected by the introduction of
glucose to carbon-starved yeast15
and phosphorylation plays a significant role in the cell cycle
and signal transduction16
Specifically O-Phosphorylation can function as a molecular switch by
142
changing the structure of a protein via alteration of the chemical nature of an amino acid for
serine threonine or tyrosine It has been reported that up to 30 of the proteome can undergo
phophorylation17
Mass spectrometry has evolved as a powerful tool to accomplish phosphosite
mapping using shotgun proteomics With available technology tens of thousands of
phosphorylation sites can be mapped to amino acids on thousands of proteins using shotgun
proteomics18-20
Mass spectrometry can offer sensitive automated non-targeted global analysis of
phosphorylation events in proteomic samples but in any large scale phosphoproteomic
investigation enrichment of phosphoproteinspeptides is required First phosphorylation is
usually a sub-stoichiometric process where only a percentage of all protein copies are
phosphorylated21
Various enrichment methods have been used for phosphopeptide enrichment
including metal oxide affinity chromatography (MOAC)22
such as TiO223
immobilized metal
affinity chromatography (IMAC)12 24 25
electrostatic repulsion-hydrophilic interaction
chromatography (ERLIC)26
and immunoaffinity of tyrosine phosphorylation27 28
After
enrichment MS analyses of phosphorylated peptides are still challenging due to ion suppression
from non-phosphorylated peptides
Even after phosphopeptide enrichment further sample preparation is needed for large
scale proteomic experiments Additional fractionation can increase protein coverage of a
sample by over ten fold such as MudPIT29
(multidimensional protein identification technology)
In online MudPIT a sample is injected onto a strong cation exchange (SCX) column coupled to
a RP column Successive salt bumps followed by low pH gradients provide the separation of
peptides in two dimensions Since the phosphate groups on phosphopeptides have a low pKa
value due to being more acidic then their unmodified counterparts they tend to elute earlier and
143
disproportionally in ion exchange chromatography like SCX Alternatively reversed-phase
reversed-phase (RP-RP) separation has been proposed as an alternative to MudPIT for offline
two dimensional (2D) separation30
One of the caveats of 2D separation is the potential for
wasted mass spectrometry time from early and late fractions having very few peptides present
all while having too much sample for middle fractions One simple method to reduce these
ldquowasted fractionsrdquo is to combine early or late fractions with the middle fractions to reduce MS
runs with little peptide content to analyze thus shortening the overall analysis time31
In addition the labile phosphorylation group has a large propensity to undergo cleavage
during collision induced dissociation (CID) producing a neutral loss The neutral loss can
produce insufficient backbone fragment ions for MSMS identification32
A solution to neutral
loss fragmentation in phosphopeptides is MS3 using CID to produce improved backbone
fragmentation13 14 33
An alternative fragmentation method to CID for fast sampling ion traps is
electron transfer dissociation (ETD)34-36
ETD produces a more uniform back-bone cleavage
where the cation peptide receives an electron from a low affinity radical anion37
The transferred
electron induces a dissociation at the N-Cα bond to produce c- and zbull-type product ions while
retaining the labile post-translational modifications (PTM) such as phosphorylation bound to the
product ions38
The Bruker amaZon ETD ion trap mass spectrometer has the potential to trigger
ETD fragmentation after a labile PTM produces a neutral loss from CID fragmentation This
method is termed neutral loss-triggered ETD fragmentation and provides a complementary
fragmentation pathway to labile poor fragmenting phosphorylated peptides
In this work we provide a qualitative comparative list of yeast phosphopeptides observed
in glucose fed vs glucose starved conditions
144
Experimental
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) ammonium hydroxide (ACS plus certified) urea (gt99)
sodium chloride (997) ammonium bicarbonate (certified) Optima LCMS grade acetonitrile
Optima LCMS grade water and EDTA disodium salt (99) were purchased from Fisher
Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) ammonium formate (ge99995) Trizma
hydrochloride (Tris HCl ge990) Triton X-100 (laboratory grade) and iron (III) chloride
hexahydrate (97) were purchased from Sigma-Aldrich (Saint Louis MO) Sodium dodecyl
sulfate (SDS) (998) was purchased from US Biological (Marblehead MA) Nickel
nitrilotriacetic acid (Ni-NTA) magnetic agarose beads were purchased from Qiagen (Valencia
CA) Autoclaved yeast peptone dextrose (YPD) media was prepared in 1 L of deionized water
using 10 g yeast extract 20 g Peptone from Becton Dickinson and Company (Sparks MD) and
20 g D-(+)-Glucose (ge995) purchased from Sigma-Aldrich (Saint Louis MO)
Modified Mary Miller Yeast Protein Isolation
The yeast culture was prepared and protein extraction was performed using a modified
Mary Miller protocol39
Briefly yeast strain s288c was inoculated with YPD media and shook
for 72 hours to allow the yeast to be glucose starved After the 72 hours the yeast culture was
partitioned into two flasks To one flask glucose was added at 2 of the final concentration and
allowed to incubate and shake for 10 minutes Then 100 optical density units (ODU) of the yeast
145
culture were collected from each flask Each yeast sample was spun down in a Beckman-Coulter
J6B centrifuge (Indianapolis IN) at 2500xg for three minutes The media was aspirated and the
tubes were flash frozen in liquid nitrogen and stored at -80oC The yeast pellets were thawed on
ice and resuspended in 1 mL of lysis buffer (50 mM Tris HCl 01 triton x-100 05 SDS
pH=80) and 1X protease and phosphatase inhibitor cocktail from Thermo Scientific (Rockford
IL) and transferred to an Eppendorf tube To the yeast 200 microL of glass beads were added and
amalgamated for 25 minutes at 4oC The sample was inverted and the Eppendorf tube was
pierced with a hot 23 gauge needle through the bottom and the tube was placed atop a 5 mL
culture tube and centrifuge in the Beckman-Coulter J6B at 2500xg for three minutes at 4oC to
collect the liquid containing the yeast cells while the glass beads remain trapped in the
Eppendorf tubes The protein lysate was then centrifuged at 3000xg for five minutes at 4oC and
the supernatant was collected and stored at -80oC
Preparation of tryptic digests
The Saccharomyces cerevisiae protein cell extract for fed or starved were analyzed with a
BCA protein assay kit (Thermo Scientific Rockford IL) and 2 mg of protein was added to four
parts -20oC acetone and allowed to precipitate the protein for 1 hour at -20
oC The samples were
then centrifuged at 14000xg for 5 minutes and the supernatant was removed and the protein
pellet was reconstituted in 360 microl of 8 M urea To each sample 20 microL of 050 M DTT was
added and allowed to incubate at 37oC for 45 minutes After incubation 54 microL of 055 M IAA
was added for carbamidomethyl of cysteine and the sample was allowed to incubate for 15
minutes in the dark To quench the IAA 20 microL of 050 M DTT was added and allowed to react
for 10 minutes To perform trypsin digestion 1400 microL of 50 mM NH4HCO3 was then added
along with 20 microg trypsin to each sample Digestion was performed overnight at 37oC and
146
quenched by addition of 25 microL of 10 formic acid For each sample the tryptic peptides were
then subjected to solid phase extraction using a Hypersep C18 100 mg solid phase extraction
(SPE) column (Thermo Scientific Bellefonte PA) Peptides were eluted with 50 ACN in
01 formic acid concentrated and reconstituted in 500 microL of 80 ACN and 01 formic acid
Phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC)
One ml of Ni-NTA was washed three times with 800 microL of H2O and then the Ni was
removed with 800 microL of 100 mM EDTA in 50 mM ammonium bicarbonate and vortexed for 30
minutes The supernatant was removed and the NTA was washed with 800 microL of H2O three
times followed by addition of 800 microL 10 mM FeCl3 hexahydrate and vortexed for 30 minutes
The Fe-NTA was then washed three times with 800 microL of H2O and once with 80 ACN in 01
formic acid before being combined with the cell extract for phosphopeptide enrichment and
vortexed for 40 minutes The sample was washed three times with 200 microL of 80 ACN in 01
formic acid before elution with two aliquots of 200 microL of 5 ammonium hydroxide in 5050
ACNH2O with 15 minutes of vortexing The enriched phosphopeptides were then dried down
with a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA) and reconstituted in 55 microL
25mM ammonium formate pH=75
First dimension neutral pH separation
Individually 50 microL of each yeast phosphopeptide enriched sample was injected onto a
Phenomenex Gemini-NX 3microm C18 110 Aring 150 x 2 mm column (Torrance CA) The Gemini
column was coupled to a Phenomenex SecurityGuard Gemini C18 2 mm guard cartridge
(Torrance CA) Mobile phase A consisted of 25 mM ammonium formate at pH=75 and mobile
phase B consisted of 25 mM ammonium formateACN with a 19 ratio respectively at pH=75
The column was operated at 50oC at a flow rate of 05 mLmin The flow profile was 2-30 B
147
over 65 minutes and then 5 min at 100 B A total of 22 fractions were collected every 3
minutes and the fractions were combined as follows 1 with 12 2 with 13 etc to 11 with 22
The combined fractions were dried down and reconstituted in 25 microL of 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL from each fraction onto an Agilent Technologies
Zorbax 300 SB-C18 5 microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5
microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm Atlantis
dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B at 250
nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) for MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
148
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions An additional mode of MSMS fragmentation electron transfer dissociation
(ETD) was triggered on the precursor ion when a neutral loss was observed in CID
fragmentation from phosphoric acid H3PO4 (Δmz of 49 and 327 for 2+ and 3+ charge states
respectively) or for metaphosphoric acid (HPO3) (Δmz of 40 and 267 for 2+ and 3+ charge
states respectively) For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms rolling average 2 acquisition range of 300-1500 mz
and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target
was set to 300000 maximum accumulation time 5000 ms two spectral averages acquisition
range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required to prevent artificial data
reduction Identification of peptides were performed using Mascot40
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt Saccharomyces
cerevisiae database (version 575) Mascot search parameters were as follows Allowed missed
cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) and phosphorylation (STY) peptide tolerance plusmn12 Da maximum
number of 13
C 1 MSMS tolerance peptide decoy (Mascot) checked plusmn05 Da instrument type
149
ESI-Trap Results from the Mascot search was exported as DAT files for analysis by Scaffold 3
and Scaffold PTM
Scaffold and Ascore data processing
Scaffold 3 (Proteome Software Portland OR)was used for MSMS proteomics data
comparison and false discovery rate analysis The DAT files were loaded into Scaffold 3 and
the fractions for the two dimensional fractionation were combined The resulting biological
triplicates for starved and glucose fed yeast were filtered with a 1 false discovery rate (FDR)
on the protein level (Supplemental Table 1) Due to the inherent poor fragmentation of
phosphopeptides a lenient FDR level may still yield biologically relevant data and thus a 54
FDR was generated for phosphopeptides (Supplemental Table 2) For a more conservative list of
phosphopeptides with a 06 FDR was generated from Scaffold 3 (Supplemental Table 3) FDR
analysis is sufficient at preventing poor data from being reported but does not prevent false
phosphosite identification in phosphopeptides To provide confidence in site identification
Scaffold PTM was used to perform Ascore41
analysis Ascore uses an algorithm to score the
probability of the phosphosite from a phosphopeptide identified by a database searching
algorithm such as Mascot Ascore can be freely accessed at httpascoremedharvardedu
Cell collection RNA isolation and microarray data analysis
All experiments were performed in biological duplicates Cell samples (10 ODU) were
taken at starved and fed states spun at 5000xg for 2 minutes at 30 oC the supernatant was
removed and the pellets snap frozen using liquid nitrogen RNA was isolated using the Epicentre
MasterPure Yeast Purification Kit (Epicentre Technologies) and quality was checked using gel
electrophoresis cRNA synthesis was carried out using the GeneChipreg Expression 3
Amplification One-Cycle Target Labeling and Control Reagents kit (Affymetrix) All
150
experiments followed the manufactures instructions cRNA samples were hybridized to
GeneChip Yeast Genome 20 Arrays for 16 hours Arrays were washed stained and scanned
according the manufactures recommendations Affymetrix CEL files were RMA normalized
with R and the Bioconductor Suite Data analysis was performed within TIGR Multiexperiment
Viewer v451 in-house Perl scripting R and Bioconductor
Results
Sample preparation for shotgun proteomics
As discussed in the introduction the purpose of this study is to provide an exploratory list
of qualitative differences in the phosphoproteome between starved vs glucose fed yeast After
yeast cell lysate production a substantial amount of sample preparation is performed to enhance
the depth of the phosphoproteome coverage A workflow of the steps following cell lysis is
outlined in Figure 1A The yeast proteins are isolated via acetone precipitation followed by
digestion with trypsin to produce peptides Phosphopeptides are intermixed with the entire
tryptic peptide sample and therefore Fe-NTA IMAC is used to enrich the phosphopeptides To
improve upon the number of phosphopeptides we then performed two dimensional separation
with high pH RP followed by low pH RP C18 and infused the eluent directly onto an ion trap
mass spectrometer Figure 1B show an improved technique for the first dimension of separation
to combine the early eluting and late eluting fractions from the first phase of separation to reduce
overall MS analysis time The combination of fractions 1 and 12 2 and 13 etc essentially
improves the duty cycle of the mass spectrometer by ensuring sufficient peptide content is
injected onto a low pH nanoLC RP C18 column
ETD-triggered mass spectrometry
151
In the present study labile phosphorylation can lead to non-informative neutral loss
During MS scanning mode the instrument will choose the 6 most abundant peaks with correct
isotopic distribution and select them for MSMS CID fragmentation During CID fragmentation
it is common for labile PTMs such as phosphorylation to undergo neutral loss and thus limited
informative b and y-type ions are formed Alternatively ETD fragmentation can be used on
specific MSMS scans which produce a neutral loss indicative of phosphorylation (98Daz or
80Daz) The subsequent ETD fragmentation on these putative phosphopeptides will lead to
uniform backbone cleavage resulting in confident identification of phosphopeptides with site-
specific localization during MSMS It is important to note that CID fragmentation still produces
very informative fragmentation for phosphorylation but ETD provides an orthogonal
fragmentation pathway to further increase the phosphoproteome coverage Additionally the
duty cycle for ETD fragmentation is roughly half that of CID therefore about half as many
potential peptides would be fragmented and sequenced if the instrument was using ETD
fragmentation exclusively
Protein Data
Even with phosphopeptide enrichment it is inevitable that non-phosphopeptides will also
be identified All data were searched with Mascot and in total over 1000 proteins were identified
with a 1 FDR rate and are listed in Supplemental Table 1 The proteins listed in Supplemental
Table 1 include both phosphoproteins and non-phosphorylated proteins In addition to the
proteins identified in the fed and starved states the unique peptides and spectral counts are also
listed for reference The phosphopeptides are specifically listed with a 54 FDR rate in
Supplemental Table 2 and comparisons between starved and fed with Ascore analysis are listed
for every phosphopeptide identified A higher confidence of phosphopeptide identification is
152
sometimes required before investing in time consuming biological experiments so a list of
phosphopeptides identified in starved and fed was taken from a setting directing Scaffold to
produce a 05 FDR which resulted in the generated FDR of 06 FDR and listed in
Supplemental Table 3
A summary of specific phosphorylation sites are listed in Table 1 with a 06 FDR and
Table 2 with a 54 FDR Each table gives totals for unique phosphopeptides identified having
an Ascore localization score ge80 without Ascore and phosphorylation events on each unique
peptides As expected the majority of phosphorylation events (over 50) occurred on serine
whereas fewer phosphorylation events (~10) occurred on tyrosine In addition the vast
majority of phosphorylation events were single phosphorylation (ge65) with very few
identifications having more than two phosphosites per peptide For specific phosphopeptide
identification and subsequent phosphoprotein identification refer to Supplemental Table 2 and 3
Discussion
Transcriptional response to glucose feeding
Yeast responds to the repletion of glucose after glucose-depletion by broad
transcriptional changes to induce growth Roughly one-third of the yeast genome is altered by at
least two fold when glucose is introduced as shown in Figure 2 Figure 2 is a heat map from a
microarray analysis showing 1601 genes up regulated and 1457 genes down regulated after
addition of glucose compared to the starved state The arbitrary cut-offs for these values were as
follows two fold induction after nutrient repletion and a false discovery rate (q-value) of 001
Red color in the heat map indicates a particular gene is up-regulated in the fed state compared to
the starved state Alternatively genes coded in green are less expressed in the fed state
compared to the starved condition The intensity of the green or red colors is indicative of the
153
intensity of the fold change in gene expression These large transcriptional changes after glucose
repletion drive and complement the current phosphoproteomic study
PKA motif analysis
One benefit of a large scale phosphoproteomics experiment is to examine the different
phosphopeptides from known motifs for specific kinases PKA and TOR are responsible for the
majority of the transcriptional response and thus PKA is a good target for motif analysis Figure
3 shows the PKA motif sequence xxxRKRKxSxxxxxx with the phosphorylation occurring on
the serine Other than F16P (fructose-16-bisphosphatase) all the proteins listed are unique to the
starved or fed samples A motif sequence will inevitably show up by random chance in any
analysis For this study the control for motif analysis uses the swissprot protein list for the
entire yeast proteome for the background Compared to background this specific PKA kinase
from Figure 3 is up-regulated by 264 fold when compared to the background One interesting
protein emerged from this motif analysis in the fed sample but not the starved sample is
Ssd1which is implicated in the control of the cell cycle in G1 phase42
Ssd1 also is
phosphorylated by Cbk1 and Cbk1 has been proposed to inhibit Ssd143
and provides an
intriguing target for future studies on starved vs glucose fed yeast growth
Localization of the phosphorylation sites
When a phosphopeptide contains any number of serine threonine or tyrosine amino
acids the localization of the phosphosite can sometimes be ambiguous Database searches used
to identify peptides like Mascot do not provide any probability for localization of correct
phosphorylation sites Ascore is an algorithm that uses the same MSMS spectra as Mascot but
instead of comparing theoretical peptide MSMS to experimental spectra Ascore searches for
informative a b or y-type peptide fragments giving evidence for phosphosite The Scaffold
154
program adds a localization probability to the Ascore values and the values are listed in
Supplemental Table 2 and 3 Figure 4 has a representative Ascore mass spectra assigning the
peaks identified and providing evidence that the phosphorylation site occurs at the threonine
instead of the serine Incorporating Ascore into this study provides a layer of validation for
putative phosphosite identification
Plasma Membrane 2-ATPase
A previous study identified and localized phosphorylation sites on plasma membrane 1-
ATPase after glucose was introduced to starved yeast15
In the current study PMA2 (plasma
membrane ATPase 2) was identified in glucose fed and not starved samples The doubly
threonine phosphorylated peptide identified in fed yeast is PMA2 with amino acid sequence
IKMLTGDAVGIAKETCR (583-599) whereas the homologous peptide in PMA1 has the exact
same amino acid sequence except for the first isoleucine substituted for valine
VKMLTGDAVGIAKETCR (554-570 not observed in data) PMA2 was observed in the 06
FDR list of phosphopeptides and has an Ascore localization probably of 100 A plant study
showed that PMA2 phosphorylation level was higher in early growth phase than when in
stationary phase44
In addition PMA2 expression in yeast permits the growth of yeast and
threonine phosphorylation has been reported on Thr-95545
The identification of PMA2 in the
fed glucose cell extract provides an interesting target for future study on the molecular
mechanisms involved in regulation growth in starved vs glucose fed yeast
Conclusion
In conclusion this work provides a qualitative comparison in the phosphoproteome
between starved and glucose fed yeast A large scale IMAC enrichment of yeast cell lysate
followed by 2-D separation provided a rich source of phosphopeptides for neutral loss triggered
155
ETD analysis using mass spectrometry A specific motif for PKA is highlighted to show the
differences in proteins identified between starved vs fed conditions In total 477 unique
phosphopeptides are identified with 06 FDR and 669 unique phosphopeptides identified with
54 FDR Phosphosite validation is performed using a localization algorithm Ascore to
provide further confidence on the site-specific characterization of these phosphopeptides The
proteins Ssd1 and PMA2 emerged as potential intriguing targets for more in-depth studies on
protein phosphorylation involved in glucose response
Supplemental Tables 1 2 and 3 are available upon request
References
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3 Slattery M G Heideman W Coordinated regulation of growth genes in
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14
6 Newcomb L L Diderich J A Slattery M G Heideman W Glucose regulation of
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9 Johnston M Feasting fasting and fermenting Glucose sensing in yeast and other cells
Trends Genet 1999 15 (1) 29-33
156
10 Warner J R The economics of ribosome biosynthesis in yeast Trends Biochem Sci
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11 Li X Gerber S A Rudner A D Beausoleil S A Haas W Villen J Elias J E
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12 Ficarro S B McCleland M L Stukenberg P T Burke D J Ross M M
Shabanowitz J Hunt D F White F M Phosphoproteome analysis by mass spectrometry and
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13 Gruhler A Olsen J V Mohammed S Mortensen P Faergeman N J Mann M
Jensen O N Quantitative phosphoproteomics applied to the yeast pheromone signaling
pathway Mol Cell Proteomics 2005 4 (3) 310-27
14 Peng J Schwartz D Elias J E Thoreen C C Cheng D Marsischky G Roelofs
J Finley D Gygi S P A proteomics approach to understanding protein ubiquitination Nat
Biotechnol 2003 21 (8) 921-6
15 Lecchi S Nelson C J Allen K E Swaney D L Thompson K L Coon J J
Sussman M R Slayman C W Tandem phosphorylation of Ser-911 and Thr-912 at the C
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Chem 2007 282 (49) 35471-81
16 Cohen P The regulation of protein function by multisite phosphorylation--a 25 year
update Trends Biochem Sci 2000 25 (12) 596-601
17 Kalume D E Molina H Pandey A Tackling the phosphoproteome tools and
strategies Curr Opin Chem Biol 2003 7 (1) 64-9
18 Nagaraj N DSouza R C Cox J Olsen J V Mann M Feasibility of large-scale
phosphoproteomics with higher energy collisional dissociation fragmentation J Proteome Res
2010 9 (12) 6786-94
19 Olsen J V Vermeulen M Santamaria A Kumar C Miller M L Jensen L J
Gnad F Cox J Jensen T S Nigg E A Brunak S Mann M Quantitative
phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis Sci
Signal 2010 3 (104) ra3
20 Breitkopf S B Asara J M Determining In Vivo Phosphorylation Sites Using Mass
Spectrometry In Current Protocols in Molecular Biology John Wiley amp Sons Inc 2012
21 Steen H Jebanathirajah J A Rush J Morrice N Kirschner M W Phosphorylation
analysis by mass spectrometry myths facts and the consequences for qualitative and
quantitative measurements Mol Cell Proteomics 2006 5 (1) 172-81
22 Kweon H K Hakansson K Metal oxide-based enrichment combined with gas-phase
ion-electron reactions for improved mass spectrometric characterization of protein
phosphorylation J Proteome Res 2008 7 (2) 749-55
23 Larsen M R Thingholm T E Jensen O N Roepstorff P Jorgensen T J Highly
selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide
microcolumns Mol Cell Proteomics 2005 4 (7) 873-86
24 Kokubu M Ishihama Y Sato T Nagasu T Oda Y Specificity of immobilized
metal affinity-based IMACC18 tip enrichment of phosphopeptides for protein phosphorylation
analysis Anal Chem 2005 77 (16) 5144-54
25 Swaney D L Wenger C D Thomson J A Coon J J Human embryonic stem cell
phosphoproteome revealed by electron transfer dissociation tandem mass spectrometry Proc
Natl Acad Sci U S A 2009 106 (4) 995-1000
157
26 Hao P Guo T Sze S K Simultaneous analysis of proteome phospho- and
glycoproteome of rat kidney tissue with electrostatic repulsion hydrophilic interaction
chromatography PLoS One 2011 6 (2) e16884
27 Rush J Moritz A Lee K A Guo A Goss V L Spek E J Zhang H Zha X
M Polakiewicz R D Comb M J Immunoaffinity profiling of tyrosine phosphorylation in
cancer cells Nat Biotechnol 2005 23 (1) 94-101
28 Ficarro S Chertihin O Westbrook V A White F Jayes F Kalab P Marto J A
Shabanowitz J Herr J C Hunt D F Visconti P E Phosphoproteome analysis of
capacitated human sperm Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3
and valosin-containing proteinp97 during capacitation J Biol Chem 2003 278 (13) 11579-89
29 Washburn M P Wolters D Yates J R 3rd Large-scale analysis of the yeast
proteome by multidimensional protein identification technology Nat Biotechnol 2001 19 (3)
242-7
30 Dowell J A Frost D C Zhang J Li L Comparison of two-dimensional
fractionation techniques for shotgun proteomics Anal Chem 2008 80 (17) 6715-23
31 Song C Ye M Han G Jiang X Wang F Yu Z Chen R Zou H Reversed-
phase-reversed-phase liquid chromatography approach with high orthogonality for
multidimensional separation of phosphopeptides Anal Chem 2010 82 (1) 53-6
32 Palumbo A M Smith S A Kalcic C L Dantus M Stemmer P M Reid G E
Tandem mass spectrometry strategies for phosphoproteome analysis Mass Spectrom Rev 2011
30 (4) 600-25
33 Beausoleil S A Jedrychowski M Schwartz D Elias J E Villen J Li J Cohn M
A Cantley L C Gygi S P Large-scale characterization of HeLa cell nuclear
phosphoproteins Proc Natl Acad Sci U S A 2004 101 (33) 12130-5
34 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and
protein sequence analysis by electron transfer dissociation mass spectrometry Proc Natl Acad
Sci U S A 2004 101 (26) 9528-33
35 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion
dependence in the partitioning between proton and electron transfer in ionion reactions
International Journal of Mass Spectrometry 2004 236 (1acirceuroldquo3) 33-42
36 Hui L Cunningham R Zhang Z Cao W Jia C Li L Discovery and
characterization of the Crustacean hyperglycemic hormone precursor related peptides (CPRP)
and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes sapidus using
multiple tandem mass spectrometry techniques J Proteome Res 2011 10 (9) 4219-29
37 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-
site identity and position on electron-transfer dissociation of polypeptide cations J Am Chem Soc
2007 129 (40) 12232-43
38 Coon J J Collisions or electrons Protein sequence analysis in the 21st century Anal
Chem 2009 81 (9) 3208-15
39 Miller M E Cross F R Distinct subcellular localization patterns contribute to
functional specificity of the Cln2 and Cln3 cyclins of Saccharomyces cerevisiae Mol Cell Biol
2000 20 (2) 542-55
40 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
158
41 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
approach for high-throughput protein phosphorylation analysis and site localization Nat
Biotechnol 2006 24 (10) 1285-92
42 Sutton A Immanuel D Arndt K T The SIT4 protein phosphatase functions in late
G1 for progression into S phase Mol Cell Biol 1991 11 (4) 2133-48
43 Jansen J M Wanless A G Seidel C W Weiss E L Cbk1 regulation of the RNA-
binding protein Ssd1 integrates cell fate with translational control Curr Biol 2009 19 (24)
2114-20
44 Kanczewska J Marco S Vandermeeren C Maudoux O Rigaud J L Boutry M
Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3
proteins converts a dimer into a hexamer Proc Natl Acad Sci U S A 2005 102 (33) 11675-80
45 Maudoux O Batoko H Oecking C Gevaert K Vandekerckhove J Boutry M
Morsomme P A plant plasma membrane H+-ATPase expressed in yeast is activated by
phosphorylation at its penultimate residue and binding of 14-3-3 regulatory proteins in the
absence of fusicoccin J Biol Chem 2000 275 (23) 17762-70
159
Figure 1 The general workflow indicating the major steps involved in sample collection
sample processing mass spectrometric data acquisition and analysis of comparative
phosphoproteomics for starved vs glucose fed yeast (A) The high pH RP separation
procedure for combining fractions to reduce low peptide containing fractions from the
UV-VIS trace is also shown (B)
160
Figure 2 Heat map for the global transcriptional response to glucose fed vs starved samples
S288C cells starved for glucose until growth was arrested and subsequently glucose was added
161
Samples for microarray analysis were taken at the starved state and 60 minutes after glucose was
added The heat map shows the fed log2 fold change for each gene relative to the starved state
across the genome performed in biological replicate (A) Black indicates no change in
expression level while red indicates higher expression for the fed relative to the starved state
Green indicates higher expression for the starved state compared to the fed state (Adapted from
Dr Michael Conways Thesis)
162
Figure 3 The motif for protein kinase A (PKA) that was observed for fed vs starved which is
xxxRKRKxSxxxxxx with the phosphorylation occurring on the serine The motif occurred at a
rate 264 fold higher than the yeast proteome used for background In addition one protein was
observed in both starved and fed with accession identification of F16P (Fructose-16-
bisphosphatase)
163
06 FDR phosphopeptide analysis
Table 1 The number of phosphopeptides identified with a 06 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
Starved Fed All
Ascore ge80 score
unique
STY 164 153 317
S 87 (530) 82 (536) 169 (533)
T 60 (366) 55 (359) 115 (363)
Y 17 (104) 16 (105) 33 (104)
Unique no Ascore
STY 242 235 477
S 131 (541) 133 (566) 264 (553)
T 86 (355) 78 (332) 164 (344)
Y 25 (103) 24 (102) 49 (103)
Phosphorylation events
on each unique peptide
1 102 113 187
2 36 40 68
3 12 11 22
4 or more 8 3 11
164
54 FDR phosphopeptide analysis
Starved Fed All
Ascore ge80 score
unique
STY 217 217 434
S 115 (530) 113 (521) 228 (525)
T 78 (359) 78 (359) 156 (359)
Y 24 (111) 26 (120) 50 (115)
Unique no Ascore
STY 337 332 669
S 193 (573) 180 (542) 373 (558)
T 111 (329) 116 (349) 227 (339)
Y
Phosphorylation events
on each unique peptide
1
2
3
4 or more
33 (98)
135
56
16
11
36 (108)
169
55
14
3
69 (103)
280
100
27
13
Table 2 The number of phosphopeptides identified with a 54 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
165
Figure 4 Ascore validation of the phosphopeptide LSLAtKK from the protein SGM1 (slow
growth on galactose and mannose protein 1) with 100 localization probability observed
in only fed but not starved yeast samples Ascorersquos algorithm identifies a b and y-type
ions and looks to identify peaks that provide evidence for a specific phosphorylation site
For this example Ascore assigns this phosphosite to threonine (T5) instead of the serine
(S2) The intense peaks at 4024 mz and 5250 mz are not identified by any a b or y-
type ions From the ladder sequence of the peptide shown numerous ions indicate the
threonine is phosphorylated while the serine is not Among these ions used for
localization are b2 y2 y5+H2O y3 y4 and y5
166
Chapter 6
Use of electron transfer dissociation for neuropeptide sequencing and
identification
Adapted from ldquoDiscovery and Characterization of the Crustacean Hyperglycemic Hormone
Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue
Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniquesrdquo Hui L
Cunningham R Zhang Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
167
Abstract
The crustacean Callinectes sapidus sinus gland (SG) is a well-defined neuroendocrine site that
produces numerous hemolymph-borne agents including the most complex class of endocrine
signaling moleculesmdashneuropeptides One such peptide is the crustacean hyperglycemic hormone
(CHH) precursor-related peptide (CPRPs) which was not previously sequenced Electron
transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and
high charge state In addition ETD was used to sequence the sulfated peptide Cholecystokinin
CCK-like Homarus americanus using a salt adduct Collectively these two examples
demonstrated the utility of ETD in sequencing neuropeptides with large molecular weight or
with labile modifications
168
Introduction
Neuropeptides are the largest and most diverse group of endocrine signaling molecules in
the nervous system They are necessary and critical for initiation and regulation of numerous
physiological processes such as feeding reproduction and development1 2
Mass spectrometry
(MS) with unique advantages such as high sensitivity high throughput chemical specificity and
the capability of de novo sequencing with limited genomic information is well suited for the
detection and sequencing of neuropeptides in endocrine glands and simultaneously provides the
potential for information on post-translational modifications such as sulfonation3-6
The sinus glands (SG) are located between the medulla interna and medulla externa of the
eyestalk in Callinectes sapidus It is a well-known neuroendocrine site that synthesizes and
secretes peptide hormones regulating various physiological activities such as molting
hemolymph glucose levels integument color changes eye pigment movements and
hydro-mineral balance7 Our labrsquos previous neuropeptidomic studies of SG in several
crustacean species including Cancer borealis8-11
Carcinus maenas12
and Homarus americanus13
14 used multiple MS strategies combining MALDI-based high resolution accurate mass profiling
biochemical derivatization and nanoscale separation coupled to tandem MS for de novo
sequencing In the current study we explore the neuropeptidome of the SG in the blue crab
Callinectes sapidus a vital species of economic importance on the seafood market worldwide In
total 70 neuropeptides are identified including 27 novel neuropeptides and among them the
crustacean hyperglycemic hormone precursor-related peptides (CPRPs) and orcokinins represent
major neuropeptide families known in the SG
The crustacean hyperglycemic hormone (CHH) and its precursor-related peptide (CPRP) are
produced concurrently during the cleavage of CHH from the CHH preprohormone protein15
The
169
CPRP peptide is located between the signal peptide and the CHH sequence and is separated from
the CHH by a dibasic cleavage site The functions of CPRPs are currently unknown16
However
the complete characterization of CPRPs provides a foundation for future experiments elucidating
their functional roles in crustaceans The cDNA of CHH preprohormone in SG of Callinectes
sapidus has been characterized17
but the direct detection of CPRP has not been reported due to
its relatively large size and possible post-translational modifications While small fragments of
CPRPs can be identified using nanoLC-ESI-Q-TOF tandem mass spectrometry the intact
peptide is difficult to detect due to the large molecular weight of CPRPs
Peptidomics traditionally uses collision induced dissociation (CID) for tandem MS
experiments for de novo sequencing Recently an alternative peptide fragmentation method has
been developed using the transfer of an electron termed electron transfer dissociation (ETD)18 19
ETD involves a radical anion with low electron affinity to be transferred to peptide cation which
results in reduced sequence discrimination and thus provides improved sequencing for larger
peptides compared to CID20
Specifically for an ion trap ETD the fluoranthene radical anion is
allowed to react with peptide cations in the three dimensional trap The resulting dissociation
cleaves at the N-Cα bond to produce c and zbull type product ions The peptidome of model
organisms like Callinectes sapidus can be further explored and expanded utilizing ETD as a
complementary fragmentation technique to CID Previous peptidomic analysis has been
completed using ETD as an additional fragmentation method21
It was observed that
enzymatically produced peptides with a higher mz produced improved sequence coverage using
ETD This observation termed decision tree analysis determined that a charge state of ge6 all
peptides endogenous or enzymatic should be fragmented via ETD22
In the present study the
highly charged peptide CPRP with an intact molecular weight of 3837 readily produces plus six
170
charge states The higher charge state of CPRP is highly amenable to fragmentation by ETD
which produces remarkably improved fragmentation and thus increased sequence coverage when
compared to CID
Sulfonation of tyrosine is a post-translational modification (PTM) that is thought to occur on
trans-membrane spanning and secreted proteins23
Cholecystokinin-8 (CCK-8) is a sulfated
peptide which has been linked to satiety24-26
and a CCK-like peptide has been observed in
lobster27
Sulfonation is an extremely labile modification and is often lost during soft
ionization such as ESI or MALDI but can be avoided by optimizing the ionization process28
One potential strategy to identify sulfopeptides is to scan for a neutral loss of 80 Da after CID
but this method does not allow for identification of site of sulfonation and has the risk to be
mistaken for phosphorylation The sulfonation PTM is a negatively charged modification on
the peptide which allows for negative ion scanning in the mass spectrometer but provides
minimal MSMS sequence coverage during CID due to preferential loss of the sulfate group
Alternatively electron-based dissociation technique enables more rapid radical driven
fragmentation where the cleavage pattern is more uniform along the peptide backbone without
initially cleaving the labile sulfated group thus preserving the site of modification These types
of dissociation techniques only work for multiply-charged ions thus a method to install a
multiply-charged cation on the target peptide is desirable It has been shown that the electron
capture dissociation (ECD) can be used to sequence sulfated peptides when a multi-charged
cation is added to the solution29
We use a similar multi-charge cation solution technique to
sequence a sulfated peptide from Homarus americanus via ETD on an ion trap mass
spectrometer Here we presented the use of the ETD fragmentation technique for the analysis
of large peptides and peptides containing labile post-translational modification
171
Experimental Section
Chemical and materials
Acetonitrile methanol magnesium chloride (ACS grade) potassium chloride (ACS grade) and
calcium chloride (ACS grade) were purchased from Fisher Scientific (Pittsburgh PA) Formic
acid was from Sigma-Aldrich (St Louis MO) Human sulfated CCK-8 and CCK-like peptide
(E(pyro)FDEY(Sulfo)GHMRFamide) were purchased from American Peptide (Sunnyvale CA)
Fused-silica capillary with 75 μm id and 360 μm od was purchased from Polymicro
Technologies (Phoenix AZ) Millipore C18 Ziptip column was used for sample cleaning and all
water used in this study was deionized water (182 MΩcm) prepared from a Milli-Q Millipore
system (Billerica MA) The physiological saline consisted of 440 mM NaCl 11mM KCl 26
mM MgCl2 13 mM CaCl2 11 mM Trizma base and 5 mM maleic acid in pH 745
Animals and dissection
Callinectes sapidus (blue crab) were obtained from commercial food market and maintained
without food in artificial sea water at 10-12 ordmC Animals were cold-anesthetized by packing on
ice for 15-30min before dissection The optic ganglia with the SG attached were dissected in
chilled (~4ordmC) physiological saline and individual SGs were isolated from the optic ganglia by
micro-dissection and immediately placed in acidified methanol (90 methanol 9 glacial acetic
acid and 1 water) and stored at -80ordmC until tissue extraction
Tissue homogenization
Acidified methanol was used during the homogenization of SGs The homogenized SGs were
immediately centrifuged at 16100 g using an Eppendorf 5415D tabletop centrifuge (Eppendorf
172
AG) The pellet was washed using acidified methanol and combined with the supernatant and
further dried in a Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation) The
resulting dried sample was re-suspended with a minimum amount (20 microL) of 01 formic acid
Fractionation of homogenates using reversed phase (RP)-HPLC
The re-suspended extracts were then vortexed and briefly centrifuged The resulting supernatants
were subsequently fractionated via high performance liquid chromatography (HPLC) HPLC
separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax
UV-D II absorbance detector (Rainin Instrument Inc Woburn MA) The mobile phases included
Solution A (deionized water containing 01 formic acid) and Solution B (acetonitrile containing
01 formic acid) About 20 μl of extract was injected onto a Macrosphere C18 column (21 mm
id x 250 mm length 5 microm particle size Alltech Assoc Inc Deerfield IL) The separation
consisted of a 120 minute gradient of 5-95 Solution B Fractions were automatically collected
every two minutes using a Rainin Dynamax FC-4 fraction collector (Rainin Instrument Inc
Woburn MA) The fractions were then concentrated using in Savant SC 110 SpeedVac
concentrator (Thermo Electron Corporation) and re-suspended with minimum amount of 01
formic acid
Nano-LC-ESI-Q-TOF MSMS
Nanoscale LC-ESI-Q-TOF MSMS was performed using a Waters nanoAcquity UPLC system
coupled to a Q-TOF Micro mass spectrometer (Waters Corp Milford MA)
Chromatographic separations were performed on a homemade C18 reversed phase capillary
column (75 microm internal diameter x100 mm length 3 microm particle size 100 Aring) The mobile phases
173
used were 01 formic acid in deionized water (A) 01 formic acid in acetonitrile (B) An
aliquot of 50 microl of a tissue extract or HPLC fraction was injected and loaded onto the trap
column (Zorbax 300SB-C18 Nano trapping column Agilent Technologies Santa Clara CA)
using 95 mobile phase A and 5 mobile phase Bat a flow rate of 10 microlmin for 10 minutes
Following this the stream select module was switched to a position at which the trap column
came in line with the analytical capillary column and a linear gradient of mobile phases A and B
was initiated For neuropeptides the linear gradient was from 5 buffer B to 45 buffer B over
90 min The nanoflow ESI source conditions were set as follows capillary voltage 3200 V
sample cone voltage 35 V extraction cone voltage 1 V source temperature 100ordmC A data
dependent acquisition was employed for the MS survey scan and the selection of three precursor
ions and subsequent MSMS of the selected parent ions The MS scan range was from mz
400-1800 and the MSMS scan was from mz 50-1800
Peptide Prediction De Novo Sequencing and Database Searching
De novo sequencing was performed using a combination of MassLynxTM
41 PepSeq software
(Waters) and manual sequencing Tandem mass spectra acquired from the Q-TOF were first
deconvoluted using MaxEnt 3 software (Waters) to convert multiply charged ions into their
singly charged forms The resulting spectra were pasted into the PepSeq window for sequencing
analysis The candidate sequences generated by the PepSeq software were compared and
evaluated for homology with previous known peptides The online program blastp (National
Center for Biotechnology Information Bethesda MD httpwwwncbinlmnihgovBLAST)
was used to search the existing NCBI crustacean protein database using the candidate peptide
sequences as queries For all searches the blastp database was set to non-redundant protein
174
sequences (ie nr) and restricted to crustacean sequences (ie taxid 6657) For each of the
proteins putatively identified via blastp the BLAST score and BLAST-generated E-value for
significant alignment are provided in the appropriate subsection of the results Peptides with
partial sequence homology were selected for further examination by comparing theoretical
MSMS fragmentation spectra generated by PepSeq with the raw MSMS spectra If the
fragmentation patterns did not match well manual sequencing was performed
NanoLC Coupled to MSMS by CID and ETD
Setup for RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN (Fisher Scientific Optima LCMS grade Fair Lawn NJ) Injections
consisted of 5 microL of prepared tissue extract onto an Agilent Technologies Zorbax 300 SB-C18 5
microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5 microLmin for 5 minutes at 95
A5 B Separation was performed on a Waters HPLC column with 3 microm Atlantis dC18 75 microm
x 150 mm (Milford MA) on a gradient from 5 to 45 mobile phase B at 250 nLmin over 90
minutes at room temperature Emitter tips were pulled from 75 microm inner diameter 360 microm
outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using a commercial
laser puller model P-2000 (Sutter Instrument Co Novato CA)
Mass Spectrometry Data Acquisitions for Collision Induced Dissociation (CID)
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany) equipped
with an on-line nanospray source was used for mass spectrometry data acquisition Hystar
(Version 32 Bruker Daltonics Bremen Germany) was used to couple and control Eksigent
175
nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for all
experiments Smart parameter setting (SPS) was set to mz 700 compound stability and trap
drive level were set at 100 Optimization of the nanospray source resulted in dry gas
temperature 125oC dry gas 40 Lmin capillary voltage -1300 V end plate offset -500 V
MSMS fragmentation amplitude 10V and SmartFragmentation set at 30-300
Data was generated in data dependent mode with strict active exclusion set after two spectra and
released after one minute MSMS was obtained via CID fragmentation for the six most
abundant MS peaks with a preference for doubly charged ions and excluded singly charged ions
For MS generation the ion charge control (ICC) target was set to 200000 maximum
accumulation time 5000 ms four spectral average acquisition range of mz 300-1500 and scan
speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target was set to
200000 maximum accumulation time 5000 ms three spectral averages acquisition range of
mz 100-2000 and scan speed (Ultrascan) of 32000 mz s-1
Mass Spectrometry Data Acquisitions for Electron Transfer Dissociation (ETD)
The amaZon ETD ion-trap mass spectrometer was operated in electron transfer dissociation for
MSMS fragmentation with the same optimized settings as reported for CID unless otherwise
stated Smart parameter setting (SPS) was set to 500 mz compound stability and trap drive
level were set at 100 MSMS was obtained via ETD fragmentation for the four most
abundant MS peaks with no preference for specifically charged ions except to exclude singly
charged ions The ETD reagent parameters were set to 400000 target ICC for the fluoranthene
radical anion The fluoranthene radical anion has an mz of 210 and therefore the 210 mz value
was removed from the resulting MSMS spectra and 160 mz was set for the MSMS low mz
cut-off
176
Direct Infusion of Sulfated Peptide Standards into the Mass Spectrometer using ETD and
CID Fragmentation
The sulfated peptide standards were infused into the mass spectrometer at a flow rate of 300
nlmin using a fused-silica capillary with 75 μm id and 360 μm od coupled to a custom pulled
tip The CCK-8 human peptide (20 microM) was infused in 5050 ACNH2O and detected in
negative ionization mode with an ICC of 70000 and fragmented with CID using the same
settings as the previous CID experiments Alternatively the CCK-like lobster sulfated peptide
(2 microM) was mixed with 30 microM of different salts (MgCl2 KCl or CaCl2) and 01 formic acid in
5050 ACNH2O The plus three charge of the CCK-like lobster peptide was selected for ETD
fragmentation in positive mode with the same setting as the previous ETD experiments The
data were then de novo sequenced for coverage and localization of the sulfation site
Data Analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen Germany)
Deviations in parameters from the default Protein Analysis in DataAnalysis were as follows
intensity threshold 1000 maximum number of compounds 1E7 and retention time window 05
minutes These parameter changes assisted in providing better quality spectra for sequencing
Identification of peptides was performed using Mascot (Version 23 Matrix Science London
UK) Searches were performed against a custom crustacean database none enzyme allow up to
1 missed cleavage amidated C-terminal as variable modifications peptide tolerance mass error
12 Da MSMS mass error tolerance is 06 Da
Results and Discussion
177
Identification and Characterization of Intact CPRPs Using ETD
Using ESI-Q-TOF 9 truncated CPRPs fragments were sequenced to cover the amino acid
sequence of the intact CPRP peptide RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE
A representative CID MSMS spectrum of ASLKSDTVTPLR is shown in Figure 1 (a) CID
using ESI-Q-TOF mainly produces fragmentation for doubly and triply charge molecules which
is adequate to sequence peptides with relatively small molecular weight (less than 2000 Da)
However CID fragmentation is insufficient to sequence the larger intact CPRP in a complex
sample whose molecular mass is 3837 Da and therefore it is extremely difficult to directly
sequence by traditional CID ESI-Q-TOF ETD is an attractive fragmentation alternative to
sequence the highly charged CPRP peptide In ETD a protonated peptide reacts with an anion
(fluoranthene radical) where the anion transfers an electron to the peptide cation and the resulting
fragmentation occurs along the N-Cα bond by free-radical-driven-cleavage The large size of
CPRP offers multiple basic amino acid sites to sequester charge and reduce the sequence
coverage from collision induced dissociate by preventing random backbone cleavage whereas
ETD does not cleave the weakest bond and provides the alternative fragmentation pathway to
obtain superior sequence coverage to larger peptides As shown in Figure 1 (cd) the
fragmentation of ETD is highly amenable to ETD compared to the CID fragmentation in Figure
1 (b) As evident from comparison ETD offers more extensive fragmentation than CID thus
providing enhanced coverage for large intact peptides such as CPRPs Specifically we observe
125 of the b- and y- cleavages for CID as compared to an average of 535 of c- and zbull-
fragments More than a four-fold increase in fragments using ETD suggests the utility of this
relatively new tandem MS fragmentation method as complementary techniques for de novo
sequencing of large intact endogenous peptides and novel neuropeptide discovery endeavors
178
Negative Mode Sulfated Peptide Identification
An accepted method for identification and quantification for sulfated peptides is negative
ionization30
CCK-8 sulfated peptide standards show intense signal in negative ionization mode
without needing to have additives added such as salts Figure 2 shows the CID MSMS
spectrum from the negative ionization and produces an intense b6 ion at 430 mz The transition
from the doubly negatively charged MS ion of 570 mz to the b6 ion provides a selected reaction
monitoring transition for quantification studies but the sequence information is limited and the
presence of the methionine produces variable oxidation In addition Figure 2 shows the
MSMS product ions with the loss of the sulfate group thus making site-specific location of
sulfation more difficult
Investigation of Cation Salt Adducts to Produce Multiply Charged Sulfated Peptides
Sulfated peptides can be isolated in positive scanning mode but usually only in a plus one
state with low signal intensity If ETD is performed on the singly charged peptide cation a
neutral is formed and is lost in the mass spectrometer and thus no sequence information can be
obtained In order to remedy this situation a technique that adding metal salts to peptides to
increase charge state for ECD used in Fourier transform ion cyclotron resonance mass
spectrometry (FTICR-MS)29
inspired the investigation of increasing charge state of targeted
peptide via metal salt adduction followed by ETD analysis in a Bruker amaZon ion trap
Various salts were investigated including MgCl2 CaCl2 and KCl each used at a concentration of
30 microM and mixed with a sulfated peptide standard at 2 microM The addition of KCl produced
mostly doubly charged ion of the lobster CCK-like peptide standard K adduct which produced
insufficient sequence information from ETD fragmentation (data not shown) In comparison
the MgCl2 and CaCl2 produced intense triply charged peptide ions but CaCl2 produced lower
179
signal intensity compared to MgCl2 (data not shown)
Cation Assisted ETD Fragmentation of CCK-like Lobster Sulfated Peptide and Future
Directions
The addition of MgCl2 to the lobster CCK-like sulfated peptide is shown in Figure 3
Except for z1 the complete z-series is obtained including the z7 ion with and without the
sulfation PTM The spectrum is complicated by the additional Mg adducts but several peaks
are shown without any Mg adduct such as z2 z3 z4 etc Clearly the method of cation
assisted ETD fragmentation can be useful using ion trap instruments and can properly sequence
sulfated peptides that are prone to neutral loss from the labile PTM One concern about future
direction in sulfopeptide isolation in non-genome sequenced species is isolation of sulfopeptides
Currently antibodies to specific sulfopeptides is the only commonly used enrichment technique
for sulfopeptides Also since metal cations are needed for this method direct infusion into an
ESI mass spectrometer is preferred to prevent running large amounts of adduct forming salts
through the LC system With direct infusion the lack of separation confounds the problem in
sulfopeptide detection because any co-eluting peptides could have ionization efficiency and thus
reduce the signal of the sulfopeptide Once discovered and sequenced a selected reaction
monitoring (SRM) method could be developed using LC retention coupled with negative
ionization mode followed by CID MSMS or possibly MSMSMS to perform quantitative
studies for sulfopeptides
Conclusions
In this study ETD was performed to improve the sequence coverage of large endogenous
neuropeptides with higher charge and bigger size The intact CPRP from Callinectes sapidus was
identified and characterized with 68 sequence coverage by MS for the first time Cation
180
assisted ETD fragmentation was also shown to be a powerful tool in de novo sequencing of
sulfated neuropeptides These endeavors into using ETD for certain neuropeptides will assist in
future analysis of large neuropeptides and PTM containing neuropeptides
181
Reference
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food intake Nature 2000 404 (6778) 661-71
2 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R
Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide
family of aplysia J Neurosci 2002 22 (17) 7797-808
3 Baggerman G Cerstiaens A De Loof A Schoofs L Peptidomics of the larval Drosophila melanogaster
central nervous system Journal of Biological Chemistry 2002 277 (43) 40368-40374
4 Desiderio D M Mass spectrometric analysis of neuropeptidergic systems in the human pituitary and
cerebrospinal fluid Journal of Chromatography B 1999 731 (1) 3-22
5 Li L J Kelley W P Billimoria C P Christie A E Pulver S R Sweedler J V Marder E Mass
spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab Cancer
borealis Journal of Neurochemistry 2003 87 (3) 642-656
6 Li L J Moroz T P Garden R W Floyd P D Weiss K R Sweedler J V Mass spectrometric survey of
interganglionically transported peptides in Aplysia Peptides 1998 19 (8) 1425-1433
7 Strand F L Neuropeptides regulators of physiological processes 1st ed ed MIT Press Cambridge Mass
1999 p 658 p
8 Fu Q Goy M F Li L J Identification of neuropeptides from the decapod crustacean sinus glands using
nanoscale liquid chromatography tandem mass spectrometry Biochemical and Biophysical Research
Communications 2005 337 (3) 765-778
9 Fu Q Christie A E Li L J Mass spectrometric characterization of crustacean hyperglycemic hormone
precursor-related peptides (CPRPs) from the sinus gland of the crab Cancer productus Peptides 2005 26 (11)
2137-2150
10 Ma M M Chen R B Ge Y He H Marshall A G Li L J Combining Bottom-Up and Top-Down Mass
Spectrometric Strategies for De Novo Sequencing of the Crustacean Hyperglycemic Hormone from Cancer borealis
Analytical Chemistry 2009 81 (1) 240-247
11 Ma M M Sturm R M Kutz-Naber K K Fu Q Li L J Immunoaffinity-based mass spectrometric
characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis Biochemical
and Biophysical Research Communications 2009 390 (2) 325-330
12 Ma M M Bors E K Dickinson E S Kwiatkowski M A Sousa G L Henry R P Smith C M Towle
D W Christie A E Li L J Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and
functional genomics General and Comparative Endocrinology 2009 161 (3) 320-334
13 Chen R B Jiang X Y Conaway M C P Mohtashemi I Hui L M Viner R Li L J Mass Spectral
Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus
Journal of Proteome Research 2010 9 (2) 818-832
14 Ma M M Chen R B Sousa G L Bors E K Kwiatkowski M A Goiney C C Goy M F Christie A
E Li L J Mass spectral characterization of peptide transmittershormones in the nervous system and
neuroendocrine organs of the American lobster Homarus americanus General and Comparative Endocrinology
2008 156 (2) 395-409
15 Ollivaux C Gallois D Amiche M Boscameric M Soyez D Molecular and cellular specificity of
post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family FEBS J 2009 276
(17) 4790-802
16 Wilcockson D C Chung J S Webster S G Is crustacean hyperglycaemic hormone precursor-related
peptide a circulating neurohormone in crabs Cell and Tissue Research 2002 307 (1) 129-138
17 Choi C Y Zheng J Watson R D Molecular cloning of a cDNA encoding a crustacean hyperglycemic
hormone from eyestalk ganglia of the blue crab Callinectes sapidus General and Comparative Endocrinology 2006
148 (3) 383-387
18 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and protein sequence analysis
by electron transfer dissociation mass spectrometry Proc Natl Acad Sci U S A 2004 101 (26) 9528-33
19 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion dependence in the partitioning
between proton and electron transfer in ionion reactions International Journal of Mass Spectrometry 2004 236
(1-3) 33-42
20 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-site identity and
position on electron-transfer dissociation of polypeptide cations J Am Chem Soc 2007 129 (40) 12232-43
182
21 Altelaar A F Mohammed S Brans M A Adan R A Heck A J Improved identification of endogenous
peptides from murine nervous tissue by multiplexed peptide extraction methods and multiplexed mass spectrometric
analysis J Proteome Res 2009 8 (2) 870-6
22 Swaney D L McAlister G C Coon J J Decision tree-driven tandem mass spectrometry for shotgun
proteomics Nat Methods 2008 5 (11) 959-64
23 Monigatti F Hekking B Steen H Protein sulfation analysis--A primer Biochim Biophys Acta 2006 1764
(12) 1904-13
24 Chandler P C Wauford P K Oswald K D Maldonado C R Hagan M M Change in CCK-8 response
after diet-induced obesity and MC34-receptor blockade Peptides 2004 25 (2) 299-306
25 Little T J Feltrin K L Horowitz M Meyer J H Wishart J Chapman I M Feinle-Bisset C A
high-fat diet raises fasting plasma CCK but does not affect upper gut motility PYY and ghrelin or energy intake
during CCK-8 infusion in lean men Am J Physiol Regul Integr Comp Physiol 2008 294 (1) R45-51
26 Blevins J E Morton G J Williams D L Caldwell D W Bastian L S Wisse B E Schwartz M W
Baskin D G Forebrain melanocortin signaling enhances the hindbrain satiety response to CCK-8 Am J Physiol
Regul Integr Comp Physiol 2009 296 (3) R476-84
27 Turrigiano G G Selverston A I A cholecystokinin-like hormone activates a feeding-related neural circuit in
lobster Nature 1990 344 (6269) 866-8
28 Wolfender J L Chu F Ball H Wolfender F Fainzilber M Baldwin M A Burlingame A L
Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB further investigation
of labile sulfo- and phosphopeptides by electrospray matrix-assisted laser desorptionionization (MALDI) and
atmospheric pressure MALDI mass spectrometry J Mass Spectrom 1999 34 (4) 447-54
29 Liu H Hakansson K Electron capture dissociation of tyrosine O-sulfated peptides complexed with divalent
metal cations Anal Chem 2006 78 (21) 7570-6
30 Young S A Julka S Bartley G Gilbert J R Wendelburg B M Hung S C Anderson W H
Yokoyama W H Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using
immunoprecipitation liquid chromatography-mass spectrometrymass spectrometry Anal Chem 2009 81 (21)
9120-8
183
Figure 1 Comparison of tandem MS spectra of truncated CPRP (ASLKSDTVTPL mz 128766)
by ESI-Q-TOF (a) and intact CPRP (MW 3837) by the amaZon ETD for both CID and ETD
fragmentation (a) MSn of precursor ion with mz 640 with charge state +6 by CID represent
loss of NH3 ordm represent loss of H2O (b) MS+6
of precursor ion with mz 640 with charge state +6
by ETD at z represent z+1 z represent z+2 (c) MS+5
of precursor ion with mz 768 with charge
state +5 by ETD z represent z+1 z represent z+2 For all labels in Figure 1 charge state +1 is
not specified
184
185
Figure 2 Negative CID spectrum of CCK-8 peptide standard (20 microM) via direct infusion show
the doubly charged b6 ion provides the most intense MSMS transition
186
Figure 3 ETD spectrum of E(pyro)FDEY(Sulfo)GHMRF-amide (2 microM) obtained on the
amaZon ETD via direct infusion with 30 microM MgCl2 The sulfated peptide can be identified
with a visible z-series of z2 to z9 and identified sulfate loss
187
Chapter 7
Investigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysis
Adapted from ldquoInvestigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J
Wellner D Li L Journal of Mass Spectrometry In Press
188
ABSTRACT
This work investigates the introduction of methanol and a salt modifier to molecular
weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide
quantities Using a neuropeptide standard bradykinin sample loss was reduced over two orders
of magnitude with and without undigested protein present Additionally a bovine serum
albumin (BSA) tryptic digestion was investigated Twenty-seven tryptic peptides were identified
from MALDI mass spectra after enriching with methanol while only two tryptic peptides were
identified after the standard MWCO protocol The strategy presented here enhances recovery
from MWCO separation for sub-microg peptide samples
INTRODUCTION
Molecular weight cut-off membrane-based centrifugal filter devices (MWCO) are
commonly used to desalt and concentrate large molecular weight proteins [1] Greeing and
Simpson recently investigated various MWCO membranes for large amounts of starting material
(~6 mg) focusing on optimal conditions for the sub 25 kDa protein fraction [2] The authors
recovered 200 μg to 29 mg of protein from multiple MWCO experiments and demonstrated that
a 1090 acetonitrile (ACN)H2O elution solvent produced optimal results [3] In addition Manza
et al provided an alternative approach to isolate proteins with a 5 kDa MWCO by using
NH4HCO3 and recovering the retained proteins [4] Alternatively the elution from MWCOs can
be collected to recover only low molecular weight peptides Multiple peptidomic studies have
utilized MWCOs for peptide isolation during the first few steps of sample preparation [5 6]
When sample amount is limited or peptide content is below 1 microg sample loss is a significant
concern when using MWCOs to isolate endogenous peptides Optimized protocols have been
189
investigated using ACN [3 7] salt [4 8] SDS [5] or native sample [6 9 10] but these
experiments primarily focused on large sample amounts rather than sub-microgram peptide
quantities
MWCOs separate large molecules from small molecules The small molecule fraction
may be rich with signaling peptides (SP) cytokines and other small molecules involved in cell-
cell signaling Signaling peptides perform various functions in the body including cell growth
cell survival and hormonal signaling between organs [11] Individual SP contribute to different
aspects of behavior such as pain (enkephalins) [12] feeding (neuropeptide Y) [13] and blood
pressure (bradykinin) [14] MWCO separations can be used to enrich biologically important SP
and explore the peptide content from biological fluids with relatively low peptide content like
blood or cerebrospinal fluid (CSF) In a recent investigation the detection of neuropeptides and
standards in crustacean hemolymph was improved when methanol and protease inhibitors were
present before performing MWCO neuropeptide isolation The impact of methanol on MWCO
sample loss was not investigated in the study [15] In another study a large-scale mass
fingerprinting protocol of endogenous peptides from CSF used a combination of salts before
MWCO fractionation but the impact of adding salts was not discussed [16] The most
commonly used brand of MWCO in the publications and in peptidomic studies is Millipore
Therefore Millipore MWCOs (using regenerated cellulose as the membrane) were used in the
present study The purpose of this work is to provide an optimized sample preparation technique
for MWCO filtering to reduce sample loss and allow sub-microg detection of peptides using MALDI
mass spectrometry
MATERIALS AND METHODS
190
Materials and Chemicals
Water acetonitrile methanol (optima LCMS grade) and sodium chloride (995) were
purchased from Fisher Scientific (Fair Lawn NJ) The α-cyano-4-hydroxy-cinnamic acid (99)
formic acid (FA) (ge98) and bovine serum albumin (ge96) were purchased from Sigma-
Aldrich (St Louis MO) Amicon Ultra 05 mL 10000 MWCO centrifugal filters and ZipTips
packed with C18 reversed-phase resin were purchased from Millipore (Billerica MA) Trypsin-
digested bovine serum albumin (BSA) was purchased from Waters (Milford MA) Bradykinin
was purchased from American Peptide Company (Sunnyvale CA)
MALDI MS Instrumentation
An AutoFLEX III MALDI TOFTOF mass spectrometer (Bruker Daltonics Billerica
MA) was operated in positive ion reflectron mode The MALDI MS instrument is equipped with
a proprietary smart beam (Bruker Daltonics Billerica MA) laser operating at 200 Hz The
instrument was internally calibrated over the mass range of mz 500minus2500 using a standard
peptide mix Two thousand laser shots were collected per sample spot at an accelerating voltage
of 19 kV and a constant laser power using random shot selection The acquired data were
analyzed using FlexAnalysis software (Bruker Daltonics Billerica MA) Mass spectrometry
data acquisition was obtained by averaging 2000 laser shots
Molecular weight cut off separation procedure
The MWCO separations were performed using Amicon Ultra 05 mL 10000 MWCO
centrifugal filters (Billerica MA) Before MWCO separation three washing steps were
performed to remove contaminants from the filter The three washes were 500 μL of 5050
H2OMeOH 500 μL of H2O and 400 μL of the solution used for MWCO separation For the
191
100 H2O solution 1 microg of BSA or bradykinin was used for separation All the other MWCO
separation experiments used 500 ng of BSA or 100 ng or less of bradykinin The MWCO filter
was then centrifuged at 14000 rcf for 5 min at room temperature in an Eppendorf 5415 D
microcentrifuge (Brinkmann Instruments Inc Westbury NY) The filtrate was concentrated in a
Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation West Palm Beach FL)
and acidified The resulting sample was desalted according to the manufacturer using C18
ZipTips from Millipore (Billerica MA) by washing the ZipTip with three times 100 ACN
three aqueous washes of 01 FA binding the peptides from the solution and one aqueous wash
of 01 FA Peptides were then eluted from the ZipTips using 15 microL of 50 ACN in 01 FA
Matrix deposition
Equal volumes of 05 μL from the 15 μL sample solution (including standards not subject
to MWCO filtering) and α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution in 50
ACN50 H2O were mixed using a dried-droplet method and spotted on a MALDI target The
resulting droplets were allowed to air dry prior to mass spectrometry acquisition
RESULTS AND DISCUSSION
Analysis of two orders of magnitude increase for bradykinin standard
Bradykinin was selected to assess the potential peptide loss in the flow-through after
performing MWCO separation As shown in Figure 1 1 microg of bradykinin standard does not
produce a detectable signal by MALDI mass spectrometry analysis after a 10 kDa MWCO
separation in water (performed in triplicate) For comparison 1 ng of bradykinin standard
diluted to 15 microL produced an intense signal on the MALDI mass spectrometer suggesting
192
significant sample loss occurs when the target analyte is low in quantity (data not shown
performed in triplicate) Figure 1 shows that the addition of a salt in this case NaCl improves
the limits of detection and decreases sample loss when 7030 watermethanol was compared to
7030 aqueous 1 M NaClmethanol The reproducible results gave a relative standard deviation
(RSD) of 6 for peak intensity Figure 1 shows that even when starting with 1 μg of bradykinin
too much sample is lost during the MWCO separation in water to detect the remainder
However an intense signal is observed for 10 ng of bradykinin after the MWCO separation when
7030 aqueous 1 M NaClmethanol is used as an elution solvent Sample loss from zip-tipping
was estimated in triplicate by calculating the decreases in peak intensity When 10 ng of
bradykinin was used sample loss was ~41 In comparison the calculated yield for 10 ng of
bradykinin after the 7030 aqueous 1 M NaClmethanol MWCO separation and zip-tip desalting
showed an estimated sample loss of 63 meaning more loss can be attributed to sample clean-
up than MWCO filtration
A series of experiments were performed to determine if 7030 aqueous 1 M
NaClmethanol is an optimal solution to recover peptides during a MWCO separation (data not
shown) A 5050 aqueous 1 M NaClmethanol and a 5050 watermethanol elution were
performed in duplicate but signal intensity of the resulting bradykinin was poor and numerous
polymer peaks were detected in the flow through A lower salt concentration 01 M NaCl was
used but also produced greatly reduced signal when compared to aqueous 1 M NaCl To assess
the optimized methodrsquos compatibility with lower sample amounts the 7030 aqueous 1 M
NaClmethanol solution was added to 1 ng of bradykinin for MWCO separation but no signal
was obtained (data not shown) Using a neuropeptide standard the addition of methanol and
NaCl salt significantly improved the sample recovery in sub-microg amounts
193
BSA tryptic peptide mixture analysis
After demonstrating the importance of using an optimized solution for MWCO
separations with an individual peptide the new method was applied to 500 ng of BSA tryptic
digest to investigate its utility with more complex peptide mixtures Table 1 lists the BSA
tryptic peptides identified in the MALDI MS analysis from different solution conditions
processed by MWCO separation As shown in Table 1 a directly spotted BSA tryptic peptide
standard in the absence of any MWCO filtration enabled identification of 39 tryptic peptides by
accurate peptide mass measurements Once again when using 100 H2O for MWCO
separations the starting amount was doubled to 1 microg (also done with 500 ng data not shown)
However many tryptic peptides were not detected due to low signal intensities and non-optimal
elution conditions Instead of H2O a 1 M NaCl solution was used for the MWCO elution but
only two tryptic peptides were identified (Table 1) The addition of 30 methanol into the
sample before MWCO filtration produced the first increase in identified BSA tryptic peptides
The remaining data from Table 1 shows improved BSA tryptic peptide identifications as the
sample (elution) conditions were further optimized Figure 2 shows the actual mass spectra
associated with the three most promising elution solutions along with 100 H2O
The BSA tryptic peptide intensities are shown in Figure 2A and the most intense tryptic
peptide YLYEIAR mz 92749 was observed in the four different solutions shown in Figure 2B
but not in the 100 H2O or 100 1 M NaCl solutions (data not shown) In Figure 2A all mass
spectra are normalized to an intensity of 7 x 104 to illustrate two points First the MWCO
filtering step still produced sample loss regardless of the solvent conditions chosen Second
there is a noticeable increase in peptide peak intensity using the optimized solvent 6040
194
aqueous 1 M NaClmethanol (Figure 2A) Figure 2C displays a zoomed-in view of a BSA
tryptic peptide signal LKECC
DKPLLEK mz 153266 (
carbamidomethyl) observed only in
the optimized solvent The detection of the mz 153266 peptide in Figure 2C highlights the
potential gain in sample and detectable peptides by using an optimized saltMeOH combination
A non-optimized saltMeOH combination will still reduce sample loss but further minimizing
sample loss during sample preparation will always be desirable in any analytical protocol
MWCO composition
The purpose of this application note is to provide evidence of sub-microg sample loss during
MWCO separations of peptide samples and a solution to overcome this limitation The
explanation of why adding MeOH and NaCl to the sample solution provides a significant
reduction in sample loss is beyond the scope of this application note Regardless Supplemental
Table S1 is an expanded version of Table 1 showing the amino acid sequence hydrophobicity
calculated using GRAVY scores and pI of the identified peptides in this study No discernible
trend was obtained from the data The membrane of commonly used MWCO in peptidomics and
for this study is comprised of chemically treated (regenerated) cellulose which is a
polysaccharide containing β (1rarr4) linked D-glucose Glucose has numerous free hydroxyl
groups which could non-specifically adsorb peptides flowing through the MWCO The addition
of MeOH has the most significant effect on signal which could be due to disrupting the
interaction between peptides and hydroxyl groups from glucose NaCl has a less significant
effect on sample recovery compared to MeOH but a detectable reduction in sample loss is noted
This improvement in sample recovery could be analogous to the use of NaCl in
195
immunodepletion protocols to reduce non-specific binding which is accomplished by adding
150 mM NaCl [17]
Analysis of bradykinin in the presence of undigested BSA
When using MWCO for peptide isolation proteins are typically present in the samples
usually in larger amounts Figure 3 shows the effect that adding BSA protein to a 10 ng
bradykinin solution before MWCO fractionation has on the resulting recovery of bradykinin
Adding 10 μg of BSA to the optimized 7030 aqueous 1 M NaClmethanol solution slightly
decreased bradykininrsquos signal with a RSD of 10 More severe signal reduction occurred after
adding 100 microg BSA with a RSD of 2 (N=2) It is not unexpected that more signal reduction
due to sample loss would occur especially in the 100 microg BSA sample because the BSA protein
has a molar ratio of 160 BSA protein molecules to one bradykinin peptide Figure 3 shows the
usefulness of the MWCO method with samples containing large amounts of proteins
RecommendationConclusion
The present work provides solutions to reduce sample loss from the use of MWCO for
sub-microg peptide isolation with or without non-digested proteins in the sample Despite its
widespread utility significant sample loss often occurs during the MWCO fractionation step
which is particularly problematic when analyzing low-abundance peptides from limited starting
material This application note aims to reduce sample loss during MWCO separations
specifically for sub-microg peptide isolation If complex samples are processed with MWCO
separation the authors recommend eluting the sample with 6040 aqueous 1 M NaClmethanol
solution as a starting point to minimize sample loss This application note provides a viable
196
alternative for sub-microg peptide MWCO separation circumventing the need to increase the starting
material by minimizing sample loss from using a MWCO membrane-based centrifugal filter
device
References
[1] HM Georgiou GE Rice MS Baker Proteomic analysis of human plasma failure of
centrifugal ultrafiltration to remove albumin and other high molecular weight proteins
Proteomics 2001 1 1503
[2] DW Greening RJ Simpson Low-molecular weight plasma proteome analysis using
centrifugal ultrafiltration Methods Mol Biol 2011 278 109
[3] DW Greening RJ Simpson A centrifugal ultrafiltration strategy for isolating the low-
molecular weight (ltor=25K) component of human plasma proteome J Proteomics 2010 73
637
[4] LL Manza SL Stamer AJ Ham SG Codreanu DC Liebler Sample preparation and
digestion for proteomic analyses using spin filters Proteomics 2005 5 1742
[5] D Theodorescu D Fliser S Wittke H Mischak R Krebs M Walden M Ross E Eltze O
Bettendorf C Wulfing A Semjonow Pilot study of capillary electrophoresis coupled to mass
spectrometry as a tool to define potential prostate cancer biomarkers in urine Electrophoresis
2005 26 2797
[6] K Antwi G Hostetter MJ Demeure BA Katchman GA Decker Y Ruiz TD Sielaff LJ
Koep DF Lake Analysis of the plasma peptidome from pancreas cancer patients connects a
peptide in plasma to overexpression of the parent protein in tumors J Proteome Res 2009 8
4722
[7] LP Aristoteli MP Molloy MS Baker Evaluation of endogenous plasma peptide extraction
methods for mass spectrometric biomarker discovery J Proteome Res 2007 6 571
[8] A Zougman B Pilch A Podtelejnikov M Kiehntopf C Schnabel C Kumar M Mann
Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7
386
[9] X Yuan DM Desiderio Human cerebrospinal fluid peptidomics J Mass Spectrom 2005 40
176
[10] X Zheng H Baker WS Hancock Analysis of the low molecular weight serum peptidome
using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer J Chromatogr A
2006 1120 173
[11] L Li JV Sweedler Peptides in the brain mass spectrometry-based measurement approaches
and challenges Annu Rev Anal Chem 2008 1 451
[12] GB Stefano G Fricchione Y Goumon T Esch Pain immunity opiate and opioid
compounds and health Med Sci Monit 2005 11 MS47
[13] J Jensen Regulatory peptides and control of food intake in non-mammalian vertebrates Comp
Biochem Physiol A Mol Integr Physiol 2001 128 471
197
[14] A Kuoppala KA Lindstedt J Saarinen PT Kovanen JO Kokkonen Inactivation of
bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma Am
J Physiol Heart Circ Physiol 2000 278 H1069
[15] R Chen M Ma L Hui J Zhang L Li Measurement of neuropeptides in crustacean
hemolymph via MALDI mass spectrometry J Am Soc Mass Spectrom 2009 20 708
[16] H Jahn S Wittke P Zurbig TJ Raedler S Arlt M Kellmann W Mullen M Eichenlaub H
Mischak K Wiedemann Peptide fingerprinting of Alzheimers disease in cerebrospinal fluid
identification and prospective evaluation of new synaptic biomarkers PLoS One 2011 6
e26540
[17] NA Cellar AS Karnoup DR Albers ML Langhorst SA Young Immunodepletion of high
abundance proteins coupled on-line with reversed-phase liquid chromatography a two-
dimensional LC sample enrichment and fractionation technique for mammalian proteomics J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877 79
198
Table 1 Identified BSA tryptic peptides from various MWCO separation conditions
BSA tryptic
peptide (MH+)
100
H2O 1microg
100
1 M NaCl
70
H2O
80
1 M NaCl
70
1 M NaCl
60
H2O
60
1 M NaCl
5083
5453
6894
7124
8985
9275
10345
10725
11385
11636
12496
12837
13057
13997
14157
14197
14398
14636
14798
15026
15118
15328
15547
15677
15768
16399
16678
16738
17248
17408
17477
17497
18809
18890
19019
19079
20450
21139
22479
Total 39 2 2 6 8 15 15 27
199
Figure 1 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard showing improvement over two orders of magnitude in detection limits Each MWCO
separation was performed at minimum in triplicate with representative spectrum selected for
each with a calculated RSD from the peak heights Three different amounts of bradykinin were
tested to assess the magnitude of sample loss under different MWCO solvent conditions The
top panel shows 1 microg of bradykinin standard after MWCO separation with 100 H2O elution
produced no signal The addition of 40 or 30 MeOH produced very low bradykinin signals
for both 100 ng (RSD of 28) and 10 ng (SNlt3 no RSD calculated) respectively In the
bottom two spectra each showed very large intensity but the 7030 aqueous 1 M NaClmethanol
10 ng bradykinin was processed with a 10kDa MWCO and zip-tipped and was reproducible with
200
a RSD of 6 The last spectrum was from 10 ng bradykinin (RSD of 3) which was diluted to
an equivalent volume as all the other experiments and directly spotted onto the MALDI plate
201
Figure 2 Representative MALDI mass spectra from MWCO separation of a BSA tryptic
peptide standard showing sample loss Stacked mass spectra from mz range 875-2150
normalized to 7 x 104 intensity representing the detection difference from a BSA tryptic peptide
standard from different MWCO separation conditions (A) It should be noted that when the
solvent for MWCO elution was 100 H2O 1 microg of BSA tryptic peptides was processed instead
of 500 ng A zoomed in view of the most abundant BSA tryptic peptide detected YLYEIAR
mz 92749 (B) Various percentages of MeOH produced significant signal but addition of a salt
(1 M NaCl) increases the signal which is closest to a directly spotted BSA tryptic peptide
standard A zoomed in view of a representative low intensity BSA tryptic peptide detected
LKECC
DKPLLEK mz 153266 (C) The optimized solution to be used for MWCO filtration
202
6040 aqueous 1 M NaClmethanol was the only procedure that enabled the detection of the
tryptic peptide in Figure 2C which was also detected in the directly spotted BSA tryptic peptide
standard All experiments were performed a minimum of two times with nearly identical results
) Carbamidomethyl amino acid modification
ordm) Tryptic peptide identified in three of the spectra in Figure 2A
dagger) Tryptic peptide identified in two of the spectra in Figure 2A
) Tryptic peptide identified in a single spectrum in Figure 2A
203
Figure 3 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard with a BSA protein present showing optimized solvent conditions minimized samples
losses Each experiment was performed in duplicate Two different amounts of BSA protein
were tested to assess the magnitude of sample loss caused by the presence of a protein The top
panel shows 10 microg of BSA protein and the second panel shows 100 microg of BSA protein added
while only 10 ng of bradykinin was added Detectable sample loss was observed when the BSA
protein was added but panel two shows that the amount of BSA protein was 1 x 104 greater
(equivalent to 160 fold molar excess) than bradykinin The last two spectra were controls using
a MWCO with the optimized solution in panel 3 and panel 4 using 10 ng bradykinin which was
diluted to an equivalent volume as all the other experiments and directly spotted onto the
MALDI plate
204
Supplemental Table 1 Expanded Table 1 including grand average of hydropathicity (GRAVY)
score theoretical pI and the sequence from the underlying amino acid sequence for the peptides
identified in the BSA digest The GRAVY and pI scores were obtained from ExPASy
Bioinformatics and modifications were not taken into consideration
(httpwebexpasyorgprotparam) Peptide assignments to the recorded peaks were done by
BSA
tryptic
peptide
(MH+)
GRAVY
score
Theoretical
pI
Sequence 100
H2O
1microg
100
1 M
NaCl
70
H2O
80
1 M
NaCl
70
1 M
NaCl
60
H2O
60
1 M
NaCl
5083 NA NA FGER
5453 0900 972 VASLR
6894 0267 979 AWSVAR
7124 -0950 647 SEIAHR
8985 0529 674 LcVLHEK
9275 -0071 600 YLYEIAR
10345 -0725 674 NEcFLSHK
10725 -0211 538 SHcIAEVEK
11385 0 599 ccTESLVNR
11636 0130 453 LVNELTEFAK
12496 -1250 545 FKDLGEEHFK
12837 0264 675 HPEYAVSVLLR
13057 -0582 532 HLVDEPQNLIK
13997 0567 437 TVMENFVAFVDK
14157 0567 437 TVmENFVAFVDK
14197 0058 530 SLHTLFGDELcK
14398 -0133 875 RHPEYAVSVLLR
14636 -0515 465 TcVADESHAGcEK
14798 0292 600 LGEYGFQNALIVR
15026 -0625 409 EYEATLEEccAK
15118 0207 597 VPQVSTPTLVEVSR
15328 -0617 617 LKEccDKPLLEK
15547 -0823 441 DDPHAcYSTVFDK
15677 -0085 437 DAFLGSFLYEYSR
15768 -0985 456 LKPDPNTLcDEFK
16399 -0067 875 KVPQVSTPTLVEVSR
16678 0064 437 MPCTEDYLSLILNR
16738 -1723 550 QEPERNEcFLSHK
17248 0064 437 MPcTEDYLSLILNR
17408 0064 437 mPcTEDYLSLILNR
17477 -0914 414 YNGVFQEccQAEDK
17497 -0621 410 EccHGDLLEcADDR
18809 -0537 606 RPcFSALTPDETYVPK
18890 -0567 674 HPYFYAPELLYYANK
19019 -1275 466 NEcFLSHKDDSPDLPK
19079 0044 454 LFTFHADIcTLPDTEK
20450 -0812 839 RHPYFYAPELLYYANK
21139 -0682 480 VHKEccHGDLLEcADDR
22479 -0458 423 EccHGDLLEcADDRADLAK
Total 39 2 2 6 8 15 15 27
205
mass matching with no tandem mass spectrometry performed Lower case amino acids indicate
a modification present in the peptide of carbamidomethyl (c) or oxidation (m)
206
Chapter 8
Conclusions and Future Directions
207
Summary
Comparative shotgun proteomics investigating numerous biological changes in various
species and sample media and peptidomic method development have been reported The
developed comparative shotgun proteomics based on label-free spectral counting with nanoLC
MSMS platform have been employed in mouse CSF rat CSF yeast culture and other biological
specimens Mass spectrometry (MS)-based peptidomic enhancements using ETD and improved
sample preparation methods for molecular weight cut-offs have been reported Together these
studies add to the Li Labs capabilities in comparative shotgun proteomics and expand available
methods for peptidomic research
Immunodepletion of CSF for comparative proteomics
Chapters 3 and 4 used similar methods to generate a list of differentially expressed
proteins providing insight into how Alexander disease (AxD) perturbs the proteome and how the
new prion model rat adapted scrapie (RAS) proteome varies from control rats In the GFAP
overexpressor mouse CSF study in Chapter 3 we have produced a panel of proteins with
significant up or down regulation in the CSF of transgenic GFAP overexpressor mice MS-based
proteomic study of this mouse model for AxD was consistent with the previous studies showing
elevation of GFAP in CSF The development of a modified IgY-14 immunodepletion technique
for low amounts of CSF with recommendations for future antibody depletion techniques to deal
with the unique challenges of mouse CSF was presented Modified proteomics protocols were
employed to profile mouse CSF with biological and technical triplicates addressing the
variability and providing quantitation with dNSAF spectral counting Validation of the data was
performed using both ELISA and RNA microarray data to provide corroboration with the
208
changes in the putative biomarkers This work presents numerous interesting targets for future
study in AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
Using the protocol developed in Chapter 3 we performed a similar study in RAS CSF
compared to control rat CSF Two differences in sample preparation for the rat CSF compared
to the mouse CSF are noted (1) rat IgY-7 immunodepletion was performed and (2) each rat
CSF sample was collected from one animal due to sufficient volume instead of pooling from
multiple animals for the mouse CSF sample After immunodepletion the CSF samples from
control and RAS (biological N=5 technical replicates N=3) were digested and separated using
one dimensional RP nanoLC separation We were able to identify 167 proteins with redundant
isoforms removed which was derived from total 512 proteins with a 1 FDR from all the CSF
samples Comparative analysis was performed using dNSAF spectral counting and 21 proteins
were significantly changed Our data were consistent with previous prion CSF studies showing
14-3-3 protein increased in CSF of prion infected animals Genomic analysis was also
performed and was used to cross-validate our proteomic data and numerous proteins were found
to be consistent including a novel marker related to prion disease RNAset2 (ribonuclease T2)
In summary this work provides a foundation for investigation of the perturbed proteome of a
new prion model RAS
Yeast Phosphoproteomic Comparison between Starved and Glucose Fed Conditions
This work presented a qualitative comparison of the phosphoproteome between starved
and glucose fed Saccharomyces cerevisiae (bakers yeast) A large scale IMAC enrichment of
yeast cell lysate followed by 2-D separations provided a rich source of phosphopeptides for CID
MSMS and neutral loss triggered ETD analysis using mass spectrometry A specific motif for
PKA was highlighted to show the differences in proteins identified between starved and glucose
209
fed conditions In total 477 unique phosphopeptides were identified with 06 FDR and 669
unique phosphopeptides identified with 54 FDR Phosphosite validation was performed using
a localization algorithm Ascore to provide further confidence on the site-specific
characterization of these phosphopeptides Two proteins Ssd1 and PMA2 emerged as potential
intriguing targets for more in-depth studies on protein phosphorylation involved in glucose
response
Methods for Peptide Sample Preparation and Sequencing
In this study ETD was performed to improve the sequence coverage of endogenous large
neuropeptides and labile tyrosine sulfation CCK-like neuropeptides found in the blue crab
Callinectes sapidus The intact CPRP from Callinectes sapidus was identified and characterized
with 68 sequence coverage by MS for the first time Cation assisted ETD fragmentation using
MgCl2 was also shown to be a powerful tool in de novo sequencing of sulfated neuropeptides
These endeavors into using ETD for certain neuropeptides will assist in future analysis of large
neuropeptides and PTM containing neuropeptides
In addition to ETD sequencing I presented a protocol on improving recovery of minute
quantities of peptides by adding methanol and a salt modifier (NaCl) to molecular weight cut-off
membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities
Despite its widespread utility significant sample loss often occurs during the MWCO
fractionation step which is particularly problematic when analyzing low-abundance peptides
from limited starting material This work presented a method to reduce sample loss during
MWCO separations specifically for sub-microg peptide isolation Using a neuropeptide standard
bradykinin sample loss was reduced by over two orders of magnitude with and without
210
undigested protein present The presence of bovine serum albumin (BSA) undigested protein
and the bradykinin standard lends evidence that sample loss occurs because of the MWCO and
not the presence of the protein Additionally a BSA tryptic digestion is presented where twenty-
seven tryptic peptides are identified from MALDI mass spectra after enriching with methanol
while only two tryptic peptides are identified after the standard MWCO protocol
Ongoing Projects and Future Directions
CSF Projects
Rat Adapted Scrapie and Time Course Study of Rat CSF
In ongoing experiments from the work described in Chapter 4 related to rat adapted
scrapie (RAS) we are working towards more complete proteome coverage of CSF and a time
course study of RAS After the promising results of the 1-D proteomic comparison between
RAS and control CSF we want to perform IgY immunodepletion on 1 mL of rat CSF followed
by 2-D high pH-low pH RP-RP LC-MSMS IgY immunodepletion is still required and
afterwards approximately 40 microg of low abundance protein would remain Following traditional
urea tryptic digestion (see Appendix 1) and solid phase extraction (SPE) clean-up the sample
would be dried down and reconstituted in mobile phase A (25 mM ammonium formate basic
pH) for high pH RP separation Fractions would be dried and subjected to MS analysis similar to
the work described in Chapter 4 The purpose of this work would be to increase the proteome
coverage from a few hundred to a thousand CSF proteins identified A time course study of RAS
is also desirable to gain insight into disease progression Rats at different stages will be
sacrificed and CSF will be collected The goal is to perform isobaric labeling between the time
courses with spectral counting being an alternative for relative protein expression We will use
the targets identified in Chapter 4 to track certain proteins for time course analysis Overall
211
these future projects will dig deeper into the proteome and how this novel prion model RAS
perturbs the proteins expressed in rats over time
Glycoproteomics and Peptidomic Analysis of CSF Samples from Individuals with
Alzheimerrsquos Disease
Alzheimerrsquos disease is an incurable progressive neurodegenerative disorder which results
in cognitive impairment Our aim is to provide additional biomarkers andor targets for drug
treatment Currently we used 500 microL of human CSF for non-membrane glycoprotein
enrichment (see Appendix 1) followed by 2-D high-pH-low-pH RP-RP amaZon ion trap LC-
MSMS analysis The initial work was a failure due to low amount of signal and significant
sample loss (data not shown) Through a comparison with Dr Xin Weirsquos PhD thesis we
estimated roughly 20 microg of proteins was obtained after glycoprotein enrichment and 1-D analysis
produced over 100 protein identifications (data not shown) but the additional off-line 2-D
separation and sample clean up resulted in low number of protein identifications for each fraction
analyzed by MS (data not shown) We have recently obtained 1 mL of human CSF samples
from healthy controls and individuals suffering from Alzheimerrsquos disease and plan to perform
the same experiments with double the starting amount and reduce the fractions collected from 2-
D separation from 11 to 6 In addition to the glycoproteomics anything not enriched will be
subjected to MWCO separation using the method presented in Chapter 7 The obtained peptide
sample will be subjected to Q-TOF nanoLC MSE data acquisition followed by de novo
sequencing using various programs including PEAKS and Mascot Collectively we feel this
project has great potential to lead to interesting targets and further expand the proteomic
knowledge of Alzheimerrsquos disease
GFAP Knock-in Mouse CSF
212
In a direct follow-up to Chapter 3 we aim to perform comparative proteomics on control
vs glial fibrillary acidic protein (GFAP) knock-in mouse CSF samples The sample preparation
protocol as presented in Chapter 3 will be used For quantitative proteomics we plan on
performing isobaric labeling followed by performing high energy collision induced dissociation
(HCD) on the Orbitrap Elite The MS acquisition will also perform a top ten scan where the top
ten MS peaks will be selected for tandem MS analysis In addition targeted quantitation of
specific proteins using multiple reaction monitoring (MRM) can be performed on potential
biomarkers and identified in this follow-up study and the work presented in Chapter 3 Also any
CSF samples with noticeable blood content cannot be used for the exploratory proteomics
experiments but can potentially be used for the MRM analysis and should be kept for such
experiments in the future
Large Scale Proteomics
Proteomics of Mouse Amniotic Fluid for Lung Maturation
The overall goal of this project is to determine what proteins are present in amniotic fluid
when the embryo is 175 weeks compared to amniotic fluid at 155 weeks The rationale behind
why these two time points matter was investigated through a lung explant culture where amniotic
fluid was added at 155 weeks and 175 weeks Visual maturation of lungs occurred with the
175 week amniotic fluid and lung maturation genes were up regulated in the mRNA in the lung
explant culture when compared to the 155 week amniotic fluid The compound which is
causing the maturation of the lungs is unknown and search for a secreted protein might provide a
clue to this process In order to test this hypothesis we carried out discovery proteomics
experiments The workflow involves tryptic digestion SPE and 1-D low pH nanoLC separation
coupled to an amaZon ETD ion trap for tandem MS analysis The resulting mass spectrometric
213
acquisition identified less than 100 proteins with a 1 FDR (data not published) To address the
lack of depth in the proteome coverage we purchased an IgY immunodepletion column to
remove the most abundant proteins in amniotic fluid similarly to Chapter 3 and 4 but on a larger
scale One abundant protein alpha-fetoprotein is present in amniotic fluid but absent or present
in very low concentration in CSF serum or plasma Alpha-fetoprotein is similar to albumin and
thus we ran amniotic fluid on an IgY immunodepletion column and observed significant
reduction in alpha-fetoprotein amount (data not published) After immunodepletion 2-D high
pH-low pH RP-RP will be performed followed by nanoLCMSMS on the amaZon ETD ion trap
We will perform mass spectrometry experiments using the 155 week amniotic fluid and 175
week amniotic fluid which have average protein contents ~2 mgmL We anticipate a minimum
of tenfold increase in the proteome coverage of amniotic fluid and provide meaningful
hypothesis driven biological experiments from this work
Phosphoproteomics of JNK Activation
c-Jun N-terminal kinase (JNK) is an important cellular mediator of stress activated
signaling Under conditions of oxidative stress JNK is activated resulting in the downstream
phosphorylation of a large number of proteins including c-Jun However the cellular response
to JNK activation is extremely complex and JNK activation can result in extremely different
physiological outcomes For example JNK is at the crossroads of cellular death and survival
and exhibits both pro-death and pro-survival activities These highly disparate outcomes of JNK
activation are highly contextual and depend on the type of stressor and duration of stress In the
brain JNK has been shown to be activated in models of Alzheimerrsquos disease Parkinsonrsquos
disease amyotrophic lateral sclerosis and stroke It is thought that JNK is activated in these
diseases as a result of oxidative stress and it is unknown whether this activation is pro-survival or
214
pro-death To elucidate JNK signaling in the brain we chose to analyze primary cortical
astrocytes under conditions of oxidative stress Since hydrogen peroxide is a physiologically
relevant oxidant in the brain and known to activate JNK we chose to use this to treat astrocytes
and then analyze changes to the phosphoproteome by mass spectrometry By doing this we
hope to elucidate important JNK-dependent targets involved in stress signaling in the brain and
that identifying these targets could lead to the identification of novel disease mechanisms and
potentially new therapeutic targets for neurodegeneration Specifically we plan on performing
stable isotope labeling by amino acids (SILAC) of astrocytes for control hydrogen peroxide
treated and hydrogen peroxide treated with JNK inhibitor The SILAC labeled astrocyte cell
lysate will be digested enriched using Fe-NTA IMAC and separated using 2-D high pH-low pH
RP-RP and infused into the amaZon ETD ion trap The protocol is similar to the yeast
comparative phosphoproteomics from Chapter 5 After analysis we plan on processing the data
using ProteoIQ to identify phosphoproteins with significant changes
Immunoprecipitation Followed by Mass Spectrometry
Stb3 Mass Spectrometry Analysis
Stb3 (Sin3-binding protein) has previously been shown to change location depending on
the presence or absence of glucose (Heideman Lab Dr Michael Conways Thesis) An
immunoprecipitation of Stb3 was performed in parallel to the work described in Chapter 5 Two
separate experiments were performed one with wild type Stb3 and another tagged with myc for
improved protein immunoprecipitation Myc tagging is a polypeptide tag which can be
recognized by an antibody and provide a better enrichment compared to using a Stb3 antibody
alone The myc tagging was done because of the low abundance of Stb3 and the limited amount
of protein that was pulled down by the antibody for Stb3 Immunopreciptation experiments were
215
performed for both starved and glucose fed samples All samples were tryptically digested
followed by low pH RP nanoLCMSMS Both CID and ETD were used for fragmentation
analysis of each sample (n=2) The Stb3 wild type had two peptide hits in one MS run which is
not surprising due to low Stb3 antibody affinity In contrast a significant amount of Stb3 was
pulled down from Myc tagged starved and glucose fed samples For the glucose starved
samples 22 unique peptides from Stb3 were identified whereas glucose fed samples yielded 21
unique Stb3 peptides identified (unpublished data) The identified Stb3 in the myc samples
allowed us to investigate what other proteins were pulled down that are not present in the wild
type samples
From previous work by our collaborator Dr Heideman it had been suggested that Stb3
forms a complex with Sin3 and Rpd3 Our proteomic data shows multiple significant peptide
hits for Sin3 and Rpd3 in myc tagged but not in wild type where Stb3 is only observed once
with a low Mascot score When looking at the unique proteins identified in myc tagged glucose
fed sample but not included in the wild type samples the myc fed sample contained eight unique
ribosomal proteins with numerous unique peptides for each The ribosomal proteins identified in
myc fed sample supports the novel hypothesis that in the glucose fed state the complex of Stb3
Rpd3 and Sin3 interact with ribosomal proteins Other proteins unique to myc tagged glucose
starved samples are glyceraldehyde-3-phosphate dehydrogenase 2 and transcriptional regulatory
protein UME6 Also three proteins were observed in both myc fed and starved but not in the
wild type samples including transposon Ty1-DR1 Gag-Pol polyprotein(YD11B) SWIRM
domain-containing protein (FUN19) and RNA polymerase II degradation factor 1 (DEF1) Our
proteomics data for the Stb3 immunoprecipitation experiments with starved vs glucose fed
216
samples provide exciting evidence to support previous observation made by the Heideman group
and highlight the utility of MS-based approach to deciphering protein-protein interactions
Conclusions
The majority of the work described in this dissertation revolves around sample
preparation for proteomics and peptidomics with a focus on generating biologically testable
hypotheses In the area of neuropeptides CPRP was fully sequenced analytical methods were
transformed for use in an ETD ion trap and sub-microg peptides can now be analyzed by mass
spectrometry after MWCO separation In the field of comparative proteomics comparisons
between mouse CSF in GFAP overexpressors and control or rat adapted scrapie model and
control CSF yeast fed vs glucose starved cell extract have all been presented Collectively this
thesis has developed new techniques for neuropeptide sample preparation and presented
numerous comparative proteomic analyses of various biological samples and how the proteomes
are dynamically perturbed by various treatments and disease conditions
217
Appendix 1
Protocols for sample preparation for mass spectrometry based
proteomics and peptidomics
218
Small Scale Urea Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 8 μL of urea solution
(400mg05mL) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Allow to digest overnight in 37degC water bath
10 Acidify with 10μL 10 formic acid
11 Perform solid phase extraction using tips dependent of sample amount
a Sub-5μg amounts ndash Millipore Ziptips
b 5-75μg amounts ndash Bond-Elut tips from Agilent (OMIX tips)
12 Dry down in Speedvac as needed for experiment
219
Small Scale ProteaseMAX Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 2 μL of 1 of
ProtesaeMAX (Promega) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Add 1 μL ProteaseMAX and let sit for 3-4 hours
10 Acidify with 2μL 10 formic acid
11 Dry down in Speedvac as needed for experiment
220
Large Scale Urea Tryptic Digestion (mg of proteins)
1 All solutions are in a 50 mM NH4HCO3 buffer (395 mgmL)
2 Add 20μL of 05M Dithiothreitol (DTT) (77mg100μL) and 160μL of urea solution
(400mg05mL) to sample
3 Allow sample to denature 45-60 minutes in a 37degC water bath
4 Add 54μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
5 Quench reaction with 20μL of DTT solution
6 Dilute with 14mL of NH4HCO3 solution
7 Add 100μg of trypsin
8 Allow to digest overnight in 37degC water bath
9 Acidify sample with 100μL of 10 formic acid
10 Perform solid phase extraction with Hypersep C18 Cartridges with 100 mg of C18
bead volume (Thermo)
11 Dry down with the Speedvac as needed for experiment
221
Fe-NTA Preparation from Ni-NTA Beads
1 Centrifuge (5 min at 140 rcf) and use a magnet to keep beads in place as supernatant
is removed
2 Wash 3 times with 800μL of H2O (using same technique of centrifuging and using
magnet to keep beads in places as supernatant is removed)
3 Add 800μL of 100mM EDTA (372mgmL) in a 50mM NH4HCO3 (395mgmL)
buffered solution (pH around 8) and vortex for 30 minutes to chelate the Ni
centrifuge and remove supernatant
4 Wash 3 times with 800μL of H2O
5 Add 800μL of 10mM FeCl3 hexahydrate (27mgmL) and vortex for 30 minutes to
bind Fe to beads centrifuge and remove supernatant
6 Wash 3 times with 800μL H2O
7 Resuspend in a storage solution of 111 ACNMeOH001 Acetic Acid (1mL total)
222
Fe-NTA IMAC Phospho-enrichment
1 Use wash solution (80ACN 01Formic Acid) to rinse beads vortex 1 minute
centrifuge and remove supernatant
2 Add sample (around 500μL in 80ACN 01Formic Acid) and vortex 40 minutes to
allow sample to bind dispose of supernatant after centrifuging
3 Wash 3 times with 200μL of wash solution discard supernatant
4 Add 200μL of elution solution (5050 ACN5Ammonium Hydroxide) vortex 15
minutes and save supernatant
5 Add 200μL of elution solution vortex 10 minutes and save supernatant
6 Wash 2 time with wash solution (collect supernatant of first wash)
7 If reusing store in 1mL solution of 111 ACNMeOH001 Acetic Acid
223
High pH Off-line Separation
1) In general a minimum of 20 microg of peptides are needed to gain any benefit
from off-line 2D fractionation It is better to inject 100 microg of peptides on
column
2) Use a Gemini column or a similar column that can handle pH=10 and for this
protocol a 21 mm x 150 mm column was used
3) Prepare ldquobuffer Ardquo 25 mM NH4 formate pH=10 Also prepare ldquobuffer Brdquo
4) Dry down desired sample and reconstitute in buffer A
5) Inject based off sample loop (current Alliance HPLC has a 50 microL sample
loop)
6) Run gradient at bottom of the page collecting fractions every 3 minutes except
for the 1st minute which is the void volume
7) Optional If you want to reduce instrument time you can combine fractions 1
with 12 2 with 13 etc until 11 with 22
Time Mobile phase A Mobile phase B Flow Rate
05mlmin
0 98 2 05 mLmin
65rsquo 70 30 05 mLmin
65rsquo1rdquo 5 95 05 mLmin
70 5 95 05 mLmin
224
Non Membrane Glycoprotein Enrichment
1 For protocols involving membrane glycoprotein enrichment see Dr Xin Weirsquos
thesis
2 Add 75 microL of Wheat germ agglutinin (WGA) bound to agarose beads and 150 microL
of Concanavalin A (ConA) to a Handee spin cup (Thermo) Wash 2 times with
lectin affinity chromatography (LAC) binding buffer (015 M NaCl 002 M Tris-
HCl 1 mM CaCl2 pH=74) centrifuging at 400 g for 30 seconds
3 Place stopper on bottom of spin cup and add 200 microg of sample (500 microL for CSF)
Bring up to 300 microL using lectin LAC binding buffer
4 Incubate for 1 hour with continuous mixing at room temperature
5 Centrifuge at 400 g for 30 seconds
6 Perform two more 300 microL LAC binding washes followed by centrifugation
7 Add 300 microL LAC eluting buffer (0075 M NaCl 001 M Tris-HCl 02 M alpha-
methyl mannoside 02 M alpha-methyl glucoside and 05 M acetyl-D-
glucosamine) vortex for 10 minutes (have stopper in place while vortexing)
centrifuge and collect
7 Add another 300 microL LAC eluting buffer centrifuge and collect
225
MWCO separation for Sub-microg peptide concentrations
1 Wash molecular weight cut-off (MWCO) with 5050 waterMeOH centrifuge at
14000 g for 5 minutes for 10 kDa filter (time will vary Millipore Amicon Ultra
filters)
2 Wash with 100 water centrifuge at 14000 g for 5 minutes
3 Add methanol to the sample to get the concentration to 30 methanol and add
salt to achieve a roughly 05 M NaCl before adding the sample to the MWCO
4 Centrifuge at 14000 for 10 minutes collect flow through
226
Immunoprecipitation
Modified from Thermo Fisher Scientific Classic IP Kit
1 10 microL Polyclonal antibody added to 1 microg protein standard in a Handee spin cup
(Thermo) diluted with 1x TBS buffer to 400 microL and incubated overnight at on
shakerend-over-end rotator
2 Wash Protein GA beads with 2x 200microL TBS buffer and added to the
antibodysample for 15 hours at 4oC
3 Centrifuge at 400 g for 30 seconds and discard flow through
4 Wash 3x with 100microL TBS buffer centrifuge at 400 g for 30 seconds and discard
flow through
5 Wash with 1x conditioning solution (neutral pH buffer) centrifuge at 400 g for 30
seconds and discard flow through
6 Eluted with elution buffer (2x100microL) centrifuge at 400 g for 30 seconds and
collect flow through
227
C18 Solid Phase Extraction (SPE)
1 Determine amount of peptides for SPE ge5 microg use Ziptips from Millipore If
between 5-75 microg use OMIX from Agilent 100 microL version and if ge75 microg use SPE
cartridges such as 100 mg Hypersep from Thermo
2 Ensure no detergents are in the sample and it is acidified
3 The three SPE procedures all use the same sets of solutions only volumes vary
4 3x Wetting solution (100 ACN) for 60 microL OMIX (20 microL ziptip and 15 mL for
100 mg cartridge)
5 3x Wash solution (01 formic acid in 100 H2O) same volumes as 4
6 Draw up the sample about 10 times minimum (generally 30-40 is preferred)
without letting the bead volume dry out
7 1X Wash solution same volumes as 4
8 Back load the eluting solution 01 formic acid in 5050 H2OACN into the
Ziptips (15 microL) or OMIX (50 microL) tips For the SPE cartridge add 700 microL of
eluting solution
9 Dry down and prepare for next step in sample preparation
228
Laser Puller Programs and Protocols
1) Obtain about two feet of Fused-silica capillary with 75 μm id and 360 μm od
2) Wash with methanol and then air dry using the bomb
3) Cut into one foot or desired length
4) Use a lighter to burn the middle portion (about an inch in length) of the tubing
5) Remove the ash by rinsing with methanol and rubbing with a Kimwipe
6) Make sure the laser puller has been on for at least 30 minutes before use to allow
for the instrument to warm up
7) Place capillary in instrument with the burnedexposed portion in the center
making sure that the length of the capillary is pulled taut
8) Enter desired program (next page) and press pull
229
Laser Puller Programs
Program 99 (default lab program)
Heat Filament Velocity Delay Pull
250 0 25 150 15
240 0 25 150 15
235 0 25 150 15
245 0 25 150 15
Program 97 (developed for larger inner diameter tips)
Heat Filament Velocity Delay Pull
230 - 25 150 -
220 - 25 150 -
215 - 25 150 8
230
On column Immunodepletion (serum plasma CSF amniotic fluid)
1) Prepare buffer A ldquoDilution Bufferrdquo 10 mM Tris-HCl pH=74 150 mM NaCl
2) Prepare buffer B ldquoStripping Bufferrdquo 01 M Glycine-HCl pH=25
3) Prepare buffer C ldquoNeutralization Bufferrdquo 100 mM Tris-HCl pH=80
4) Determine loading amount (LC1=~1000 ug of serum or plasma less for CSF due
to the increased amount of albumin percentage in CSF)
5) Add Dilution buffer to sample before injection and ensure the sample is proper
pH (~7)
6) Use gradient below
Time A B C Flow Rate
(mLmin)
0rsquo 100 0 0 02
4rsquo59rdquo 100 0 0 02
5rsquo 100 0 0 05
8rsquo59rdquo 100 0 0 05
9rsquo 0 100 0 05
22rsquo 0 100 0 05
22rsquo1rdquo 0 0 100 05
39rsquo 0 0 100 05
7) Store the column in 1x dilution buffer until next use
231
Small Scale Immunodepletion (100 microL of CSF)
1) Use 75 microg (per 100 microL CSF) of Sigma Seppro IgY-14 or IgY-7 bead slurry
2) Add an equal volume of 2x dilution buffer (20 mM Tris-HCl pH=74 300 mM
NaCl) to the starting amount of CSF
3) Add to a spin cup with a filter and allow to mix for 30 minutes
4) Centrifuge at 400 g for 30 seconds and collect the flow through
5) Add 50 microL of 1x dilution buffer mix for 5 minutes centrifuge at 400 g for 30
seconds and collect the flow through
6) Use 1x stripping washes (01 M Glycine-HCl pH=25) centrifuge as before and
discard Repeat four times
7) Use 1x neutralization buffer (100 mM Tris-HCl pH=80) centrifuge as before
and discard Repeat two times
8) Store the beads in the spin column in 1x dilution buffer until next use
232
Alliance Maintenance Protocol
Seal Wash
10 methanol no acetonitrile This wash cleans behind the pump-head seals to
ensure proper lubrication Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime SealWsh gt Start
2 Press Stop after 5 minutes
Prime Injector
10 methanol for maintenance high organic solvent for dirty runs (eg 95
acetonitrile) Done before injecting any real samples to ensure no bubbles are
present in the injector Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime NdlWsh gt Start
2 After completion press Close
Purge Injector
Solvent is dependent on run Run this protocol at beginning of experiments each day
Minimum once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Navigate Direct Function gt 4 Purge Injector gt OK
3 Set Sample loop volumes 60 leave Compression Check unchecked gt OK
Prime Solvent Pumps
Solvent is dependent on run If solvents are changed run this protocol Minimum
once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys choose composition A type 100 Enter x4
3 Navigate Direct Function gt 3 Wet Prime gt OK
4 Set Flow Rate 7000 mLmin Time 100 min gt OK
5 Repeat for all changedactive solvent pumps
Condition Column
Dependent on user Use starting conditions for run
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys type starting solvent compositions for run
3 Navigate Direct Function gt 6 Condition Column gt OK
4 Set Time as desired
233
Appendix 2
List of Publications and Presentations
234
PUBLICATIONS
ldquoDiscovery and characterization of the crustacean hyperglycemic hormone precursor related
peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes
sapidus using multiple tandem mass spectrometry techniquesrdquo Hui L Cunningham R Zhang
Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
ldquoInvestigation and reduction of sub-microgram peptide loss using molecular weight cut-off
fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J Wellner D Li L
Journal of Mass Spectrometry In Press
ldquoMass spectrometry-based proteomics and peptidomics for systems biology and biomarker
discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse models of
Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P Messing A Li
L Journal of Proteome Research Submitted
ldquoInvestigation of the differences in the phosphoproteome between starved vs glucose fed
Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M Heideman
W Li L In preparation
ldquoIdentification of astrocytic JNK targets by phosphoproteomics using mass spectrometryrdquo
Cunningham R Dowell J Wang J Wellner D Li L Milhelm M In preparation
ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A Cunningham R Wellner
D Wang J Ma D Li L Aiken J In preparation
235
INVITED SEMINARS AND CONFERENCE PRESENTATIONS
Robert Cunningham Davenne Mayour and Shoshanna Coon ldquoMorphology and Thermal
Stability of Monolayers on Porous Siliconrdquo The 231th
ACS National Meeting 2006 Atlanta
GA
Robert Cunningham Xin Wei Paige Jany Albee Messing and Lingjun Li ldquoMass
Spectrometry-Based Analysis of Cerebrospinal Fluid Peptidome and Proteome for Biomarker
Discovery in Alexander Diseaserdquo The 57th
ASMS Conference 2009 Philadelphia PA
Robert Cunningham ldquoWhat to Expect in a PhD Graduate Schoolrdquo Invited seminar University
of Northern Iowa 2010 Cedar Falls IA
Robert Cunningham Dustin Frost Albee Messing and Lingjun Li ldquoInvestigation of an
Optimized ProteaseMAX Assisted Trypsin Digestion of Human CSF for Pseudo-SRM
Quantification of GFAP and Identification of Biomarkersrdquo The 58th
ASMS Conference 2010
Salt Lake City UT
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo Oral presentation at Pittcon Conference and Expo 2011 2011 Atlanta
GA
Robert Cunningham Michael Conway Daniel Wellner Douglas Grunwald Warren
Heideman and Lingjun Li ldquoDevelopment of optimized phosphopeptide enrichment methods for
comparison of starved and glucose fed yeast Saccharomyces cerevisiaerdquo The 59th
ASMS
Conference 2011 Denver CO
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo 2011 Wisconsin Human Proteomics Symposium 2011 Madison WI
iii
Table of Contents
Page
________________________________________________________________________
Acknowledgements i
Table of Contents iii
Abstract iv
Chapter 1 Introduction brief background and research summary 1
Chapter 2 Mass spectrometry-based proteomics and peptidomics for
biomarker discovery and the current state of the field 15
Chapter 3 Protein changes in immunodepleted cerebrospinal fluid from
transgenic mouse models of Alexander disease detected
using mass spectrometry 73
Chapter 4 Genomic and proteomic profiling of rat adapted scrapie 110
Chapter 5 Investigation of the differences in the phosphoproteome
between starved vs glucose fed Saccharomyces cerevisiae 139
Chapter 6 Use of electron transfer dissociation for neuropeptide
sequencing and identification 166
Chapter 7 Investigation and reduction of sub-microgram peptide loss
using molecular weight cut-off fractionation prior to
mass spectrometric analysis 187
Chapter 8 Conclusions and future directions 206
Appendix 1 Protocols for sample preparation for mass spectrometry
based proteomics and peptidomics 217
Appendix 2 Publications and presentations 233
_______________________________________________________________________
iv
Mass Spectrometry Applications for Comparative Proteomics and
Peptidomic Discovery
Robert Stewart Cunningham
Under the supervision of Professor Lingjun Li
At the University of Wisconsin-Madison
Abstract
In this thesis multiple biological samples from various diseases models or
treatments are investigated using shotgun proteomics and improved methods are
developed to enable extended characterization and detection of neuropeptides In general
this thesis aims to expand upon the rapidly evolving field of mass spectrometry (MS)-
based proteomics and peptidomics by primarily enhancing small scale sample analysis
A review of the current status and progress in the field of biomarker discovery in
peptidomics and proteomics is presented To this rapidly expanding body of literature
our critical review offers new insights into MS-based biomarker studies investigating
numerous biological samples methods for post-translational modifications quantitative
proteomics and biomarker validation Methods are developed and presented including
immunodepletion for small volume cerebrospinal fluid (CSF) samples for comparison of
the CSF proteomes between an Alexander disease transgenic mouse model with
overexpression of the glial fibrillary acidic protein and a control animal This thesis also
covers the application of the small scale immunodepletion of CSF for comparative
proteomic analysis of a novel rat adapted scrapie (RAS) model for prion disease and
v
compares the RAS CSF proteome to control rat CSF using MS Large scale
phosphoproteomics of starved vs glucose fed yeast is presented to better understand the
phosphoproteome changes that occur during glucose feeding Method development for
neuropeptide analysis is expanded upon using electron transfer dissociation (ETD)
fragmentation to successfully sequence for the first time the crustacean hyperglycemic
hormone precursor-related peptide (CPRP) from the blue crab Callinectes sapidus In
addition a method for ETD sequencing of sulfonated neuropeptides using a magnesium
salt adduct in an ion trap mass spectrometer is reported This thesis also reports on a
method for sub-microg peptide isolation when using a molecular weight cut-off filtration
device to improve sample recovery by over 2 orders of magnitude All the protocols used
throughout the work are provided in an easy to use step-by-step format in the Appendix
Collectively this body of work extends the capabilities of mass spectrometry as a
bioanalytical tool for shotgun proteomics and expands upon methods for neuropeptide
discovery and analysis
1
Chapter 1
Introduction Brief Background and Research Summary
2
Abstract
Mass spectrometry based comparative proteomics and improved methods for
neuropeptide discovery have been reported in this thesis A very brief introduction of crustacean
neuropeptidomics is covered in this chapter followed by Chapter 2 which covers in great detail
comparative proteomics using mass spectrometry with an emphasis on biomarker discovery
Chapter 3 reports a novel immunodepletion technique used for comparative proteomics between
glial fibrillary acidic protein (GFAP) overexpressor and control mouse cerebrospinal fluid (CSF)
Chapter 4 involves rat CSF comparative proteomics between rat adapted scrapie and control
animals Chapter 5 details comparisons of phosphoproteomes in Saccharomyces cerevisiae
(Bakerrsquos yeast) between starved vs glucose fed conditions Chapter 6 investigates the use of
electron transfer dissociation (ETD) for kDa sized neuropeptides and neuropeptides with tyrosine
sulfonation Chapter 7 presents a molecular weight cut-off (MWCO) protocol to allow sub-microg
peptides to be recovered Chapter 8 provides a conclusion to this thesis and outlines future
directions for certain projects
3
Background
Mass spectrometry (MS) requires gas phase ions for experimental measurement and
intact large nonvolatile biomolecules proved difficult to be analyzed by electron ionization or
chemical ionization until the invention of two soft ionization techniques matrix-assisted laser
desorptionionization (MALDI)1 and electrospray ionization (ESI)
2 ESI and MALDI are the
two most common soft ionization techniques for mass spectrometry Once ionized molecules
such as peptides or proteins can be separated by their mass to charge ratios (mz) using various
mass analyzers manipulating electric andor magnetic fields ESI and MALDI mass
spectrometric techniques have become central analytical methods in biological sciences because
they are suitable for nonvolatile thermally labile analytes in femtomole quantities3 ESI allows
the coupling of high pressure liquid chromatography and the constant flow of solvent is
electrically charged at the outlet to produce a Taylor cone4 During desolvation the Raleigh
limit is reached and a coulombic explosion occurs commonly producing multiply charged ions
A modification of ESI is nanoLC-ESI which operates at nLmin flow rates to analyze low sample
amounts such as proteins or peptides in biological samples5 NanoLC-ESI uses less solvent as
the source because of the reduced flow rate of standard LCMS with an ESI source and nanoLC-
ESI is the predominate inlet for shotgun proteomic ESI MS experiments6 Conversely MALDI
can use sub-microL volume of sample and be co-crystallized with an ultraviolet absorbing organic
matrix that is obliterated using a laser at appropriate wavelength to generate gas phase ions
Alternatively MALDI has the unique capability to work with tissue samples and ionize in the
solid state instead of liquid like ESI
4
Mass analyzers require an operating pressure between 10-4
-10-10
Torr to allow proper ion
transfer and separationisolation of the ion based upon its mz Numerous mass analyzers are
currently available and each have their own strengths and weaknesses as shown in Figure 1 The
biomolecules are separated by the mass analyzers and detected without fragmentation which is
termed MS analysis Following MS detection tandem mass spectrometry (MSMS) of the
original precursor ion can be performed to provide additional structural information such as a
ladder sequence of amino acids for peptides Numerous fragmentation techniques are available
for proteomics such as collision induced dissociation (CID) electron transfer dissociation (ETD)
or high energy collision induced dissociation (HCD) Each of these fragmentation techniques
have their own benefits in proteomics experiments and are discussed in depth in Chapter 2 The
background and current status for comparative proteomics with specific emphasis on biomarker
analysis are covered in Chapter 2
Neuropeptidomic Method Development in the Crustacean Model System
Utilizing Mass Spectrometry
Chapter 6 and 7 focus on sample preparation and ETD fragmentation techniques to
characterize neuropeptides in the neuroendocrine organs in the crustacean nervous system
Neuropeptides are short chains of amino acids which are a diverse class of cell-cell signaling
molecules in the nervous system Neuropeptides have been investigated for being involved in
numerous physiological processes such as memory7 learning
8 depression
9 pain
10 reward
11
reproduction12
sleep-wake cycles13
homeostasis14
and feeding15-17
Figure 2 depicts how
neuropeptides are synthesized as pre-prohormones in the rough endoplasmic reticulum and
5
packaged in the Golgi apparatus After being packaged these pre-prohormones are processed
into bioactive peptides within the vesicle which is occurring during vesicular transport down an
axon and released into the synaptic cleft Secreted neuropeptides stimulate the post synaptic
neurons by interacting with G-protein coupled receptors at the chemical synapse
The crustacean model nervous system is well-defined neural network which has been
used in neurobiology and neuroendocrine (neurosecretory) research and thus lends itself for
studying neuromodulation18-22
Figure 3 shows the locations of several neuroendocrine organs in
the crustacean Callinectes sapidus including the sinus gland (SG) which is studied in Chapter 6
The Lingjun Li lab has focused on neuropeptide discovery and function in crustacean
neuroendocrine organs using mass spectrometry23-25
The work presented in Chapters 6 and 7
expand on sample preparation and analytical tools to further investigate the neuropeptidome
Research Overview
Comparative Proteomics of Biological Samples
Chapter 2 provides a thorough review of comparative proteomics and biomarker analysis
using mass spectrometry The scientific community has shown great interest in the field of mass
spectrometry-based proteomics and peptidomics for its applications in biology Proteomics
technologies have evolved to generate large datasets of proteins or peptides involved in various
biological and disease progression processes producing testable hypotheses for complex
biological questions This chapter provides an introduction and insight into relevant topics in
proteomics and peptidomics including biological material selection sample preparation
separation techniques peptide fragmentation post-translational modifications quantification
6
bioinformatics and biomarker discovery and validation In addition current literature and
remaining challenges and emerging technologies for proteomics and peptidomics are discussed
Chapter 3 investigates comparative proteomics between a GFAP overexpressor mouse
model and a control mouses cerebrospinal fluid (CSF) CSF is a low protein content biological
fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in
direct contact with the brain but consist of very abundant proteins similar to serum which require
removal A modified IgY-14 immunodepletion treatment is presented to remove abundant
proteins such as albumin to enhance analysis of the low volumes of CSF that are obtainable
from mice These GFAP overexpressor mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we present the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates are performed to address animal variability as well as reproducibility in mass
spectrometric analysis Relative quantitation is performed using distributive normalized spectral
abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins with
significant changes in the CSF of GFAP transgenic mice are identified with validation from
ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
Chapter 4 explores the CSF proteomics of a novel prion model called rat adapted scrapie
(RAS) A similar IgY immunodepletion technique is presented as in Chapter 3 and is similarly
used to reduce the abundant proteins in CSF CSF from control and RAS (biological N=5
technical replicates N=3) were digested and separated using one dimensional reversed-phase
nanoLC separation In total 512 proteins 167 non-redundant protein groups and 1032 unique
peptides are identified with a 1 FDR Comparative analysis was done using dNSAF spectral
7
counting and 21 proteins were significantly up or down-regulated The proteins are compared to
the 1048 differentially regulated genes and additionally compared to previously published
proteins showing changes consistent with other prion animal models Of particular interest is
RAS specific proteins like RNAseT2 which is not up-regulated in mouse prion disease but is
designated as upregulated in both the genomic and proteomics data for RAS
Chapter 5 explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Previous work by the
Heideman lab investigated the transcriptional response to fresh glucose in yeast26
Kinases such
as protein kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose
response so we described a large scale phosphoproteomic MS based study in this chapter
Yeast cell extract was digested and phosphopeptides were enriched by immobilized metal
affinity chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase
(RP)-RP separation The low pH separation was infused directly into an ion trap mass
spectrometer with neutral loss triggered ETDfragmentation Specifically the CID fragmentation
can cause a neutral loss of 98z from the H3PO4 and can produce an incomplete fragmentation
pattern and lead to ambiguous identification so when the neutral loss is detected ETD MSMS
fragmentation is performed The neutral loss triggered ETD fragmentation is included in this
study to improve phosphopeptide identifications In total 477 phosphopeptides are identified
with 06 FDR and 669 phosphopeptides identified with 54 FDR Motif analysis of PKA and
phosphosite validation are performed as well
8
The future of comparative proteomics investigating small sample amounts or PTMs is
promising Further advances in enrichment separations science mass spectrometry
analyzersdetectors and bioinformatics will continue to create more powerful tools that enable
digging deeper in proteomics with both small (mouse CSF) and large (cell extracts) sample
amounts
Methods for Neuropeptide Analysis Using ETD fragmentation and Sample
Preparation
Chapter 6 investigates the utility of ETD fragmentation to sequence endogenous large
neuropeptides and labile tyrosine sulfation in the crustacean Callinectes sapidus The sinus
gland (SG) of the crustacean is a well-defined neuroendocrine site that produces numerous
hemolymph-borne agents including the most complex class of endocrine signaling moleculesmdash
neuropeptides One such peptide is the crustacean hyperglycemic hormone (CHH) precursor-
related peptide (CPRPs) which was not previously sequenced Electron transfer dissociation
(ETD) is used for sequencing of intact CPRPs due to their large size and high charge state In
addition ETD is used to sequence a sulfated peptide cholecystokinin (CCK)-like peptide in the
lobster Homarus americanus using a salt adduct Collectively this chapter presents two
examples of the utility of ETD in sequencing neuropeptides with large molecular weight or with
labile modifications
Chapter 7 reports a protocol on improving recovery of minute quantities of peptides by
adding methanol and a salt modifier (NaCl) to molecular weight cut-off membrane-based
centrifugal filters (MWCO) to enrich sub-microgram peptide quantities In some biological
9
fluids such as CSF the endogenous peptide content is very low and using pure water to perform
the MWCO separation produces too much sample loss Using a neuropeptide standard
bradykinin sample loss is reduced over two orders of magnitude with and without undigested
protein present The presence of bovine serum albumin (BSA) undigested protein and the
bradykinin standard lends evidence that sample loss occurs because of the MWCO and not the
presence of the protein Additionally a BSA tryptic digestion is presented where twenty-seven
tryptic peptides are identified from MALDI mass spectra after enriching with methanol while
only two tryptic peptides are identified after the standard MWCO protocol The strategy
presented in Chapter 7 greatly enhances recovery from MWCO separation for sub-microg peptide
samples
10
References
1 Tanaka K Waki H Ido Y Akita S Yoshida Y Yoshida T Matsuo T Protein and polymer analyses up to mz 100 000 by laser ionization time-of-flight mass spectrometry Rapid Communications in Mass Spectrometry 1988 2 (8) 151-153
2 Fenn J B Mann M Meng C K Wong S F Whitehouse C M Electrospray ionization for mass spectrometry of large biomolecules Science 1989 246 (4926) 64-71
3 Domon B Aebersold R Mass spectrometry and protein analysis Science 2006 312 (5771) 212-7
4 Whitehouse C M Dreyer R N Yamashita M Fenn J B Electrospray interface for liquid chromatographs and mass spectrometers Anal Chem 1985 57 (3) 675-9
5 Wilm M Mann M Analytical properties of the nanoelectrospray ion source Anal Chem 1996 68 (1) 1-8
6 Karas M Bahr U Dulcks T Nano-electrospray ionization mass spectrometry addressing analytical problems beyond routine Fresenius J Anal Chem 2000 366 (6-7) 669-76
7 Gulpinar M Yegen B The physiology of learning and memory Role of peptides and stress Curr Protein Pept Sci 2004 5 (6) 457-473
8 Ogren S O Kuteeva E Elvander-Tottie E Hokfelt T Neuropeptides in learning and memory processes with focus on galanin In Eur J Pharmacol 2010 Vol 626 pp 9-17
9 Strand F Neuropeptides general characteristics and neuropharmaceutical potential in treating CNS disorders Prog Drug Res 2003 61 1-37
10 Li W Chang M Peng Y-L Gao Y-H Zhang J-N Han R-W Wang R Neuropeptide S produces antinociceptive effects at the supraspinal level in mice Regul Pept 2009 156 (1-3) 90-95
11 Wu C-H Tao P-L Huang E Y-K Distribution of neuropeptide FF (NPFF) receptors in correlation with morphine-induced reward in the rat brain Peptides 2010 31 (7) 1374-1382
12 Tsutsui K Bentley G E Kriegsfeld L J Osugi T Seong J Y Vaudry H Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin new key neuropeptides controlling reproduction J Neuroendocrinol 2010 22 (7) 716-727
13 Piper D C Upton N Smith M I Hunter A J The novel brain neuropeptide orexin-A modulates the sleep-wake cycle of rats Eur J Neurosci 2000 12 (2) 726-730
14 Tsuneki H Wada T Sasaoka T Role of orexin in the regulation of glucose homeostasis - Tsuneki - 2009 - Acta Physiologica - Wiley Online Library Acta Physiol 2010
15 Kageyama H Takenoya F Shiba K Shioda S Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding Neuropeptides 2010 44 (2) 133-138
16 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide family of aplysia J Neurosci 2002 22 (17) 7797-7808
11
17 Jensen J Regulatory peptides and control of food intake in non-mammalian vertebrates Comp Biochem Physiol Part A Mol Integr Physiol 2001 128 (3) 469-477
18 Billimoria C P Li L Marder E Profiling of neuropeptides released at the stomatogastric ganglion of the crab Cancer borealis with mass spectrometry J Neurochem 2005 95 (1) 191-199
19 Cape S S Rehm K J Ma M Marder E Li L Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster Homarus americanus J Neurochem 2008 105 (3) 690-702
20 Cruz Bermuacutedez N D Fu Q Kutz Naber K K Christie A E Li L Marder E Mass
spectrometric characterization and physiological actions of GAHKNYLRFamide a novel FMRFamide‐like peptide from crabs of the genus Cancer J Neurochem 2006 97 (3) 784-799
21 Grashow R Brookings T Marder E Reliable neuromodulation from circuits with variable underlying structure Proc Natl Acad Sci USA 2009 106 (28) 11742-11746
22 Ma M Szabo T M Jia C Marder E Li L Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins Peptides 2009 30 (9) 1660-1668
23 Chen R Hui L Cape S S Wang J Li L Comparative Neuropeptidomic Analysis of Food Intake via a Multi-faceted Mass Spectrometric Approach ACS Chem Neurosci 2010 1 (3) 204-214
24 Li L Sweedler J V Peptides in the brain mass spectrometry-based measurement approaches and challenges Annu Rev Anal Chem 2008 1 451-483
25 Ma M Wang J Chen R Li L Expanding the crustacean neuropeptidome using a multifaceted mass spectrometric approach J Proteome Res 2009 8 (5) 2426-2437
26 Slattery M G Heideman W Coordinated regulation of growth genes in Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
12
Figure 1 Capabilities and performances of certain mass analyzers Check marks indicate
availability check marks in parentheses indicate optional + ++ and +++ indicate possible or
moderate goodhigh and excellentvery high respectively Adapted with permission from
reference 3
13
Figure 2 Synthesis processing and release of neuropeptides (a) Cartoon showing two
interacting neurons (b) The synthesis of neuropeptides in the neuronal organelles and their
transport down the axon to the presynaptic terminal is depicted (c) Shows neuropeptide release
and interaction with the dendrite of the post-synaptic cell (Adapted from PhD thesis by Dr
Stephanie Cape)
14
Figure 3 Schematic showing location of the neuronal tissues being analyzed in the MS studies
of neuropeptides in Callinectes sapidus The SGs (sinus glands located in the eyestalks of the
crab) and the POs (pericardial organs located in the chamber surrounding the heart) release
neurohormones into the hemolymph (crab circulatory fluid) The brain is near the STNS
(stomatogastric nervous system neural network that controls the motion of the gut and foregut)
which has direct connections to the STG (stomatogastric ganglion) The STG is located in an
artery so it is continually in contact with hemolymph (Adapted from PhD thesis by Dr Robert
Sturm)
15
Chapter 2
Mass Spectrometry-based Proteomics and Peptidomics for Biomarker
Discovery and the Current State of the Field
Adapted from ldquoMass spectrometry-based proteomics and peptidomics for systems biology and
biomarker discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
16
Abstract
The scientific community has shown great interest in the field of mass spectrometry-based
proteomics and peptidomics for its applications in biology Proteomics technologies have
evolved to produce large datasets of proteins or peptides involved in various biological and
disease progression processes producing testable hypothesis for complex biological questions
This review provides an introduction and insight to relevant topics in proteomics and
peptidomics including biological material selection sample preparation separation techniques
peptide fragmentation post-translation modifications quantification bioinformatics and
biomarker discovery and validation In addition current literature and remaining challenges and
emerging technologies for proteomics and peptidomics are presented
17
Introduction
The field of proteomics has seen a huge expansion in the last two decades Multiple factors have
contributed to the rapid expansion of this field including the ever evolving mass spectrometry
instrumentation new sample preparation methods genomic sequencing of numerous model
organisms allowing database searching of proteomes improved quantitation capabilities and
availability of bioinformatic tools The ability to investigate the proteomes of numerous
biological samples and the ability to generate future hypothesis driven experiments makes
proteomics and biomarker studies exceedingly popular in biological studies today In addition
the advances in post-translational modification (PTM) analysis and quantification ability further
enhance the utility of mass spectrometry (MS)-based proteomics A subset of proteomics
research is devoted to profiling and quantifying neurologically related proteins and endogenous
peptides which has progressed rapidly in the past decade This review provides a general
overview as outlined in Figure 1 of proteomics technology including methodological and
conceptual improvements with a focus on recent studies and neurological biomarker studies
Biological Material Selection
The choice of biological matrix is an important first step in any proteomics analysis The
ease of sample collection (eg urine plasma or saliva) versus usefulness or localization of
sample (eg specific tissue or proximity fluid) needs to be evaluated early on in a study design
Plasma derived by centrifugation of blood to remove whole cells is a very popular
choice in proteomics due to the high protein content (~65 mg ml1) and the ubiquitous nature of
blood in the body and the ability to obtain large sample amounts or various time points without
the need to sacrifice the animal or to perform invasive techniques Plasma is centrifuged
18
immediately after sample collection unlike serum where coagulation needs to occur first To
obtain plasma blood is collected in a tube with an anticoagulant added (ETDA heparin or
citrate) and centrifuged but previous reports have shown variable results when heparin has been
used as an anticoagulant2 Human Proteome Organization (HUPO) specifically recommends the
anticoagulants EDTA or citrate to treat plasma3 4
One of the primary concerns with plasma is
degradation of the protein content via endogenous proteases found in the sample5 One way to
address this problem is the use of protease inhibitors In addition freezethaw cycles need to be
minimized to prevent protein degradation and variability6 7
Plasma proteomics has seen
extensive coordinated efforts to start assessing the diagnostic needs using plasma8 HUPO also
has established a public human database for plasma and serum proteomics from 35 collaborating
labratories9 Large dynamic range studies have been performed on plasma with a starting sample
amount of 2625 microl (1575 mg) resulting in 3654 proteins identified with a sub 5 false
discovery rate10
The large dynamic range spanning across eleven orders of magnitude as visualized in
Figure 2 is one of the biggest obstacles in plasma proteomics Figure 2 also shows that as lower
abundance proteins are investigated the origins of those identified proteins are more diverse than
the most abundant proteins Recent mining of the plasma proteome showed an ability to search
for disease biomarker applications across seven orders of magnitude In addition the tissue of
origin for the identified plasma proteins were identified and its origin was more diverse as the
protein concentration decreased11
Plasma has been used as a source for biomarker studies such
as colorectal cancer12 13
cardiovascular disease14
and abdominal aortic aneurysm15
Even
though the blood brain barrier prevents direct blood to brain interaction neurological disorders
such as Alzheimerrsquos disease (AD) have had their proteomes studied using plasma16
19
An alternative sample derived from blood is serum which is plasma allowed to coagulate
instead of adding anti-coagulates The time for coagulation is usually 30 minutes and during that
time significant and random degradation from endogenous proteases can occur The additional
variability caused from the coagulation process can change the concentration of multiple
potentially valuable biomarkers As biodiversity between samples or organisms is a challenging
endeavor additional sample variability due to serum generation may be undesirable but serum is
still currently being used for biomarker disease studies17
Serum has been used to compare the
proteome differences in neurological diseases such as AD Parkinsonrsquos disease and amyotrophic
lateral sclerosis and a review can be found elsewhere discussing the subject18
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy of brain or spinal cord
in evaluating diseases of the central nervous system and has been used for studies in neurological
disorders due to being a rich source of neuro-related proteins and peptides19
The protein
composition of the most abundant proteins in CSF is well defined and numerous studies exist to
broaden the proteins identified20-22
CSF has an exceedingly low protein content (~04 μgμL)
which is ~100 times lower than serum or plasma and over 60 of the total protein content in
CSF consists of a single protein albumin23-25
In addition the variable concentrations of proteins
span up to twelve orders of magnitude further complicating analysis and masking biologically
relevant proteins to any given study26
One of the highest number of identified proteins is from
Schutzer et al with 2630 non-redundant proteins from 14 mL of pooled human CSF This study
involved the removal of highly abundant proteins by performing IgY-14 immunodepletion
followed by two dimensional (2D) liquid chromatography (LC) separation27
Studies have also
been performed to characterize individual biomarkers or complex patterns of biomarkers in
various diseases in the CSF28 29
One potential pitfall of CSF proteomic analysis is
20
contamination from blood which can be identified by counting red blood cells present or
examining surrogate markers from blood contamination other than hemoglobin such as
peroxiredoxin catalase and carbonic anhydrase30
A proof of principle CSF peptidomics study
identified numerous endogenous peptides associated with the central nervous system which can
be used as a bank for neurological disorder studies31
Numerous recent reports highlighted the
utility of CSF analysis for biomarker studies in AD32 33
medulloblastoma34
both post-mortem
and ante-mortem35
Cellular lysates offer the distinct advantage to work with a cell line yeast or bacteria
with large amounts of proteins available for analysis36 37
with Saccharomyces cerevisiae being
the most common cell lysate38 39
Other cell lines are also used including HeLa40
and E coli41
The ability to obtain milligrams of proteins easily to scale up experiments without animal
sacrifice offers a clear advantage in biological sample selection Current literature supports
cellular lysate as a valued and sought after source of proteins for large scale proteomics
experiments because of the ability to assess treatments conditions and testable hypotheses42-44
Cellular lysate from rat B104 neuroblastoma cell line was used as an in vitro model for cerebral
ischemia and showed abundance changes in multiple proteins involved in various neurological
disorders45
Other Sources of Biological Samples
Urine
The urine proteome appears to be another attractive reservoir for biomarker discovery
due to the relatively low complexity compared with the plasma proteome and the noninvasive
collection of urine Urine is often considered as an ideal source to identify biomarkers for renal
21
diseases due to the fact that in healthy adults approximately 70 of the urine proteome originate
from the kidney and the urinary tract 46
thus the use of urine to identify neurological disorders is
neglected However strong evidence have shown that proteins that are associated with
neurodegenerative diseases can be excreted in the urine47-49
indicating the application of urine
proteomics could be a useful approach to the discovery of biomarkers and development of
diagnostic assays for neurodegenerative diseases However the current view of urine proteome
is still limited by factors such as sample preparation techniques and sensitivity of the mass
spectrometers There has been a tremendous drive to increase the coverage of urine proteome
In a recent study Court et al compared and evaluated several different sample preparation
methods with the objective of developing a standardized robust and scalable protocol that could
be used in biomarkers development by shotgun proteomics50
In another study Marimuthu et al
reported the largest catalog of proteins in urine identified in a single study to date The
proteomic analysis of urine samples pooled from healthy individuals was conducted by using
high-resolution Fourier transform mass spectrometry A total of 1823 proteins were identified
of which 671 proteins have not been previously reported in urine 51
Saliva
For diagnosis purposes saliva collection has the advantage of being an easy and non-
invasive technique The recent studies on saliva proteins that are critically involved in AD and
Parkinsonrsquos diseases suggested that saliva could be a potentially important sample source to
identify biomarkers for neurodegenerative diseases Bermejo-Pareja et al reported the level of
salivary Aβ42 in patients with mild AD was noticeably increased compared to a group of
controls 52
In another study Devic et al identified two of the most important Parkinsons
22
disease related proteinsmdash α-synuclein (α-Syn) and DJ-1 in human saliva 53
They observed that
salivary α-Syn levels tended to decrease while DJ-1 levels tended to increase in Parkinsons
disease The published results from this study also suggest that α-Syn might correlate with the
severity of motor symptoms in Parkinsons disease Due in part to recent advancements in MS-
based proteomics has provided promising results in utilizing saliva to explore biomarkers for
both local and systemic diseases 54 55
the further profiling of saliva proteome will provide
valuable biomarker discovery source for neurodegenerative diseases
Tissue
Compared to body fluids such as plasma serum and urine where the proteomic analysis
is complicated by the wide dynamic range of protein concentration the analysis of tissue
homogenates using the well-established and conventional proteomic analysis techniques has the
advantage of reduced dynamic range However the homogenization and extraction process may
suffer from the caveat that spatial information is lost which would be inadequate for the
detection of biomarkers whose localization and distribution play important roles in disease
development and progression Matrix-assisted laser desorptionionization (MALDI) imaging
mass spectrometry (IMS) is a method that allows the investigation of a wide range of molecules
including proteins peptides lipids drugs and metabolites directly in thin slices of tissue 56-59
Because this technology allows for identification and simultaneous localization of biomolecules
of interests in tissue sections linking the spatial expression of molecules to histopathology
MALDI-IMS has been utilized as a powerful tool for the discovery of new cancer biomarker
candidates as well as other clinical applications60 61
The utilization of MALDI-IMS for human
or animal brain tissue to identify or map the distribution of molecules related to
neurodegenerative diseases were also recently reported62 63
23
Secretome
There has been an increasing interest in the study of proteins secreted by various cells
(the secretomes) from tissue-proximal fluids or conditioned media as a potential source of
biomarkers Cell secretomes mainly comprise proteins that are secreted or are shed from the cell
surface and these proteins can play important role in both physiological processes (eg cell
signaling communication and migration) and pathological processes including tumor
angiogenesis differentiation invasion and metastasis In particular the study of cancer cell
secretomes by MS based proteomics has offered new opportunities for cancer biomarker
discovery as tumor proteins may be secreted or shed into the bloodstream and could be used as
noninvasive biomarkers The latest advances and challenges of sample preparation sample
concentration and separation techniques used specifically for secretome analysis and its clinical
applications in the discovery of disease specific biomarkers have been comprehensively
reviewed64 65
Here we only highlight the proteomic profiling of neural cells secretome that has
been applied to neurosciences for a better understanding of the roles secreted proteins play in
response to brain injury and neurological diseases The LC-MS shotgun identification of
proteins released by astrocytes has been recently reported66-68
In these studies the changes
observed in the astrocyte secretomes induced by inflammatory cytokines or cholinergic
stimulation were investigated6667
Alternatively our group performed 2D-LC separation and
included cytoplasmic protein extract from astrocytes as a control to identify cytoplasmic protein
contaminants which are not actively secreted from cells68
Sample Preparation
24
Proteomic analysis and biomarker discovery research in biological samples such as body
fluids tissues and cells are often hampered by the vast complexity and large dynamic range of
the proteins Because disease identifying biomarkers are more likely to be low-abundance
proteins it is imperative to remove the high-abundance proteins or apply enrichment techniques
to allow detection and better coverage of the low-abundance proteins for MS analysis Several
strategies including depletion and protein equalizer approach have been used during sample
preparation to reduce sample complexity69 70
and the latest advances of these methods have been
reviewed by Selvaraju et al 71
Alternatively the complexity of biological samples can be
reduced by capturing a specific subproteome that may have the biological information of interest
The latter strategy is especially useful in the biomarker discovery where the changes in the
proteome are not solely reflected through the concentration level of specific proteins but also
through changes in the post-translational modifications (PTMs) Here we will mainly discuss
the enrichment of phosphoproteinpeptides glycoproteinpeptides and sample preparation for
peptidomics and membrane proteins
Phosphoproteomics
Phosphorylation can act as a molecular switch on a protein by turning it on or off within
the cell It is thought that up to 30 of the proteins can be phosphorylated72
and it plays
significant roles in such biological processes as the cell cycle and signal transduction73
Currently tens of thousands of phosphorylation sites can be proposed using analytical methods
available today74 75
The amino acids that are targeted for phosphorylation studies are serine
threonine and tyrosine with the abundance of detection decreasing typically in that order Other
25
amino acids have been reported to be phosphorylated but traditional phosphoproteomics
experiments ignore these rare events76
In a typical large-scale phosphoproteomics experiment the sample size is usually in
milligram amounts to account for the low stoichiometry of phosphorylated proteins The large
amount of protein is then digested typically with trypsin but alternatively experiments have
been performed with Lys-C digestion to produce large enzymatic peptides The larger peptides
produced from Lys-C render higher charged peptides during electrospray ionization (ESI) and
allow improved electron-based fragmentation to determine specific sites of phosphorylation77
From the pool of peptides phosphopeptides must be enriched otherwise they will be masked by
the vast number and higher ionization efficiency of non-phosphorylated peptides The two most
common enrichment techniques are immobilized metal ion affinity chromatography (IMAC) and
metal oxide affinity chromatography (MOAC) TiO2 being the most common oxide used for this
purpose A recent study reported that phosphorylation of neuronal intermediate filament proteins
in neurofibrillary tangles are involved in Alzheimerrsquos disease78
Glycoproteomics
Protein glycosylation is one of the most common and complicated forms of PTM Types
of protein glycosylation in eukaryotes are categorized as either N-linked where glycans are
attached to asparagine residues in a consensus sequence N-X-ST (X can be any amino acid
except proline) via an N-acetylglucosamine (N-GlcNAc) residue or the O-glycosylation where
the glycans are attached to serine or threonine Glycosylation plays a fundamental role in
numerous biological processes and aberrant alterations in protein glycosylation are associated
with neurodegenerative disease states such as Creutzfeld-Jakob Disease (CJD) and AD79 80
26
Due to the low abundance of glycosylated forms of proteins compared to non-glycosylated
proteins it is essential to enrich glycoproteins or glycopeptides in complex biological samples
prior to MS analysis Two of the most common enrichment methods used in glycoproteomics are
lectin affinity chromatography (LAC) and hydrazide chemistry The detailed methodologies of
LAC hydrazide chemistry and other enrichment methods in glycoproteomics have been
extensively reviewed in the past81 82
In particular LAC is of great interest in studies of
glycosylation alterations as markers of AD and other neurodegenerative diseases due to its recent
applications in brain glycoproteomics83
Our group has utilized multi-lectin affinity
chromatography containing concanavalin A (ConA) and wheat germ agglutinin (WGA) to enrich
N-linked glycoproteins in control and prion-infected mouse plasma84
This method enabled us to
identify a low-abundance glycoprotein serum amyloid P-component (SAP) PNGase F digestion
and Western blotting validation confirmed that the glycosylated form of SAP was significantly
elevated in mice with early prion infection and it could be potentially used as a diagnostic
biomarker for prion diseases
Membrane proteins
Membrane proteins play an indispensable role in maintaining cellular integrity of their
structure and perform many important functions including signaling transduction intercellular
communication vesicle trafficking ion transport and protein translocationintegration85
However due to being relatively insoluble in water and low abundance it is challenging to
analyze membrane proteins by traditional MS-based proteomics approaches Numerous efforts
have been made to improve the solubility and enrichment of membrane proteins during sample
preparation Several comprehensive studies recently covered the commonly used technologies in
27
membrane proteomics and different strategies that circumvent technical issues specific to the
membrane 86-90
Recently Sun et al reported using 1-butyl-3-methyl imidazolium
tetrafluoroborate (BMIM BF4) an ionic liquid (IL) as a sample preparation buffer for the
analysis of integral membrane proteins (IMPs) by microcolumn reversed phase liquid
chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MSMS)
The authors compared BMIM BF4 to the other commonly used solvents such as sodium dodecyl
sulfate methanol Rapigest and urea but they found that the number of identified IMPs from rat
brain extracted by ILs was significantly increased The improved identifications could be due to
the fact that BMIM BF4 has higher thermal stability and thus offered higher solubilizing ability
for IMPs which provided better compatibility for tryptic digestion than traditionally used solvent
systems38
In addition to characterization of membrane proteome the investigation of PTMs on
membrane proteins is equally important for characterization of disease markers and drug
treatment targets Phosphorylations and glycosylations are the two most important PTMs for
membrane proteins In many membrane protein receptors the cytoplasmic domains can be
phosphorylated reversibly and function as signal transducers whereas the receptor activities of
the extracellular domains are mediated via N-linked glycosylation Wiśniewski et al provides an
informative summary on recent advances in proteomic technology for the identification and
characterization of these modifications91
Our group has pioneered the development of detergent
assisted lectin affinity chromatography (DALAC) for the enrichment of hydrophobic
glycoproteins using mouse brain extract92
We compared the binding efficiency of lectin affinity
chromatography in the presence of four commonly used detergents and determined that under
certain concentrations detergents can minimize the nonspecific bindings and facilitate the
elution of hydrophobic glycoproteins In summary NP-40 was suggested as the most suitable
28
detergent for DALAC due to the higher membrane protein recovery glycoprotein recovery and
membranous glycoprotein identifications compared to other detergents tested In a different
study on mouse brain membrane proteome Zhang et al reported an optimized protocol using
electrostatic repulsion hydrophilic interaction chromatography (ERLIC) for the simultaneous
enrichment of glyco- and phosphopeptides from mouse brain membrane protein preparation93
Using this protocol they successfully identified 544 unique glycoproteins and 922 glycosylation
sites which were significantly higher than those using the hydrazide chemistry method
Additionally a total of 383 phosphoproteins and 915 phosphorylation sites were identified
suggesting that the ERLIC separation has the potential for simultaneous analysis of both glyco-
and phosphoproteomes
Peptidomics
Peptidomics can be loosely defined as the study of the low molecular weight fraction of
proteins encompassing biologically active endogenous peptides protein fragments from
endogenous protein degradation products or other small proteins such as cytokines and signaling
peptides Studies can involve endogenous peptides94
peptidomic profiling33
and de novo
sequencing of peptides95 96
Neuropeptidomics focuses on biologically active short segments of
peptides and have been investigated in numerous species including Rattus97 98
Mus musculus99
100 Bovine taurus
101 Japanese quail diencephalon
102 and invertebrates
103-106 The isolation of
peptides is typically performed through molecular weight cut-offs from either biofluids such as
CSF plasma or tissue extracts If the protein and peptide content is high such as for tissue or cell
lysates protein precipitation can be done via high organic solvents and the resulting supernatant
can be analyzed for extracted peptides where extraction solvent and conditions could have a
29
significant effect on what endogenous peptides are extracted from tissue107
A comparative
peptidomic study of human cell lines highlights the utility of finding peptide signatures as
potential biomarkers108
A thorough review of endogenous peptides and neuropeptides is beyond
the scope of this review and an excellent review on this topic is available elsewhere109
Fractionation and Separation
The mass spectrometer has a limited duty cycle and data dependent analysis can only
scan a limited number of mz peaks at any given time In addition significant ion suppression
can occur if there is a difference in concentration between co-eluting peptides or if too many
peptides co-elute Therefore one of the biggest challenges in biomarker discovery is the
complexity of the sample and the presence of high-abundance proteins in body fluids such as
CSF serum and plasma In addition to the removal of the most abundant proteins by
immunodepletion the reduction of the complexity of the sample by further fractionation is
indispensable to facilitate the characterization of unidentified biomarkers from the low
abundance proteins Traditionally used techniques for complex protein analysis include gel
based fractionation methods such as two-dimensional gel electrophoresis (2D-GE) and its
variation two-dimensional differential gel electrophoresis (2D-DIGE) or non-gel based such as
one- or multidimensional liquid chromatography (LC) and microscale separation techniques
such as capillary electrophoresis (CE)
2D-GE MS has been widely used as a powerful tool to separate proteins and identify
differentially expressed proteins ever since 2D gels were coupled to mass spectrometry In 2D-
GE MS thousands of proteins can be separated on a single gel according to pI and molecular
weight Individual protein spots that show differences in abundance between different samples
30
can then be excised from the gel digested into peptides and analyzed by MALDI MS or by
liquid chromatography tandem mass spectrometry (LC-MSMS) for protein identification The
introduction of 2D-DIGE adds a quantitative strategy to gel electrophoresis by enabling multiple
protein extracts to be separated on the same 2D gel thus providing comparative analysis of
proteomes in complex samples In 2D-DIGE protein extracts from two different conditions and
an internal standard can be labeled with fluorescent dyes for example Cy3 Cy5 and Cy2
respectively prior to two-dimensional gel electrophoresis Compared to traditional 2D-GE 2D-
DIGE provides the clear advantage of overcoming the inter-gel variation problem 110
Proteomic
profiling of CSF by 2D-GE and 2D-DIGE has led to the identification of putative biomarkers in
multiple neurological disorders For example Brechlin et al reported an optimized 2-DIGE
protocol profiled CSF from 36 CJD patients The applicability of their approach was proven by
the detection of known CJD biomarkers such as 14-3-3 protein neuron-specific enolase lactate
dehydrogenase and other proteins that are potentially relevant to CJD 111
In another study to
identify novel CSF biomarkers for multiple sclerosis CSF from 112 multiple sclerosis patients
and control individuals were analyzed by 2D-GE MS for comparative proteomics Ten potential
multiple sclerosis biomarkers were selected for validation by immunoassay 112
These
methodologies sample preparation techniques and applications of 2D-DIGE in
neuroproteomics were reviewed by Diez et al113
Although 2D gel provides excellent resolving
power and capability to visualize abundance changes there are some limitations to the method
For example gel based separation is not suitable for low abundance proteins extremely basic or
acidic proteins very small or large proteins and hydrophobic proteins114 115
Complementary to gel-based approaches shotgun proteomics coupled to LC have
become increasingly popular in proteomic research because they are reproducible highly
31
automated and capable of detecting low abundance proteins Furthermore another advantage of
LC-MS shotgun proteomics is the suitability for isotope labeling for protein quantification which
is reviewed in a later section In shotgun proteomics a protein mixture is digested and resulting
peptides are separated by LC prior to tandem MS fragmentation to identify different proteins by
peptide sequencing The most common separation for shotgun proteomics peptidomics or top-
down proteomics experiments use low-pH reversed phase (RP) C18 C8 or C4 columns RPLC
is well established which provides high resolution desalts the sample which can interfere with
ionization and the mobile phase is compatible with ESI Nanoscale C18 columns allow for
separation and introduction of sub microgram samples If larger amounts of sample are
available two dimensional separations are usually preferred to greatly enhance the coverage of
the investigated proteome which will be discussed in depth later It is preferable to have an
orthogonal separation method and since RP separates via hydrophobicity strong cation exchange
(SCX) was the original choice due to its separation by charge MudPIT (multidimensional
protein identification technology) usually refers to the use of SCX as the first phase of separation
and is a well-established platform116
SCX has the advantage over RP separation technologies to
effectively remove interfering detergents from the sample SCX separation is not based solely
off charge and hydrophobicity contributes to elution therefore a small amount of organic
modifier usually 10-15 is added to lessen the hydrophobicity effects117
The addition of
organic modifiers needs to be minimized otherwise binding to the C18 trap cartridge or C18
column will be reduced if performed on-line SCX can be used for PTMs and offers specific
applications for proteomic studies and an excellent current review is offered on this subject
elsewhere118
An alternative MudPIT separation scheme employing high pH RPLC as the first
phase of separation and low pH RPLC in the second dimension (RP-RP) has been successfully
32
applied to the proteomic analysis of complex biological samples119 120
The advantage of using
RP as the first dimension is the higher resolution for separation and better compatibility with
down-stream MS detection by eliminating salt Song et al reported a phosphoproteome analysis
based on this 2D RP-RP coupling scheme121
Hydrophilic interaction chromatography (HILIC) employs distinct separation modality
where the retention of peptides is increased with increasing polarity122
The loading of sample is
done by high organic and eluted by increasing the percentage of the aqueous phase or polarity of
the mobile phase opposite from RPLC thus establishing orthogonality of the two separation
modes123
HILIC has quickly become a very useful method and is actively used for proteomic
experiments124
for increased sensitivity125
phosphoproteomics126
glycoproteins127
and
quantification studies128
An alternative and modification to HILIC is ERLIC which adds an
additional mode of separation by electrostatic attraction An earlier study using ERLIC
demonstrated the ability to separate phosphopeptides from non-phosphorylated peptides at
pH=2129
A recent study looking into changes in the phosphoproteome of Marekrsquos Disease
applied ERLIC to chicken embryonic fibroblast lysate identifying only 13 phosphopeptides
out of all the identified peptides Due to the lack of isolation of phosphopeptides from ERLIC
the investigators performed immobilized metal affinity chromatography (IMAC) enrichment on
the fractions increasing identification of phosphopeptides over 50 fold130
A comparative study
of ERLIC to HILIC and SCX following TiO2 phospho-enrichment reported that
SCXgtERLICgtHILIC for phosphopeptide identifications126
Recent developments in instrumentation to combine LC with ion mobility spectrometry
(IMS) and MS (LC-IMS-MS) offered more advantages than conventional LC due to the rapid
high-resolution separations of analytes based on their charge mass and shape as reflected by
33
mobility in a given buffer gas The mobility of an ion in a buffer gas is determined by the ionrsquos
charge and its collision cross-section with the buffer gas The methodologies of IMS separations
and the application of LC-IMS-MS for the proteomics analysis of complex systems including
human plasma have been reviewed by Clemmerrsquos group131-133
They proposed a method that
employs intrinsic amino acid size parameters to obtain ion mobility predictions which can be
used to rank candidate peptide ion assignments and significantly improve peptide identification
134
Although 2D gel and LC are routinely used as separation techniques in MS-based
proteomics capillary electrophoresis (CE) has received increasing attention as a promising
alternative due to the fast and high-resolution separation it offers CE has a wide variety of
operation modes among which capillary zone electrophoresis (CZE) and capillary isoelectric
focusing (CIEF) have the greatest potential applications in MS-based proteomics thus will be
highlighted here CZE separates analytes by their charge-to-size ratios in buffers under a high
electrical field and is often used as the final dimension prior to MS analysis while the separation
feature of CIEF is based on isoelectric point and this technique is more suitable to be used as the
first dimension separation Detailed description of different CEndashMS interfaces sample
preconcentration and capillary coating to minimize analyte adsorption could be found in several
reviews135-141
CE technique is complementary to conventional LC in that it is suitable for the
analysis of polar and chargeable compounds Dovichirsquos group conducted proteomic analysis of
the secreted protein fraction of Mycobacterium marinum which has intermediate protein
complexity142
The tryptic digests were either analyzed by UPLC-ESI-MSMS in triplicates or
prefactionated by RPLC followed by CZE-ESI-MSMS It was demonstrated that the two
methods identified similar numbers of peptides and proteins within similar analysis times
34
However CZE-ESI-MSMS analysis of the prefractionated sample tended to identify more
peptides that are basic and have lower mz values than those identified by UPLC-ESI-MSMS
This analysis also presented the largest number of protein identifications by using CE-MSMS
suggesting the effectiveness of prefractionation of complex samples by LC method prior to CZE-
ESI-MSMS The use of CIEF as the first dimension of separation provides both sample
concentration and excellent resolving power The combination of CIEF and RPLC separation
has been applied to the proteomic analyses where the amount of protein sample is limited and
cannot meet the requirement of minimal load amount for 2D LC-MSMS143 144
So far CE-MS
has been widely applied to the proteomic analysis of various biological samples such as urine145
146 CSF
147 blood
148 frozen tissues
149 and the formalin-fixed and paraffin-embedded (FFPE)
tissue samples150
The recent CEndashMS applications to clinical proteomics have been summarized
in several reviews135 151 152
Protein Quantification
In 2D gel electrophoresis the quantitative analysis of protein mixtures is performed on
the gel by comparing the intensity of the protein stain The development of 2D-DIGE eliminated
the gel-to-gel variation and greatly improved the quantitative capability and reliability of 2D gel
methodology110
However the accuracy of 2D gel based protein quantification suffers from the
limitations that a seemingly single gel spot often contains multiple proteins and the difficulty of
detecting proteins with extreme molecular weights and pI values as well as highly hydrophobic
proteins such as membrane proteins Therefore non-gel based shotgun proteomics technology is
more suitable for accurate and large-scale protein identification and quantification in complex
samples Briefly the quantification in non-gel based shotgun proteomics can be categorized into
35
two major approaches stable isotope labeling-based and label-free methods The common
strategies for quantitative proteomic analysis are reviewed and summarized in Table 1
Isotope labeling methods
Because stable isotope-labeled peptides have the same chemical properties as their
unlabeled counterparts the two peptides within a mixture should exhibit identical behaviors in
MS ionization The mass difference introduced by isotope labeling enables the detection of a
pair of two distinct peptide masses by MS within the mixture and allowing for the measurement
of the relative abundance differences between two peptides Depending on how isotopes are
incorporated into the protein or peptide these labeling methods can be divided into two groups
In vitro chemical derivatization techniques which incorporate a label or tag into the peptide or
protein during sample preparation metabolic labeling techniques which introduce the isotope
label directly into the organism via isotope-enriched nutrients from food or media
1 In vitro derivatization techniques
There are multiple methods to introduce heavy isotopes into proteins or peptides in vitro
The commonly used strategies include 18
O 16
O enzymatic labeling Isotope-Coded Affinity Tag
(ICAT) Tandem Mass Tags (TMTs) and Isobaric Tags for Relative and Absolute Quantification
(iTRAQ) The 18
O labeling method enzymatically cleaves the peptide bond with trypsin in the
presence of 18
O-enriched H2O and introduces 4-Da mass shift in the tryptic peptides153
The
advantages of this method include 18
O-enriched water is extremely stable tryptic peptides will
be labeled with the same mass shift secondary reactions inherent to other chemical labeling can
be avoided Conversely widespread use of 18
O-labeling has been hindered due to the difficulty
of attaining complete 18
O incorporation and the lack of robustness154 155
Currently ICAT
36
TMTs and iTRAQ methods are extensively used in quantitative proteomics In ICAT cysteine
residues are specifically derivatized with a reagent containing either zero or eight deuterium
atoms as well as a biotin group for affinity purification of cysteine-containing peptides156 157
The advantage of ICAT is that the affinity purification via biotin moiety can facilitate the
detection of low-abundance cysteine-containing peptides In addition the mass difference
introduced by labeling increases mass spectral complexity with quantification from the different
precursor masses done by MS and peptide identification being achieved through tandem MS
(MSMS) This added complexity from different peptide masses was addressed by using isobaric
labeling methods such as TMTs and iTRAQ 158 159
where the same peptides in different samples
are isobaric after tagging and appear as single mz in MS scans thus enhancing the peptide limit
of detection and reducing the MS scan complexity Isobaric labeling reagents are composed of a
primary amine reactive group and an isotopic reporter group linked by an isotopic balancer group
for the normalization of the total mass of the tags The reporter group serves for quantification
purpose since it is cleaved during collision-induced dissociation (CID) to yield a characteristic
isotope-encoded fragment Moreover isobaric labeling methods allow the comparison of
multiple samples within a single experiment Recently a 6-plex version of TMTs was
reported160
and iTRAQ enables up to eight samples to be labeled and relatively quantified in a
single experiment161
8-plex iTRAQ reagents have been used for the comparison of complicated
biological samples such as CSF in the studies of neurodegenerative diseases 162
Recently our
group developed a novel N N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2)
tagging reagents with high quantitation efficacy DiLeu has the advantage of synthetic simplicity
and greatly reduced synthesis cost compared to TMTs and iTRAQ163
Xiang et al demonstrated
that DiLeu produced comparable iTRAQ ability for protein sequence coverage (~43) and
37
quantitation accuracy (lt15) for tryptically digested proteins More importantly DiLeu
reagents could promote enhanced fragmentation of labeled peptides thus allowing more
confident peptide and protein identifications
2 In Vivo Metabolic Labeling
Metabolic processes can also be employed for the incorporation of stable-isotope labels
into the proteins or organisms by enriching culture media or food with light or heavy versions of
isotope labels (2H
13C
15N) The advantage of in vivo labeling is that metabolic labeling does
not suffer from incomplete labeling which is an inherent drawback for in vitro derivatization
techniques In addition metabolic labeling occurs from the start of the experiment and proteins
with light or heavy labels are simultaneously extracted thus reducing the error and variability of
quantification introduced during sample preparation The most widely used strategy for
metabolic labeling is known as stable-isotope labeling of amino acids in cell culture (SILAC)
which was introduced by Mann and co-workers164 165
In SILAC one cell population is grown
in normal or light media while the other is grown in heavy media enriched with a heavy
isotope-encoded (typically 13
C or 15
N) amino acid such as arginine or leucine Cells from the
two populations are then combined proteins are extracted digested and analyzed by MS The
relative protein expression differences are then determined from the extracted ion
chromatograms from both the light and heavy peptide forms SILAC has been shown to be a
powerful tool for the study of intracellular signal transduction In addition this technique has
recently been applied to the quantitative analysis of phosphotyrosine (pTyr) proteomes to
characterize pTyr-dependent signaling pathways166 167
38
Labe-free quantification
Although various isotope labeling methods have provided powerful tools for quantitative
proteomics several limitations of these approaches are noted Labeling increases the cost and
complexity of sample preparation introduces potential errors during the labeling reaction It also
requires a higher sample concentration and complicates data processing and interpretation In
addition so far only TMTs and iTRAQ allow the comparison of multiple (up to eight) samples
simultaneously The comparison of more than eight samples in a single experiment cannot be
achieved by isotope labeling In order to address these concerns there has been significant
interest in the development of label-free quantitative approaches Current label-free
quantification methods for MS-based proteomics were developed based on the observation that
the chromatographic peak area of a peptide168 169
or frequency of MSMS spectra170
correlating
to the protein or peptide concentration Therefore the two most common label-free
quantification approaches are conducted by comparing (i) area under the curve (AUC) of any
given peptides171 172
or (ii) by frequency measurements of MSMS spectra assigned to a protein
commonly referred to as spectral counting173
Several recent reviews provided detailed and
comprehensive knowledge comparing label-free methods with labeling methods data processing
and commercially available software for label-free quantitative proteomics174-177
Dissociation Techniques
The vast majority of proteomic experiments have proteins or peptides being identified by
two critical pieces of data obtained from the mass spectrometer The first is the precursor ion
identified by its mz which is informative to the mass of the peptide being analyzed The second
is the use of tandem mass spectrometry to fragment or dissociate the precursor ion and record the
39
generated fragment ion pattern to discern the amino acid sequence The three most popular
dissociation or fragmentation techniques for peptides are CID electron-transfer dissociation
(ETD) and high-energy collision dissociation (HCD) A recent study on the human plasma
proteome demonstrated that combined fragmentation techniques enhance coverage by providing
complementary information for identifications CID enabled the greatest number of protein
identifications while HCD identified an additional 25 proteins and ETD contributed an
additional 13 protein identifications178
ETDECD
Electron capture dissociation (ECD) 179
preceded ETD but ECD was developed for use
in a Penning trap for Fourier transform ion cyclotron resonance (FTICR) mass spectrometers
ECD requires the ion of interest to be in contact with near-thermal electrons and for the electron
capture event to occur on the millisecond time scale but the time scale is inadequate for electron
trapping in Paul traps or quadrupoles in the majority of mass spectrometers180
ETD involves a
radical anion like fluoranthene with low electron affinity to be transferred to peptide cation
which results in more uniform cleavage along the peptide backbone The cation accepts an
electron and the newly formed odd-electron protonated peptide undergoes fragmentation by
cleavage of the N-Cα bond which results in fragmentation ions consisting of c- and zbull-type
product ions The uniformed cleavage results in reduced sequence discrimination to labile bonds
such as PTMs and also provides improved sequencing for larger peptides compared to CID181
The realization that larger peptides produced better MSMS quality spectra compared to CID led
to a decision tree analysis strategy where peptide charge states and size determined whether the
precursor peptide would be fragmented with CID or ETD182
One of the main benefits of
ETDECD is the ability to sequence peptides with labile PTMs such as phosphorylation77 183
40
sulfation184
glycosylation185
ubiquitination186
and histone modifications187
ETD also has the
benefit of providing better sequence information on larger neuropeptides when compared to
CID188
However a thorough analysis suggested that CID still yielded more peptideprotein
identifications than ETD in large scale proteoimcs189
HCD
High energy collision dissociation (HCD)190
is an emerging fragmentation technique that
offers improved detection of small reporter ions from iTRAQ-based studies191 192
HCD is
performed at a higher energy in a collision cell instead of an ion trap like CID thus HCD does
not suffer from the low-mass cutoff limitation Furthermore HCD offers enhanced
fragmentation efficiency assisting in MSMS spectra interpretation and protein identification193
A major drawback for HCD is that the spectral acquisition times are up to two-fold longer due to
increased ion requirement for Fourier transform detection in the orbitrap194
HCD has been
reported to increase phosphopeptide identifications over CID74
but in a different study CID was
reported to offer more phosphopeptide identifications over HCD194
Work has also been done to
transfer the decision tree analysis for HCD which basically switches CID with HCD claiming
better quality data determined by higher Mascot scores with more peptide identifications195
MSE
Data dependent acquisition (DDA) is the most commonly used ion selection process in
mass spectrometers for proteomic experiments An alternative process which does not have ion
selection nor switch between MS and MSMS modes is termed MSE MS
E is a data independent
mode and does not require precursor ions of a significant intensity to be selected for MSMS
analysis196
A data independent mode decouples the mass spectrometer choosing which
precursor ions to fragment and when the ions are fragmented MSE works by a low or high
41
energy scan and no ion isolation is occurring The low energy scan is where the precursor ion is
not fragmented and the high energy scan allows fragmentation The resulting mix of precursor
and fragmentation ions is then detected simultaneously197
The data will then need to be
deconvoluted using a proprietary time-aligned algorithm that is discussed elsewhere198
The
continuous data independent acquisition allows multiple MSMS spectra to be collected during
the natural analyte peak broadening observed in chromatography which provides more data
points for AUC label-free quantification In addition lower abundance peptides can be
sequenced as more MSMS spectra are collected throughout the elution of an LC peak allowing
better signal averaging for smaller analyte peak of interest during coelution and reducing
sampling bias in typical DDA experiments where only more abundant peaks can be selected for
fragmentation
A comparison of spiked internal protein standards into a complex protein digest provided
evidence that MSE was comparable to DDA analysis in LC-MS
199 MS
E has been used for label
free proteomics of immunodepleted serum in large scale proteomics samples200
In addition
MSE was performed for the characterization of human cerebellum and primary visual cortex
proteomes Hundreds of proteins were identified including many previously reported in
neurological disorders201
MSE is quickly becoming a versatile data acquisition method recently
used in such studies as cancer cells202
schizophrenia203
and pituitary proteome discovery204
The usefulness of MSE as an unbiased data acquisition method is being assimilated into multiple
proteomics studies including studies involving neurological disorders
Data Analysis
42
One of the major bottlenecks in non-targeted proteomic experiments is how to handle the
enormous amount of data obtained Database searches biostatistical analysis de novo
sequencing PTM validation all have their place and multiple available platforms are available
If the organism being studied has had its genome sequenced databases can be created
with a list of proteins in the FASTA format to be used in database searching There are
numerous database searching algorithms for sequence identification of MSMS data including
Mascot205
Sequest206
Xtandem207
OMSSA208
and PEAKS209
These searching algorithms are
performed by matching MSMS spectra and precursor mass to sequences found within proteins
How well the actual spectra match the theoretical spectra determines a score which is unique to
the searching algorithm and usually can be extrapolated to the probability of a random hit
Recently a database has been developed for PTM analysis by the use of the program SIMS210
Specifically for phosphopeptides Ascorersquos algorithm scans the MSMS data to determine the
likelihood of correct phosphosite identification from the presence of site identifying product
ions211
If the organism that is being analyzed has not had its genome sequenced and no (or very
limited) FASTA database is available a homology search can be performed using SPIDER212
available with PEAKS software Alternatively individual MSMS spectrum can be de novo
sequenced but software is available to perform automated de novo sequencing of numerous
spectra (PEAKS208
DeNovoX and PepSeq)
For large-scale protein identifications the false discovery rate (FDR) must be established
by the searching algorithm and that is accomplished by re-searching the data with a false
database created by reversing or scrambling the amino acid sequence of the original database
used for the protein search Any hits from the false database will contribute to the FDR and this
value can be adjusted usually around 1 An additional layer of confidence in the obtained data
43
can be achieved in shotgun proteomics experiments by removing all the proteins that are
identified by only one peptide
Once a set of confident proteins or peptides have been generated from database
searching bioinformatic analysis or biostatistical analysis is needed Numerous software
packages are available for different purposes FLEXIQuant is an example for absolute
quantitation of isotopically labeled protein or peptides of interest213
FDR analysis of
phosphopeptides or other specific PTMs can be adjusted with such software as Scaffold
providing data consisting only of a specific modification214
Bioinformatic tools such as
Scaffold or ProteoIQ also include gene ontology (GO) analysis which can classify identified
proteins by three categories cellular component molecular function or biological process
Custom bioinformatics programs can also be developed and are often useful in various proteomic
studies including biomarker discovery in neurological diseases215
More detailed review of
bioinformatics in peptidomics216
and proteomics217
can be found elsewhere
Validation of Biomarkers by Targeted Proteomics
The validation of putative biomarkers identified by MS-based proteomic analysis is often
required to provide orthogonal analysis to rule out a false positive by MS and providing
additional evidence for the biomarker candidate(s) from the study for future potential clinical
assays At present antibody-based assays such as Western blotting ELISA and
immunochemistry are the most widely used methods for biomarker validation Although accurate
and well established these methods rely on protein specific antibodies for the measurement of
the putative biomarker and could be difficult for large-scale validation of all or even a subset of a
long list of putative protein biomarkers typically obtained by MS-based comparative proteomic
44
analysis Large scale validation is impractical due to the cost for each antibody the labor to
develop a publishable Western blot or ELISA and the antibody availability for certain proteins
As an alternative strategy quantitative assays based on multiple-reaction monitoring (MRM) MS
using a triple quadrupole mass spectrometer have been employed in biomarker verification
MRM is the most common use of MSMS for absolute quantitation It is a hypothesis
driven experiment where the peptide of interest and its subsequent fragmentation pattern must be
known prior to the quantitative MRM experiments MRM involves selecting a specific mz (first
quadrupole) to be isolated for fragmentation (second quadrupole) followed by one or more of
the most intense fragment ions (third quadrupole) being monitored The ability to quantitate and
thus validate the proteins or peptides as potential biomarkers is achieved by performing MRM on
isotopically labeled reference peptide for targeted peptideprotein of interest The main obstacle
for quantification of peptides is interference and ion suppression effects from co-eluting
substances Since the isotopically labeled and native peptide will co-elute the same interference
and ion suppression will occur for both peptides and thus correcting these interfering effects
Peptides need to be systematically chosen for a highly sensitive and reproducible MRM
experiment to ensure proper validation of putative biomarkers Peptides require certain intrinsic
properties which include an mz within the practical mass detection range for the instrument and
high ionization efficiency If the desired peptide to be quantified is derived from a digestion
then peptides that have detectable incomplete digestion or missed cleavage site can be a major
source of variability Peptides with a methionine and to a lesser extent tryptophan are
traditionally removed from consideration from MRM quantitative experiments due to the
variable nature of the oxidation that can occur In addition if chromatographic separation is
performed the retention behavior of the peptide must be well behaved with little tailing effects
45
eluting late causing broadening of the peak and even irreversible binding to the column As an
example hydrophilic peptides being eluted off a C18 column may exhibit the previously
described concerns and a different chromatographic separation will need to be explored for
improved limits of detection quantitation and validation To determine consistent peptide
detection or usefulness of certain peptides databases such as Proteomics Database218
PRIDE219
PeptideAtlas220
have been developed to compile proteomic data repositories from initial
discovery experiments
After the peptide is selected for analysis the proper MRM transitions need to be selected
to optimize the sensitivity and selectivity of the experiment It is common for investigators to
select two or three of the most intense transitions for the proposed experiment It is imperative
that the same instrument is used for the determination of transition ions as different mass
spectrometers may have a bias towards different fragment ions
MRM experiments are still highly popular experiments for hypothesis directed
experiments221
biomarker analysis222
and validation223
Validation of putative biomarkers is
increasingly becoming a necessary step when performing large scale non-hypothesis driven
proteomics experiments The traditional validation techniques of ELISA Western blotting and
immunohistochemistry are still used but MRM experiments are becoming an attractive
alternative for validation of putative biomarkers due to its enhanced throughput and specificity
Current work is still being performed to both expand the linear dynamic range224
and
sensitivity225
of MRM A recent endeavor to increase the sensitivity for MRM experiments was
accomplished by ldquoPulsed MRMrdquo via the use of an ion funnel trap to enhance confinement and
accumulation of ions The authors claimed an increase by 5-fold for peak amplitude and a 2-3
fold reduction in chemical background225
46
Remaining Challenges and Emerging Technologies
Large sample numbers for mass spectrometry analysis
Multiple conventional studies in proteomics have been performed on a single or a few
biological samples As bio-variability can be exceedingly high the need for larger sample sizes
is currently being investigated Prentice et al used a starting point of 3200 patient samples
from the Womenrsquos Health Institute (WHI) to probe the plasma proteome using MS for
biomarkers The study did not test the 3200 patient samples by MS because even a simple one
hour one dimensional RP analysis on a mass spectrometer would take months of instrument time
for uninterrupted analysis Instead the authors pooled 100 samples together to bring the total
number of pooled samples to 32 To provide relevant plasma biomarkers the samples were then
subjected to immunodepletion 2-D protein separation (96 fractions total) and then 1-D RPLC of
tryptic peptide separation on-line interface to a mass spectrometer The large sample cohorts
help address bio-variability that can be a concern from small sample size proteomic experiments
and provide ample sample amounts to investigate the low abundance proteins226
Hemoglobin-derived neuropeptides and non-classical neuropeptides
Neuropeptides such as neuropeptide Y and enkephalin are short chains of amino acids
that are secreted from a range of neuronal cells that signal nearby cells In contrast non-classical
neuropeptides are termed as neuropeptides or ldquomicroproteinsrdquo which are derived from
intracellular protein fragments and synthesized from the cytosol227
MS was recently used to
determine that hemopressins which are hemoglobin-derived peptides are upregulated in Cpefatfat
mice brains Gelman et al designed an MS experiment to compare hemoglobin-derived
47
peptides comparing the brain blood and heart peptidome in mice The authors provided data
that specific hemoglobin peptides were produced in the brain and were not produced in the
blood Certain alpha and beta hemoglobin peptides were also up regulated in the brain for
Cpefatfat
mice and bind to CB1 cannabinoid receptors228
As discussed earlier in the review
peptidomics and specifically neuropeptidomics are popular fields of study utilizing MS and non-
classical neuropeptides is an exciting emerging area of research that could further expand the
diversity of cell-cell signaling molecules
Ultrasensitive mass spectrometry for single cell analysis
In addition to large scale analysis MS-based proteomics and peptidomics are making
progress into ultrasensitive single cell analysis The most successful MS-based techniques for
single cell analysis was performed with MALDI and studies that have been performed on
relatively large neurons are reviewed elsewhere229
The ultrasensitive MS analysis is currently
directed towards single cell analysis of smaller cells including cancer cells The first challenge
in single cell analysis is the isolation and further sample preparation to yield relevant data
Collection and isolation of a cell type can be accomplished using antibodies for fluorescence
activated cell sorting (FACS) and immune magnetic separation FACS works by flow cytometry
sorting cells by a laser that excites a fluorescent tag that is attached to an antibody Immune
magnetic separation allows separation by antibodies with magnetic properties such as
Dynabeads230
One exciting study combining FACS and MS termed mass cytometry This
technology works by infusing a droplet into an inductively coupled plasma mass spectrometer
(ICP-MS) containing a single cell bound to antibodies chelated to transition elements allowing a
quantifying response between single cells231
Clearly the future of single cell analysis for
48
biomarker analysis and proteomics is encouraging and has the potential to be an emerging field
in MS-based proteomics and peptidomics
Laserspray ionization (LSI)
Laserspray ionization (LSI) is an exciting new method to produce multiply charged mass
spectra from MALDI that is nearly identical to ESI232-234
Recently it has been reported that LSI
can be performed in lieu of matrix to produce a total solvent-free analysis234
The benefits of
being able to generate multiply charged peptides without any solvent may offer advantages
including MS analysis of insoluble membrane proteins or hydrophobic peptides avoidance of
chemical reactions while in solvents a reduction of sample loss due to liquid sample preparation
and ability to avoid diffusion effects from tissue imaging studies234
The multiply charged peptide and protein ions produced by LSI expand the mass range
for tissue imaging analysis More importantly the multiply-charged peptide ions are amenable
for electron-based fragmentation methods such as ETD or ECD which can be employed in
conjunction with tissue imaging experiments to yield in situ sequencing and identification of
peptides of interest235
Paper spray ionization
Paper spray (PS) is an ambient ionization method which was first reported using
chromatography paper allowing detection of metabolites from dried blood spots The original
method used a cut out piece of paper with a voltage clipped on the back while applying 10 microL of
methanolH2O236
Improvements have been made to this technology to enhance analysis
efficiency with a new solvent 91 dichloromethaneisopropanol (vv) and the use of silica paper
49
over chromatography paper237
Interesting applications or modifications have been made to PS
including direct analysis of biological tissue238
and leaf spray for direct analysis of plant
materials239
but both detect metabolites instead of proteins or peptides Paper spray ionization
was previously shown to enable detection of cytochrome c and bradykinin [2-9] standards in a
proof of principle study240
Clearly the utility of PS analysis in proteomics and peptidomics is
yet to be explored
niECD
New fragmentation techniques have been investigated for their utility in proteomics and
peptidomics including a recently reported negative-ion electron capture dissociation (niECD)
Acidic peptides which usually contain PTMs such as phosphorylation or sulfonation are often
difficult to be detected as multiply charged peptides in the positive ion mode As discussed
earlier multiply charged peptides are required for ECDETD fragmentation The fragmentation
of niECD is accomplished by a multiply negatively charged peptide adding an electron The
resulting fragmentation of multiply sulfated and phosphorylated peptide and protein standards
showed no sulfate loss and preserved phosphorylation site The resulting fragmentation pattern
from niECD was also improved in the peptide anions and provides a new strategy for de novo
sequencing with PTM localization241
Conclusions and Perspectives
Proteomics methodologies have produced large datasets of proteins involved in various
biological and disease progression processes Numerous mass spectrometry-based proteomics
and peptidomics tools have been developed and are continuously being improved in both
50
chromatographic or electrophoretic separation and MS hardware and software However several
important issues that remain to be addressed rely on further technical advances in proteomics
analysis When large proteomes consisting of thousands of proteins are analyzed and quantified
dynamic range is still limited with more abundant proteins being preferentially detected
Development and optimization of chemical tagging reagents that target specific protein classes
maybe necessary to help enrich important signaling proteins and assess cellular and molecular
heterogeneity of the proteome and peptidome Furthermore a significant bottleneck in
usefulness of proteomics research is the ability to validate the results and provide clear
significant biological relevance to the results The idea of P4 medicine242 243
is an attractive
concept where the four Prsquos stand for predictive preventive personalized and participatory
Proteomics is one of the critical ldquoomicsrdquo fields and has led to the development of enabling
innovative strategies to P4 medicine244
A goal of P4 medicine is to assess both early disease
detection and disease progression in a person A simplified example of how proteomics fits into
P4 medicine is that certain brain-specific proteins could be used for diagnosis with
presymptomatic prion disease244
The concept of proteomic experiments providing an individual
biomarker is becoming more obsolete with the revised vision being a biomolecular barcode that
could potentially be ldquoscannedrdquo or be a fingerprint for a specific disease or early onset to that
disease being closer to reality An excellent review on what biomarker analysis can do for true
patients is available245
Proteomics can also generate new hypothesis that can be tested by classical biochemical
approaches If a disease has an unknown pathogenesis proteomics is a good starting point to try
to assemble putative markers that can lead to further hypothesis for evaluation If a particular
protein or PTM is associated with a disease state either qualitatively or quantitatively potential
51
treatments could target that protein of interest or investigators could monitor that protein or
PTM during potential treatments of the disease Proteomics has expanded greatly over the last
few decades with the goal of providing revealing insights to some of the most complex
biological problems currently facing the scientific community
Acknowledgements
Preparation of this manuscript was supported in part by the University of Wisconsin Graduate
School Wisconsin Alzheimerrsquos Disease Research Center Pilot Grant and a Department of
Defense Pilot Award LL acknowledges an H I Romnes Faculty Fellowship
52
Figure 1 A summary of general workflows of biomarker discovery pipeline by MS-based
proteomic approaches
Biological sample (CSF blood urine saliva cell
lysate tissue homogenates secreted proteins etc)
Protein extraction Sample pretreatment
2D-GE2D-DIGE MS 1D or 2D LC-MSMS
MALDI-IMS
Identification of
differentially
expressed proteins
Protein identification
Potential biomarkers
Biomarker validation
- Antibody based immunoassays
- MRM
Quantitative analysis
- Isotope labeling
- Label free
Identification and
localization of
differentially expressed
biomolecules
Intact tissue
Sample preparation Slice frozen tissues
thaw-mounted on plate
Apply Matrix
53
Figure 2 Tissue expression and dynamic range of the human plasma proteome A pie chart
representing the tissue of origin for the high abundance proteins shows that the majority of
proteins come from the liver (A) Conversely the lower abundance plasma proteins have a much
more diverse tissue of origin (B and C) The large dynamic range of plasma proteins is presented
and the proteins can be grouped into three categories (classical plasma proteins tissue leakage
products interleukinscytokines) (D) Adapted from Zhang et al11
and Schiess et al246
with
permission
54
55
Table 1 A summary of the common strategies applied to MS-based quantitative proteomic
analysis
Gel based Stable isotope labeling Label free
2D-GE
2D-DIGE 110
In vitro derivatization
18O
16O
153
ICAT 156
TMT 159
iTRAQ 158
Formaldehyde 247
ICPL 248
In vivo metabolic labeling
14N
15N
249
SILAC 164
AUC measurement 169 172
Spectral counting 173
AUC Area Under Curve ICAT Isotope-Coded Affinity Tag TMT Tandem Mass Tags iTRAQ Isobaric Tags for
Relative and Absolute Quantification ICPL Isotope Coded Protein Labeling SILAC Stable Isotope Labeling by
Amino Acids in Cell Culture Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
56
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Anal Chem 2008 80 (20) 7846-54
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211 Beausoleil S A Villen J Gerber S A Rush J Gygi S P A probability-based
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storing protein identification data J Proteome Res 2004 3 (6) 1234-42
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Hermjakob H PRIDE new developments and new datasets Nucleic Acids Res 2008 36
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Mass Spectrometry for Quantification of Heat Shock Proteins Anal Chem 2012
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validation in blood specimens by selected reaction monitoring mass spectrometry of N-
glycosites Methods Mol Biol 2011 728 179-94
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natural isotopologue transitions Talanta 2011 87 307-10
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Smith R D Pulsed multiple reaction monitoring approach to enhancing sensitivity of a tandem
quadrupole mass spectrometer Anal Chem 2011 83 (6) 2162-71
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McIntosh M Wang P Buson Busald T Hsia J Jackson R D Rossouw J E Manson J
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228 Gelman J S Sironi J Castro L M Ferro E S Fricker L D Hemopressins and
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profiling Trends Biotechnol 2000 18 (4) 151-60
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spectrometry Anal Chem 2009 81 (16) 6813-22
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atmospheric pressure Anal Chem 2011 83 (11) 4076-84
235 Inutan E D Richards A L Wager-Miller J Mackie K McEwen C N Trimpin
S Laserspray ionization a new method for protein analysis directly from tissue at atmospheric
pressure with ultrahigh mass resolution and electron transfer dissociation Mol Cell Proteomics
2010 10 (2) M110 000760
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substrate for paper-spray analysis of therapeutic drugs in dried blood spots Anal Chem 84 (2)
931-8
238 Wang H Manicke N E Yang Q Zheng L Shi R Cooks R G Ouyang Z
Direct analysis of biological tissue by paper spray mass spectrometry Anal Chem 83 (4) 1197-
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243 Hood L Friend S H Predictive personalized preventive participatory (P4) cancer
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(2) 111-21
72
245 Belda-Iniesta C de Castro J Perona R Translational proteomics what can you do for
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N-metabolic labelingmass
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Rapid Commun Mass Spectrom 2002 16 (14) 1389-97
73
Chapter 3
Protein changes in immunodepleted cerebrospinal fluid from transgenic
mouse models of Alexander disease detected using mass spectrometry
Adapted from ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse
models of Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P
Messing A Li L Submitted
74
ABSTRACT
Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range
spanning at least nine orders of magnitude in protein content and is in direct contact with the
brain A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the
low volumes of CSF that are obtainable from mice As a model system in which to test this
approach we utilized transgenic mice that over-express the intermediate filament glial fibrillary
acidic protein (GFAP) These mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we report the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates were performed to address animal variability as well as reproducibility in
mass spectrometric analysis Relative quantitation was performed using distributive normalized
spectral abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins
with significant changes in the CSF of GFAP transgenic mice has been identified with validation
from ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
75
INTRODUCTION
Alexander disease (AxD) is a fatal neurogenetic disorder caused by heterozygous point
mutations in the coding region of GFAP (encoding glial fibrillary acidic protein)1 The hallmark
diagnostic feature of this disease is the accumulation of astrocytic cytoplasmic inclusions known
as Rosenthal fibers containing GFAP Hsp27 αB-crystallin and other components2-5
Although
several potential treatment strategies6-8
are under investigation clinical trial design is hampered
by the absence of a standardized clinical scoring system or means to quantify lesions in MRI
that could serve to monitor severity and progression of disease One solution to this problem
would be the identification of biomarkers in readily sampled body fluids as indirect indicators of
disease
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy site for brain or spinal
cord in evaluating diseases of the central nervous system The protein composition of CSF is
well defined at least for the most abundant species of proteins and numerous studies exist that
characterize individual biomarkers or complex patterns of biomarkers in various diseases9 10
GFAP itself is present in CSF (albeit at much lower levels than in brain parenchyma) and in one
study of three Alexander disease patients its levels were markedly increased11
Whether an
increase in CSF GFAP will be a consistent finding in Alexander disease or whether other useful
biomarkers for this disease could be identified through an unbiased analysis of the CSF
proteome is not yet known
The rarity of Alexander disease makes analysis of human samples difficult However
mouse models exist that replicate key features of the disease such as formation of Rosenthal
fibers Unfortunately mouse CSF poses particular problems for proteomic studies and there is
76
an urgent need for technical improvements for dealing with this fluid For instance collection
from an adult mouse typically yields ~10 μL of CSF often with some contamination by blood12
To further complicate analysis CSF has an exceedingly low protein content (~04 μgμL) with
over 60 of the total protein content consisting of a single protein albumin13 14
A number of
techniques have been developed to remove albumin from biological samples including Cibacron
Blue15
IgG immunodepletion16
and IgY immunodepletion17-19
IgY which is avian in origin
offers reduced non-specific binding and increased avidity when compared to IgG antibodies from
rabbits goats and mice20-23
One widely used IgY cocktail is IgY-14 which contains fourteen
specific antibodies specific for albumin IgG transferrin fibrinogen α1-antitrypsin IgA IgM
α2-macroglobulin haptoglobin apolipoproteins A-I A-II and B complement C3 and α1-acid
glycoprotein Since existing protocols for IgY-14 depletion are optimized for use with large
volumes of serum new protocols must be developed to permit its use with the low volumes of a
low protein fluid represented by mouse CSF
Various improvements have also taken place in the field of proteomic analysis that could
facilitate analysis of mouse CSF Data dependent tandem mass spectrometry followed by
quantification of proteins is used in standard shotgun proteomics24-29
Several methods now exist
for introducing quantitation into mass spectrometry including stable isotope labeling30-32
isobaric tandem mass tags33 34
and spectral counting35 36
Spectral counting which is a
frequency measurement that uses MSMS counts of identified peptides as the metric to enable
protein quantitation is attractive because it is label-free and requires no additional sample
preparation Finally recent advances in spectral counting has produced a data refinement
strategy termed normalized spectral abundance factor (NSAF)37 38
and further developed into
distributive NSAF (dNSAF) to address issues with peptides shared by multiple proteins39
77
To identify potential biomarkers in AxD we report a novel scaled-down version of IgY
antibody depletion strategy to reduce the complexity and remove high abundance proteins in
mouse CSF samples The generated spectral counts data were then subjected to dNSAF natural
log data transformation and t-test analysis to determine which proteins differ in abundance when
comparing GFAP transgenics and controls with multiple biological and technical replicates
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) urea (gt99) sodium chloride (997) and ammonium
bicarbonate (certified) were purchased from Fisher Scientific (Fair Lawn NJ) Deionized water
(182 MΩcm) was prepared with a Milli-Q Millipore system (Billerica MA) Optima LCMS
grade acetonitrile and water were purchased from Fisher Scientific (Fair Lawn NJ) DL-
Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega
(Madison WI) Formic acid (ge98) was obtained from Fluka (Buchs Switzerland)
Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris ge999 ) ammonium formate
(ge99995) glycine (ge985) and IgY-14 antibodies were purchased from Sigma-Aldrich
(Saint Louis MO)
Mice
Transgenic mice over-expressing the human GFAP gene (Tg737 line) were maintained
as hemizygotes in the FVBN background and genotyped via PCR on DNA prepared from tail
samples as described previously40
The mice were housed on a 14-10 light-dark cycle with ad
libitum access to food and water All procedures were conducted using protocols approved by
the UW-Madison IACUC
78
CSF collection
CSF was collected from mice as described previously12
Briefly mice were anesthetized
with avertin (400-600 mgkg ip) A midline sagittal incision was made over the dorsal aspect
of the hindbrain and three muscle layers carefully peeled back to expose the cisterna magna The
membrane covering the cisterna magna was pierced with a 30 gauge needle and CSF was
collected immediately using a flexible plastic pipette Approximately ten microliters of CSF was
collected per animal All samples used for MS analysis showed no visible contamination of
blood
Enzyme-linked immunosorbent assay (ELISA)
A sandwich ELISA was used to quantify GFAP8 Briefly a microtiter plate was coated
with a cocktail of monoclonal antibodies (Covance SMI-26R) Plates were blocked with 5
milk before addition of sample or standards diluted in PBS with Triton-X and BSA A rabbit
polyclonal antibody (DAKO Z334) was used to detect the GFAP followed by a peroxidase
conjugated anti-rabbit IgG antibody (Sigma A6154) secondary antibody The peroxidase activity
was detected with SuperSignal ELISA Pico Chemiluminescent Substrate (PIERCE) and
quantified with a GloRunner Microplate Luminometer Values below the biological limit of
detection (16ngL) were given the value 16ngL before multiplying by the dilution factor
Immunodepletion of abundant proteins
Currently there are no commercial immunodepletion products available for use with CSF
and to address the low protein content of this fluid (~04 microgmicroL) Therefore 100 microL of
purchased IgY-14 resin was coupled with a Pierce Spin Cup using a paper filter from Thermo
Scientific (Waltham MA) CSF samples from either transgenics or controls were first pooled to
100 microL and then added to 100 microL of 20 mM Tris-HCl 300 mM pH 74 (2x dilution buffer) and
79
allowed to mix with the custom IgY-14 depletion spin column on an end-over-end rotor for 30
minutes at 4oC The sample was then centrifuged at 04 rcf for 45 seconds with an Eppendorf
Centrifuge 5415D (Hamburg Germany) The antibodies were then washed with 50 microL 1x
dilution buffer vortexed for 5 minutes centrifuged at 04 rcf for 45 seconds and the flow through
was collected for tryptic digestion The antibodies were then stripped of the bound proteins with
four 025 mL washes of 01 M glycine pH 25 neutralized with two 025 mL washes of 01 M
Tris-HCl pH 80 and washed once with 025mL of 1x dilution buffer The immunodepletion
protocol was repeated for each transgenic or control pooled 100 microL sample (N=3)
Preparation of tryptic digests
The immunodepleted pooled mouse CSF samples (200 microL total volume) were
concentrated to 10 microL using a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA)
To each sample 8 microL of 133 M urea and 1 microL of 050 M DTT were added and allowed to
incubate at 37oC for one hour After incubation 27 microL of 055 M IAA was added for
carboxymethylation and the sample was allowed to incubate for 15 minutes in the dark To
quench the IAA 1 microL of 050 M DTT was added and allowed to react for 10 minutes To
perform trypsin digestion 70 microL of 50 mM NH4HCO3 was then added along with 025 microg
trypsin which had previously been dissolved in 50 mM acetic acid at a concentration of 05
microgmicroL Digestion was performed overnight at 37oC and quenched by addition of 25 microL of 10
formic acid The tryptic peptides were then subjected to solid phase extraction using a Varian
Omix Tip C18 100 microL (Palo Alto CA) Peptides were eluted with 50 ACN in 01 formic
acid concentrated and reconstituted in 30 microL H2O in 01 formic acid
RP nanoLC separation
80
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto an Agilent
Technologies Zorbax 300 SB-C18 5 microm 5x03 mm trap cartridge (Santa Clara CA) at a flow
rate of 5 microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm
Atlantis dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B
at 250 nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
81
range of 300-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot41
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt mus musculus
(house mouse) database (version 575) False positive analyses42
were calculated using an
automatic decoy option of Mascot Results from the Mascot results were reported using
Proteinscape 21 and technical replicates were combined and reported as a protein compilation
using ProteinExtractor (Bruker Daltonics Bremen Germany)
Mascot search parameters were as follows Allowed missed cleavages 2 enzyme
trypsin variable modifications carboxymethylation (C) and oxidation (M) peptide tolerance
plusmn12 Da maximum number of 13
C 1 MSMS tolerance plusmn05 Da instrument type ESI-Trap
Reported proteins required a minimum of one peptide with a Mascot score ge300 with bold red
characterization Spectral counts were determined from the number of MSMS spectra identified
from accepted proteins A bold red peptide combines a bold peptide which represents the first
query result from a submitted MSMS spectrum with the red peptide which indicates the top
peptide for the identified protein Requiring one bold red peptide assists in removal of
homologous redundant proteins and further improves protein results In addition requiring one
82
peptide to be identified by a score gt300 removes the ability for proteins to be identified by
multiple low Mascot scoring peptides
Each immunodepleted biological replicate had technical triplicates performed and the
technical triplicates were summed together by ProteinExtractor Peptide spectral counts were
then summed for each protein and subjected to dNSAF analysis Details for this method can be
found elsewhere37 39
but briefly peptide spectral counts are summed per protein (SpC) based on
unique peptides and a weighted distribution of any shared peptides with homologous proteins
ProteinScape removed 83 homologous proteins found in the current study to bring the total
number of proteins identified to 266 but some non-unique homologous peptides which are
shared by multiple proteins are still present in the resulting 266 remaining proteins To address
these non-unique homologous peptides distributive spectral counting was performed as
described elsewhere39
The dSpC is divided by the proteinrsquos length (L) and then divided by the
summation of the dSpCL from all proteins in the experiment (N) to produce each proteinrsquos
specific dNSAF value
N
i
i
kk
LdSpC
LdSpCdNSAF
1
)(
)()(
The resulting data were then transformed by taking the natural log of the dNSAF value The
means for each protein were calculated using the ln(dNSAF) from N=3 biological replicates and
the whole data set was subjected to the Shapiro-Wilk test for normalityGaussian distribution
performed on the software PAST (Version 198 University of Oslo Norway Osla) The
Shapiro-Wilk test was repeated multiple times to determine a non-zero value for zero spectral
83
counts A non-zero value is required to alleviate the errors of dividing by zero which was
experimentally determined to be 043 The Gaussian data were then subjected to the t-test to
identify statistically significant changes in protein expression
RESULTS AND DISCUSSION
General workflow
Individual CSF samples were manually inspected and samples were only selected that
showed no visual blood contamination Preliminary experiments showed that the maximum
degree of blood contamination estimated from counts of red blood cells in the CSF that was not
visible to the eye was less than 005 by total mass Approximately 10 individual mouse CSF
samples were pooled to achieve the desired 100 μL volume for a single biological replicate The
CSF samples were IgY immunodepleted followed by digestion with trypsin The resulting
digested samples were then de-salted concentrated reconstituted in 30 μL of 01 formic acid
and 5 μL was loaded onto a reversed phase nanoflow HPLC column subjected to a 90 minute
gradient and infused into the amaZon ETD ion trap mass spectrometer The workflow for
mouse CSF analysis is shown in Figure 1 The remainder of the desalted sample was saved for
technical replicates
Immunodepletion for CSF
Currently there are no immunodepletion techniques specifically designed for CSF
Nonetheless the protein profiles between CSF and serum are similar enough to use currently
available immunodepletion techniques designed for serum as a starting point The smallest
commercially available IgY-14 immunodepletion kit is for 15 μL of serum which is equivalent in
protein content to ~15 mL of CSF Therefore for 100 μL CSF one fifteenth of the total IgY-14
84
beads would be used for the equivalent immunodepletion which is 66 μL of proprietary bead
slurry The potential for irreversible binding of abundant proteins to their respective IgY
antibody even after an extra stripping wash and low amounts of total beads made using 66 μL
of IgY-14 bead slurry unrealistic In practice almost doubling the amount of IgY-14 beads (100
μL) used was not sufficient and resulted in albumin and transferrin peptides to be observed in
high abundance (data not shown) The most important protein to immunodeplete is albumin and
it has been reported to be a greater percentage of total CSF protein content (~60) than serum
(~49) in humans14
The difference in albumin percentage supports the results that proprietary
blends of immunodepletion beads for high abundance proteins such as albumin cannot be
scaled down on a strict protein scale and further modifications to the serum immunodepletion
protocol need to be made
Since IgY-14 beads were developed for use with serum all of its protocols need to be
taken into account to modify the protocol for CSF Serum samples should be diluted fifty times
before mixing with the IgY-14 beads but CSF protein concentration is at least one hundred times
lower than serum Therefore CSF is below half the recommended diluted protein concentration
for IgY immunodepletion Consequently multiple steps have been devised to address this
limitation First the binding time between the proteins targeted for removal from the CSF and
IgY-14 beads was doubled during the end-over-end rotor depletion step from the recommended
15 minutes to 30 minutes Second to prevent non-specific protein binding to the antibodies the
CSF samples were diluted with an equal amount (100 μL) of 2X dilution buffer The dilution
buffer of NaCl and Tris-HCl was needed to assist in reducing non-specific binding of proteins to
the 14 antibodies and ensuring the sample is held at physiological pH In addition to these
modifications a doubling of the starting IgY-14 bead slurry to 200 μL provided the desired
85
results Overall this modified protocol results in effective depletion of CSF abundant proteins
using only one-fifth of the antibodies provided by the smallest commercially available platform
Data Analysis
Spectral counting technique for relative quantitation provides numerous benefits for the
study of mouse CSF due to its label-free advantage In contrast isotopic labeling method often
involves additional sample processing that could cause sample loss which is highly undesirable
for low protein content and low volume samples Labeling methods also require a mixing of two
sets of isotopically labeled samples which would effectively increase the sample complexity and
reduce the amount of sample that can be loaded onto the nanoLC column by half In addition
more than two sets of samples can be compared by label-free methods The use of label-free
spectral counting method does not lead to an increase in sample complexity or interference in
quantitation from peptides in the mz window selected for tandem MS Using spectral counting
for relative quantitation however is dependent on reproducible HPLC separation and careful
mass spectrometry operation to minimize technical variability during the study To address
concerns of analytical reliability and run to run deviations base peak chromatograms from two
transgenic IgY-14 immunodepleted biological replicates including two technical replicates of
each were shown to be highly reproducible (Figure 2)
Each biological sample was analyzed in triplicate with the same protocols on the amaZon
ETD with three control and three transgenic samples From the three technical replicates for
each biological replicate the spectral counts of the peptides for the proteins identified were
summed The results from these mouse CSF biological triplicates are shown in Figure 3A for
GFAP overexpressor and Figure 3B for control The summation of spectral counts for each
biological replicate was performed to remove the inherent bias related to data dependent analysis
86
for protein identification One concern in grouping technical replicates is a potential loss of
information regarding analytical variability Figure 4 provides a graphical representation of
variability of technical replicates illustrating the standard deviation of technical replicates with
error bars Figure 4 shows that a statistically changed protein creatine kinase M (A) and an
unchanged protein kininogen-1 (B) have similar variability within (technical replicates) and
between samples (biological replicates) for each protein In addition Figure 4B illustrates that
even with the variability of kininogen-1 the resulting mean shown by the dashed line of control
and transgenic samples were almost equal whereas Figure 4A shows significantly different
expression level of creatine kinase M Performing replicate analysis of each biological sample
(n=3) helps correct for systematic errors deriving from sample handling and pooling of samples
helps reduce random error during the CSF sample collection process
Protein Identification and Spectral Counting Analysis
The data for dNSAF analysis like any mass spectrometry proteomics experiment
requires multiple layers of verification to ensure reliable data Our initial protein identifications
were subjected to a database search using a decoy database from Mascot which resulted in an
average false positive rate below 1 for all the experimental data collected Representative
MSMS spectra between control and GFAP transgenic samples are illustrated in Figure 5
Overall 266 proteins were identified in a combination of control and transgenic samples
(Supplemental Table 1) A total of 349 proteins were initially identified but 83 proteins were
isoforms of previously identified proteins and automatically excluded by ProteinExtractor The
next level of quality control was to only include ln(dNSAF) values from proteins identified by 2
or more unique peptides having a Mascot score of ge300 and observed in two out of three
biological replicates These selection parameters resulted in 106 proteins remaining after
87
dNSAF analysis (Supplemental Table 2) The resulting spectral counts were then subjected to
dSpC in order to account and correct for the systematic error of peptides shared by multiple
proteins (Supplemental Table 3)
It is inevitable in large scale and complex proteomics experiments that some proteins will
be seen in some samples and not others In addition when controls were compared to transgenic
samples as shown in Figure 3C 127 proteins were unique to either control or GFAP transgenic
mice and 139 proteins were seen in both samples From dNSAF equation if the spectral count
is zero the numerator is zero and the value will not be normalized between runs In order to
circumvent the zero spectral counts in dNSAF analysis zero spectral counts must be replaced by
an experimentally determined non-zero value determined to be 043 The 043 spectral counts
for zero spectral counts was calculated by serial Shapiro-Wilk tests to determine the lowest value
(043) for zero spectral counts and maintain a Gaussian distribution for all data sets The 043
value for zero spectral counts in the current study was higher than the 016 reported value for
zero spectral counts in the original NSAF spectral counting study37
Our study may have a
higher zero spectral count value than the previous study because the spectral counting data were
an addition of three technical replicates and three times 016 is close to 043 The normalized
Gaussian data were then subjected to t-test analysis and P-values below 005 are considered as
statistically significant and are presented in Table 1 The proteins with significant up or down
regulation from Table 1 can be further evaluated as how close significant proteins were to a p-
value of 005 such as ganglioside GM2 activator serine protease inhibitor A3N and collagen
alpha-2(I) chain having t-test scores of 0045 0047 and 0043 respectively Proteins exhibiting
a P-value close to 005 were more likely to be highly variable proteins or have smaller fold
changes between control and transgenic samples and thus provide less biological relevancy to
88
future studies The modest change in antithrobin-III which is reduced by 14-fold in transgenic
is included due a low pooled standard deviation in spectral counts
Spectral counting has been analyzed with fold changes derived directly from the average
spectral counts from the technical replicates and then the average of the three biological
replicates We decided to perform additional analysis using fold changes to dig deeper into
proteins that statistically support the null hypothesis from the dNSAF analysis To only pull out
highly confident protein identifications we used the same strict cut-off of two unique peptides
identified per protein as in dNSAF analysis We only accepted proteins with greater than three-
fold change in total spectral counts listed in Table 2 Two proteins contactin-1 (cntn1) and
cathepsin B (CB) illustrate the potential biomarkers missed by dNSAF analysis Cntn1 had zero
spectral count in the transgenic sample and had an average spectral count of 41 in control
samples The lack of any spectral counts in one biological control for cntn1 resulted in a large
standard deviation in the ln(dNSAF) means for the control which resulted in the t-test supporting
the null hypothesis Another example is CB which was detected by numerous spectral counts in
every GFAP transgenic biological replicate with an average spectral count of 97 (Table 2) The
presence of CB in one biological control sample (23 average spectral counts) resulted in a high
standard deviation in the mean of the control samples These examples exhibit a limitation of
dNSAF analysis which could cause a loss of potentially useful information
Previously Identified Proteins with Expression Changes
Previously three proteins have been described as increased in CSF from individual(s)
suffering from AxD In one study a single patientrsquos CSF was found to have elevated levels of
αβ-crystallin and HSP2744
In a second study three patients were reported to have elevated
levels of GFAP with concentrations from 4760 ngL to 30000 ngL (compared to lt175 ngL for
89
controls)11
GFAP was detected in our current study however the other two proteins were not
detected One possibility is that αB-crystallin and Hsp-27 levels in mouse CSF are too low for
detection by MS analysis In addition while the transgenic mice display the hallmark
pathological feature of AxD in the form of Rosenthal fibers they do not have any evident
leukodystrophy and thus may not display the full range of changes in CSF as might be found in
human patients
Creatine Kinase M
Creatine kinase M (M-CK) is a 43 kDa protein whose role is to reversibly catalyze
phosphate transfer between ATP and energy storage compounds M-CK has been primarily
found in muscle tissue and for humans creatine kinase M is diagnostically analyzed in the blood
for myocardial infarction (heart attack) In addition M-CK is also found in Purkinje neurons of
the cerebellum45 46
A related protein creatine kinase B (B-CK) also exhibited an apparent 21
fold increase in transgenic CSF over control but this difference was not statistically different
B-CK concentration is known to be elevated in CSF following head trauma47
or cerebral
infarction48
but decreased in astrocytes in individuals affected by multiple sclerosis49
Cathepsin
The data showed multiple cathepsins were up regulated in the CSF of transgenic mice
when compared to control mice The up regulated cathepsins were S L1 and B isoforms which
are all cysteine proteases Cathepsin S (CS) was never observed in control samples but
observed with an average of 73 spectral counts per analysis Cathepsin L1 (CL1) was up
regulated by 94 times in transgenic mice These two cathepsins exhibited significant changes
using dNSAF and t-test statistics as shown in Table 1 and Cathepsin B (CB) showed a 42 fold
increase in transgenic CSF as shown in Table 2
90
Cathepsins regulate apoptosis in cells50
which is the major mechanism for elimination of
cells deemed by the organism to be dangerous damaged or expendable CL and CB are
redundant in the system as thiol proteases and inhibition of CB by CA-047 causes a diminished
apoptosis response in multiple cell lines51
Intriguingly increased levels of CB or CL are
correlated with poor prognosis for cancer patients and shorter disease-free intervals It is
believed that these proteases degrade the extracellular membrane which allows tumor cells to
invade adjacent tissue and metastasize52
With regards to AxD the up regulation of these
cathepsins may be indicative of the bodyrsquos natural defense and response to Rosenthal fibers
Thus stimulation of these cathepsins may provide a further protective stress response but the
positive correlation between these proteases and cancer highlights the multiple roles of these
proteins in pathological response Alternatively it has been shown that increased CB is involved
with the tumor necrosis factor α (TNFα) induced apoptosis cascade53
The activation of the
TNFα could produce oligodendrocyte toxicity54
with the expression of TNFα being elevated in
tissue samples from mouse models and AxD patients55
The potential for a positive or a negative
effect in increasing or decreasing cathepsins warrant future research with cathepsins and AxD
Contactin-1
Contactin-1 (Cntn1) is a 133 kDa cell surface protein that is highly glycosylated and
belongs to a family of immunoglobulin domain-containing cell adhesion molecues56
Table 2
shows that Cntn1 is down regulated in transgenic mice since spectral counts were only observed
in control mice Cntn1 is expressed in neurons and oligodendrocytes and higher levels were
observed during brain development57
In addition Cntn1 leads to activation of Notch1 which
mediates differentiation and maturation of oligodendrocyte precursor cells (OPC) Although the
mechanism leading to a reduction of Cntn1 in CSF is not known it may reflect a change in
91
astrocyte interactions with either neurons or oligodendrocytes to alter their expression of this
protein
Validation of putative biomarkers and MS proteomics data using ELISA and RNA
microarray data
To further validate the relative protein expression data obtained via MS-based spectral
counting techniques orthogonal immunological and molecular biological approaches have been
examined As GFAP was shown to be elevated in the CSF of transgenic animals we used a
well-defined GFAP ELISA protocol to test its CSF concentration CSF from 8 week old male
mice was collected from both transgenic and control animals Five samples of transgenic CSF
was prepared by pooling four to eight animals for the GFAP ELISA In the case of controls
each sample represents a single animal GFAP concentrations observed by both the MS and
ELISA showed significant increases of GFAP in the CSF when comparing transgenic to control
animals
Another validation of MS spectral counts is observed in a microarray analysis performed
on transgenic mouse olfactory bulb tissue 55
In this paper nine of the proteins found by MS
showed similar gene expression in the microarray (Table 3) It is not surprising that all the genes
observed in the microarray are not the same as the proteins observed by MS analysis Gene
expression and protein synthesis and expression are not always correlated but the similarities
and overlapping trends observed with these two assays are encouraging As shown in Table 3
gene expression analysis also revealed the up regulation of several cathepsin proteins GFAP
and down regulation of contactin-1 in CSF collected from transgenic mice corroborating the
MS-based proteomics results
92
CONCLUSIONS
In this study we have produced a panel of proteins with significant up or down regulation
in the CSF of transgenic GFAP overexpressor mice This mouse model for AxD was consistent
with the previous studies showing elevation of GFAP in CSF The development of a modified
IgY-14 immunodepletion technique for low amounts of CSF was presented This improved
protocol is useful for future investigations to deal with the unique challenges of mouse CSF
analysis Modified proteomics protocols were employed to profile mouse CSF with biological
and technical triplicates addressing the variability and providing quantitation with dNSAF
spectral counting Validation of the MS-based proteomics data were performed using both
ELISA and RNA microarray data to provide further confidence in the changes in the putative
protein biomarkers This study presents three classes of interesting targets for future study in
AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
93
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94
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2412
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preparation and analysis Proteomics 2005 5 (13) 3314-28
18 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag
L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep
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19 Huang L Fang X Immunoaffinity fractionation of plasma proteins by chicken IgY
antibodies Methods Mol Biol 2008 425 41-51
20 Greunke K Braren I Alpers I Blank S Sodenkamp J Bredehorst R Spillner E
Recombinant IgY for improvement of immunoglobulin-based analytical applications Clin
Biochem 2008 41 (14-15) 1237-44
21 Xiao Y Gao X Taratula O Treado S Urbas A Holbrook R D Cavicchi R E
Avedisian C T Mitra S Savla R Wagner P D Srivastava S He H Anti-HER2 IgY
antibody-functionalized single-walled carbon nanotubes for detection and selective destruction
of breast cancer cells BMC Cancer 2009 9 351
22 Liu T Qian W J Mottaz H M Gritsenko M A Norbeck A D Moore R J
Purvine S O Camp D G 2nd Smith R D Evaluation of multiprotein immunoaffinity
subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry
Mol Cell Proteomics 2006 5 (11) 2167-74
23 Hinerfeld D Innamorati D Pirro J Tam S W SerumPlasma depletion with
chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species
J Biomol Tech 2004 15 (3) 184-90
24 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D
Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in
the discovery of candidate protein biomarkers in a diabetes autoantibody standardization
program sample subset J Proteome Res 2008 7 (2) 698-707
25 Ru Q C Zhu L A Silberman J Shriver C D Label-free semiquantitative peptide
feature profiling of human breast cancer and breast disease sera via two-dimensional liquid
chromatography-mass spectrometry Mol Cell Proteomics 2006 5 (6) 1095-104
26 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S
Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-
dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of
Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66
27 Rao P V Reddy A P Lu X Dasari S Krishnaprasad A Biggs E Roberts C T
Nagalla S R Proteomic identification of salivary biomarkers of type-2 diabetes J Proteome
Res 2009 8 (1) 239-45
28 Yu K H Barry C G Austin D Busch C M Sangar V Rustgi A K Blair I A
Stable isotope dilution multidimensional liquid chromatography-tandem mass spectrometry for
pancreatic cancer serum biomarker discovery J Proteome Res 2009 8 (3) 1565-76
29 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422
(6928) 198-207
95
30 Ong S E Blagoev B Kratchmarova I Kristensen D B Steen H Pandey A
Mann M Stable isotope labeling by amino acids in cell culture SILAC as a simple and
accurate approach to expression proteomics Mol Cell Proteomics 2002 1 (5) 376-86
31 Hsu J L Huang S Y Chow N H Chen S H Stable-isotope dimethyl labeling for
quantitative proteomics Anal Chem 2003 75 (24) 6843-52
32 Liu H Sadygov R G Yates J R 3rd A model for random sampling and estimation
of relative protein abundance in shotgun proteomics Anal Chem 2004 76 (14) 4193-201
33 Xiang F Ye H Chen R Fu Q Li L NN-dimethyl leucines as novel isobaric
tandem mass tags for quantitative proteomics and peptidomics Anal Chem 82 (7) 2817-25
34 Ross P L Huang Y N Marchese J N Williamson B Parker K Hattan S
Khainovski N Pillai S Dey S Daniels S Purkayastha S Juhasz P Martin S Bartlet-
Jones M He F Jacobson A Pappin D J Multiplexed protein quantitation in
Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol Cell Proteomics
2004 3 (12) 1154-69
35 Zybailov B Coleman M K Florens L Washburn M P Correlation of relative
abundance ratios derived from peptide ion chromatograms and spectrum counting for
quantitative proteomic analysis using stable isotope labeling Anal Chem 2005 77 (19) 6218-
24
36 Old W M Meyer-Arendt K Aveline-Wolf L Pierce K G Mendoza A Sevinsky
J R Resing K A Ahn N G Comparison of label-free methods for quantifying human
proteins by shotgun proteomics Mol Cell Proteomics 2005 4 (10) 1487-502
37 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M
P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J
Proteome Res 2006 5 (9) 2339-47
38 Mosley A L Florens L Wen Z Washburn M P A label free quantitative
proteomic analysis of the Saccharomyces cerevisiae nucleus J Proteomics 2009 72 (1) 110-20
39 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome
quantitation how to deal with peptides shared by multiple proteins Anal Chem 82 (6) 2272-81
40 Messing A Head M W Galles K Galbreath E J Goldman J E Brenner M
Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice Am J Pathol 1998
152 (2) 391-8
41 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
42 Elias J E Gygi S P Target-decoy search strategy for increased confidence in large-
scale protein identifications by mass spectrometry Nat Methods 2007 4 (3) 207-14
43 You J S Gelfanova V Knierman M D Witzmann F A Wang M Hale J E The
impact of blood contamination on the proteome of cerebrospinal fluid Proteomics 2005 5 (1)
290-6
44 Takanashi J Sugita K Tanabe Y Niimi H Adolescent case of Alexander disease
MR imaging and MR spectroscopy Pediatr Neurol 1998 18 (1) 67-70
45 Hemmer W Wallimann T Functional Aspects of Creatine Kinase in Brain
Developmental Neuroscience 1993 15 (3-5) 249-260
46 Hemmer W Zanolla E Furter-Graves E M Eppenberger H M Wallimann T
Creatine kinase isoenzymes in chicken cerebellum specific localization of brain-type creatine
96
kinase in Bergmann glial cells and muscle-type creatine kinase in Purkinje neurons Eur J
Neurosci 1994 6 (4) 538-49
47 Nordby H K Tveit B Ruud I Creatine kinase and lactate dehydrogenase in the
cerebrospinal fluid in patients with head injuries Acta Neurochirurgica 1975 32 (3) 209-217
48 Bell R D Alexander G M Nguyen T Albin M S Quantification of cerebral
infarct size by creatine kinase BB isoenzyme Stroke 1986 17 (2) 254-60
49 Steen C Wilczak N Hoogduin J M Koch M De Keyser J Reduced Creatine
Kinase B Activity in Multiple Sclerosis Normal Appearing White Matter PLoS ONE 5 (5)
e10811
50 Chwieralski C Welte T Buumlhling F Cathepsin-regulated apoptosis Apoptosis 2006
11 (2) 143-149
51 Droga-Mazovec G Bojic L Petelin A Ivanova S Romih R Repnik U Salvesen
G S Stoka V Turk V Turk B Cysteine cathepsins trigger caspase-dependent cell death
through cleavage of bid and antiapoptotic Bcl-2 homologues J Biol Chem 2008 283 (27)
19140-50
52 Duffy M J Proteases as prognostic markers in cancer Clin Cancer Res 1996 2 (4)
613-8
53 Guicciardi M E Deussing J Miyoshi H Bronk S F Svingen P A Peters C
Kaufmann S H Gores G J Cathepsin B contributes to TNF-alpha-mediated hepatocyte
apoptosis by promoting mitochondrial release of cytochrome c J Clin Invest 2000 106 (9)
1127-37
54 Butt A M Jenkins H G Morphological changes in oligodendrocytes in the intact
mouse optic nerve following intravitreal injection of tumour necrosis factor J Neuroimmunol
1994 51 (1) 27-33
55 Hagemann T L Gaeta S A Smith M A Johnson D A Johnson J A Messing
A Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal
fibers reveals a stress response followed by glial activation and neuronal dysfunction Hum Mol
Genet 2005 14 (16) 2443-58
56 Falk J Bonnon C Girault J A Faivre-Sarrailh C F3contactin a neuronal cell
adhesion molecule implicated in axogenesis and myelination Biol Cell 2002 94 (6) 327-34
57 Eckerich C Zapf S Ulbricht U Muumlller S Fillbrandt R Westphal M Lamszus
K Contactin is expressed in human astrocytic gliomas and mediates repulsive effects Glia
2006 53 (1) 1-12
97
Table 1 Statistically changed proteins between transgenic and control mouse CSF using
dNSAF analysis
Accession Protein Pa SC
b Fold
Changec
Control
dSpCd
Transgenic
dSpCd
KCRM_MOUSE Creatine kinase M-type 0034 425 30 182 541
HEXB_MOUSE Βeta-hexosaminidase subunit β 0012 183 uarr 0 59
CMGA_MOUSE Chromogranin-A 000078 153 darr 44 0
ANT3_MOUSE Antithrombin-III 0017 176 -14 67 47
SAP3_MOUSE Ganglioside GM2 activator 0045 415 darr 28 0
SPA3N_MOUSE Serine protease inhibitor A3N 0047 170 42 10 42
CO1A2_MOUSE Collagen alpha-2(I) chain 0043 56 darr 19 0
BLVRB_MOUSE Flavin reductase 00054 204 uarr 0 12
CATS_MOUSE Cathepsin S 00032 232 uarr 0 73
GFAP_MOUSE Glial fibrillary acidic protein 0021 107 uarr 0 21
RET4_MOUSE Retinol-binding protein 4 0040 189 darr 13 0
CBPE_MOUSE Carboxypeptidase E 0043 139 darr 11 0
CATL1_MOUSE Cathepsin L1 0015 87 94 02 19
The statistics are performed using the t-test from the ln(dNSAF) Gaussian data
a P p-value of the t-test where the null hypothesis states that there was no change in expression between
control and transgenic GFAP overexpressor b SC percentage of sequence coverage of the listed protein from
sequenced tryptic peptides c Fold Change positive values are indicative of a fold change for transgenic CSF
negative values are fold changes for control CSF (ie down-regulated in transgenic CSF) uarr indicates the protein
was only detected in transgenic CSF and darr indicates the protein was only detected in control CSF d dSpC
distributive spectral counts which represent the average spectral counts observed per run analysis on the mass
spectrometer and corrected using distributive analysis for peptides shared by more than one protein
98
Table 2 Proteins showing greater than three-fold changes with at least two unique
peptides identified for each protein
Accession Protein SC ()a Fold
Change b
Control
dSpC c
Transgenic
dSpC c
MUG1_MOUSE Murinoglobulin-1 precursor 147 42 155 37
CO4B_MOUSE Complement C4-B 113 54 22 118
PRDX6_MOUSE Peroxiredoxin-6 576 46 14 64
CNTN1_MOUSE Contactin-1 65 darr 41 0
CATB_MOUSE Cathepsin B 263 42 23 97
CAH3_MOUSE Carbonic anhydrase 3 396 76 11 84
APOH_MOUSE Beta-2-glycoprotein 1 128 44 44 1
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
PGK1_MOUSE Phosphoglycerate kinase 1 355 36 17 61
NDKB_MOUSE Nucleoside diphosphate kinase B 408 34 13 44
FHL1_MOUSE
Four and a half LIM domains
protein 1 243 39 13 51
NELL2_MOUSE
Protein kinase C-binding protein
NELL2 45 -43 13 03
MDHM_MOUSE
Malate dehydrogenase
mitochondrial 385 41 12 49
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
a SC percentage of sequence coverage of the listed protein from sequenced tryptic peptides b Fold
Change positive values are indicative of a fold change for transgenic CSF negative values are fold changes for
control CSF and darr indicates the protein was only detected in control CSF c dSpC distributive spectral counts
which represent the average spectral counts observed per run analysis on the mass spectrometer and corrected using
distributive analysis for peptides shared by more than one protein
99
Table 3 Validation of changes in proteins revealed by MS-based spectral counting
consistent with previously published microarray data
Consistent changes in RNA and proteomic data
uarr regulated in transgenic darr regulated in transgenic
Cathepsin S Contactin-1
Cathepsin B Carboxypeptidase E
Cathepsin L1
Peroxiredoxin-6
Complement C4-B
Glial fibrillary acidic protein
Serine protease inhibitor A3N
Note Validation of putative biomarkers from the current proteomics dataset by previously
published RNA microarray data55
Both up and down regulated proteins were consistent with the
RNA microarray data
_
100
___________________________________________
SUPPLEMENTAL INFORMATION (Available upon request)
Table S1 Compilation list of proteins identified from all the control and transgenic biological
replicates
Table S2 Distributive spectral counting calculations performed for proteins observed to share
identified peptides
Table S3 Proteins with dNSAF spectral counting performed including t-test analysis for a
comparison between transgenic and control CSF
101
FIGURE LEGENDS
Figure 1 The general workflow indicating the major steps involved in sample collection sample
processing mass spectrometric data acquisition and analysis of mouse CSF samples
Figure 2 Assessment of run to run variability of the base peak chromatograms within and
between two biological and technical replicates The peak profile and intensity scale is
consistent between the four chromatograms The four panels show two biological replicates (Tg
4 and Tg5) with two technical replicates for each biological sample
Figure 3 A Venn-diagram of the biological triplicates (1 2 and 3) of transgenic mouse
CSF showing 78 proteins identified in all three experiments B Venn-diagram of the biological
triplicates (1 2 and 3) of control mouse CSF having 61 proteins identified by all three
replicates C The overlap between control and transgenic CSF proteomic analysis showing 139
proteins identified by both groups and 73 and 54 uniquely identified by respective groups
Figure 4 Assessment of technical replicate variability between biological replicates The error
bars in both A and B are the standard deviation derived from the technical triplicates for each
biological replicate Panel A shows creatine kinase M having more or equal variability in the
biological triplicates than each technical triplicate The means of the biological triplicates are
illustrated by the dashed line Panel B represents kininogen-1 which is unchanged between
control (Ctl) and transgenic (Tg) samples The biological variability coupled with the technical
replicates provides a barely noticeable difference in the pooled mean between control and
102
transgenic spectral counts The difference in means is contrasted with the three fold change
observed from creatine kinase M (A)
Figure 5 MSMS profile of the tryptic peptide LSVEALNSLTGEFK from creatine kinase M
(A) The top MSMS is from a control sample with an elution at 463 minutes (B) The bottom
MSMS is from the GFAP transgenic sample with an elution at 46 minutes These tandem MS
spectra show instrument reliability and consistent fragmentation patterns which are necessary for
spectral counting analysis
Figure 6 Validation of MS spectral counts using a GFAP ELISA GFAP content (ngL)
measured within mouse CSF from both transgenic and control animals The data represents the
average with standard deviation (n=5 each sample represents pooling of 1 to 8 animals) The
statistics are performed using a student t-test plt00001
103
Figure 1
104
Figure 2
105
Figure3
106
Figure 4
107
Figure 5
108
Figure 6
Ctl Tg
100
1000
10000
100000
Mouse CSF Sample
GF
AP
(n
gL
)
109
Table of Contents Summary
Cerebrospinal fluid (CSF) was obtained from transgenic mouse models of Alexander disease as
well as healthy controls We immunodepleted pooled CSF from mice by a modified IgY-14
protocol Shotgun proteomics utilizing trypsin digestion 1-D nanoRP separation CID tandem
mass spectrometry analysis Mascot database searching and relative quantitation via distributive
normalized spectral abundance factor resulted in the identification of 266 proteins and 27
putative biomarkers
110
Chapter 4
Genomic and proteomic profiling of rat adapted scrapie
Adapted from ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A
Cunningham R Wang J Wellner D Ma D Li L Aiken J In preparation
111
Abstract
A novel model of prion disease using rats called Rat Adapted Scrapie (RAS) was
developed with the genomics from brain and proteomics from cerebrospinal fluid (CSF) profiled
The CSF proteome from control and RAS (biological N=5 technical replicates N=3) were
digested and separated using one dimensional reversed-phase nanoLC coupled to data-
dependent tandem MS acquisition on an ion trap mass spectrometer In total 512 proteins 167
non-redundant protein groups and 1032 unique peptides were identified with a 1 false
discovery rate (FDR) Of the proteins identified 21 were statistically up-regulated (plt005) and
7 statistically down-regulated in the RAS CSF We also identified 1048 genes which were
differentially regulated in rat prion disease and upon mapping these changes to mouse gene
expression however only 22 of these genes were in common with mRNAs responding to
prion infection in mice suggesting that the molecular pathology observed in mice may not be
applicable to other species The proteins are compared to the differentially regulated genes as
well as to previously published proteins showing changes consistent with other prion animal
models
112
Introduction
Prion diseases are an unusual class of fatal transmissible neurodegenerative disorders
that affect the mammalian central nervous system They are caused by the accumulation of an
abnormal conformation of the normal host encoded cellular prion protein PrPC This
conformational rearrangement of PrPC is brought about by template directed misfolding wherein
seed molecules of the abnormal isoform PrPScrapie
PrPSc
convert PrPC into new PrP
Sc molecules
Scrapie in sheep and goats is the prototypic prion disease and is so named because clinically
affected animals scrape their wool off on pen and stall walls Laboratory investigation of prion
diseases typically relies upon rodents which can be infected with natural isolates of scrapie1
albeit with some difficulty as ovid scrapie isolates need time to adapt to rodents This adaptation
is characteristic of prion disease interspecies transmissions and properly reflects the molecular
adaptation that must occur to allow interaction between exogenous foreign PrPSc
and host PrPC
molecules selecting for conformers which exhibit template directed misfolding In some cases
no conformational solution is found reflecting a species barrier to disease transmission
In recent years advances in genomics and proteomics technologies have allowed
unprecedented examination of the biomolecules that are altered upon exposure to prion agents
These studies2 3
have relied upon analysis of gene and protein expression changes in response to
prion infection with the aim of trying to identify pathways that might underlie the mechanism of
prion-induced neurotoxicity A second important aim has been to identify signature molecules
that might act as surrogate biomarkers for these diseases as there are significant analytical
challenges associated with sensitively detecting and specifically distinguishing disease-induced
conformational changes (PrPSc
) of the prion protein from normal host conformations (PrPC)
113
Mass spectrometry (MS)-based proteomics has become a promising tool for biomarker
discovery from biological fluids such as CSF blood and urine4-6
Two-dimensional gel
electrophoresis MS (2D-GE MS) and two-dimensional differential gel electrophoresis (2D-DIGE
MS) are the most widely used methods for proteomic profiling of prion infected CSF so far due
to the advantage of ready separation and quantification of proteins in complex biological samples
Studies using 2D-GE MS or 2D-DIGE MS to profile proteins in CSF have led to the
identifications of currently accepted biomarker for sCJD 14-3-3 protein and other potential
biomarkers for prion diseases7-9
However the application of this method in biomarker
discovery is limited by insufficient sensitivity and potential bias against certain classes of
proteins as gel-based separation does not work well for the low abundance proteins very basic
or acidic proteins very small or large proteins and hydrophobic proteins 10 11
In contrast to 2D-
GE or 2D-DIGE shotgun proteomics employs enzymatic digestion of the protein samples
followed by chromatographic separation prior to introduction into a mass spectrometer for
tandem MS analyses Shotgun proteomics has become increasingly popular in proteomic
research because these methods are reproducible highly automated and have a greater
likelihood of detecting low abundance proteins12 13
Due to the sample complexity in CSF and
because albumin comprises over half of the protein content in CSF removal of high-abundance
proteins including albumin is necessary to improve proteomic coverage and identify low-
abundance proteins One method is IgY immunodepletion14 15
which is performed prior to LC-
MSMS and uses species specific antibodies from chicken yolk to remove abundant proteins in
biological samples such as CSF In the present work CSF from control and rat adapted scrapie
animal models (biological N=5 technical N=3 replicates) were analyzed by LC-MSMS and we
114
indentified 512 proteins and 167 non-redundant protein isoforms (referred to as protein groups)
with 21 proteins being statistically up-regulated (p lt 005) and 7 statistically down-regulated
By and large this work has been performed using laboratory mice for the gene
expression studies and human cerebrospinal fluid for proteomics mice do not provide sufficient
volumes of CSF for proteomic studies Both approaches are exceedingly valuable as the mouse
model allows cross-sectional time course experiments to be performed including the important
pre-clinical phase of disease Critically however the relevance and generalizability of mouse
prion responses to other prion diseases especially human disease is unknown Human proteomic
studies on CSF guarantee relevant protein identifications but are limited to the clinical phase of
the disease when apparent markers may reflect gross neurodegeneration covering up subtle but
more specific responses To address these issues we have adapted mouse RML prions into rats
with the aim of expanding the knowledge of prion disease responses addressing the limitations
of mice and opening up the possibility of preclinical proteomic profiling in a laboratory rodent
In the present work CSF samples from control and rat adapted scrapie were analyzed by system
biology approaches Here we show that Rat Adapted Scrapie (RAS) is a powerful tool for an -
omics based approach to decipher the molecular impact of prion disease in vivo with
applicability to the molecular mechanisms of disease and biomarker discovery We identified
1048 genes which were differentially regulated in rat prion disease Astonishingly on the whole
mouse orthologs of these genes did not change in mouse adapted scrapie and vice versa
questioning the universality of previous mouse gene expression profiles These RAS gene
expression changes were identified in the CSF proteome where we detected 512 proteins and 167
protein groups with 21 proteins being statistically up-regulated (plt005) and 7 statistically down-
115
regulated in the CSF of prion diseased rats Many of the proteins detected have previously been
observed in human CSF from CJD patients
Materials and Methods
Ethics Statement
This study was carried out in accordance with the recommendations in the NIH Guide for Care
and Use of Laboratory Animals and the guidelines of the Canadian Council on Animal Care The
protocols used were approved by the Institutional Animal Care and Use Committees at the
University of Wisconsin and University of Alberta
Chemicals
Acetonitrile (HPLC grade) and ammonium bicarbonate (certified) were purchased from
Fisher Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) Optima LCMS grade acetonitrile and water were purchased
from Fisher Scientific (Fair Lawn NJ) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris
ge999 ) ammonium formate (ge99995) glycine (ge985) and IgY-7 antibodies were
purchased from Sigma-Aldrich (Saint Louis MO)
Rat Transmission and Adaptation
Prion agents used were the rocky mountain lab RML strain of mouse adapted scrapie
Stetsonville transmissible mink encephalopathy16
(TME) Hyper (Hy) strain of Hamster TME 17
1st passage Skunk adapted TME prepared as described and C from genetically defined
transmissions18
116
Brains from animals clinically affected with prion disease were aseptically removed and
prepared as 10 wv homogenates in sterile water 50 microL of 10 brain homogenate was
inoculated into weanling wistar rats intracranially After one year of incubation preclinical rats
from RML infections were euthanized by CO2 inhalation and the brain excised homogenized
and re-inoculated into naive animals Subsequent serial passages were from rats clinically
affected with rat adapted scrapie
Brains from rat passages were aseptically removed and bisected sagittally Brain halves
were reserved for immunohistochemistry (IHC) in formalin or frozen for immunoblotting RNA
isolation or subsequent passage For IHC tissues were first fixed in 10 buffered formalin
followed by antigen retrieval at 121 oC 21 bar in 10mM citrate buffer for 2 min After cooling
to room temperature sections were treated with 88 formic acid for 30 min and 4M guanidine
thiocyanate for 2 h Endogenous peroxidases were inactivated by 003 hydrogen peroxide and
tissue was blocked with 1 normal mouse serum Mouse anti-PrP mAB SAF83 (Cayman
Chemical) was biotinylated and applied at a 1250 dilution in blocking buffer overnight at 4 oC
Washes were performed in 10mM PBS with 005 tween-20 Streptavidin-peroxidase
(Invitrogen) was added for Diaminobenzidine (DAB) color reaction Anti-GFAP
immunohistochemistry was performed as above except that formic acid and guanidine treatment
steps were excluded Mouse anti-GFAP mab G-A-5 (Sigma) was used at a 1400 dilution
Brain samples for immunoblot were treated with or without Proteinase K (Roche) at a
ratio of 35 microg PK 100 ug protein at 50 microg mL for 30 minutes at 37 oC Phosphotungstenic acid
enrichments were performed as described14 19
Bis-Tris SDS-PAGE was performed on 12
polyacrylamide gels (Invitrogen) and transferred to PVDF Immunoblotting was performed using
117
mABs SAF83 (Cayman Chemical) 6H4 (Prionics) or 3F10 (a kind gift from Yong-Sun Kim) all
at a 120000 dilution
Gene Expression Profiling
RNA was extracted from frozen brain halves from clinically affected and control animals with
the QIAshredder and RNeasy mini kit (Qiagen Valencia CA) in accordance with the
manufacturerrsquos recommendations Due to the relatively large mass of rat brain an initial
homogenization was performed with a needle and syringe in 5mL of buffer RLT before further
diluting an aliquot in accordance with the manufacturers protocol Total RNA was amplified and
labeled in preparation for chemical fragmentation and hybridization with the MessageAmp
Premier RNA amplification and labeling kit (Life Technologies Grand Island NY) Amplified
and labeled cRNAs were hybridized on Affymetrix (Santa Clara CA) rat genome 230 20 high
density oligonucleotide arrays in accordance with the manufacturers recommendations
Differentially Expressed Genes were identified using Arraystar 50 (DNAStar Madison WI)
Robust multi-array normalization using the quantile approach was used to normalize all
microarray data A moderated T-test with a multiple comparison adjustment20
was used to reduce
the false discovery rate yet preserve a meaningful number of genes for pathway analysis
Pathway analysis was performed using the DAVID Bioinformatics database21
Comparative
analysis of genes induced by prions in mouse22
and rat disease was performed on genes
exhibiting at least a 2-fold change in expression Orthologs of rat and mouse genes were
identified using ENSEMBLE biomart release 6823
CSF Proteomic Profiling
118
CSF was collected from rats anaesthetized with isoflorane via puncture of the cisterna
magna A volume of 100-200 microL was routinely obtained CSF was touch spun for 30s at 2000xg
on a benchtop nano centrifuge to identify any blood contamination by the presence of a red
pellet CSF with blood contamination was not used for proteomic analysis CSF was prepared
for profiling by first depleting abundant proteins with an antibody based immunopartitioning
column IgY-R7 (Sigma-Aldrich Saint Louis MO) The manufacturers recommendations were
followed with a few deviations 40 microg of protein (~100 microL CSF) was bound to 100 microL of IgY
bead slurry in total volume of 600 microL formulated in 150mM ammonium bicarbonate The flow
through was collected and denatured by the addition of 10 microL of 8 M guanidine thiocyanate and
lyophilized in a speed-vac apparatus The sample was then reconstituted in 10 microL of water and 1
microL of 050 M DTT were added and allowed to incubate at 37oC for 30 minutes After incubation
27 microL of 055 M IAA was added for carboxymethylation and the sample was allowed to
incubate in the dark for 15 minutes After that 1 microL of 050 M DTT was added and allowed to
sit at room temperature for 10 minutes To perform trypsin digestion 70 microL of 50 mM
NH4HCO3 was then added along with 025 microg trypsin Digestion was performed overnight at
37oC and quenched by addition of 5 microL of 10 formic acid The tryptic peptides were then
subjected to solid phase extraction using an Agilent OMIX Tip C18 100 microL (Santa Clara CA)
Peptides were eluted with 50 ACN in 01 formic acid concentrated and reconstituted in 30
microL H2O with 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of a nanoAcquity (Milford MA) with a 10 microL injection
loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B consisted of
ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto a Waters 5 microm
119
Symmetry C18 180 microm x 20 mm trap cartridge (Milford MA) at a flow rate of 75 microLmin for 5
minutes at 95 A 5 B Separation was performed on a Waters 17 microm BEH130 C18 75 microm x
100 mm (Milford MA) with a gradient of 5 to 15 B over 10 minutes followed by 15 to
40 B over 80 minutes at room temperature
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with a Bruker CaptiveSpray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Waters Acquity console software to perform MS acquisitions for all experiments Smart
parameter setting (SPS) was set to 700 mz compound stability and trap drive level was set at
100 Optimization of the CaptiveSpray source resulted in dry gas temperature of 160oC dry
gas flow of 30 Lmin capillary voltage of -1325 V end plate offset of -500 V MSMS
fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
range of 350-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
120
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot24
(Version 24 Matrix
Science London UK) Database searching was performed against a forward and reversed
concatenated SwissProt Rattus rattus Mascot search parameters were as follows Allowed
missed cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) peptide tolerance plusmn12 Da maximum number of 13
C 1 MSMS
tolerance plusmn05 Da instrument type ESI-Trap Data was then transformed into Dat file formats
and transferred to ProteoIQ (NuSep Bogart GA) False positive analyses were calculated using
ProteoIQ and set at 1
Results
Development of Rat Adapted Scrapie
To develop a rat adapted strain of prion disease we introduced 6 different prion agents RML
TME Hy Skunk Adapted TME and Chronic Wasting Disease (CWD) from wild type 96G and
96S deer16-18
into 6 rats (Fig 1) Of these primary transmissions only RML induced the
accumulation of Proteinase K resistant PrP after one year of incubation as determined by western
blotting on 10 brain homogenates and PrPSc
enriched phoshotungstenic acid precipitated brain
homogenates Second passage of rat adapted RML scrapie resulted in clinically affected rats at
565 days post inoculation Serial passages of TME or CWD agents were not performed Clinical
symptoms consisted of ataxia lethargy wasting kyphosis and myoclonus Prion affected rats
121
also showed low level porphyrin staining around their head Subsequent serial passage decreased
incubation time to 215 days
Proteinase K resistant prion protein was observed from all clinically affected animals both by
immunoblot (Fig 2) and by immunohistochemistry (Fig 3) Di- and mono-glycosylated bands
were the most abundant isoforms of PrPSc
PrPSc
was extensively deposited in the cerebral cortex
hippocampus thalamus inferior colliculus and granular layer of the cerebellum GFAP
expressing activated astrocytes were found throughout the brain particularly in the white matter
of the hippocampus thalamus and cerebellum Spongiform change was a dominant feature of
clinical rat
Gene expression Profiling
In total 1048 genes were differentially regulated within a 95 confidence interval
(Supplementary Table 1) and 305 genes were differentially expressed by greater than 2-fold (Fig
4) The 1048 genes that were statistically significant were used for pathway analysis using
DAVID Pathway analysis suggested that the gene expression profile was consistent with
immune activation and maturation as well as inflammation (Supplementary Table 2) a likely
interpretation given the observable astrocytosis (Fig 3) and gliosis associated with prion disease
Other pathways highlighted by the analysis included increases in transcription of genes involved
in lysosomes and endosomes
To further probe the gene expression data we compared genes which were differentially
expressed in rat adapted scrapie against their orthologs expression in mouse scrapie and vice
versa (Figs 5 and 6) We did not observe a high degree of correlation between expression fold
changes For example GFAP a gene whose up-regulation in prion disease is well known was
122
increased 25 fold in rat adapted scrapie but up-regulated 93 fold in mouse scrapie A
qualitative analysis of expression of orthologs in prion disease suggests that many genes
deregulated at least 2-fold in mouse or rat do not have orthologs that are differentially expressed
For example the gene RNAseT2 was up-regulated greater than threefold in rat adapted scrapie
but was not significantly up-regulated in mouse Similarly three genes important in metals
homeostatsis metallothionein 1a and 2a and ceruloplasmin were up-regulated in rat 46 43 and
3 fold respectively but were not differentially expressed in mouse prion disease
CSF Proteomics
Each immunodepleted biological replicate (N=5 for each control and RAS) had technical
triplicates performed The triplicates were summed together using ProteoIQ Peptide spectral
counts were then summed for each protein and subjected to dNSAF analysis using ProteoIQ
internal algorithms Details for this method can be found elsewhere25 26
but briefly peptide
spectral counts are summed per protein (SpC) based on unique peptides and a weighted
distribution of any shared peptides with homologous proteins T-tests were used to identify
significant changes in protein expression 1032 unique peptides which identify 512 proteins and
167 protein groups were found Of these 512 proteins 437 were identified in both RAS and
control CSF samples (Fig 7) The complete list of all 512 proteins can be viewed in
Supplemental Table 3 Using the ProteoIQ the most likely isoform was selected such as 14-3-3
protein gamma
From Table 1 we observe five proteins that agree with the genomic data for up
regulations in RAS granulin macrophage colony-stimulating factor 1 receptor cathepsin D
complement factor H and ribonuclease T2 In addition serine protease inhibitor A3N was not
123
detected as up regulated in the RAS genomic data but was found to be up-regulated in previous
genomic profiling of the mouse prion model22
One interesting trend from the data in Table 1 is
that the majority of proteins found to be up-regulated in the RAS model were not detected in the
control samples The absence of the detection of those proteins such as ribonuclease T2 in the
control CSF does not necessarily suggest the absence of the protein nonetheless it is below the
detection limits for this current proteomics protocol and instrumentation
Discussion
Mice have been the preferred laboratory rodent for prion diseases research because they
can be inexpensively housed and are amenable to transgenesis which allows for short incubation
periods as well as hypothesis testing upon targeted pathways Pursuant to the determination of
the mouse genome and the development of high density transcriptional arrays for measurements
of gene expression profiling mice have been used extensively to examine the molecular
pathology of prion disease probing the impact of disease and animal strain In order to expand
upon this foundation we adapted mouse prions to infect rats We reasoned that by taking a
comparative approach to the molecular pathology of prion disease inferences could be obtained
into the variability of the molecular response to prion diseases and that understanding this
variability might suggest whether human prion disease responses are more or less similar to
mouse responses A second rationale is the desire to identify surrogate markers of prion disease
While this approach has been taken before using gene expression profiling a more direct
approach has been to use proteomic approaches on cerebral spinal fluid (CSF) identifying
proteins that are increase in abundance with disease A rat prion disease is valuable for this
because the rat proteome is established and rats allow for the collection of relatively large
volumes of CSF (~100 microL per animal) which allows for robust analytical separations enhancing
124
detection of biomarkers Finally rats unlike humans can be used in a time course study of prion
disease This allows for the identification of early transcriptional and proteomic responses to
prion infection responses which are particularly valuable for the identification of surrogate
disease biomarkers
To initiate the development of a rat prion disease we attempted to adapt six different
prion disease agents PrPres
molecules to rat via intracranial inoculation of weanling animals
(Figure 1) Of these six agents only mouse RML prions were able to surmount the species
barrier to prion disease transmission Mouse PrP differs from rat by eight amino acid changes
six in the mature protein and four in the N-terminal octa-peptide repeat region (Supplementary
Fig 1) As rat shares the most prion protein sequence homology with mouse it is perhaps not
surprising that it transmitted whereas the other did not confirming that the primary prion protein
sequence is the most important determinant for interspecies transmission We conclude that there
is a large molecular species barrier preventing conversion of rat PrPc into PrP
res
The transmission of mouse RML into rats was characterized by a shortening of the
incubation period following each passage This is indicative of agent adaption to the new host
and increases in the titer present in end-stage brain Overall our adaptation of mouse prion
disease into rats resulted in a similar agent to that observed by Kimberlin27
The differences in
incubation period at second passage are largely due to our collecting the animals at 365 days post
inoculation upon first passage whereas Kimberlin and Walker allowed the first passage animals
to reach end-stage clinical rats
Rat adapted scrapie is in many ways similar to other prion diseases The clinical period of
disease is marked by a progressive neurological dysfunction with prominent ataxia kyphosis and
125
wasting The protease resistant PrP observed is typical as is the widespread deposition of PrPSc
in
the brain Spongiosis and reactive astrogliosis are as expected of a prion disease
Gene expression profiles from rats clinically affected with prion disease revealed a strong
neuronal inflammation associated with proliferated and activated astrocytes This is perhaps best
observed through the up-regulation of GFAP GFAP positive astrocytes were omnipresent
throughout the brain and GFAP mRNA was up-regulated 25 fold The up-regulation of GFAP is
a hallmark of the molecular response to prion infection and has been routinely observed Our
comparative analysis of gene expression changes in mouse RML versus rat adapted scrapie
suggest substantial differences in gene expression in response to prion disease despite the fact
that the overall response is neuro-inflammatory This suggests that the potential overlap between
mouse expression profiles and a putative human CJD expression profile could be quite different
at the level of individual transcripts that might be expected to be changed
CSF Proteomics
CSF proteomics can be exceedingly challenging due to the small sample available large
dynamic range in proteins and abundant proteins limiting sample loading onto nanoscale
columns Dynamic range reduction in the CSF sample was achieved using a custom amount of
IgY-7 antibodies to abundant proteins such as albumin After immunodepletion the total
protein content was reduced by ~90 limiting the proteomics analysis to one dimensional
separation Label free quantitation spectral counting was performed because it requires less
protein and does not increase sample complexity The proteins identified from the affected and
control N=5 are shown in Supplemental Table 2 Consistent detection of proteins in CSF from
both control and infected rats was observed (Fig 7C) Only two proteins were identified in
126
controls that were not observed in RAS and only 10 proteins were only observed in RAS Some
of these proteins that were only identified in RAS are significantly changed (Supplemental Table
3) One concern in proteomics data is the variability from run to run and the possibility that
certain proteins are identified from different unique peptides Figure 7A shows that the vast
majority of the unique peptides detected (783) were identified with a 1 FDR in both RAS and
control CSF samples highlighting the analytical reproducibility of our methodology
Proteomic analysis of the infected rat CSF provides a reasonable approach to cross
validate biomarkers detected by gene expression profiling For example RNAseT2 a secreted
ribonuclease was up-regulated 3-fold at the mRNA level and was also enriched in CSF from
infected rats (Table 1) Similarly coordinated increases in detection of colony stimulating factor
1 receptor complement factor H granulin and cathepsin D were also observed Conversely
proteomic analysis of CSF also allows for the observation of post-transcriptional responses to
prion disease that might be absent in gene expression profiling The 14-3-3 proteins and neuron
specific enolase both known markers for CJD are only detected by proteomic analysis Thus
gene expression profiling and proteomic detection serve to increase confidence in the
observation of up-regulation enhancing the likelihood that proteins detected by both
methodologies are specific and perhaps may be more sensitive at earlier time points
Comparison to human CSF prion disease proteome
In our comparative analysis 21 proteins were found to be up-regulated while 7 proteins
down-regulated in prion infected rats Included in the up-regulated proteins were the 14-3-3
proteins epsilon and gamma 14-3-3 zetadelta was also up-regulated but not statistically
significant Enolase 2 or neuron specific enolase (NSE) was also up-regulated in CSF of infected
127
rats These proteins are all in agreement with results from previous proteomic profiling of human
CSF from patients with CJD8 9
The detection of 14-3-3 protein is included in the diagnostic
criteria approved by World Health Organization for the pre-mortem diagnosis of clinically
suspected cases of sCJD28
although its application in large-scale screening of CJD is still
debated due to high false positive rate where elevated 14-3-3 protein levels were also reported in
other conditions associated with acute neuronal damage29 30
It was suggested that other brain-
derived proteins that are also specific to CJD can be used in conjunction with 14-3-3 protein to
increase diagnosis accuracy and specificity31
NSE is present in high concentration in neurons
and in central and peripheral neuroendocrine cells NSE serves as another valuable biomarker in
diagnosis of CJD as the elevated level of NSE in CSF was found in early and middle stages of
CJD 32
Other proteins detected in CSF included cystatin C and serpina3N although both of
these were not statistically changed These proteins were both previously identified as being
putative biomarkers for CJD33 34
Utility for biomarker discovery with emphasis on onset of biomarker appearance in CSF
The investigation of the protein changes in CSF from RAS compared to control rats
provides a solid foundation when investigating potential biomarkers with prion disease onset
The cross-validation of the genomic and proteomics data further emphasizes the targets for
consideration during disease onset Biomarker discovery provides the potential to determine if
animals such as cows in bovine spongiform encephalopathy (BSE) are affected instead of
having to be euthanized when BSE is expected In addition CSF collection in mice or hamsters
Prion models is extremely difficult and limited alternatively with the advent of the RAS model
CSF can be collected in sufficient amounts for CSF proteomics CSF collection for mice or
hamsters can be ~10 microL and could be contaminated with blood further complicating proteomic
128
analysis unlike rats which over 10 times more CSF can be collected per animal35
Due to the
amount of CSF available time course studies analyzing CSF proteomics is possible in RAS due
to animal numbers that are manageable and reasonable The RAS model further allows
investigators to bypass working with highly infections CJD CSF samples to investigate the CSF
proteome changes
Conclusion
In this study we have described the gene and protein expression changes in brain and
spinal fluid from a transmission of mouse prions into rats We find that while the overall gene
expression profile in rats is similar to that in mice the specific genes that make up that profile
are different suggesting that genes that change in response to prion disease in different species
may not be as conserved Analysis of CSF from rat adapted scrapie identified similar protein
changes as known in human CJD The rat will be a useful model to identify surrogate markers
that appear prior to the onset of clinical disease and thus may be of higher specificity and
sensitivity
Supplemental Information Available Upon Request
1 Chandler R L Encephalopathy in mice produced by inoculation with scrapie brain material Lancet 1961 1 (7191) 1378-9 2 Silva C J Onisko B C Dynin I Erickson M L Requena J R Carter J M Utility of Mass Spectrometry in the Diagnosis of Prion Diseases Anal Chem 2011 3 Wei X Herbst A Ma D Aiken J Li L A quantitative proteomic approach to prion disease biomarker research delving into the glycoproteome J Proteome Res 2011 10 (6) 2687-702 4 Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C Mann M Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7 (1) 386-99 5 Sjodin M O Bergquist J Wetterhall M Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers J Chromatogr B Analyt Technol Biomed Life Sci 2003 878 (22) 2003-12 6 Cunningham R Ma D Li L Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery Frontiers in Biology 2012 7 (4) 313-335
129
7 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66 8 Harrington M G Merril C R Asher D M Gajdusek D C Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease N Engl J Med 1986 315 (5) 279-83 9 Piubelli C Fiorini M Zanusso G Milli A Fasoli E Monaco S Righetti P G Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping Proteomics 2006 6 Suppl 1 S256-61 10 Lilley K S Razzaq A Dupree P Two-dimensional gel electrophoresis recent advances in sample preparation detection and quantitation Curr Opin Chem Biol 2002 6 (1) 46-50 11 Oh-Ishi M Maeda T Separation techniques for high-molecular-mass proteins J Chromatogr B Analyt Technol Biomed Life Sci 2002 771 (1-2) 49-66 12 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset J Proteome Res 2008 7 (2) 698-707 13 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422 (6928) 198-207 14 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample preparation and analysis Proteomics 2005 5 (13) 3314-28 15 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep Biochem Biotechnol 2009 39 (3) 221-47 16 Marsh R F Bessen R A Lehmann S Hartsough G R Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy J Gen Virol 1991 72 ( Pt 3) 589-94 17 Bessen R A Marsh R F Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent J Virol 1992 66 (4) 2096-101 18 Johnson C J Herbst A Duque-Velasquez C Vanderloo J P Bochsler P Chappell R McKenzie D Prion protein polymorphisms affect chronic wasting disease progression PLoS One 2011 6 (3) e17450 19 Safar J Wille H Itri V Groth D Serban H Torchia M Cohen F E Prusiner S B Eight prion strains have PrP(Sc) molecules with different conformations Nat Med 1998 4 (10) 1157-65 20 Benjamini Y Hochberg Y Controlling the False Discovery Rate A Practical and Powerful Approach to Multiple Testing Journal of the Royal Statistical Society Series B (Methodological) 1995 57 (1) 289-300 21 Huang da W Sherman B T Lempicki R A Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat Protoc 2009 4 (1) 44-57 22 Moody L R Herbst A J Aiken J M Upregulation of interferon-gamma-induced genes during prion infection J Toxicol Environ Health A 2009 74 (2-4) 146-53 23 Flicek P Amode M R Barrell D Beal K Brent S Carvalho-Silva D Clapham P Coates G Fairley S Fitzgerald S Gil L Gordon L Hendrix M Hourlier T Johnson N Kahari A K Keefe D Keenan S Kinsella R Komorowska M Koscielny G Kulesha E Larsson P Longden I McLaren W Muffato M Overduin B Pignatelli M Pritchard B Riat H S Ritchie G R Ruffier M Schuster M Sobral D Tang Y A Taylor K Trevanion S Vandrovcova J White S Wilson M Wilder S P Aken B L Birney E Cunningham F Dunham I Durbin R Fernandez-Suarez X M Harrow J Herrero J
130
Hubbard T J Parker A Proctor G Spudich G Vogel J Yates A Zadissa A Searle S M Ensembl 2012 Nucleic Acids Res 2012 40 (Database issue) D84-90 24 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 1999 20 (18) 3551-67 25 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J Proteome Res 2006 5 (9) 2339-47 26 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome quantitation how to deal with peptides shared by multiple proteins Anal Chem 2010 82 (6) 2272-81 27 Kimberlin R H Cole S Walker C A Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters J Gen Virol 1987 68 ( Pt 7) 1875-81 28 World Health Organization Global surveillance diagnosis and therapy of human transmissible spongiform encephalopathies report from a WHO consultation 1998 World Health Organization Geneva Switzerland 9-11 February 1998 1-26 29 Bartosik-Psujek H Archelos J J Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG J Neurol 2004 251 (4) 414-20 30 Saiz A Graus F Dalmau J Pifarre A Marin C Tolosa E Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders Ann Neurol 1999 46 (5) 774-7 31 Sanchez-Juan P Green A Ladogana A Cuadrado-Corrales N Saanchez-Valle R Mitrovaa E Stoeck K Sklaviadis T Kulczycki J Hess K Bodemer M Slivarichova D Saiz A Calero M Ingrosso L Knight R Janssens A C van Duijn C M Zerr I CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease Neurology 2006 67 (4) 637-43 32 Zerr I Bodemer M Racker S Grosche S Poser S Kretzschmar H A Weber T Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease Lancet 1995 345 (8965) 1609-10 33 Sanchez J C Guillaume E Lescuyer P Allard L Carrette O Scherl A Burgess J Corthals G L Burkhard P R Hochstrasser D F Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease Proteomics 2004 4 (8) 2229-33 34 Miele G Seeger H Marino D Eberhard R Heikenwalder M Stoeck K Basagni M Knight R Green A Chianini F Wuthrich R P Hock C Zerr I Aguzzi A Urinary alpha1-antichymotrypsin a biomarker of prion infection PLoS One 2008 3 (12) e3870 35 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
131
Figure 1 Interspecies transmission of prion disease to rats End-stage clinical brain homogenates
were used to passage prion disease After one year of incubation animals were euthanized to
determine the extent of PrPres
accumulation Protease resistance PrP was only observed in those
animals infected with RML scrapie prions This material was serially passaged for two more
incubations before becoming rat-adapted as indicated by the shortening of the incubation period
132
Table 1 A list of selected proteins and mRNAs up-regulated in RAS CSF and brain tissue If
the protein is detected in the RAS CSF but not the control CSF the CSF expression is reported
with a infin If there is no change or data on certain genes related to an up regulated protein nd is
noted The mouse genomic data presented here was previously published22
Gene Protein Symbol Accession CSF
Expression
Rat
GEX
Mouse
GEX
14-3-3 protein zetadelta Ywhaz NP_037143 infin nd nd
14-3-3 protein epsilon Ywhae NP_113791 infin nd nd
14-3-3 protein gamma Ywhag NP_062249 infin nd nd
serine protease inhibitor A3N Serpin A3N NP_113719 54 nd 975
enolase 2 (gamma neuronal) Eno2 NP_647541 infin nd nd
granulin GRN NP_058809 62 364 184
macrophage colony-stimulating
factor 1 receptor
Csf1r NP_001025072 infin 293 205
cathepsin D CTSD NP_599161 infin 255 299
complement factor H Cfh NP_569093 376 234 nd
ribonuclease T2 RNAset2 NP_001099680 infin 302 nd
133
Figure 2 Accumulation of PrPSc
in rat adapted scrapie First second and third passage brain
homogenates were assayed for the presence of protease resistant PrP by immunoblot PrPSc
was
observed following phosphotungstenic acid enrichment at first passage By clinical disease in 2nd
and 3rd
passage rats PrPSc
had substantially accumulated
134
Figure 3 Histological appearance of rat adapted scrapie in the hippocampus at clinical disease
Infected animals showed intense immuno-staining for deposits of PrPSc
and GFAP expressing
astrocytes Spongiform change is an abundant feature in rat adapted scrapie
135
Figure 4 Scatter plot of gene expression profiling of rat adapted scrapie The expression of
individual genes from uninfected and infected animals were plotted to display up and down
regulation The dashed green line is no change Solid green lines are 2-fold changes in gene
expression
136
Figure 5 Quantitative relationship between orthologous pairs of two-fold up-regulated genes in
mouse and rat scrapie Genes up-regulated more than two fold were mapped to their orthologs
and the fold change was plotted Expression is log2 transformed
137
Figure 6 Comparison of genes upregulated in rat and mouse prion diseases Genes up-regulated
two fold in rodent scrapie were identified and the expression of their orthologs was determined
138
Figure 7 Venn diagram comparison of the CSF proteomics data between rat adapted scrapie
(RAS) model and control rats (A) Unique peptides from tryptic peptides produced for the
proteins identified (B) The total proteins identified including all isoforms within the protein
groups (C) The protein groups comparing only the top protein hit of the protein isoforms
showing very consistent protein identifications between RAS and control
139
Chapter 5
Investigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiae
Adapted from ldquoInvestigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M
Heideman W Li L In preparation
140
Abstract
This work explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Kinases such as protein
kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose response
Thus performing a large scale phosphoproteomic MS study can be highly beneficial to map the
signaling networks and cascades involved in glucose-dependent stimulated yeast growth Yeast
cell extract was digested and phosphopeptides were enriched by immobilized metal affinity
chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase (RP)-RP
separation The low pH separation was infused directly into an ion trap mass spectrometer with
neutral loss electron-transfer dissociation (ETD)-triggered fragmentation to improve
phosphopeptide identifications In total 477 unique phosphopeptides are identified with 06
false discovery rate (FDR) and 669 unique phosphopeptides identified with 54 FDR This
study includes comparative phosphoproteins for one specific motif of PKA xxxKRKRxSxxxx
which is presented and differences between starved vs glucose fed are highlighted Phosphosite
validation is performed using a localization algorithm Ascore to provide more confident and
site-specific characterization of phosphopeptides
141
Introduction
Microorganisms such as Saccharomyces cerevisiae (yeast) cycle between growth when
nutrients are available and quiescence when nutrients are scarce When glucose is limited yeast
go into growth arrest state but when glucose is added growth quickly resumes Kinases such as
protein kinase A (PKA) and target of rapamycin (TOR) regulate growth in response to nutrient
conditions and have been well studied through transcriptional control1-4
Yeast execute large
transcriptome alterations in response to changing environmental growth conditions5 6
Gene
regulation by glucose introduction in yeast has been studied including genes used for growth on
alternative carbon sources and activation of genes coding for glucose transport and protein
synthesis7-10
Response to nutrients for survival is not limited to yeast biology and indeed all
living creatures both prokaryotes and multicellular eukaryotes like mammals require nutrient
responsiveness and coordinating cellular functions to survive
With regulation of certain genes well studied by glucose introduction the mechanism and
global protein modulation caused by glucose introduction remain unknown6 Large-scale
phosphorylation analysis has been previously performed in yeast using mass spectrometry11-14
Mapping the phosphoproteome in yeast transitioning from quiescence to growth conditions to
better understand the roles of phosphorylation in orchestrating growth is needed The
phosphorylation state during growth or starvation could be used to engineer cells to drive ectopic
activity (or inhibition) to promote growth and ethanol production on non-native sugars like
xylose
It has been reported that the phosphorylation state can be affected by the introduction of
glucose to carbon-starved yeast15
and phosphorylation plays a significant role in the cell cycle
and signal transduction16
Specifically O-Phosphorylation can function as a molecular switch by
142
changing the structure of a protein via alteration of the chemical nature of an amino acid for
serine threonine or tyrosine It has been reported that up to 30 of the proteome can undergo
phophorylation17
Mass spectrometry has evolved as a powerful tool to accomplish phosphosite
mapping using shotgun proteomics With available technology tens of thousands of
phosphorylation sites can be mapped to amino acids on thousands of proteins using shotgun
proteomics18-20
Mass spectrometry can offer sensitive automated non-targeted global analysis of
phosphorylation events in proteomic samples but in any large scale phosphoproteomic
investigation enrichment of phosphoproteinspeptides is required First phosphorylation is
usually a sub-stoichiometric process where only a percentage of all protein copies are
phosphorylated21
Various enrichment methods have been used for phosphopeptide enrichment
including metal oxide affinity chromatography (MOAC)22
such as TiO223
immobilized metal
affinity chromatography (IMAC)12 24 25
electrostatic repulsion-hydrophilic interaction
chromatography (ERLIC)26
and immunoaffinity of tyrosine phosphorylation27 28
After
enrichment MS analyses of phosphorylated peptides are still challenging due to ion suppression
from non-phosphorylated peptides
Even after phosphopeptide enrichment further sample preparation is needed for large
scale proteomic experiments Additional fractionation can increase protein coverage of a
sample by over ten fold such as MudPIT29
(multidimensional protein identification technology)
In online MudPIT a sample is injected onto a strong cation exchange (SCX) column coupled to
a RP column Successive salt bumps followed by low pH gradients provide the separation of
peptides in two dimensions Since the phosphate groups on phosphopeptides have a low pKa
value due to being more acidic then their unmodified counterparts they tend to elute earlier and
143
disproportionally in ion exchange chromatography like SCX Alternatively reversed-phase
reversed-phase (RP-RP) separation has been proposed as an alternative to MudPIT for offline
two dimensional (2D) separation30
One of the caveats of 2D separation is the potential for
wasted mass spectrometry time from early and late fractions having very few peptides present
all while having too much sample for middle fractions One simple method to reduce these
ldquowasted fractionsrdquo is to combine early or late fractions with the middle fractions to reduce MS
runs with little peptide content to analyze thus shortening the overall analysis time31
In addition the labile phosphorylation group has a large propensity to undergo cleavage
during collision induced dissociation (CID) producing a neutral loss The neutral loss can
produce insufficient backbone fragment ions for MSMS identification32
A solution to neutral
loss fragmentation in phosphopeptides is MS3 using CID to produce improved backbone
fragmentation13 14 33
An alternative fragmentation method to CID for fast sampling ion traps is
electron transfer dissociation (ETD)34-36
ETD produces a more uniform back-bone cleavage
where the cation peptide receives an electron from a low affinity radical anion37
The transferred
electron induces a dissociation at the N-Cα bond to produce c- and zbull-type product ions while
retaining the labile post-translational modifications (PTM) such as phosphorylation bound to the
product ions38
The Bruker amaZon ETD ion trap mass spectrometer has the potential to trigger
ETD fragmentation after a labile PTM produces a neutral loss from CID fragmentation This
method is termed neutral loss-triggered ETD fragmentation and provides a complementary
fragmentation pathway to labile poor fragmenting phosphorylated peptides
In this work we provide a qualitative comparative list of yeast phosphopeptides observed
in glucose fed vs glucose starved conditions
144
Experimental
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) ammonium hydroxide (ACS plus certified) urea (gt99)
sodium chloride (997) ammonium bicarbonate (certified) Optima LCMS grade acetonitrile
Optima LCMS grade water and EDTA disodium salt (99) were purchased from Fisher
Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) ammonium formate (ge99995) Trizma
hydrochloride (Tris HCl ge990) Triton X-100 (laboratory grade) and iron (III) chloride
hexahydrate (97) were purchased from Sigma-Aldrich (Saint Louis MO) Sodium dodecyl
sulfate (SDS) (998) was purchased from US Biological (Marblehead MA) Nickel
nitrilotriacetic acid (Ni-NTA) magnetic agarose beads were purchased from Qiagen (Valencia
CA) Autoclaved yeast peptone dextrose (YPD) media was prepared in 1 L of deionized water
using 10 g yeast extract 20 g Peptone from Becton Dickinson and Company (Sparks MD) and
20 g D-(+)-Glucose (ge995) purchased from Sigma-Aldrich (Saint Louis MO)
Modified Mary Miller Yeast Protein Isolation
The yeast culture was prepared and protein extraction was performed using a modified
Mary Miller protocol39
Briefly yeast strain s288c was inoculated with YPD media and shook
for 72 hours to allow the yeast to be glucose starved After the 72 hours the yeast culture was
partitioned into two flasks To one flask glucose was added at 2 of the final concentration and
allowed to incubate and shake for 10 minutes Then 100 optical density units (ODU) of the yeast
145
culture were collected from each flask Each yeast sample was spun down in a Beckman-Coulter
J6B centrifuge (Indianapolis IN) at 2500xg for three minutes The media was aspirated and the
tubes were flash frozen in liquid nitrogen and stored at -80oC The yeast pellets were thawed on
ice and resuspended in 1 mL of lysis buffer (50 mM Tris HCl 01 triton x-100 05 SDS
pH=80) and 1X protease and phosphatase inhibitor cocktail from Thermo Scientific (Rockford
IL) and transferred to an Eppendorf tube To the yeast 200 microL of glass beads were added and
amalgamated for 25 minutes at 4oC The sample was inverted and the Eppendorf tube was
pierced with a hot 23 gauge needle through the bottom and the tube was placed atop a 5 mL
culture tube and centrifuge in the Beckman-Coulter J6B at 2500xg for three minutes at 4oC to
collect the liquid containing the yeast cells while the glass beads remain trapped in the
Eppendorf tubes The protein lysate was then centrifuged at 3000xg for five minutes at 4oC and
the supernatant was collected and stored at -80oC
Preparation of tryptic digests
The Saccharomyces cerevisiae protein cell extract for fed or starved were analyzed with a
BCA protein assay kit (Thermo Scientific Rockford IL) and 2 mg of protein was added to four
parts -20oC acetone and allowed to precipitate the protein for 1 hour at -20
oC The samples were
then centrifuged at 14000xg for 5 minutes and the supernatant was removed and the protein
pellet was reconstituted in 360 microl of 8 M urea To each sample 20 microL of 050 M DTT was
added and allowed to incubate at 37oC for 45 minutes After incubation 54 microL of 055 M IAA
was added for carbamidomethyl of cysteine and the sample was allowed to incubate for 15
minutes in the dark To quench the IAA 20 microL of 050 M DTT was added and allowed to react
for 10 minutes To perform trypsin digestion 1400 microL of 50 mM NH4HCO3 was then added
along with 20 microg trypsin to each sample Digestion was performed overnight at 37oC and
146
quenched by addition of 25 microL of 10 formic acid For each sample the tryptic peptides were
then subjected to solid phase extraction using a Hypersep C18 100 mg solid phase extraction
(SPE) column (Thermo Scientific Bellefonte PA) Peptides were eluted with 50 ACN in
01 formic acid concentrated and reconstituted in 500 microL of 80 ACN and 01 formic acid
Phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC)
One ml of Ni-NTA was washed three times with 800 microL of H2O and then the Ni was
removed with 800 microL of 100 mM EDTA in 50 mM ammonium bicarbonate and vortexed for 30
minutes The supernatant was removed and the NTA was washed with 800 microL of H2O three
times followed by addition of 800 microL 10 mM FeCl3 hexahydrate and vortexed for 30 minutes
The Fe-NTA was then washed three times with 800 microL of H2O and once with 80 ACN in 01
formic acid before being combined with the cell extract for phosphopeptide enrichment and
vortexed for 40 minutes The sample was washed three times with 200 microL of 80 ACN in 01
formic acid before elution with two aliquots of 200 microL of 5 ammonium hydroxide in 5050
ACNH2O with 15 minutes of vortexing The enriched phosphopeptides were then dried down
with a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA) and reconstituted in 55 microL
25mM ammonium formate pH=75
First dimension neutral pH separation
Individually 50 microL of each yeast phosphopeptide enriched sample was injected onto a
Phenomenex Gemini-NX 3microm C18 110 Aring 150 x 2 mm column (Torrance CA) The Gemini
column was coupled to a Phenomenex SecurityGuard Gemini C18 2 mm guard cartridge
(Torrance CA) Mobile phase A consisted of 25 mM ammonium formate at pH=75 and mobile
phase B consisted of 25 mM ammonium formateACN with a 19 ratio respectively at pH=75
The column was operated at 50oC at a flow rate of 05 mLmin The flow profile was 2-30 B
147
over 65 minutes and then 5 min at 100 B A total of 22 fractions were collected every 3
minutes and the fractions were combined as follows 1 with 12 2 with 13 etc to 11 with 22
The combined fractions were dried down and reconstituted in 25 microL of 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL from each fraction onto an Agilent Technologies
Zorbax 300 SB-C18 5 microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5
microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm Atlantis
dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B at 250
nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) for MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
148
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions An additional mode of MSMS fragmentation electron transfer dissociation
(ETD) was triggered on the precursor ion when a neutral loss was observed in CID
fragmentation from phosphoric acid H3PO4 (Δmz of 49 and 327 for 2+ and 3+ charge states
respectively) or for metaphosphoric acid (HPO3) (Δmz of 40 and 267 for 2+ and 3+ charge
states respectively) For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms rolling average 2 acquisition range of 300-1500 mz
and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target
was set to 300000 maximum accumulation time 5000 ms two spectral averages acquisition
range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required to prevent artificial data
reduction Identification of peptides were performed using Mascot40
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt Saccharomyces
cerevisiae database (version 575) Mascot search parameters were as follows Allowed missed
cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) and phosphorylation (STY) peptide tolerance plusmn12 Da maximum
number of 13
C 1 MSMS tolerance peptide decoy (Mascot) checked plusmn05 Da instrument type
149
ESI-Trap Results from the Mascot search was exported as DAT files for analysis by Scaffold 3
and Scaffold PTM
Scaffold and Ascore data processing
Scaffold 3 (Proteome Software Portland OR)was used for MSMS proteomics data
comparison and false discovery rate analysis The DAT files were loaded into Scaffold 3 and
the fractions for the two dimensional fractionation were combined The resulting biological
triplicates for starved and glucose fed yeast were filtered with a 1 false discovery rate (FDR)
on the protein level (Supplemental Table 1) Due to the inherent poor fragmentation of
phosphopeptides a lenient FDR level may still yield biologically relevant data and thus a 54
FDR was generated for phosphopeptides (Supplemental Table 2) For a more conservative list of
phosphopeptides with a 06 FDR was generated from Scaffold 3 (Supplemental Table 3) FDR
analysis is sufficient at preventing poor data from being reported but does not prevent false
phosphosite identification in phosphopeptides To provide confidence in site identification
Scaffold PTM was used to perform Ascore41
analysis Ascore uses an algorithm to score the
probability of the phosphosite from a phosphopeptide identified by a database searching
algorithm such as Mascot Ascore can be freely accessed at httpascoremedharvardedu
Cell collection RNA isolation and microarray data analysis
All experiments were performed in biological duplicates Cell samples (10 ODU) were
taken at starved and fed states spun at 5000xg for 2 minutes at 30 oC the supernatant was
removed and the pellets snap frozen using liquid nitrogen RNA was isolated using the Epicentre
MasterPure Yeast Purification Kit (Epicentre Technologies) and quality was checked using gel
electrophoresis cRNA synthesis was carried out using the GeneChipreg Expression 3
Amplification One-Cycle Target Labeling and Control Reagents kit (Affymetrix) All
150
experiments followed the manufactures instructions cRNA samples were hybridized to
GeneChip Yeast Genome 20 Arrays for 16 hours Arrays were washed stained and scanned
according the manufactures recommendations Affymetrix CEL files were RMA normalized
with R and the Bioconductor Suite Data analysis was performed within TIGR Multiexperiment
Viewer v451 in-house Perl scripting R and Bioconductor
Results
Sample preparation for shotgun proteomics
As discussed in the introduction the purpose of this study is to provide an exploratory list
of qualitative differences in the phosphoproteome between starved vs glucose fed yeast After
yeast cell lysate production a substantial amount of sample preparation is performed to enhance
the depth of the phosphoproteome coverage A workflow of the steps following cell lysis is
outlined in Figure 1A The yeast proteins are isolated via acetone precipitation followed by
digestion with trypsin to produce peptides Phosphopeptides are intermixed with the entire
tryptic peptide sample and therefore Fe-NTA IMAC is used to enrich the phosphopeptides To
improve upon the number of phosphopeptides we then performed two dimensional separation
with high pH RP followed by low pH RP C18 and infused the eluent directly onto an ion trap
mass spectrometer Figure 1B show an improved technique for the first dimension of separation
to combine the early eluting and late eluting fractions from the first phase of separation to reduce
overall MS analysis time The combination of fractions 1 and 12 2 and 13 etc essentially
improves the duty cycle of the mass spectrometer by ensuring sufficient peptide content is
injected onto a low pH nanoLC RP C18 column
ETD-triggered mass spectrometry
151
In the present study labile phosphorylation can lead to non-informative neutral loss
During MS scanning mode the instrument will choose the 6 most abundant peaks with correct
isotopic distribution and select them for MSMS CID fragmentation During CID fragmentation
it is common for labile PTMs such as phosphorylation to undergo neutral loss and thus limited
informative b and y-type ions are formed Alternatively ETD fragmentation can be used on
specific MSMS scans which produce a neutral loss indicative of phosphorylation (98Daz or
80Daz) The subsequent ETD fragmentation on these putative phosphopeptides will lead to
uniform backbone cleavage resulting in confident identification of phosphopeptides with site-
specific localization during MSMS It is important to note that CID fragmentation still produces
very informative fragmentation for phosphorylation but ETD provides an orthogonal
fragmentation pathway to further increase the phosphoproteome coverage Additionally the
duty cycle for ETD fragmentation is roughly half that of CID therefore about half as many
potential peptides would be fragmented and sequenced if the instrument was using ETD
fragmentation exclusively
Protein Data
Even with phosphopeptide enrichment it is inevitable that non-phosphopeptides will also
be identified All data were searched with Mascot and in total over 1000 proteins were identified
with a 1 FDR rate and are listed in Supplemental Table 1 The proteins listed in Supplemental
Table 1 include both phosphoproteins and non-phosphorylated proteins In addition to the
proteins identified in the fed and starved states the unique peptides and spectral counts are also
listed for reference The phosphopeptides are specifically listed with a 54 FDR rate in
Supplemental Table 2 and comparisons between starved and fed with Ascore analysis are listed
for every phosphopeptide identified A higher confidence of phosphopeptide identification is
152
sometimes required before investing in time consuming biological experiments so a list of
phosphopeptides identified in starved and fed was taken from a setting directing Scaffold to
produce a 05 FDR which resulted in the generated FDR of 06 FDR and listed in
Supplemental Table 3
A summary of specific phosphorylation sites are listed in Table 1 with a 06 FDR and
Table 2 with a 54 FDR Each table gives totals for unique phosphopeptides identified having
an Ascore localization score ge80 without Ascore and phosphorylation events on each unique
peptides As expected the majority of phosphorylation events (over 50) occurred on serine
whereas fewer phosphorylation events (~10) occurred on tyrosine In addition the vast
majority of phosphorylation events were single phosphorylation (ge65) with very few
identifications having more than two phosphosites per peptide For specific phosphopeptide
identification and subsequent phosphoprotein identification refer to Supplemental Table 2 and 3
Discussion
Transcriptional response to glucose feeding
Yeast responds to the repletion of glucose after glucose-depletion by broad
transcriptional changes to induce growth Roughly one-third of the yeast genome is altered by at
least two fold when glucose is introduced as shown in Figure 2 Figure 2 is a heat map from a
microarray analysis showing 1601 genes up regulated and 1457 genes down regulated after
addition of glucose compared to the starved state The arbitrary cut-offs for these values were as
follows two fold induction after nutrient repletion and a false discovery rate (q-value) of 001
Red color in the heat map indicates a particular gene is up-regulated in the fed state compared to
the starved state Alternatively genes coded in green are less expressed in the fed state
compared to the starved condition The intensity of the green or red colors is indicative of the
153
intensity of the fold change in gene expression These large transcriptional changes after glucose
repletion drive and complement the current phosphoproteomic study
PKA motif analysis
One benefit of a large scale phosphoproteomics experiment is to examine the different
phosphopeptides from known motifs for specific kinases PKA and TOR are responsible for the
majority of the transcriptional response and thus PKA is a good target for motif analysis Figure
3 shows the PKA motif sequence xxxRKRKxSxxxxxx with the phosphorylation occurring on
the serine Other than F16P (fructose-16-bisphosphatase) all the proteins listed are unique to the
starved or fed samples A motif sequence will inevitably show up by random chance in any
analysis For this study the control for motif analysis uses the swissprot protein list for the
entire yeast proteome for the background Compared to background this specific PKA kinase
from Figure 3 is up-regulated by 264 fold when compared to the background One interesting
protein emerged from this motif analysis in the fed sample but not the starved sample is
Ssd1which is implicated in the control of the cell cycle in G1 phase42
Ssd1 also is
phosphorylated by Cbk1 and Cbk1 has been proposed to inhibit Ssd143
and provides an
intriguing target for future studies on starved vs glucose fed yeast growth
Localization of the phosphorylation sites
When a phosphopeptide contains any number of serine threonine or tyrosine amino
acids the localization of the phosphosite can sometimes be ambiguous Database searches used
to identify peptides like Mascot do not provide any probability for localization of correct
phosphorylation sites Ascore is an algorithm that uses the same MSMS spectra as Mascot but
instead of comparing theoretical peptide MSMS to experimental spectra Ascore searches for
informative a b or y-type peptide fragments giving evidence for phosphosite The Scaffold
154
program adds a localization probability to the Ascore values and the values are listed in
Supplemental Table 2 and 3 Figure 4 has a representative Ascore mass spectra assigning the
peaks identified and providing evidence that the phosphorylation site occurs at the threonine
instead of the serine Incorporating Ascore into this study provides a layer of validation for
putative phosphosite identification
Plasma Membrane 2-ATPase
A previous study identified and localized phosphorylation sites on plasma membrane 1-
ATPase after glucose was introduced to starved yeast15
In the current study PMA2 (plasma
membrane ATPase 2) was identified in glucose fed and not starved samples The doubly
threonine phosphorylated peptide identified in fed yeast is PMA2 with amino acid sequence
IKMLTGDAVGIAKETCR (583-599) whereas the homologous peptide in PMA1 has the exact
same amino acid sequence except for the first isoleucine substituted for valine
VKMLTGDAVGIAKETCR (554-570 not observed in data) PMA2 was observed in the 06
FDR list of phosphopeptides and has an Ascore localization probably of 100 A plant study
showed that PMA2 phosphorylation level was higher in early growth phase than when in
stationary phase44
In addition PMA2 expression in yeast permits the growth of yeast and
threonine phosphorylation has been reported on Thr-95545
The identification of PMA2 in the
fed glucose cell extract provides an interesting target for future study on the molecular
mechanisms involved in regulation growth in starved vs glucose fed yeast
Conclusion
In conclusion this work provides a qualitative comparison in the phosphoproteome
between starved and glucose fed yeast A large scale IMAC enrichment of yeast cell lysate
followed by 2-D separation provided a rich source of phosphopeptides for neutral loss triggered
155
ETD analysis using mass spectrometry A specific motif for PKA is highlighted to show the
differences in proteins identified between starved vs fed conditions In total 477 unique
phosphopeptides are identified with 06 FDR and 669 unique phosphopeptides identified with
54 FDR Phosphosite validation is performed using a localization algorithm Ascore to
provide further confidence on the site-specific characterization of these phosphopeptides The
proteins Ssd1 and PMA2 emerged as potential intriguing targets for more in-depth studies on
protein phosphorylation involved in glucose response
Supplemental Tables 1 2 and 3 are available upon request
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19 Olsen J V Vermeulen M Santamaria A Kumar C Miller M L Jensen L J
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20 Breitkopf S B Asara J M Determining In Vivo Phosphorylation Sites Using Mass
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Activation of the plant plasma membrane H+-ATPase by phosphorylation and binding of 14-3-3
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45 Maudoux O Batoko H Oecking C Gevaert K Vandekerckhove J Boutry M
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159
Figure 1 The general workflow indicating the major steps involved in sample collection
sample processing mass spectrometric data acquisition and analysis of comparative
phosphoproteomics for starved vs glucose fed yeast (A) The high pH RP separation
procedure for combining fractions to reduce low peptide containing fractions from the
UV-VIS trace is also shown (B)
160
Figure 2 Heat map for the global transcriptional response to glucose fed vs starved samples
S288C cells starved for glucose until growth was arrested and subsequently glucose was added
161
Samples for microarray analysis were taken at the starved state and 60 minutes after glucose was
added The heat map shows the fed log2 fold change for each gene relative to the starved state
across the genome performed in biological replicate (A) Black indicates no change in
expression level while red indicates higher expression for the fed relative to the starved state
Green indicates higher expression for the starved state compared to the fed state (Adapted from
Dr Michael Conways Thesis)
162
Figure 3 The motif for protein kinase A (PKA) that was observed for fed vs starved which is
xxxRKRKxSxxxxxx with the phosphorylation occurring on the serine The motif occurred at a
rate 264 fold higher than the yeast proteome used for background In addition one protein was
observed in both starved and fed with accession identification of F16P (Fructose-16-
bisphosphatase)
163
06 FDR phosphopeptide analysis
Table 1 The number of phosphopeptides identified with a 06 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
Starved Fed All
Ascore ge80 score
unique
STY 164 153 317
S 87 (530) 82 (536) 169 (533)
T 60 (366) 55 (359) 115 (363)
Y 17 (104) 16 (105) 33 (104)
Unique no Ascore
STY 242 235 477
S 131 (541) 133 (566) 264 (553)
T 86 (355) 78 (332) 164 (344)
Y 25 (103) 24 (102) 49 (103)
Phosphorylation events
on each unique peptide
1 102 113 187
2 36 40 68
3 12 11 22
4 or more 8 3 11
164
54 FDR phosphopeptide analysis
Starved Fed All
Ascore ge80 score
unique
STY 217 217 434
S 115 (530) 113 (521) 228 (525)
T 78 (359) 78 (359) 156 (359)
Y 24 (111) 26 (120) 50 (115)
Unique no Ascore
STY 337 332 669
S 193 (573) 180 (542) 373 (558)
T 111 (329) 116 (349) 227 (339)
Y
Phosphorylation events
on each unique peptide
1
2
3
4 or more
33 (98)
135
56
16
11
36 (108)
169
55
14
3
69 (103)
280
100
27
13
Table 2 The number of phosphopeptides identified with a 54 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
165
Figure 4 Ascore validation of the phosphopeptide LSLAtKK from the protein SGM1 (slow
growth on galactose and mannose protein 1) with 100 localization probability observed
in only fed but not starved yeast samples Ascorersquos algorithm identifies a b and y-type
ions and looks to identify peaks that provide evidence for a specific phosphorylation site
For this example Ascore assigns this phosphosite to threonine (T5) instead of the serine
(S2) The intense peaks at 4024 mz and 5250 mz are not identified by any a b or y-
type ions From the ladder sequence of the peptide shown numerous ions indicate the
threonine is phosphorylated while the serine is not Among these ions used for
localization are b2 y2 y5+H2O y3 y4 and y5
166
Chapter 6
Use of electron transfer dissociation for neuropeptide sequencing and
identification
Adapted from ldquoDiscovery and Characterization of the Crustacean Hyperglycemic Hormone
Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue
Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniquesrdquo Hui L
Cunningham R Zhang Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
167
Abstract
The crustacean Callinectes sapidus sinus gland (SG) is a well-defined neuroendocrine site that
produces numerous hemolymph-borne agents including the most complex class of endocrine
signaling moleculesmdashneuropeptides One such peptide is the crustacean hyperglycemic hormone
(CHH) precursor-related peptide (CPRPs) which was not previously sequenced Electron
transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and
high charge state In addition ETD was used to sequence the sulfated peptide Cholecystokinin
CCK-like Homarus americanus using a salt adduct Collectively these two examples
demonstrated the utility of ETD in sequencing neuropeptides with large molecular weight or
with labile modifications
168
Introduction
Neuropeptides are the largest and most diverse group of endocrine signaling molecules in
the nervous system They are necessary and critical for initiation and regulation of numerous
physiological processes such as feeding reproduction and development1 2
Mass spectrometry
(MS) with unique advantages such as high sensitivity high throughput chemical specificity and
the capability of de novo sequencing with limited genomic information is well suited for the
detection and sequencing of neuropeptides in endocrine glands and simultaneously provides the
potential for information on post-translational modifications such as sulfonation3-6
The sinus glands (SG) are located between the medulla interna and medulla externa of the
eyestalk in Callinectes sapidus It is a well-known neuroendocrine site that synthesizes and
secretes peptide hormones regulating various physiological activities such as molting
hemolymph glucose levels integument color changes eye pigment movements and
hydro-mineral balance7 Our labrsquos previous neuropeptidomic studies of SG in several
crustacean species including Cancer borealis8-11
Carcinus maenas12
and Homarus americanus13
14 used multiple MS strategies combining MALDI-based high resolution accurate mass profiling
biochemical derivatization and nanoscale separation coupled to tandem MS for de novo
sequencing In the current study we explore the neuropeptidome of the SG in the blue crab
Callinectes sapidus a vital species of economic importance on the seafood market worldwide In
total 70 neuropeptides are identified including 27 novel neuropeptides and among them the
crustacean hyperglycemic hormone precursor-related peptides (CPRPs) and orcokinins represent
major neuropeptide families known in the SG
The crustacean hyperglycemic hormone (CHH) and its precursor-related peptide (CPRP) are
produced concurrently during the cleavage of CHH from the CHH preprohormone protein15
The
169
CPRP peptide is located between the signal peptide and the CHH sequence and is separated from
the CHH by a dibasic cleavage site The functions of CPRPs are currently unknown16
However
the complete characterization of CPRPs provides a foundation for future experiments elucidating
their functional roles in crustaceans The cDNA of CHH preprohormone in SG of Callinectes
sapidus has been characterized17
but the direct detection of CPRP has not been reported due to
its relatively large size and possible post-translational modifications While small fragments of
CPRPs can be identified using nanoLC-ESI-Q-TOF tandem mass spectrometry the intact
peptide is difficult to detect due to the large molecular weight of CPRPs
Peptidomics traditionally uses collision induced dissociation (CID) for tandem MS
experiments for de novo sequencing Recently an alternative peptide fragmentation method has
been developed using the transfer of an electron termed electron transfer dissociation (ETD)18 19
ETD involves a radical anion with low electron affinity to be transferred to peptide cation which
results in reduced sequence discrimination and thus provides improved sequencing for larger
peptides compared to CID20
Specifically for an ion trap ETD the fluoranthene radical anion is
allowed to react with peptide cations in the three dimensional trap The resulting dissociation
cleaves at the N-Cα bond to produce c and zbull type product ions The peptidome of model
organisms like Callinectes sapidus can be further explored and expanded utilizing ETD as a
complementary fragmentation technique to CID Previous peptidomic analysis has been
completed using ETD as an additional fragmentation method21
It was observed that
enzymatically produced peptides with a higher mz produced improved sequence coverage using
ETD This observation termed decision tree analysis determined that a charge state of ge6 all
peptides endogenous or enzymatic should be fragmented via ETD22
In the present study the
highly charged peptide CPRP with an intact molecular weight of 3837 readily produces plus six
170
charge states The higher charge state of CPRP is highly amenable to fragmentation by ETD
which produces remarkably improved fragmentation and thus increased sequence coverage when
compared to CID
Sulfonation of tyrosine is a post-translational modification (PTM) that is thought to occur on
trans-membrane spanning and secreted proteins23
Cholecystokinin-8 (CCK-8) is a sulfated
peptide which has been linked to satiety24-26
and a CCK-like peptide has been observed in
lobster27
Sulfonation is an extremely labile modification and is often lost during soft
ionization such as ESI or MALDI but can be avoided by optimizing the ionization process28
One potential strategy to identify sulfopeptides is to scan for a neutral loss of 80 Da after CID
but this method does not allow for identification of site of sulfonation and has the risk to be
mistaken for phosphorylation The sulfonation PTM is a negatively charged modification on
the peptide which allows for negative ion scanning in the mass spectrometer but provides
minimal MSMS sequence coverage during CID due to preferential loss of the sulfate group
Alternatively electron-based dissociation technique enables more rapid radical driven
fragmentation where the cleavage pattern is more uniform along the peptide backbone without
initially cleaving the labile sulfated group thus preserving the site of modification These types
of dissociation techniques only work for multiply-charged ions thus a method to install a
multiply-charged cation on the target peptide is desirable It has been shown that the electron
capture dissociation (ECD) can be used to sequence sulfated peptides when a multi-charged
cation is added to the solution29
We use a similar multi-charge cation solution technique to
sequence a sulfated peptide from Homarus americanus via ETD on an ion trap mass
spectrometer Here we presented the use of the ETD fragmentation technique for the analysis
of large peptides and peptides containing labile post-translational modification
171
Experimental Section
Chemical and materials
Acetonitrile methanol magnesium chloride (ACS grade) potassium chloride (ACS grade) and
calcium chloride (ACS grade) were purchased from Fisher Scientific (Pittsburgh PA) Formic
acid was from Sigma-Aldrich (St Louis MO) Human sulfated CCK-8 and CCK-like peptide
(E(pyro)FDEY(Sulfo)GHMRFamide) were purchased from American Peptide (Sunnyvale CA)
Fused-silica capillary with 75 μm id and 360 μm od was purchased from Polymicro
Technologies (Phoenix AZ) Millipore C18 Ziptip column was used for sample cleaning and all
water used in this study was deionized water (182 MΩcm) prepared from a Milli-Q Millipore
system (Billerica MA) The physiological saline consisted of 440 mM NaCl 11mM KCl 26
mM MgCl2 13 mM CaCl2 11 mM Trizma base and 5 mM maleic acid in pH 745
Animals and dissection
Callinectes sapidus (blue crab) were obtained from commercial food market and maintained
without food in artificial sea water at 10-12 ordmC Animals were cold-anesthetized by packing on
ice for 15-30min before dissection The optic ganglia with the SG attached were dissected in
chilled (~4ordmC) physiological saline and individual SGs were isolated from the optic ganglia by
micro-dissection and immediately placed in acidified methanol (90 methanol 9 glacial acetic
acid and 1 water) and stored at -80ordmC until tissue extraction
Tissue homogenization
Acidified methanol was used during the homogenization of SGs The homogenized SGs were
immediately centrifuged at 16100 g using an Eppendorf 5415D tabletop centrifuge (Eppendorf
172
AG) The pellet was washed using acidified methanol and combined with the supernatant and
further dried in a Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation) The
resulting dried sample was re-suspended with a minimum amount (20 microL) of 01 formic acid
Fractionation of homogenates using reversed phase (RP)-HPLC
The re-suspended extracts were then vortexed and briefly centrifuged The resulting supernatants
were subsequently fractionated via high performance liquid chromatography (HPLC) HPLC
separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax
UV-D II absorbance detector (Rainin Instrument Inc Woburn MA) The mobile phases included
Solution A (deionized water containing 01 formic acid) and Solution B (acetonitrile containing
01 formic acid) About 20 μl of extract was injected onto a Macrosphere C18 column (21 mm
id x 250 mm length 5 microm particle size Alltech Assoc Inc Deerfield IL) The separation
consisted of a 120 minute gradient of 5-95 Solution B Fractions were automatically collected
every two minutes using a Rainin Dynamax FC-4 fraction collector (Rainin Instrument Inc
Woburn MA) The fractions were then concentrated using in Savant SC 110 SpeedVac
concentrator (Thermo Electron Corporation) and re-suspended with minimum amount of 01
formic acid
Nano-LC-ESI-Q-TOF MSMS
Nanoscale LC-ESI-Q-TOF MSMS was performed using a Waters nanoAcquity UPLC system
coupled to a Q-TOF Micro mass spectrometer (Waters Corp Milford MA)
Chromatographic separations were performed on a homemade C18 reversed phase capillary
column (75 microm internal diameter x100 mm length 3 microm particle size 100 Aring) The mobile phases
173
used were 01 formic acid in deionized water (A) 01 formic acid in acetonitrile (B) An
aliquot of 50 microl of a tissue extract or HPLC fraction was injected and loaded onto the trap
column (Zorbax 300SB-C18 Nano trapping column Agilent Technologies Santa Clara CA)
using 95 mobile phase A and 5 mobile phase Bat a flow rate of 10 microlmin for 10 minutes
Following this the stream select module was switched to a position at which the trap column
came in line with the analytical capillary column and a linear gradient of mobile phases A and B
was initiated For neuropeptides the linear gradient was from 5 buffer B to 45 buffer B over
90 min The nanoflow ESI source conditions were set as follows capillary voltage 3200 V
sample cone voltage 35 V extraction cone voltage 1 V source temperature 100ordmC A data
dependent acquisition was employed for the MS survey scan and the selection of three precursor
ions and subsequent MSMS of the selected parent ions The MS scan range was from mz
400-1800 and the MSMS scan was from mz 50-1800
Peptide Prediction De Novo Sequencing and Database Searching
De novo sequencing was performed using a combination of MassLynxTM
41 PepSeq software
(Waters) and manual sequencing Tandem mass spectra acquired from the Q-TOF were first
deconvoluted using MaxEnt 3 software (Waters) to convert multiply charged ions into their
singly charged forms The resulting spectra were pasted into the PepSeq window for sequencing
analysis The candidate sequences generated by the PepSeq software were compared and
evaluated for homology with previous known peptides The online program blastp (National
Center for Biotechnology Information Bethesda MD httpwwwncbinlmnihgovBLAST)
was used to search the existing NCBI crustacean protein database using the candidate peptide
sequences as queries For all searches the blastp database was set to non-redundant protein
174
sequences (ie nr) and restricted to crustacean sequences (ie taxid 6657) For each of the
proteins putatively identified via blastp the BLAST score and BLAST-generated E-value for
significant alignment are provided in the appropriate subsection of the results Peptides with
partial sequence homology were selected for further examination by comparing theoretical
MSMS fragmentation spectra generated by PepSeq with the raw MSMS spectra If the
fragmentation patterns did not match well manual sequencing was performed
NanoLC Coupled to MSMS by CID and ETD
Setup for RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN (Fisher Scientific Optima LCMS grade Fair Lawn NJ) Injections
consisted of 5 microL of prepared tissue extract onto an Agilent Technologies Zorbax 300 SB-C18 5
microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5 microLmin for 5 minutes at 95
A5 B Separation was performed on a Waters HPLC column with 3 microm Atlantis dC18 75 microm
x 150 mm (Milford MA) on a gradient from 5 to 45 mobile phase B at 250 nLmin over 90
minutes at room temperature Emitter tips were pulled from 75 microm inner diameter 360 microm
outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using a commercial
laser puller model P-2000 (Sutter Instrument Co Novato CA)
Mass Spectrometry Data Acquisitions for Collision Induced Dissociation (CID)
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany) equipped
with an on-line nanospray source was used for mass spectrometry data acquisition Hystar
(Version 32 Bruker Daltonics Bremen Germany) was used to couple and control Eksigent
175
nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for all
experiments Smart parameter setting (SPS) was set to mz 700 compound stability and trap
drive level were set at 100 Optimization of the nanospray source resulted in dry gas
temperature 125oC dry gas 40 Lmin capillary voltage -1300 V end plate offset -500 V
MSMS fragmentation amplitude 10V and SmartFragmentation set at 30-300
Data was generated in data dependent mode with strict active exclusion set after two spectra and
released after one minute MSMS was obtained via CID fragmentation for the six most
abundant MS peaks with a preference for doubly charged ions and excluded singly charged ions
For MS generation the ion charge control (ICC) target was set to 200000 maximum
accumulation time 5000 ms four spectral average acquisition range of mz 300-1500 and scan
speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target was set to
200000 maximum accumulation time 5000 ms three spectral averages acquisition range of
mz 100-2000 and scan speed (Ultrascan) of 32000 mz s-1
Mass Spectrometry Data Acquisitions for Electron Transfer Dissociation (ETD)
The amaZon ETD ion-trap mass spectrometer was operated in electron transfer dissociation for
MSMS fragmentation with the same optimized settings as reported for CID unless otherwise
stated Smart parameter setting (SPS) was set to 500 mz compound stability and trap drive
level were set at 100 MSMS was obtained via ETD fragmentation for the four most
abundant MS peaks with no preference for specifically charged ions except to exclude singly
charged ions The ETD reagent parameters were set to 400000 target ICC for the fluoranthene
radical anion The fluoranthene radical anion has an mz of 210 and therefore the 210 mz value
was removed from the resulting MSMS spectra and 160 mz was set for the MSMS low mz
cut-off
176
Direct Infusion of Sulfated Peptide Standards into the Mass Spectrometer using ETD and
CID Fragmentation
The sulfated peptide standards were infused into the mass spectrometer at a flow rate of 300
nlmin using a fused-silica capillary with 75 μm id and 360 μm od coupled to a custom pulled
tip The CCK-8 human peptide (20 microM) was infused in 5050 ACNH2O and detected in
negative ionization mode with an ICC of 70000 and fragmented with CID using the same
settings as the previous CID experiments Alternatively the CCK-like lobster sulfated peptide
(2 microM) was mixed with 30 microM of different salts (MgCl2 KCl or CaCl2) and 01 formic acid in
5050 ACNH2O The plus three charge of the CCK-like lobster peptide was selected for ETD
fragmentation in positive mode with the same setting as the previous ETD experiments The
data were then de novo sequenced for coverage and localization of the sulfation site
Data Analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen Germany)
Deviations in parameters from the default Protein Analysis in DataAnalysis were as follows
intensity threshold 1000 maximum number of compounds 1E7 and retention time window 05
minutes These parameter changes assisted in providing better quality spectra for sequencing
Identification of peptides was performed using Mascot (Version 23 Matrix Science London
UK) Searches were performed against a custom crustacean database none enzyme allow up to
1 missed cleavage amidated C-terminal as variable modifications peptide tolerance mass error
12 Da MSMS mass error tolerance is 06 Da
Results and Discussion
177
Identification and Characterization of Intact CPRPs Using ETD
Using ESI-Q-TOF 9 truncated CPRPs fragments were sequenced to cover the amino acid
sequence of the intact CPRP peptide RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE
A representative CID MSMS spectrum of ASLKSDTVTPLR is shown in Figure 1 (a) CID
using ESI-Q-TOF mainly produces fragmentation for doubly and triply charge molecules which
is adequate to sequence peptides with relatively small molecular weight (less than 2000 Da)
However CID fragmentation is insufficient to sequence the larger intact CPRP in a complex
sample whose molecular mass is 3837 Da and therefore it is extremely difficult to directly
sequence by traditional CID ESI-Q-TOF ETD is an attractive fragmentation alternative to
sequence the highly charged CPRP peptide In ETD a protonated peptide reacts with an anion
(fluoranthene radical) where the anion transfers an electron to the peptide cation and the resulting
fragmentation occurs along the N-Cα bond by free-radical-driven-cleavage The large size of
CPRP offers multiple basic amino acid sites to sequester charge and reduce the sequence
coverage from collision induced dissociate by preventing random backbone cleavage whereas
ETD does not cleave the weakest bond and provides the alternative fragmentation pathway to
obtain superior sequence coverage to larger peptides As shown in Figure 1 (cd) the
fragmentation of ETD is highly amenable to ETD compared to the CID fragmentation in Figure
1 (b) As evident from comparison ETD offers more extensive fragmentation than CID thus
providing enhanced coverage for large intact peptides such as CPRPs Specifically we observe
125 of the b- and y- cleavages for CID as compared to an average of 535 of c- and zbull-
fragments More than a four-fold increase in fragments using ETD suggests the utility of this
relatively new tandem MS fragmentation method as complementary techniques for de novo
sequencing of large intact endogenous peptides and novel neuropeptide discovery endeavors
178
Negative Mode Sulfated Peptide Identification
An accepted method for identification and quantification for sulfated peptides is negative
ionization30
CCK-8 sulfated peptide standards show intense signal in negative ionization mode
without needing to have additives added such as salts Figure 2 shows the CID MSMS
spectrum from the negative ionization and produces an intense b6 ion at 430 mz The transition
from the doubly negatively charged MS ion of 570 mz to the b6 ion provides a selected reaction
monitoring transition for quantification studies but the sequence information is limited and the
presence of the methionine produces variable oxidation In addition Figure 2 shows the
MSMS product ions with the loss of the sulfate group thus making site-specific location of
sulfation more difficult
Investigation of Cation Salt Adducts to Produce Multiply Charged Sulfated Peptides
Sulfated peptides can be isolated in positive scanning mode but usually only in a plus one
state with low signal intensity If ETD is performed on the singly charged peptide cation a
neutral is formed and is lost in the mass spectrometer and thus no sequence information can be
obtained In order to remedy this situation a technique that adding metal salts to peptides to
increase charge state for ECD used in Fourier transform ion cyclotron resonance mass
spectrometry (FTICR-MS)29
inspired the investigation of increasing charge state of targeted
peptide via metal salt adduction followed by ETD analysis in a Bruker amaZon ion trap
Various salts were investigated including MgCl2 CaCl2 and KCl each used at a concentration of
30 microM and mixed with a sulfated peptide standard at 2 microM The addition of KCl produced
mostly doubly charged ion of the lobster CCK-like peptide standard K adduct which produced
insufficient sequence information from ETD fragmentation (data not shown) In comparison
the MgCl2 and CaCl2 produced intense triply charged peptide ions but CaCl2 produced lower
179
signal intensity compared to MgCl2 (data not shown)
Cation Assisted ETD Fragmentation of CCK-like Lobster Sulfated Peptide and Future
Directions
The addition of MgCl2 to the lobster CCK-like sulfated peptide is shown in Figure 3
Except for z1 the complete z-series is obtained including the z7 ion with and without the
sulfation PTM The spectrum is complicated by the additional Mg adducts but several peaks
are shown without any Mg adduct such as z2 z3 z4 etc Clearly the method of cation
assisted ETD fragmentation can be useful using ion trap instruments and can properly sequence
sulfated peptides that are prone to neutral loss from the labile PTM One concern about future
direction in sulfopeptide isolation in non-genome sequenced species is isolation of sulfopeptides
Currently antibodies to specific sulfopeptides is the only commonly used enrichment technique
for sulfopeptides Also since metal cations are needed for this method direct infusion into an
ESI mass spectrometer is preferred to prevent running large amounts of adduct forming salts
through the LC system With direct infusion the lack of separation confounds the problem in
sulfopeptide detection because any co-eluting peptides could have ionization efficiency and thus
reduce the signal of the sulfopeptide Once discovered and sequenced a selected reaction
monitoring (SRM) method could be developed using LC retention coupled with negative
ionization mode followed by CID MSMS or possibly MSMSMS to perform quantitative
studies for sulfopeptides
Conclusions
In this study ETD was performed to improve the sequence coverage of large endogenous
neuropeptides with higher charge and bigger size The intact CPRP from Callinectes sapidus was
identified and characterized with 68 sequence coverage by MS for the first time Cation
180
assisted ETD fragmentation was also shown to be a powerful tool in de novo sequencing of
sulfated neuropeptides These endeavors into using ETD for certain neuropeptides will assist in
future analysis of large neuropeptides and PTM containing neuropeptides
181
Reference
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food intake Nature 2000 404 (6778) 661-71
2 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R
Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide
family of aplysia J Neurosci 2002 22 (17) 7797-808
3 Baggerman G Cerstiaens A De Loof A Schoofs L Peptidomics of the larval Drosophila melanogaster
central nervous system Journal of Biological Chemistry 2002 277 (43) 40368-40374
4 Desiderio D M Mass spectrometric analysis of neuropeptidergic systems in the human pituitary and
cerebrospinal fluid Journal of Chromatography B 1999 731 (1) 3-22
5 Li L J Kelley W P Billimoria C P Christie A E Pulver S R Sweedler J V Marder E Mass
spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab Cancer
borealis Journal of Neurochemistry 2003 87 (3) 642-656
6 Li L J Moroz T P Garden R W Floyd P D Weiss K R Sweedler J V Mass spectrometric survey of
interganglionically transported peptides in Aplysia Peptides 1998 19 (8) 1425-1433
7 Strand F L Neuropeptides regulators of physiological processes 1st ed ed MIT Press Cambridge Mass
1999 p 658 p
8 Fu Q Goy M F Li L J Identification of neuropeptides from the decapod crustacean sinus glands using
nanoscale liquid chromatography tandem mass spectrometry Biochemical and Biophysical Research
Communications 2005 337 (3) 765-778
9 Fu Q Christie A E Li L J Mass spectrometric characterization of crustacean hyperglycemic hormone
precursor-related peptides (CPRPs) from the sinus gland of the crab Cancer productus Peptides 2005 26 (11)
2137-2150
10 Ma M M Chen R B Ge Y He H Marshall A G Li L J Combining Bottom-Up and Top-Down Mass
Spectrometric Strategies for De Novo Sequencing of the Crustacean Hyperglycemic Hormone from Cancer borealis
Analytical Chemistry 2009 81 (1) 240-247
11 Ma M M Sturm R M Kutz-Naber K K Fu Q Li L J Immunoaffinity-based mass spectrometric
characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis Biochemical
and Biophysical Research Communications 2009 390 (2) 325-330
12 Ma M M Bors E K Dickinson E S Kwiatkowski M A Sousa G L Henry R P Smith C M Towle
D W Christie A E Li L J Characterization of the Carcinus maenas neuropeptidome by mass spectrometry and
functional genomics General and Comparative Endocrinology 2009 161 (3) 320-334
13 Chen R B Jiang X Y Conaway M C P Mohtashemi I Hui L M Viner R Li L J Mass Spectral
Analysis of Neuropeptide Expression and Distribution in the Nervous System of the Lobster Homarus americanus
Journal of Proteome Research 2010 9 (2) 818-832
14 Ma M M Chen R B Sousa G L Bors E K Kwiatkowski M A Goiney C C Goy M F Christie A
E Li L J Mass spectral characterization of peptide transmittershormones in the nervous system and
neuroendocrine organs of the American lobster Homarus americanus General and Comparative Endocrinology
2008 156 (2) 395-409
15 Ollivaux C Gallois D Amiche M Boscameric M Soyez D Molecular and cellular specificity of
post-translational aminoacyl isomerization in the crustacean hyperglycaemic hormone family FEBS J 2009 276
(17) 4790-802
16 Wilcockson D C Chung J S Webster S G Is crustacean hyperglycaemic hormone precursor-related
peptide a circulating neurohormone in crabs Cell and Tissue Research 2002 307 (1) 129-138
17 Choi C Y Zheng J Watson R D Molecular cloning of a cDNA encoding a crustacean hyperglycemic
hormone from eyestalk ganglia of the blue crab Callinectes sapidus General and Comparative Endocrinology 2006
148 (3) 383-387
18 Syka J E Coon J J Schroeder M J Shabanowitz J Hunt D F Peptide and protein sequence analysis
by electron transfer dissociation mass spectrometry Proc Natl Acad Sci U S A 2004 101 (26) 9528-33
19 Coon J J Syka J E P Schwartz J C Shabanowitz J Hunt D F Anion dependence in the partitioning
between proton and electron transfer in ionion reactions International Journal of Mass Spectrometry 2004 236
(1-3) 33-42
20 Xia Y Gunawardena H P Erickson D E McLuckey S A Effects of cation charge-site identity and
position on electron-transfer dissociation of polypeptide cations J Am Chem Soc 2007 129 (40) 12232-43
182
21 Altelaar A F Mohammed S Brans M A Adan R A Heck A J Improved identification of endogenous
peptides from murine nervous tissue by multiplexed peptide extraction methods and multiplexed mass spectrometric
analysis J Proteome Res 2009 8 (2) 870-6
22 Swaney D L McAlister G C Coon J J Decision tree-driven tandem mass spectrometry for shotgun
proteomics Nat Methods 2008 5 (11) 959-64
23 Monigatti F Hekking B Steen H Protein sulfation analysis--A primer Biochim Biophys Acta 2006 1764
(12) 1904-13
24 Chandler P C Wauford P K Oswald K D Maldonado C R Hagan M M Change in CCK-8 response
after diet-induced obesity and MC34-receptor blockade Peptides 2004 25 (2) 299-306
25 Little T J Feltrin K L Horowitz M Meyer J H Wishart J Chapman I M Feinle-Bisset C A
high-fat diet raises fasting plasma CCK but does not affect upper gut motility PYY and ghrelin or energy intake
during CCK-8 infusion in lean men Am J Physiol Regul Integr Comp Physiol 2008 294 (1) R45-51
26 Blevins J E Morton G J Williams D L Caldwell D W Bastian L S Wisse B E Schwartz M W
Baskin D G Forebrain melanocortin signaling enhances the hindbrain satiety response to CCK-8 Am J Physiol
Regul Integr Comp Physiol 2009 296 (3) R476-84
27 Turrigiano G G Selverston A I A cholecystokinin-like hormone activates a feeding-related neural circuit in
lobster Nature 1990 344 (6269) 866-8
28 Wolfender J L Chu F Ball H Wolfender F Fainzilber M Baldwin M A Burlingame A L
Identification of tyrosine sulfation in Conus pennaceus conotoxins alpha-PnIA and alpha-PnIB further investigation
of labile sulfo- and phosphopeptides by electrospray matrix-assisted laser desorptionionization (MALDI) and
atmospheric pressure MALDI mass spectrometry J Mass Spectrom 1999 34 (4) 447-54
29 Liu H Hakansson K Electron capture dissociation of tyrosine O-sulfated peptides complexed with divalent
metal cations Anal Chem 2006 78 (21) 7570-6
30 Young S A Julka S Bartley G Gilbert J R Wendelburg B M Hung S C Anderson W H
Yokoyama W H Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using
immunoprecipitation liquid chromatography-mass spectrometrymass spectrometry Anal Chem 2009 81 (21)
9120-8
183
Figure 1 Comparison of tandem MS spectra of truncated CPRP (ASLKSDTVTPL mz 128766)
by ESI-Q-TOF (a) and intact CPRP (MW 3837) by the amaZon ETD for both CID and ETD
fragmentation (a) MSn of precursor ion with mz 640 with charge state +6 by CID represent
loss of NH3 ordm represent loss of H2O (b) MS+6
of precursor ion with mz 640 with charge state +6
by ETD at z represent z+1 z represent z+2 (c) MS+5
of precursor ion with mz 768 with charge
state +5 by ETD z represent z+1 z represent z+2 For all labels in Figure 1 charge state +1 is
not specified
184
185
Figure 2 Negative CID spectrum of CCK-8 peptide standard (20 microM) via direct infusion show
the doubly charged b6 ion provides the most intense MSMS transition
186
Figure 3 ETD spectrum of E(pyro)FDEY(Sulfo)GHMRF-amide (2 microM) obtained on the
amaZon ETD via direct infusion with 30 microM MgCl2 The sulfated peptide can be identified
with a visible z-series of z2 to z9 and identified sulfate loss
187
Chapter 7
Investigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysis
Adapted from ldquoInvestigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J
Wellner D Li L Journal of Mass Spectrometry In Press
188
ABSTRACT
This work investigates the introduction of methanol and a salt modifier to molecular
weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide
quantities Using a neuropeptide standard bradykinin sample loss was reduced over two orders
of magnitude with and without undigested protein present Additionally a bovine serum
albumin (BSA) tryptic digestion was investigated Twenty-seven tryptic peptides were identified
from MALDI mass spectra after enriching with methanol while only two tryptic peptides were
identified after the standard MWCO protocol The strategy presented here enhances recovery
from MWCO separation for sub-microg peptide samples
INTRODUCTION
Molecular weight cut-off membrane-based centrifugal filter devices (MWCO) are
commonly used to desalt and concentrate large molecular weight proteins [1] Greeing and
Simpson recently investigated various MWCO membranes for large amounts of starting material
(~6 mg) focusing on optimal conditions for the sub 25 kDa protein fraction [2] The authors
recovered 200 μg to 29 mg of protein from multiple MWCO experiments and demonstrated that
a 1090 acetonitrile (ACN)H2O elution solvent produced optimal results [3] In addition Manza
et al provided an alternative approach to isolate proteins with a 5 kDa MWCO by using
NH4HCO3 and recovering the retained proteins [4] Alternatively the elution from MWCOs can
be collected to recover only low molecular weight peptides Multiple peptidomic studies have
utilized MWCOs for peptide isolation during the first few steps of sample preparation [5 6]
When sample amount is limited or peptide content is below 1 microg sample loss is a significant
concern when using MWCOs to isolate endogenous peptides Optimized protocols have been
189
investigated using ACN [3 7] salt [4 8] SDS [5] or native sample [6 9 10] but these
experiments primarily focused on large sample amounts rather than sub-microgram peptide
quantities
MWCOs separate large molecules from small molecules The small molecule fraction
may be rich with signaling peptides (SP) cytokines and other small molecules involved in cell-
cell signaling Signaling peptides perform various functions in the body including cell growth
cell survival and hormonal signaling between organs [11] Individual SP contribute to different
aspects of behavior such as pain (enkephalins) [12] feeding (neuropeptide Y) [13] and blood
pressure (bradykinin) [14] MWCO separations can be used to enrich biologically important SP
and explore the peptide content from biological fluids with relatively low peptide content like
blood or cerebrospinal fluid (CSF) In a recent investigation the detection of neuropeptides and
standards in crustacean hemolymph was improved when methanol and protease inhibitors were
present before performing MWCO neuropeptide isolation The impact of methanol on MWCO
sample loss was not investigated in the study [15] In another study a large-scale mass
fingerprinting protocol of endogenous peptides from CSF used a combination of salts before
MWCO fractionation but the impact of adding salts was not discussed [16] The most
commonly used brand of MWCO in the publications and in peptidomic studies is Millipore
Therefore Millipore MWCOs (using regenerated cellulose as the membrane) were used in the
present study The purpose of this work is to provide an optimized sample preparation technique
for MWCO filtering to reduce sample loss and allow sub-microg detection of peptides using MALDI
mass spectrometry
MATERIALS AND METHODS
190
Materials and Chemicals
Water acetonitrile methanol (optima LCMS grade) and sodium chloride (995) were
purchased from Fisher Scientific (Fair Lawn NJ) The α-cyano-4-hydroxy-cinnamic acid (99)
formic acid (FA) (ge98) and bovine serum albumin (ge96) were purchased from Sigma-
Aldrich (St Louis MO) Amicon Ultra 05 mL 10000 MWCO centrifugal filters and ZipTips
packed with C18 reversed-phase resin were purchased from Millipore (Billerica MA) Trypsin-
digested bovine serum albumin (BSA) was purchased from Waters (Milford MA) Bradykinin
was purchased from American Peptide Company (Sunnyvale CA)
MALDI MS Instrumentation
An AutoFLEX III MALDI TOFTOF mass spectrometer (Bruker Daltonics Billerica
MA) was operated in positive ion reflectron mode The MALDI MS instrument is equipped with
a proprietary smart beam (Bruker Daltonics Billerica MA) laser operating at 200 Hz The
instrument was internally calibrated over the mass range of mz 500minus2500 using a standard
peptide mix Two thousand laser shots were collected per sample spot at an accelerating voltage
of 19 kV and a constant laser power using random shot selection The acquired data were
analyzed using FlexAnalysis software (Bruker Daltonics Billerica MA) Mass spectrometry
data acquisition was obtained by averaging 2000 laser shots
Molecular weight cut off separation procedure
The MWCO separations were performed using Amicon Ultra 05 mL 10000 MWCO
centrifugal filters (Billerica MA) Before MWCO separation three washing steps were
performed to remove contaminants from the filter The three washes were 500 μL of 5050
H2OMeOH 500 μL of H2O and 400 μL of the solution used for MWCO separation For the
191
100 H2O solution 1 microg of BSA or bradykinin was used for separation All the other MWCO
separation experiments used 500 ng of BSA or 100 ng or less of bradykinin The MWCO filter
was then centrifuged at 14000 rcf for 5 min at room temperature in an Eppendorf 5415 D
microcentrifuge (Brinkmann Instruments Inc Westbury NY) The filtrate was concentrated in a
Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation West Palm Beach FL)
and acidified The resulting sample was desalted according to the manufacturer using C18
ZipTips from Millipore (Billerica MA) by washing the ZipTip with three times 100 ACN
three aqueous washes of 01 FA binding the peptides from the solution and one aqueous wash
of 01 FA Peptides were then eluted from the ZipTips using 15 microL of 50 ACN in 01 FA
Matrix deposition
Equal volumes of 05 μL from the 15 μL sample solution (including standards not subject
to MWCO filtering) and α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution in 50
ACN50 H2O were mixed using a dried-droplet method and spotted on a MALDI target The
resulting droplets were allowed to air dry prior to mass spectrometry acquisition
RESULTS AND DISCUSSION
Analysis of two orders of magnitude increase for bradykinin standard
Bradykinin was selected to assess the potential peptide loss in the flow-through after
performing MWCO separation As shown in Figure 1 1 microg of bradykinin standard does not
produce a detectable signal by MALDI mass spectrometry analysis after a 10 kDa MWCO
separation in water (performed in triplicate) For comparison 1 ng of bradykinin standard
diluted to 15 microL produced an intense signal on the MALDI mass spectrometer suggesting
192
significant sample loss occurs when the target analyte is low in quantity (data not shown
performed in triplicate) Figure 1 shows that the addition of a salt in this case NaCl improves
the limits of detection and decreases sample loss when 7030 watermethanol was compared to
7030 aqueous 1 M NaClmethanol The reproducible results gave a relative standard deviation
(RSD) of 6 for peak intensity Figure 1 shows that even when starting with 1 μg of bradykinin
too much sample is lost during the MWCO separation in water to detect the remainder
However an intense signal is observed for 10 ng of bradykinin after the MWCO separation when
7030 aqueous 1 M NaClmethanol is used as an elution solvent Sample loss from zip-tipping
was estimated in triplicate by calculating the decreases in peak intensity When 10 ng of
bradykinin was used sample loss was ~41 In comparison the calculated yield for 10 ng of
bradykinin after the 7030 aqueous 1 M NaClmethanol MWCO separation and zip-tip desalting
showed an estimated sample loss of 63 meaning more loss can be attributed to sample clean-
up than MWCO filtration
A series of experiments were performed to determine if 7030 aqueous 1 M
NaClmethanol is an optimal solution to recover peptides during a MWCO separation (data not
shown) A 5050 aqueous 1 M NaClmethanol and a 5050 watermethanol elution were
performed in duplicate but signal intensity of the resulting bradykinin was poor and numerous
polymer peaks were detected in the flow through A lower salt concentration 01 M NaCl was
used but also produced greatly reduced signal when compared to aqueous 1 M NaCl To assess
the optimized methodrsquos compatibility with lower sample amounts the 7030 aqueous 1 M
NaClmethanol solution was added to 1 ng of bradykinin for MWCO separation but no signal
was obtained (data not shown) Using a neuropeptide standard the addition of methanol and
NaCl salt significantly improved the sample recovery in sub-microg amounts
193
BSA tryptic peptide mixture analysis
After demonstrating the importance of using an optimized solution for MWCO
separations with an individual peptide the new method was applied to 500 ng of BSA tryptic
digest to investigate its utility with more complex peptide mixtures Table 1 lists the BSA
tryptic peptides identified in the MALDI MS analysis from different solution conditions
processed by MWCO separation As shown in Table 1 a directly spotted BSA tryptic peptide
standard in the absence of any MWCO filtration enabled identification of 39 tryptic peptides by
accurate peptide mass measurements Once again when using 100 H2O for MWCO
separations the starting amount was doubled to 1 microg (also done with 500 ng data not shown)
However many tryptic peptides were not detected due to low signal intensities and non-optimal
elution conditions Instead of H2O a 1 M NaCl solution was used for the MWCO elution but
only two tryptic peptides were identified (Table 1) The addition of 30 methanol into the
sample before MWCO filtration produced the first increase in identified BSA tryptic peptides
The remaining data from Table 1 shows improved BSA tryptic peptide identifications as the
sample (elution) conditions were further optimized Figure 2 shows the actual mass spectra
associated with the three most promising elution solutions along with 100 H2O
The BSA tryptic peptide intensities are shown in Figure 2A and the most intense tryptic
peptide YLYEIAR mz 92749 was observed in the four different solutions shown in Figure 2B
but not in the 100 H2O or 100 1 M NaCl solutions (data not shown) In Figure 2A all mass
spectra are normalized to an intensity of 7 x 104 to illustrate two points First the MWCO
filtering step still produced sample loss regardless of the solvent conditions chosen Second
there is a noticeable increase in peptide peak intensity using the optimized solvent 6040
194
aqueous 1 M NaClmethanol (Figure 2A) Figure 2C displays a zoomed-in view of a BSA
tryptic peptide signal LKECC
DKPLLEK mz 153266 (
carbamidomethyl) observed only in
the optimized solvent The detection of the mz 153266 peptide in Figure 2C highlights the
potential gain in sample and detectable peptides by using an optimized saltMeOH combination
A non-optimized saltMeOH combination will still reduce sample loss but further minimizing
sample loss during sample preparation will always be desirable in any analytical protocol
MWCO composition
The purpose of this application note is to provide evidence of sub-microg sample loss during
MWCO separations of peptide samples and a solution to overcome this limitation The
explanation of why adding MeOH and NaCl to the sample solution provides a significant
reduction in sample loss is beyond the scope of this application note Regardless Supplemental
Table S1 is an expanded version of Table 1 showing the amino acid sequence hydrophobicity
calculated using GRAVY scores and pI of the identified peptides in this study No discernible
trend was obtained from the data The membrane of commonly used MWCO in peptidomics and
for this study is comprised of chemically treated (regenerated) cellulose which is a
polysaccharide containing β (1rarr4) linked D-glucose Glucose has numerous free hydroxyl
groups which could non-specifically adsorb peptides flowing through the MWCO The addition
of MeOH has the most significant effect on signal which could be due to disrupting the
interaction between peptides and hydroxyl groups from glucose NaCl has a less significant
effect on sample recovery compared to MeOH but a detectable reduction in sample loss is noted
This improvement in sample recovery could be analogous to the use of NaCl in
195
immunodepletion protocols to reduce non-specific binding which is accomplished by adding
150 mM NaCl [17]
Analysis of bradykinin in the presence of undigested BSA
When using MWCO for peptide isolation proteins are typically present in the samples
usually in larger amounts Figure 3 shows the effect that adding BSA protein to a 10 ng
bradykinin solution before MWCO fractionation has on the resulting recovery of bradykinin
Adding 10 μg of BSA to the optimized 7030 aqueous 1 M NaClmethanol solution slightly
decreased bradykininrsquos signal with a RSD of 10 More severe signal reduction occurred after
adding 100 microg BSA with a RSD of 2 (N=2) It is not unexpected that more signal reduction
due to sample loss would occur especially in the 100 microg BSA sample because the BSA protein
has a molar ratio of 160 BSA protein molecules to one bradykinin peptide Figure 3 shows the
usefulness of the MWCO method with samples containing large amounts of proteins
RecommendationConclusion
The present work provides solutions to reduce sample loss from the use of MWCO for
sub-microg peptide isolation with or without non-digested proteins in the sample Despite its
widespread utility significant sample loss often occurs during the MWCO fractionation step
which is particularly problematic when analyzing low-abundance peptides from limited starting
material This application note aims to reduce sample loss during MWCO separations
specifically for sub-microg peptide isolation If complex samples are processed with MWCO
separation the authors recommend eluting the sample with 6040 aqueous 1 M NaClmethanol
solution as a starting point to minimize sample loss This application note provides a viable
196
alternative for sub-microg peptide MWCO separation circumventing the need to increase the starting
material by minimizing sample loss from using a MWCO membrane-based centrifugal filter
device
References
[1] HM Georgiou GE Rice MS Baker Proteomic analysis of human plasma failure of
centrifugal ultrafiltration to remove albumin and other high molecular weight proteins
Proteomics 2001 1 1503
[2] DW Greening RJ Simpson Low-molecular weight plasma proteome analysis using
centrifugal ultrafiltration Methods Mol Biol 2011 278 109
[3] DW Greening RJ Simpson A centrifugal ultrafiltration strategy for isolating the low-
molecular weight (ltor=25K) component of human plasma proteome J Proteomics 2010 73
637
[4] LL Manza SL Stamer AJ Ham SG Codreanu DC Liebler Sample preparation and
digestion for proteomic analyses using spin filters Proteomics 2005 5 1742
[5] D Theodorescu D Fliser S Wittke H Mischak R Krebs M Walden M Ross E Eltze O
Bettendorf C Wulfing A Semjonow Pilot study of capillary electrophoresis coupled to mass
spectrometry as a tool to define potential prostate cancer biomarkers in urine Electrophoresis
2005 26 2797
[6] K Antwi G Hostetter MJ Demeure BA Katchman GA Decker Y Ruiz TD Sielaff LJ
Koep DF Lake Analysis of the plasma peptidome from pancreas cancer patients connects a
peptide in plasma to overexpression of the parent protein in tumors J Proteome Res 2009 8
4722
[7] LP Aristoteli MP Molloy MS Baker Evaluation of endogenous plasma peptide extraction
methods for mass spectrometric biomarker discovery J Proteome Res 2007 6 571
[8] A Zougman B Pilch A Podtelejnikov M Kiehntopf C Schnabel C Kumar M Mann
Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7
386
[9] X Yuan DM Desiderio Human cerebrospinal fluid peptidomics J Mass Spectrom 2005 40
176
[10] X Zheng H Baker WS Hancock Analysis of the low molecular weight serum peptidome
using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer J Chromatogr A
2006 1120 173
[11] L Li JV Sweedler Peptides in the brain mass spectrometry-based measurement approaches
and challenges Annu Rev Anal Chem 2008 1 451
[12] GB Stefano G Fricchione Y Goumon T Esch Pain immunity opiate and opioid
compounds and health Med Sci Monit 2005 11 MS47
[13] J Jensen Regulatory peptides and control of food intake in non-mammalian vertebrates Comp
Biochem Physiol A Mol Integr Physiol 2001 128 471
197
[14] A Kuoppala KA Lindstedt J Saarinen PT Kovanen JO Kokkonen Inactivation of
bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma Am
J Physiol Heart Circ Physiol 2000 278 H1069
[15] R Chen M Ma L Hui J Zhang L Li Measurement of neuropeptides in crustacean
hemolymph via MALDI mass spectrometry J Am Soc Mass Spectrom 2009 20 708
[16] H Jahn S Wittke P Zurbig TJ Raedler S Arlt M Kellmann W Mullen M Eichenlaub H
Mischak K Wiedemann Peptide fingerprinting of Alzheimers disease in cerebrospinal fluid
identification and prospective evaluation of new synaptic biomarkers PLoS One 2011 6
e26540
[17] NA Cellar AS Karnoup DR Albers ML Langhorst SA Young Immunodepletion of high
abundance proteins coupled on-line with reversed-phase liquid chromatography a two-
dimensional LC sample enrichment and fractionation technique for mammalian proteomics J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877 79
198
Table 1 Identified BSA tryptic peptides from various MWCO separation conditions
BSA tryptic
peptide (MH+)
100
H2O 1microg
100
1 M NaCl
70
H2O
80
1 M NaCl
70
1 M NaCl
60
H2O
60
1 M NaCl
5083
5453
6894
7124
8985
9275
10345
10725
11385
11636
12496
12837
13057
13997
14157
14197
14398
14636
14798
15026
15118
15328
15547
15677
15768
16399
16678
16738
17248
17408
17477
17497
18809
18890
19019
19079
20450
21139
22479
Total 39 2 2 6 8 15 15 27
199
Figure 1 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard showing improvement over two orders of magnitude in detection limits Each MWCO
separation was performed at minimum in triplicate with representative spectrum selected for
each with a calculated RSD from the peak heights Three different amounts of bradykinin were
tested to assess the magnitude of sample loss under different MWCO solvent conditions The
top panel shows 1 microg of bradykinin standard after MWCO separation with 100 H2O elution
produced no signal The addition of 40 or 30 MeOH produced very low bradykinin signals
for both 100 ng (RSD of 28) and 10 ng (SNlt3 no RSD calculated) respectively In the
bottom two spectra each showed very large intensity but the 7030 aqueous 1 M NaClmethanol
10 ng bradykinin was processed with a 10kDa MWCO and zip-tipped and was reproducible with
200
a RSD of 6 The last spectrum was from 10 ng bradykinin (RSD of 3) which was diluted to
an equivalent volume as all the other experiments and directly spotted onto the MALDI plate
201
Figure 2 Representative MALDI mass spectra from MWCO separation of a BSA tryptic
peptide standard showing sample loss Stacked mass spectra from mz range 875-2150
normalized to 7 x 104 intensity representing the detection difference from a BSA tryptic peptide
standard from different MWCO separation conditions (A) It should be noted that when the
solvent for MWCO elution was 100 H2O 1 microg of BSA tryptic peptides was processed instead
of 500 ng A zoomed in view of the most abundant BSA tryptic peptide detected YLYEIAR
mz 92749 (B) Various percentages of MeOH produced significant signal but addition of a salt
(1 M NaCl) increases the signal which is closest to a directly spotted BSA tryptic peptide
standard A zoomed in view of a representative low intensity BSA tryptic peptide detected
LKECC
DKPLLEK mz 153266 (C) The optimized solution to be used for MWCO filtration
202
6040 aqueous 1 M NaClmethanol was the only procedure that enabled the detection of the
tryptic peptide in Figure 2C which was also detected in the directly spotted BSA tryptic peptide
standard All experiments were performed a minimum of two times with nearly identical results
) Carbamidomethyl amino acid modification
ordm) Tryptic peptide identified in three of the spectra in Figure 2A
dagger) Tryptic peptide identified in two of the spectra in Figure 2A
) Tryptic peptide identified in a single spectrum in Figure 2A
203
Figure 3 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard with a BSA protein present showing optimized solvent conditions minimized samples
losses Each experiment was performed in duplicate Two different amounts of BSA protein
were tested to assess the magnitude of sample loss caused by the presence of a protein The top
panel shows 10 microg of BSA protein and the second panel shows 100 microg of BSA protein added
while only 10 ng of bradykinin was added Detectable sample loss was observed when the BSA
protein was added but panel two shows that the amount of BSA protein was 1 x 104 greater
(equivalent to 160 fold molar excess) than bradykinin The last two spectra were controls using
a MWCO with the optimized solution in panel 3 and panel 4 using 10 ng bradykinin which was
diluted to an equivalent volume as all the other experiments and directly spotted onto the
MALDI plate
204
Supplemental Table 1 Expanded Table 1 including grand average of hydropathicity (GRAVY)
score theoretical pI and the sequence from the underlying amino acid sequence for the peptides
identified in the BSA digest The GRAVY and pI scores were obtained from ExPASy
Bioinformatics and modifications were not taken into consideration
(httpwebexpasyorgprotparam) Peptide assignments to the recorded peaks were done by
BSA
tryptic
peptide
(MH+)
GRAVY
score
Theoretical
pI
Sequence 100
H2O
1microg
100
1 M
NaCl
70
H2O
80
1 M
NaCl
70
1 M
NaCl
60
H2O
60
1 M
NaCl
5083 NA NA FGER
5453 0900 972 VASLR
6894 0267 979 AWSVAR
7124 -0950 647 SEIAHR
8985 0529 674 LcVLHEK
9275 -0071 600 YLYEIAR
10345 -0725 674 NEcFLSHK
10725 -0211 538 SHcIAEVEK
11385 0 599 ccTESLVNR
11636 0130 453 LVNELTEFAK
12496 -1250 545 FKDLGEEHFK
12837 0264 675 HPEYAVSVLLR
13057 -0582 532 HLVDEPQNLIK
13997 0567 437 TVMENFVAFVDK
14157 0567 437 TVmENFVAFVDK
14197 0058 530 SLHTLFGDELcK
14398 -0133 875 RHPEYAVSVLLR
14636 -0515 465 TcVADESHAGcEK
14798 0292 600 LGEYGFQNALIVR
15026 -0625 409 EYEATLEEccAK
15118 0207 597 VPQVSTPTLVEVSR
15328 -0617 617 LKEccDKPLLEK
15547 -0823 441 DDPHAcYSTVFDK
15677 -0085 437 DAFLGSFLYEYSR
15768 -0985 456 LKPDPNTLcDEFK
16399 -0067 875 KVPQVSTPTLVEVSR
16678 0064 437 MPCTEDYLSLILNR
16738 -1723 550 QEPERNEcFLSHK
17248 0064 437 MPcTEDYLSLILNR
17408 0064 437 mPcTEDYLSLILNR
17477 -0914 414 YNGVFQEccQAEDK
17497 -0621 410 EccHGDLLEcADDR
18809 -0537 606 RPcFSALTPDETYVPK
18890 -0567 674 HPYFYAPELLYYANK
19019 -1275 466 NEcFLSHKDDSPDLPK
19079 0044 454 LFTFHADIcTLPDTEK
20450 -0812 839 RHPYFYAPELLYYANK
21139 -0682 480 VHKEccHGDLLEcADDR
22479 -0458 423 EccHGDLLEcADDRADLAK
Total 39 2 2 6 8 15 15 27
205
mass matching with no tandem mass spectrometry performed Lower case amino acids indicate
a modification present in the peptide of carbamidomethyl (c) or oxidation (m)
206
Chapter 8
Conclusions and Future Directions
207
Summary
Comparative shotgun proteomics investigating numerous biological changes in various
species and sample media and peptidomic method development have been reported The
developed comparative shotgun proteomics based on label-free spectral counting with nanoLC
MSMS platform have been employed in mouse CSF rat CSF yeast culture and other biological
specimens Mass spectrometry (MS)-based peptidomic enhancements using ETD and improved
sample preparation methods for molecular weight cut-offs have been reported Together these
studies add to the Li Labs capabilities in comparative shotgun proteomics and expand available
methods for peptidomic research
Immunodepletion of CSF for comparative proteomics
Chapters 3 and 4 used similar methods to generate a list of differentially expressed
proteins providing insight into how Alexander disease (AxD) perturbs the proteome and how the
new prion model rat adapted scrapie (RAS) proteome varies from control rats In the GFAP
overexpressor mouse CSF study in Chapter 3 we have produced a panel of proteins with
significant up or down regulation in the CSF of transgenic GFAP overexpressor mice MS-based
proteomic study of this mouse model for AxD was consistent with the previous studies showing
elevation of GFAP in CSF The development of a modified IgY-14 immunodepletion technique
for low amounts of CSF with recommendations for future antibody depletion techniques to deal
with the unique challenges of mouse CSF was presented Modified proteomics protocols were
employed to profile mouse CSF with biological and technical triplicates addressing the
variability and providing quantitation with dNSAF spectral counting Validation of the data was
performed using both ELISA and RNA microarray data to provide corroboration with the
208
changes in the putative biomarkers This work presents numerous interesting targets for future
study in AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
Using the protocol developed in Chapter 3 we performed a similar study in RAS CSF
compared to control rat CSF Two differences in sample preparation for the rat CSF compared
to the mouse CSF are noted (1) rat IgY-7 immunodepletion was performed and (2) each rat
CSF sample was collected from one animal due to sufficient volume instead of pooling from
multiple animals for the mouse CSF sample After immunodepletion the CSF samples from
control and RAS (biological N=5 technical replicates N=3) were digested and separated using
one dimensional RP nanoLC separation We were able to identify 167 proteins with redundant
isoforms removed which was derived from total 512 proteins with a 1 FDR from all the CSF
samples Comparative analysis was performed using dNSAF spectral counting and 21 proteins
were significantly changed Our data were consistent with previous prion CSF studies showing
14-3-3 protein increased in CSF of prion infected animals Genomic analysis was also
performed and was used to cross-validate our proteomic data and numerous proteins were found
to be consistent including a novel marker related to prion disease RNAset2 (ribonuclease T2)
In summary this work provides a foundation for investigation of the perturbed proteome of a
new prion model RAS
Yeast Phosphoproteomic Comparison between Starved and Glucose Fed Conditions
This work presented a qualitative comparison of the phosphoproteome between starved
and glucose fed Saccharomyces cerevisiae (bakers yeast) A large scale IMAC enrichment of
yeast cell lysate followed by 2-D separations provided a rich source of phosphopeptides for CID
MSMS and neutral loss triggered ETD analysis using mass spectrometry A specific motif for
PKA was highlighted to show the differences in proteins identified between starved and glucose
209
fed conditions In total 477 unique phosphopeptides were identified with 06 FDR and 669
unique phosphopeptides identified with 54 FDR Phosphosite validation was performed using
a localization algorithm Ascore to provide further confidence on the site-specific
characterization of these phosphopeptides Two proteins Ssd1 and PMA2 emerged as potential
intriguing targets for more in-depth studies on protein phosphorylation involved in glucose
response
Methods for Peptide Sample Preparation and Sequencing
In this study ETD was performed to improve the sequence coverage of endogenous large
neuropeptides and labile tyrosine sulfation CCK-like neuropeptides found in the blue crab
Callinectes sapidus The intact CPRP from Callinectes sapidus was identified and characterized
with 68 sequence coverage by MS for the first time Cation assisted ETD fragmentation using
MgCl2 was also shown to be a powerful tool in de novo sequencing of sulfated neuropeptides
These endeavors into using ETD for certain neuropeptides will assist in future analysis of large
neuropeptides and PTM containing neuropeptides
In addition to ETD sequencing I presented a protocol on improving recovery of minute
quantities of peptides by adding methanol and a salt modifier (NaCl) to molecular weight cut-off
membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities
Despite its widespread utility significant sample loss often occurs during the MWCO
fractionation step which is particularly problematic when analyzing low-abundance peptides
from limited starting material This work presented a method to reduce sample loss during
MWCO separations specifically for sub-microg peptide isolation Using a neuropeptide standard
bradykinin sample loss was reduced by over two orders of magnitude with and without
210
undigested protein present The presence of bovine serum albumin (BSA) undigested protein
and the bradykinin standard lends evidence that sample loss occurs because of the MWCO and
not the presence of the protein Additionally a BSA tryptic digestion is presented where twenty-
seven tryptic peptides are identified from MALDI mass spectra after enriching with methanol
while only two tryptic peptides are identified after the standard MWCO protocol
Ongoing Projects and Future Directions
CSF Projects
Rat Adapted Scrapie and Time Course Study of Rat CSF
In ongoing experiments from the work described in Chapter 4 related to rat adapted
scrapie (RAS) we are working towards more complete proteome coverage of CSF and a time
course study of RAS After the promising results of the 1-D proteomic comparison between
RAS and control CSF we want to perform IgY immunodepletion on 1 mL of rat CSF followed
by 2-D high pH-low pH RP-RP LC-MSMS IgY immunodepletion is still required and
afterwards approximately 40 microg of low abundance protein would remain Following traditional
urea tryptic digestion (see Appendix 1) and solid phase extraction (SPE) clean-up the sample
would be dried down and reconstituted in mobile phase A (25 mM ammonium formate basic
pH) for high pH RP separation Fractions would be dried and subjected to MS analysis similar to
the work described in Chapter 4 The purpose of this work would be to increase the proteome
coverage from a few hundred to a thousand CSF proteins identified A time course study of RAS
is also desirable to gain insight into disease progression Rats at different stages will be
sacrificed and CSF will be collected The goal is to perform isobaric labeling between the time
courses with spectral counting being an alternative for relative protein expression We will use
the targets identified in Chapter 4 to track certain proteins for time course analysis Overall
211
these future projects will dig deeper into the proteome and how this novel prion model RAS
perturbs the proteins expressed in rats over time
Glycoproteomics and Peptidomic Analysis of CSF Samples from Individuals with
Alzheimerrsquos Disease
Alzheimerrsquos disease is an incurable progressive neurodegenerative disorder which results
in cognitive impairment Our aim is to provide additional biomarkers andor targets for drug
treatment Currently we used 500 microL of human CSF for non-membrane glycoprotein
enrichment (see Appendix 1) followed by 2-D high-pH-low-pH RP-RP amaZon ion trap LC-
MSMS analysis The initial work was a failure due to low amount of signal and significant
sample loss (data not shown) Through a comparison with Dr Xin Weirsquos PhD thesis we
estimated roughly 20 microg of proteins was obtained after glycoprotein enrichment and 1-D analysis
produced over 100 protein identifications (data not shown) but the additional off-line 2-D
separation and sample clean up resulted in low number of protein identifications for each fraction
analyzed by MS (data not shown) We have recently obtained 1 mL of human CSF samples
from healthy controls and individuals suffering from Alzheimerrsquos disease and plan to perform
the same experiments with double the starting amount and reduce the fractions collected from 2-
D separation from 11 to 6 In addition to the glycoproteomics anything not enriched will be
subjected to MWCO separation using the method presented in Chapter 7 The obtained peptide
sample will be subjected to Q-TOF nanoLC MSE data acquisition followed by de novo
sequencing using various programs including PEAKS and Mascot Collectively we feel this
project has great potential to lead to interesting targets and further expand the proteomic
knowledge of Alzheimerrsquos disease
GFAP Knock-in Mouse CSF
212
In a direct follow-up to Chapter 3 we aim to perform comparative proteomics on control
vs glial fibrillary acidic protein (GFAP) knock-in mouse CSF samples The sample preparation
protocol as presented in Chapter 3 will be used For quantitative proteomics we plan on
performing isobaric labeling followed by performing high energy collision induced dissociation
(HCD) on the Orbitrap Elite The MS acquisition will also perform a top ten scan where the top
ten MS peaks will be selected for tandem MS analysis In addition targeted quantitation of
specific proteins using multiple reaction monitoring (MRM) can be performed on potential
biomarkers and identified in this follow-up study and the work presented in Chapter 3 Also any
CSF samples with noticeable blood content cannot be used for the exploratory proteomics
experiments but can potentially be used for the MRM analysis and should be kept for such
experiments in the future
Large Scale Proteomics
Proteomics of Mouse Amniotic Fluid for Lung Maturation
The overall goal of this project is to determine what proteins are present in amniotic fluid
when the embryo is 175 weeks compared to amniotic fluid at 155 weeks The rationale behind
why these two time points matter was investigated through a lung explant culture where amniotic
fluid was added at 155 weeks and 175 weeks Visual maturation of lungs occurred with the
175 week amniotic fluid and lung maturation genes were up regulated in the mRNA in the lung
explant culture when compared to the 155 week amniotic fluid The compound which is
causing the maturation of the lungs is unknown and search for a secreted protein might provide a
clue to this process In order to test this hypothesis we carried out discovery proteomics
experiments The workflow involves tryptic digestion SPE and 1-D low pH nanoLC separation
coupled to an amaZon ETD ion trap for tandem MS analysis The resulting mass spectrometric
213
acquisition identified less than 100 proteins with a 1 FDR (data not published) To address the
lack of depth in the proteome coverage we purchased an IgY immunodepletion column to
remove the most abundant proteins in amniotic fluid similarly to Chapter 3 and 4 but on a larger
scale One abundant protein alpha-fetoprotein is present in amniotic fluid but absent or present
in very low concentration in CSF serum or plasma Alpha-fetoprotein is similar to albumin and
thus we ran amniotic fluid on an IgY immunodepletion column and observed significant
reduction in alpha-fetoprotein amount (data not published) After immunodepletion 2-D high
pH-low pH RP-RP will be performed followed by nanoLCMSMS on the amaZon ETD ion trap
We will perform mass spectrometry experiments using the 155 week amniotic fluid and 175
week amniotic fluid which have average protein contents ~2 mgmL We anticipate a minimum
of tenfold increase in the proteome coverage of amniotic fluid and provide meaningful
hypothesis driven biological experiments from this work
Phosphoproteomics of JNK Activation
c-Jun N-terminal kinase (JNK) is an important cellular mediator of stress activated
signaling Under conditions of oxidative stress JNK is activated resulting in the downstream
phosphorylation of a large number of proteins including c-Jun However the cellular response
to JNK activation is extremely complex and JNK activation can result in extremely different
physiological outcomes For example JNK is at the crossroads of cellular death and survival
and exhibits both pro-death and pro-survival activities These highly disparate outcomes of JNK
activation are highly contextual and depend on the type of stressor and duration of stress In the
brain JNK has been shown to be activated in models of Alzheimerrsquos disease Parkinsonrsquos
disease amyotrophic lateral sclerosis and stroke It is thought that JNK is activated in these
diseases as a result of oxidative stress and it is unknown whether this activation is pro-survival or
214
pro-death To elucidate JNK signaling in the brain we chose to analyze primary cortical
astrocytes under conditions of oxidative stress Since hydrogen peroxide is a physiologically
relevant oxidant in the brain and known to activate JNK we chose to use this to treat astrocytes
and then analyze changes to the phosphoproteome by mass spectrometry By doing this we
hope to elucidate important JNK-dependent targets involved in stress signaling in the brain and
that identifying these targets could lead to the identification of novel disease mechanisms and
potentially new therapeutic targets for neurodegeneration Specifically we plan on performing
stable isotope labeling by amino acids (SILAC) of astrocytes for control hydrogen peroxide
treated and hydrogen peroxide treated with JNK inhibitor The SILAC labeled astrocyte cell
lysate will be digested enriched using Fe-NTA IMAC and separated using 2-D high pH-low pH
RP-RP and infused into the amaZon ETD ion trap The protocol is similar to the yeast
comparative phosphoproteomics from Chapter 5 After analysis we plan on processing the data
using ProteoIQ to identify phosphoproteins with significant changes
Immunoprecipitation Followed by Mass Spectrometry
Stb3 Mass Spectrometry Analysis
Stb3 (Sin3-binding protein) has previously been shown to change location depending on
the presence or absence of glucose (Heideman Lab Dr Michael Conways Thesis) An
immunoprecipitation of Stb3 was performed in parallel to the work described in Chapter 5 Two
separate experiments were performed one with wild type Stb3 and another tagged with myc for
improved protein immunoprecipitation Myc tagging is a polypeptide tag which can be
recognized by an antibody and provide a better enrichment compared to using a Stb3 antibody
alone The myc tagging was done because of the low abundance of Stb3 and the limited amount
of protein that was pulled down by the antibody for Stb3 Immunopreciptation experiments were
215
performed for both starved and glucose fed samples All samples were tryptically digested
followed by low pH RP nanoLCMSMS Both CID and ETD were used for fragmentation
analysis of each sample (n=2) The Stb3 wild type had two peptide hits in one MS run which is
not surprising due to low Stb3 antibody affinity In contrast a significant amount of Stb3 was
pulled down from Myc tagged starved and glucose fed samples For the glucose starved
samples 22 unique peptides from Stb3 were identified whereas glucose fed samples yielded 21
unique Stb3 peptides identified (unpublished data) The identified Stb3 in the myc samples
allowed us to investigate what other proteins were pulled down that are not present in the wild
type samples
From previous work by our collaborator Dr Heideman it had been suggested that Stb3
forms a complex with Sin3 and Rpd3 Our proteomic data shows multiple significant peptide
hits for Sin3 and Rpd3 in myc tagged but not in wild type where Stb3 is only observed once
with a low Mascot score When looking at the unique proteins identified in myc tagged glucose
fed sample but not included in the wild type samples the myc fed sample contained eight unique
ribosomal proteins with numerous unique peptides for each The ribosomal proteins identified in
myc fed sample supports the novel hypothesis that in the glucose fed state the complex of Stb3
Rpd3 and Sin3 interact with ribosomal proteins Other proteins unique to myc tagged glucose
starved samples are glyceraldehyde-3-phosphate dehydrogenase 2 and transcriptional regulatory
protein UME6 Also three proteins were observed in both myc fed and starved but not in the
wild type samples including transposon Ty1-DR1 Gag-Pol polyprotein(YD11B) SWIRM
domain-containing protein (FUN19) and RNA polymerase II degradation factor 1 (DEF1) Our
proteomics data for the Stb3 immunoprecipitation experiments with starved vs glucose fed
216
samples provide exciting evidence to support previous observation made by the Heideman group
and highlight the utility of MS-based approach to deciphering protein-protein interactions
Conclusions
The majority of the work described in this dissertation revolves around sample
preparation for proteomics and peptidomics with a focus on generating biologically testable
hypotheses In the area of neuropeptides CPRP was fully sequenced analytical methods were
transformed for use in an ETD ion trap and sub-microg peptides can now be analyzed by mass
spectrometry after MWCO separation In the field of comparative proteomics comparisons
between mouse CSF in GFAP overexpressors and control or rat adapted scrapie model and
control CSF yeast fed vs glucose starved cell extract have all been presented Collectively this
thesis has developed new techniques for neuropeptide sample preparation and presented
numerous comparative proteomic analyses of various biological samples and how the proteomes
are dynamically perturbed by various treatments and disease conditions
217
Appendix 1
Protocols for sample preparation for mass spectrometry based
proteomics and peptidomics
218
Small Scale Urea Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 8 μL of urea solution
(400mg05mL) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Allow to digest overnight in 37degC water bath
10 Acidify with 10μL 10 formic acid
11 Perform solid phase extraction using tips dependent of sample amount
a Sub-5μg amounts ndash Millipore Ziptips
b 5-75μg amounts ndash Bond-Elut tips from Agilent (OMIX tips)
12 Dry down in Speedvac as needed for experiment
219
Small Scale ProteaseMAX Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 2 μL of 1 of
ProtesaeMAX (Promega) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Add 1 μL ProteaseMAX and let sit for 3-4 hours
10 Acidify with 2μL 10 formic acid
11 Dry down in Speedvac as needed for experiment
220
Large Scale Urea Tryptic Digestion (mg of proteins)
1 All solutions are in a 50 mM NH4HCO3 buffer (395 mgmL)
2 Add 20μL of 05M Dithiothreitol (DTT) (77mg100μL) and 160μL of urea solution
(400mg05mL) to sample
3 Allow sample to denature 45-60 minutes in a 37degC water bath
4 Add 54μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
5 Quench reaction with 20μL of DTT solution
6 Dilute with 14mL of NH4HCO3 solution
7 Add 100μg of trypsin
8 Allow to digest overnight in 37degC water bath
9 Acidify sample with 100μL of 10 formic acid
10 Perform solid phase extraction with Hypersep C18 Cartridges with 100 mg of C18
bead volume (Thermo)
11 Dry down with the Speedvac as needed for experiment
221
Fe-NTA Preparation from Ni-NTA Beads
1 Centrifuge (5 min at 140 rcf) and use a magnet to keep beads in place as supernatant
is removed
2 Wash 3 times with 800μL of H2O (using same technique of centrifuging and using
magnet to keep beads in places as supernatant is removed)
3 Add 800μL of 100mM EDTA (372mgmL) in a 50mM NH4HCO3 (395mgmL)
buffered solution (pH around 8) and vortex for 30 minutes to chelate the Ni
centrifuge and remove supernatant
4 Wash 3 times with 800μL of H2O
5 Add 800μL of 10mM FeCl3 hexahydrate (27mgmL) and vortex for 30 minutes to
bind Fe to beads centrifuge and remove supernatant
6 Wash 3 times with 800μL H2O
7 Resuspend in a storage solution of 111 ACNMeOH001 Acetic Acid (1mL total)
222
Fe-NTA IMAC Phospho-enrichment
1 Use wash solution (80ACN 01Formic Acid) to rinse beads vortex 1 minute
centrifuge and remove supernatant
2 Add sample (around 500μL in 80ACN 01Formic Acid) and vortex 40 minutes to
allow sample to bind dispose of supernatant after centrifuging
3 Wash 3 times with 200μL of wash solution discard supernatant
4 Add 200μL of elution solution (5050 ACN5Ammonium Hydroxide) vortex 15
minutes and save supernatant
5 Add 200μL of elution solution vortex 10 minutes and save supernatant
6 Wash 2 time with wash solution (collect supernatant of first wash)
7 If reusing store in 1mL solution of 111 ACNMeOH001 Acetic Acid
223
High pH Off-line Separation
1) In general a minimum of 20 microg of peptides are needed to gain any benefit
from off-line 2D fractionation It is better to inject 100 microg of peptides on
column
2) Use a Gemini column or a similar column that can handle pH=10 and for this
protocol a 21 mm x 150 mm column was used
3) Prepare ldquobuffer Ardquo 25 mM NH4 formate pH=10 Also prepare ldquobuffer Brdquo
4) Dry down desired sample and reconstitute in buffer A
5) Inject based off sample loop (current Alliance HPLC has a 50 microL sample
loop)
6) Run gradient at bottom of the page collecting fractions every 3 minutes except
for the 1st minute which is the void volume
7) Optional If you want to reduce instrument time you can combine fractions 1
with 12 2 with 13 etc until 11 with 22
Time Mobile phase A Mobile phase B Flow Rate
05mlmin
0 98 2 05 mLmin
65rsquo 70 30 05 mLmin
65rsquo1rdquo 5 95 05 mLmin
70 5 95 05 mLmin
224
Non Membrane Glycoprotein Enrichment
1 For protocols involving membrane glycoprotein enrichment see Dr Xin Weirsquos
thesis
2 Add 75 microL of Wheat germ agglutinin (WGA) bound to agarose beads and 150 microL
of Concanavalin A (ConA) to a Handee spin cup (Thermo) Wash 2 times with
lectin affinity chromatography (LAC) binding buffer (015 M NaCl 002 M Tris-
HCl 1 mM CaCl2 pH=74) centrifuging at 400 g for 30 seconds
3 Place stopper on bottom of spin cup and add 200 microg of sample (500 microL for CSF)
Bring up to 300 microL using lectin LAC binding buffer
4 Incubate for 1 hour with continuous mixing at room temperature
5 Centrifuge at 400 g for 30 seconds
6 Perform two more 300 microL LAC binding washes followed by centrifugation
7 Add 300 microL LAC eluting buffer (0075 M NaCl 001 M Tris-HCl 02 M alpha-
methyl mannoside 02 M alpha-methyl glucoside and 05 M acetyl-D-
glucosamine) vortex for 10 minutes (have stopper in place while vortexing)
centrifuge and collect
7 Add another 300 microL LAC eluting buffer centrifuge and collect
225
MWCO separation for Sub-microg peptide concentrations
1 Wash molecular weight cut-off (MWCO) with 5050 waterMeOH centrifuge at
14000 g for 5 minutes for 10 kDa filter (time will vary Millipore Amicon Ultra
filters)
2 Wash with 100 water centrifuge at 14000 g for 5 minutes
3 Add methanol to the sample to get the concentration to 30 methanol and add
salt to achieve a roughly 05 M NaCl before adding the sample to the MWCO
4 Centrifuge at 14000 for 10 minutes collect flow through
226
Immunoprecipitation
Modified from Thermo Fisher Scientific Classic IP Kit
1 10 microL Polyclonal antibody added to 1 microg protein standard in a Handee spin cup
(Thermo) diluted with 1x TBS buffer to 400 microL and incubated overnight at on
shakerend-over-end rotator
2 Wash Protein GA beads with 2x 200microL TBS buffer and added to the
antibodysample for 15 hours at 4oC
3 Centrifuge at 400 g for 30 seconds and discard flow through
4 Wash 3x with 100microL TBS buffer centrifuge at 400 g for 30 seconds and discard
flow through
5 Wash with 1x conditioning solution (neutral pH buffer) centrifuge at 400 g for 30
seconds and discard flow through
6 Eluted with elution buffer (2x100microL) centrifuge at 400 g for 30 seconds and
collect flow through
227
C18 Solid Phase Extraction (SPE)
1 Determine amount of peptides for SPE ge5 microg use Ziptips from Millipore If
between 5-75 microg use OMIX from Agilent 100 microL version and if ge75 microg use SPE
cartridges such as 100 mg Hypersep from Thermo
2 Ensure no detergents are in the sample and it is acidified
3 The three SPE procedures all use the same sets of solutions only volumes vary
4 3x Wetting solution (100 ACN) for 60 microL OMIX (20 microL ziptip and 15 mL for
100 mg cartridge)
5 3x Wash solution (01 formic acid in 100 H2O) same volumes as 4
6 Draw up the sample about 10 times minimum (generally 30-40 is preferred)
without letting the bead volume dry out
7 1X Wash solution same volumes as 4
8 Back load the eluting solution 01 formic acid in 5050 H2OACN into the
Ziptips (15 microL) or OMIX (50 microL) tips For the SPE cartridge add 700 microL of
eluting solution
9 Dry down and prepare for next step in sample preparation
228
Laser Puller Programs and Protocols
1) Obtain about two feet of Fused-silica capillary with 75 μm id and 360 μm od
2) Wash with methanol and then air dry using the bomb
3) Cut into one foot or desired length
4) Use a lighter to burn the middle portion (about an inch in length) of the tubing
5) Remove the ash by rinsing with methanol and rubbing with a Kimwipe
6) Make sure the laser puller has been on for at least 30 minutes before use to allow
for the instrument to warm up
7) Place capillary in instrument with the burnedexposed portion in the center
making sure that the length of the capillary is pulled taut
8) Enter desired program (next page) and press pull
229
Laser Puller Programs
Program 99 (default lab program)
Heat Filament Velocity Delay Pull
250 0 25 150 15
240 0 25 150 15
235 0 25 150 15
245 0 25 150 15
Program 97 (developed for larger inner diameter tips)
Heat Filament Velocity Delay Pull
230 - 25 150 -
220 - 25 150 -
215 - 25 150 8
230
On column Immunodepletion (serum plasma CSF amniotic fluid)
1) Prepare buffer A ldquoDilution Bufferrdquo 10 mM Tris-HCl pH=74 150 mM NaCl
2) Prepare buffer B ldquoStripping Bufferrdquo 01 M Glycine-HCl pH=25
3) Prepare buffer C ldquoNeutralization Bufferrdquo 100 mM Tris-HCl pH=80
4) Determine loading amount (LC1=~1000 ug of serum or plasma less for CSF due
to the increased amount of albumin percentage in CSF)
5) Add Dilution buffer to sample before injection and ensure the sample is proper
pH (~7)
6) Use gradient below
Time A B C Flow Rate
(mLmin)
0rsquo 100 0 0 02
4rsquo59rdquo 100 0 0 02
5rsquo 100 0 0 05
8rsquo59rdquo 100 0 0 05
9rsquo 0 100 0 05
22rsquo 0 100 0 05
22rsquo1rdquo 0 0 100 05
39rsquo 0 0 100 05
7) Store the column in 1x dilution buffer until next use
231
Small Scale Immunodepletion (100 microL of CSF)
1) Use 75 microg (per 100 microL CSF) of Sigma Seppro IgY-14 or IgY-7 bead slurry
2) Add an equal volume of 2x dilution buffer (20 mM Tris-HCl pH=74 300 mM
NaCl) to the starting amount of CSF
3) Add to a spin cup with a filter and allow to mix for 30 minutes
4) Centrifuge at 400 g for 30 seconds and collect the flow through
5) Add 50 microL of 1x dilution buffer mix for 5 minutes centrifuge at 400 g for 30
seconds and collect the flow through
6) Use 1x stripping washes (01 M Glycine-HCl pH=25) centrifuge as before and
discard Repeat four times
7) Use 1x neutralization buffer (100 mM Tris-HCl pH=80) centrifuge as before
and discard Repeat two times
8) Store the beads in the spin column in 1x dilution buffer until next use
232
Alliance Maintenance Protocol
Seal Wash
10 methanol no acetonitrile This wash cleans behind the pump-head seals to
ensure proper lubrication Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime SealWsh gt Start
2 Press Stop after 5 minutes
Prime Injector
10 methanol for maintenance high organic solvent for dirty runs (eg 95
acetonitrile) Done before injecting any real samples to ensure no bubbles are
present in the injector Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime NdlWsh gt Start
2 After completion press Close
Purge Injector
Solvent is dependent on run Run this protocol at beginning of experiments each day
Minimum once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Navigate Direct Function gt 4 Purge Injector gt OK
3 Set Sample loop volumes 60 leave Compression Check unchecked gt OK
Prime Solvent Pumps
Solvent is dependent on run If solvents are changed run this protocol Minimum
once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys choose composition A type 100 Enter x4
3 Navigate Direct Function gt 3 Wet Prime gt OK
4 Set Flow Rate 7000 mLmin Time 100 min gt OK
5 Repeat for all changedactive solvent pumps
Condition Column
Dependent on user Use starting conditions for run
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys type starting solvent compositions for run
3 Navigate Direct Function gt 6 Condition Column gt OK
4 Set Time as desired
233
Appendix 2
List of Publications and Presentations
234
PUBLICATIONS
ldquoDiscovery and characterization of the crustacean hyperglycemic hormone precursor related
peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes
sapidus using multiple tandem mass spectrometry techniquesrdquo Hui L Cunningham R Zhang
Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
ldquoInvestigation and reduction of sub-microgram peptide loss using molecular weight cut-off
fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J Wellner D Li L
Journal of Mass Spectrometry In Press
ldquoMass spectrometry-based proteomics and peptidomics for systems biology and biomarker
discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse models of
Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P Messing A Li
L Journal of Proteome Research Submitted
ldquoInvestigation of the differences in the phosphoproteome between starved vs glucose fed
Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M Heideman
W Li L In preparation
ldquoIdentification of astrocytic JNK targets by phosphoproteomics using mass spectrometryrdquo
Cunningham R Dowell J Wang J Wellner D Li L Milhelm M In preparation
ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A Cunningham R Wellner
D Wang J Ma D Li L Aiken J In preparation
235
INVITED SEMINARS AND CONFERENCE PRESENTATIONS
Robert Cunningham Davenne Mayour and Shoshanna Coon ldquoMorphology and Thermal
Stability of Monolayers on Porous Siliconrdquo The 231th
ACS National Meeting 2006 Atlanta
GA
Robert Cunningham Xin Wei Paige Jany Albee Messing and Lingjun Li ldquoMass
Spectrometry-Based Analysis of Cerebrospinal Fluid Peptidome and Proteome for Biomarker
Discovery in Alexander Diseaserdquo The 57th
ASMS Conference 2009 Philadelphia PA
Robert Cunningham ldquoWhat to Expect in a PhD Graduate Schoolrdquo Invited seminar University
of Northern Iowa 2010 Cedar Falls IA
Robert Cunningham Dustin Frost Albee Messing and Lingjun Li ldquoInvestigation of an
Optimized ProteaseMAX Assisted Trypsin Digestion of Human CSF for Pseudo-SRM
Quantification of GFAP and Identification of Biomarkersrdquo The 58th
ASMS Conference 2010
Salt Lake City UT
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo Oral presentation at Pittcon Conference and Expo 2011 2011 Atlanta
GA
Robert Cunningham Michael Conway Daniel Wellner Douglas Grunwald Warren
Heideman and Lingjun Li ldquoDevelopment of optimized phosphopeptide enrichment methods for
comparison of starved and glucose fed yeast Saccharomyces cerevisiaerdquo The 59th
ASMS
Conference 2011 Denver CO
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo 2011 Wisconsin Human Proteomics Symposium 2011 Madison WI
iv
Mass Spectrometry Applications for Comparative Proteomics and
Peptidomic Discovery
Robert Stewart Cunningham
Under the supervision of Professor Lingjun Li
At the University of Wisconsin-Madison
Abstract
In this thesis multiple biological samples from various diseases models or
treatments are investigated using shotgun proteomics and improved methods are
developed to enable extended characterization and detection of neuropeptides In general
this thesis aims to expand upon the rapidly evolving field of mass spectrometry (MS)-
based proteomics and peptidomics by primarily enhancing small scale sample analysis
A review of the current status and progress in the field of biomarker discovery in
peptidomics and proteomics is presented To this rapidly expanding body of literature
our critical review offers new insights into MS-based biomarker studies investigating
numerous biological samples methods for post-translational modifications quantitative
proteomics and biomarker validation Methods are developed and presented including
immunodepletion for small volume cerebrospinal fluid (CSF) samples for comparison of
the CSF proteomes between an Alexander disease transgenic mouse model with
overexpression of the glial fibrillary acidic protein and a control animal This thesis also
covers the application of the small scale immunodepletion of CSF for comparative
proteomic analysis of a novel rat adapted scrapie (RAS) model for prion disease and
v
compares the RAS CSF proteome to control rat CSF using MS Large scale
phosphoproteomics of starved vs glucose fed yeast is presented to better understand the
phosphoproteome changes that occur during glucose feeding Method development for
neuropeptide analysis is expanded upon using electron transfer dissociation (ETD)
fragmentation to successfully sequence for the first time the crustacean hyperglycemic
hormone precursor-related peptide (CPRP) from the blue crab Callinectes sapidus In
addition a method for ETD sequencing of sulfonated neuropeptides using a magnesium
salt adduct in an ion trap mass spectrometer is reported This thesis also reports on a
method for sub-microg peptide isolation when using a molecular weight cut-off filtration
device to improve sample recovery by over 2 orders of magnitude All the protocols used
throughout the work are provided in an easy to use step-by-step format in the Appendix
Collectively this body of work extends the capabilities of mass spectrometry as a
bioanalytical tool for shotgun proteomics and expands upon methods for neuropeptide
discovery and analysis
1
Chapter 1
Introduction Brief Background and Research Summary
2
Abstract
Mass spectrometry based comparative proteomics and improved methods for
neuropeptide discovery have been reported in this thesis A very brief introduction of crustacean
neuropeptidomics is covered in this chapter followed by Chapter 2 which covers in great detail
comparative proteomics using mass spectrometry with an emphasis on biomarker discovery
Chapter 3 reports a novel immunodepletion technique used for comparative proteomics between
glial fibrillary acidic protein (GFAP) overexpressor and control mouse cerebrospinal fluid (CSF)
Chapter 4 involves rat CSF comparative proteomics between rat adapted scrapie and control
animals Chapter 5 details comparisons of phosphoproteomes in Saccharomyces cerevisiae
(Bakerrsquos yeast) between starved vs glucose fed conditions Chapter 6 investigates the use of
electron transfer dissociation (ETD) for kDa sized neuropeptides and neuropeptides with tyrosine
sulfonation Chapter 7 presents a molecular weight cut-off (MWCO) protocol to allow sub-microg
peptides to be recovered Chapter 8 provides a conclusion to this thesis and outlines future
directions for certain projects
3
Background
Mass spectrometry (MS) requires gas phase ions for experimental measurement and
intact large nonvolatile biomolecules proved difficult to be analyzed by electron ionization or
chemical ionization until the invention of two soft ionization techniques matrix-assisted laser
desorptionionization (MALDI)1 and electrospray ionization (ESI)
2 ESI and MALDI are the
two most common soft ionization techniques for mass spectrometry Once ionized molecules
such as peptides or proteins can be separated by their mass to charge ratios (mz) using various
mass analyzers manipulating electric andor magnetic fields ESI and MALDI mass
spectrometric techniques have become central analytical methods in biological sciences because
they are suitable for nonvolatile thermally labile analytes in femtomole quantities3 ESI allows
the coupling of high pressure liquid chromatography and the constant flow of solvent is
electrically charged at the outlet to produce a Taylor cone4 During desolvation the Raleigh
limit is reached and a coulombic explosion occurs commonly producing multiply charged ions
A modification of ESI is nanoLC-ESI which operates at nLmin flow rates to analyze low sample
amounts such as proteins or peptides in biological samples5 NanoLC-ESI uses less solvent as
the source because of the reduced flow rate of standard LCMS with an ESI source and nanoLC-
ESI is the predominate inlet for shotgun proteomic ESI MS experiments6 Conversely MALDI
can use sub-microL volume of sample and be co-crystallized with an ultraviolet absorbing organic
matrix that is obliterated using a laser at appropriate wavelength to generate gas phase ions
Alternatively MALDI has the unique capability to work with tissue samples and ionize in the
solid state instead of liquid like ESI
4
Mass analyzers require an operating pressure between 10-4
-10-10
Torr to allow proper ion
transfer and separationisolation of the ion based upon its mz Numerous mass analyzers are
currently available and each have their own strengths and weaknesses as shown in Figure 1 The
biomolecules are separated by the mass analyzers and detected without fragmentation which is
termed MS analysis Following MS detection tandem mass spectrometry (MSMS) of the
original precursor ion can be performed to provide additional structural information such as a
ladder sequence of amino acids for peptides Numerous fragmentation techniques are available
for proteomics such as collision induced dissociation (CID) electron transfer dissociation (ETD)
or high energy collision induced dissociation (HCD) Each of these fragmentation techniques
have their own benefits in proteomics experiments and are discussed in depth in Chapter 2 The
background and current status for comparative proteomics with specific emphasis on biomarker
analysis are covered in Chapter 2
Neuropeptidomic Method Development in the Crustacean Model System
Utilizing Mass Spectrometry
Chapter 6 and 7 focus on sample preparation and ETD fragmentation techniques to
characterize neuropeptides in the neuroendocrine organs in the crustacean nervous system
Neuropeptides are short chains of amino acids which are a diverse class of cell-cell signaling
molecules in the nervous system Neuropeptides have been investigated for being involved in
numerous physiological processes such as memory7 learning
8 depression
9 pain
10 reward
11
reproduction12
sleep-wake cycles13
homeostasis14
and feeding15-17
Figure 2 depicts how
neuropeptides are synthesized as pre-prohormones in the rough endoplasmic reticulum and
5
packaged in the Golgi apparatus After being packaged these pre-prohormones are processed
into bioactive peptides within the vesicle which is occurring during vesicular transport down an
axon and released into the synaptic cleft Secreted neuropeptides stimulate the post synaptic
neurons by interacting with G-protein coupled receptors at the chemical synapse
The crustacean model nervous system is well-defined neural network which has been
used in neurobiology and neuroendocrine (neurosecretory) research and thus lends itself for
studying neuromodulation18-22
Figure 3 shows the locations of several neuroendocrine organs in
the crustacean Callinectes sapidus including the sinus gland (SG) which is studied in Chapter 6
The Lingjun Li lab has focused on neuropeptide discovery and function in crustacean
neuroendocrine organs using mass spectrometry23-25
The work presented in Chapters 6 and 7
expand on sample preparation and analytical tools to further investigate the neuropeptidome
Research Overview
Comparative Proteomics of Biological Samples
Chapter 2 provides a thorough review of comparative proteomics and biomarker analysis
using mass spectrometry The scientific community has shown great interest in the field of mass
spectrometry-based proteomics and peptidomics for its applications in biology Proteomics
technologies have evolved to generate large datasets of proteins or peptides involved in various
biological and disease progression processes producing testable hypotheses for complex
biological questions This chapter provides an introduction and insight into relevant topics in
proteomics and peptidomics including biological material selection sample preparation
separation techniques peptide fragmentation post-translational modifications quantification
6
bioinformatics and biomarker discovery and validation In addition current literature and
remaining challenges and emerging technologies for proteomics and peptidomics are discussed
Chapter 3 investigates comparative proteomics between a GFAP overexpressor mouse
model and a control mouses cerebrospinal fluid (CSF) CSF is a low protein content biological
fluid with dynamic range spanning at least nine orders of magnitude in protein content and is in
direct contact with the brain but consist of very abundant proteins similar to serum which require
removal A modified IgY-14 immunodepletion treatment is presented to remove abundant
proteins such as albumin to enhance analysis of the low volumes of CSF that are obtainable
from mice These GFAP overexpressor mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we present the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates are performed to address animal variability as well as reproducibility in mass
spectrometric analysis Relative quantitation is performed using distributive normalized spectral
abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins with
significant changes in the CSF of GFAP transgenic mice are identified with validation from
ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
Chapter 4 explores the CSF proteomics of a novel prion model called rat adapted scrapie
(RAS) A similar IgY immunodepletion technique is presented as in Chapter 3 and is similarly
used to reduce the abundant proteins in CSF CSF from control and RAS (biological N=5
technical replicates N=3) were digested and separated using one dimensional reversed-phase
nanoLC separation In total 512 proteins 167 non-redundant protein groups and 1032 unique
peptides are identified with a 1 FDR Comparative analysis was done using dNSAF spectral
7
counting and 21 proteins were significantly up or down-regulated The proteins are compared to
the 1048 differentially regulated genes and additionally compared to previously published
proteins showing changes consistent with other prion animal models Of particular interest is
RAS specific proteins like RNAseT2 which is not up-regulated in mouse prion disease but is
designated as upregulated in both the genomic and proteomics data for RAS
Chapter 5 explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Previous work by the
Heideman lab investigated the transcriptional response to fresh glucose in yeast26
Kinases such
as protein kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose
response so we described a large scale phosphoproteomic MS based study in this chapter
Yeast cell extract was digested and phosphopeptides were enriched by immobilized metal
affinity chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase
(RP)-RP separation The low pH separation was infused directly into an ion trap mass
spectrometer with neutral loss triggered ETDfragmentation Specifically the CID fragmentation
can cause a neutral loss of 98z from the H3PO4 and can produce an incomplete fragmentation
pattern and lead to ambiguous identification so when the neutral loss is detected ETD MSMS
fragmentation is performed The neutral loss triggered ETD fragmentation is included in this
study to improve phosphopeptide identifications In total 477 phosphopeptides are identified
with 06 FDR and 669 phosphopeptides identified with 54 FDR Motif analysis of PKA and
phosphosite validation are performed as well
8
The future of comparative proteomics investigating small sample amounts or PTMs is
promising Further advances in enrichment separations science mass spectrometry
analyzersdetectors and bioinformatics will continue to create more powerful tools that enable
digging deeper in proteomics with both small (mouse CSF) and large (cell extracts) sample
amounts
Methods for Neuropeptide Analysis Using ETD fragmentation and Sample
Preparation
Chapter 6 investigates the utility of ETD fragmentation to sequence endogenous large
neuropeptides and labile tyrosine sulfation in the crustacean Callinectes sapidus The sinus
gland (SG) of the crustacean is a well-defined neuroendocrine site that produces numerous
hemolymph-borne agents including the most complex class of endocrine signaling moleculesmdash
neuropeptides One such peptide is the crustacean hyperglycemic hormone (CHH) precursor-
related peptide (CPRPs) which was not previously sequenced Electron transfer dissociation
(ETD) is used for sequencing of intact CPRPs due to their large size and high charge state In
addition ETD is used to sequence a sulfated peptide cholecystokinin (CCK)-like peptide in the
lobster Homarus americanus using a salt adduct Collectively this chapter presents two
examples of the utility of ETD in sequencing neuropeptides with large molecular weight or with
labile modifications
Chapter 7 reports a protocol on improving recovery of minute quantities of peptides by
adding methanol and a salt modifier (NaCl) to molecular weight cut-off membrane-based
centrifugal filters (MWCO) to enrich sub-microgram peptide quantities In some biological
9
fluids such as CSF the endogenous peptide content is very low and using pure water to perform
the MWCO separation produces too much sample loss Using a neuropeptide standard
bradykinin sample loss is reduced over two orders of magnitude with and without undigested
protein present The presence of bovine serum albumin (BSA) undigested protein and the
bradykinin standard lends evidence that sample loss occurs because of the MWCO and not the
presence of the protein Additionally a BSA tryptic digestion is presented where twenty-seven
tryptic peptides are identified from MALDI mass spectra after enriching with methanol while
only two tryptic peptides are identified after the standard MWCO protocol The strategy
presented in Chapter 7 greatly enhances recovery from MWCO separation for sub-microg peptide
samples
10
References
1 Tanaka K Waki H Ido Y Akita S Yoshida Y Yoshida T Matsuo T Protein and polymer analyses up to mz 100 000 by laser ionization time-of-flight mass spectrometry Rapid Communications in Mass Spectrometry 1988 2 (8) 151-153
2 Fenn J B Mann M Meng C K Wong S F Whitehouse C M Electrospray ionization for mass spectrometry of large biomolecules Science 1989 246 (4926) 64-71
3 Domon B Aebersold R Mass spectrometry and protein analysis Science 2006 312 (5771) 212-7
4 Whitehouse C M Dreyer R N Yamashita M Fenn J B Electrospray interface for liquid chromatographs and mass spectrometers Anal Chem 1985 57 (3) 675-9
5 Wilm M Mann M Analytical properties of the nanoelectrospray ion source Anal Chem 1996 68 (1) 1-8
6 Karas M Bahr U Dulcks T Nano-electrospray ionization mass spectrometry addressing analytical problems beyond routine Fresenius J Anal Chem 2000 366 (6-7) 669-76
7 Gulpinar M Yegen B The physiology of learning and memory Role of peptides and stress Curr Protein Pept Sci 2004 5 (6) 457-473
8 Ogren S O Kuteeva E Elvander-Tottie E Hokfelt T Neuropeptides in learning and memory processes with focus on galanin In Eur J Pharmacol 2010 Vol 626 pp 9-17
9 Strand F Neuropeptides general characteristics and neuropharmaceutical potential in treating CNS disorders Prog Drug Res 2003 61 1-37
10 Li W Chang M Peng Y-L Gao Y-H Zhang J-N Han R-W Wang R Neuropeptide S produces antinociceptive effects at the supraspinal level in mice Regul Pept 2009 156 (1-3) 90-95
11 Wu C-H Tao P-L Huang E Y-K Distribution of neuropeptide FF (NPFF) receptors in correlation with morphine-induced reward in the rat brain Peptides 2010 31 (7) 1374-1382
12 Tsutsui K Bentley G E Kriegsfeld L J Osugi T Seong J Y Vaudry H Discovery and evolutionary history of gonadotrophin-inhibitory hormone and kisspeptin new key neuropeptides controlling reproduction J Neuroendocrinol 2010 22 (7) 716-727
13 Piper D C Upton N Smith M I Hunter A J The novel brain neuropeptide orexin-A modulates the sleep-wake cycle of rats Eur J Neurosci 2000 12 (2) 726-730
14 Tsuneki H Wada T Sasaoka T Role of orexin in the regulation of glucose homeostasis - Tsuneki - 2009 - Acta Physiologica - Wiley Online Library Acta Physiol 2010
15 Kageyama H Takenoya F Shiba K Shioda S Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding Neuropeptides 2010 44 (2) 133-138
16 Sweedler J V Li L Rubakhin S S Alexeeva V Dembrow N C Dowling O Jing J Weiss K R Vilim F S Identification and characterization of the feeding circuit-activating peptides a novel neuropeptide family of aplysia J Neurosci 2002 22 (17) 7797-7808
11
17 Jensen J Regulatory peptides and control of food intake in non-mammalian vertebrates Comp Biochem Physiol Part A Mol Integr Physiol 2001 128 (3) 469-477
18 Billimoria C P Li L Marder E Profiling of neuropeptides released at the stomatogastric ganglion of the crab Cancer borealis with mass spectrometry J Neurochem 2005 95 (1) 191-199
19 Cape S S Rehm K J Ma M Marder E Li L Mass spectral comparison of the neuropeptide complement of the stomatogastric ganglion and brain in the adult and embryonic lobster Homarus americanus J Neurochem 2008 105 (3) 690-702
20 Cruz Bermuacutedez N D Fu Q Kutz Naber K K Christie A E Li L Marder E Mass
spectrometric characterization and physiological actions of GAHKNYLRFamide a novel FMRFamide‐like peptide from crabs of the genus Cancer J Neurochem 2006 97 (3) 784-799
21 Grashow R Brookings T Marder E Reliable neuromodulation from circuits with variable underlying structure Proc Natl Acad Sci USA 2009 106 (28) 11742-11746
22 Ma M Szabo T M Jia C Marder E Li L Mass spectrometric characterization and physiological actions of novel crustacean C-type allatostatins Peptides 2009 30 (9) 1660-1668
23 Chen R Hui L Cape S S Wang J Li L Comparative Neuropeptidomic Analysis of Food Intake via a Multi-faceted Mass Spectrometric Approach ACS Chem Neurosci 2010 1 (3) 204-214
24 Li L Sweedler J V Peptides in the brain mass spectrometry-based measurement approaches and challenges Annu Rev Anal Chem 2008 1 451-483
25 Ma M Wang J Chen R Li L Expanding the crustacean neuropeptidome using a multifaceted mass spectrometric approach J Proteome Res 2009 8 (5) 2426-2437
26 Slattery M G Heideman W Coordinated regulation of growth genes in Saccharomyces cerevisiae Cell Cycle 2007 6 (10) 1210-9
12
Figure 1 Capabilities and performances of certain mass analyzers Check marks indicate
availability check marks in parentheses indicate optional + ++ and +++ indicate possible or
moderate goodhigh and excellentvery high respectively Adapted with permission from
reference 3
13
Figure 2 Synthesis processing and release of neuropeptides (a) Cartoon showing two
interacting neurons (b) The synthesis of neuropeptides in the neuronal organelles and their
transport down the axon to the presynaptic terminal is depicted (c) Shows neuropeptide release
and interaction with the dendrite of the post-synaptic cell (Adapted from PhD thesis by Dr
Stephanie Cape)
14
Figure 3 Schematic showing location of the neuronal tissues being analyzed in the MS studies
of neuropeptides in Callinectes sapidus The SGs (sinus glands located in the eyestalks of the
crab) and the POs (pericardial organs located in the chamber surrounding the heart) release
neurohormones into the hemolymph (crab circulatory fluid) The brain is near the STNS
(stomatogastric nervous system neural network that controls the motion of the gut and foregut)
which has direct connections to the STG (stomatogastric ganglion) The STG is located in an
artery so it is continually in contact with hemolymph (Adapted from PhD thesis by Dr Robert
Sturm)
15
Chapter 2
Mass Spectrometry-based Proteomics and Peptidomics for Biomarker
Discovery and the Current State of the Field
Adapted from ldquoMass spectrometry-based proteomics and peptidomics for systems biology and
biomarker discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
16
Abstract
The scientific community has shown great interest in the field of mass spectrometry-based
proteomics and peptidomics for its applications in biology Proteomics technologies have
evolved to produce large datasets of proteins or peptides involved in various biological and
disease progression processes producing testable hypothesis for complex biological questions
This review provides an introduction and insight to relevant topics in proteomics and
peptidomics including biological material selection sample preparation separation techniques
peptide fragmentation post-translation modifications quantification bioinformatics and
biomarker discovery and validation In addition current literature and remaining challenges and
emerging technologies for proteomics and peptidomics are presented
17
Introduction
The field of proteomics has seen a huge expansion in the last two decades Multiple factors have
contributed to the rapid expansion of this field including the ever evolving mass spectrometry
instrumentation new sample preparation methods genomic sequencing of numerous model
organisms allowing database searching of proteomes improved quantitation capabilities and
availability of bioinformatic tools The ability to investigate the proteomes of numerous
biological samples and the ability to generate future hypothesis driven experiments makes
proteomics and biomarker studies exceedingly popular in biological studies today In addition
the advances in post-translational modification (PTM) analysis and quantification ability further
enhance the utility of mass spectrometry (MS)-based proteomics A subset of proteomics
research is devoted to profiling and quantifying neurologically related proteins and endogenous
peptides which has progressed rapidly in the past decade This review provides a general
overview as outlined in Figure 1 of proteomics technology including methodological and
conceptual improvements with a focus on recent studies and neurological biomarker studies
Biological Material Selection
The choice of biological matrix is an important first step in any proteomics analysis The
ease of sample collection (eg urine plasma or saliva) versus usefulness or localization of
sample (eg specific tissue or proximity fluid) needs to be evaluated early on in a study design
Plasma derived by centrifugation of blood to remove whole cells is a very popular
choice in proteomics due to the high protein content (~65 mg ml1) and the ubiquitous nature of
blood in the body and the ability to obtain large sample amounts or various time points without
the need to sacrifice the animal or to perform invasive techniques Plasma is centrifuged
18
immediately after sample collection unlike serum where coagulation needs to occur first To
obtain plasma blood is collected in a tube with an anticoagulant added (ETDA heparin or
citrate) and centrifuged but previous reports have shown variable results when heparin has been
used as an anticoagulant2 Human Proteome Organization (HUPO) specifically recommends the
anticoagulants EDTA or citrate to treat plasma3 4
One of the primary concerns with plasma is
degradation of the protein content via endogenous proteases found in the sample5 One way to
address this problem is the use of protease inhibitors In addition freezethaw cycles need to be
minimized to prevent protein degradation and variability6 7
Plasma proteomics has seen
extensive coordinated efforts to start assessing the diagnostic needs using plasma8 HUPO also
has established a public human database for plasma and serum proteomics from 35 collaborating
labratories9 Large dynamic range studies have been performed on plasma with a starting sample
amount of 2625 microl (1575 mg) resulting in 3654 proteins identified with a sub 5 false
discovery rate10
The large dynamic range spanning across eleven orders of magnitude as visualized in
Figure 2 is one of the biggest obstacles in plasma proteomics Figure 2 also shows that as lower
abundance proteins are investigated the origins of those identified proteins are more diverse than
the most abundant proteins Recent mining of the plasma proteome showed an ability to search
for disease biomarker applications across seven orders of magnitude In addition the tissue of
origin for the identified plasma proteins were identified and its origin was more diverse as the
protein concentration decreased11
Plasma has been used as a source for biomarker studies such
as colorectal cancer12 13
cardiovascular disease14
and abdominal aortic aneurysm15
Even
though the blood brain barrier prevents direct blood to brain interaction neurological disorders
such as Alzheimerrsquos disease (AD) have had their proteomes studied using plasma16
19
An alternative sample derived from blood is serum which is plasma allowed to coagulate
instead of adding anti-coagulates The time for coagulation is usually 30 minutes and during that
time significant and random degradation from endogenous proteases can occur The additional
variability caused from the coagulation process can change the concentration of multiple
potentially valuable biomarkers As biodiversity between samples or organisms is a challenging
endeavor additional sample variability due to serum generation may be undesirable but serum is
still currently being used for biomarker disease studies17
Serum has been used to compare the
proteome differences in neurological diseases such as AD Parkinsonrsquos disease and amyotrophic
lateral sclerosis and a review can be found elsewhere discussing the subject18
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy of brain or spinal cord
in evaluating diseases of the central nervous system and has been used for studies in neurological
disorders due to being a rich source of neuro-related proteins and peptides19
The protein
composition of the most abundant proteins in CSF is well defined and numerous studies exist to
broaden the proteins identified20-22
CSF has an exceedingly low protein content (~04 μgμL)
which is ~100 times lower than serum or plasma and over 60 of the total protein content in
CSF consists of a single protein albumin23-25
In addition the variable concentrations of proteins
span up to twelve orders of magnitude further complicating analysis and masking biologically
relevant proteins to any given study26
One of the highest number of identified proteins is from
Schutzer et al with 2630 non-redundant proteins from 14 mL of pooled human CSF This study
involved the removal of highly abundant proteins by performing IgY-14 immunodepletion
followed by two dimensional (2D) liquid chromatography (LC) separation27
Studies have also
been performed to characterize individual biomarkers or complex patterns of biomarkers in
various diseases in the CSF28 29
One potential pitfall of CSF proteomic analysis is
20
contamination from blood which can be identified by counting red blood cells present or
examining surrogate markers from blood contamination other than hemoglobin such as
peroxiredoxin catalase and carbonic anhydrase30
A proof of principle CSF peptidomics study
identified numerous endogenous peptides associated with the central nervous system which can
be used as a bank for neurological disorder studies31
Numerous recent reports highlighted the
utility of CSF analysis for biomarker studies in AD32 33
medulloblastoma34
both post-mortem
and ante-mortem35
Cellular lysates offer the distinct advantage to work with a cell line yeast or bacteria
with large amounts of proteins available for analysis36 37
with Saccharomyces cerevisiae being
the most common cell lysate38 39
Other cell lines are also used including HeLa40
and E coli41
The ability to obtain milligrams of proteins easily to scale up experiments without animal
sacrifice offers a clear advantage in biological sample selection Current literature supports
cellular lysate as a valued and sought after source of proteins for large scale proteomics
experiments because of the ability to assess treatments conditions and testable hypotheses42-44
Cellular lysate from rat B104 neuroblastoma cell line was used as an in vitro model for cerebral
ischemia and showed abundance changes in multiple proteins involved in various neurological
disorders45
Other Sources of Biological Samples
Urine
The urine proteome appears to be another attractive reservoir for biomarker discovery
due to the relatively low complexity compared with the plasma proteome and the noninvasive
collection of urine Urine is often considered as an ideal source to identify biomarkers for renal
21
diseases due to the fact that in healthy adults approximately 70 of the urine proteome originate
from the kidney and the urinary tract 46
thus the use of urine to identify neurological disorders is
neglected However strong evidence have shown that proteins that are associated with
neurodegenerative diseases can be excreted in the urine47-49
indicating the application of urine
proteomics could be a useful approach to the discovery of biomarkers and development of
diagnostic assays for neurodegenerative diseases However the current view of urine proteome
is still limited by factors such as sample preparation techniques and sensitivity of the mass
spectrometers There has been a tremendous drive to increase the coverage of urine proteome
In a recent study Court et al compared and evaluated several different sample preparation
methods with the objective of developing a standardized robust and scalable protocol that could
be used in biomarkers development by shotgun proteomics50
In another study Marimuthu et al
reported the largest catalog of proteins in urine identified in a single study to date The
proteomic analysis of urine samples pooled from healthy individuals was conducted by using
high-resolution Fourier transform mass spectrometry A total of 1823 proteins were identified
of which 671 proteins have not been previously reported in urine 51
Saliva
For diagnosis purposes saliva collection has the advantage of being an easy and non-
invasive technique The recent studies on saliva proteins that are critically involved in AD and
Parkinsonrsquos diseases suggested that saliva could be a potentially important sample source to
identify biomarkers for neurodegenerative diseases Bermejo-Pareja et al reported the level of
salivary Aβ42 in patients with mild AD was noticeably increased compared to a group of
controls 52
In another study Devic et al identified two of the most important Parkinsons
22
disease related proteinsmdash α-synuclein (α-Syn) and DJ-1 in human saliva 53
They observed that
salivary α-Syn levels tended to decrease while DJ-1 levels tended to increase in Parkinsons
disease The published results from this study also suggest that α-Syn might correlate with the
severity of motor symptoms in Parkinsons disease Due in part to recent advancements in MS-
based proteomics has provided promising results in utilizing saliva to explore biomarkers for
both local and systemic diseases 54 55
the further profiling of saliva proteome will provide
valuable biomarker discovery source for neurodegenerative diseases
Tissue
Compared to body fluids such as plasma serum and urine where the proteomic analysis
is complicated by the wide dynamic range of protein concentration the analysis of tissue
homogenates using the well-established and conventional proteomic analysis techniques has the
advantage of reduced dynamic range However the homogenization and extraction process may
suffer from the caveat that spatial information is lost which would be inadequate for the
detection of biomarkers whose localization and distribution play important roles in disease
development and progression Matrix-assisted laser desorptionionization (MALDI) imaging
mass spectrometry (IMS) is a method that allows the investigation of a wide range of molecules
including proteins peptides lipids drugs and metabolites directly in thin slices of tissue 56-59
Because this technology allows for identification and simultaneous localization of biomolecules
of interests in tissue sections linking the spatial expression of molecules to histopathology
MALDI-IMS has been utilized as a powerful tool for the discovery of new cancer biomarker
candidates as well as other clinical applications60 61
The utilization of MALDI-IMS for human
or animal brain tissue to identify or map the distribution of molecules related to
neurodegenerative diseases were also recently reported62 63
23
Secretome
There has been an increasing interest in the study of proteins secreted by various cells
(the secretomes) from tissue-proximal fluids or conditioned media as a potential source of
biomarkers Cell secretomes mainly comprise proteins that are secreted or are shed from the cell
surface and these proteins can play important role in both physiological processes (eg cell
signaling communication and migration) and pathological processes including tumor
angiogenesis differentiation invasion and metastasis In particular the study of cancer cell
secretomes by MS based proteomics has offered new opportunities for cancer biomarker
discovery as tumor proteins may be secreted or shed into the bloodstream and could be used as
noninvasive biomarkers The latest advances and challenges of sample preparation sample
concentration and separation techniques used specifically for secretome analysis and its clinical
applications in the discovery of disease specific biomarkers have been comprehensively
reviewed64 65
Here we only highlight the proteomic profiling of neural cells secretome that has
been applied to neurosciences for a better understanding of the roles secreted proteins play in
response to brain injury and neurological diseases The LC-MS shotgun identification of
proteins released by astrocytes has been recently reported66-68
In these studies the changes
observed in the astrocyte secretomes induced by inflammatory cytokines or cholinergic
stimulation were investigated6667
Alternatively our group performed 2D-LC separation and
included cytoplasmic protein extract from astrocytes as a control to identify cytoplasmic protein
contaminants which are not actively secreted from cells68
Sample Preparation
24
Proteomic analysis and biomarker discovery research in biological samples such as body
fluids tissues and cells are often hampered by the vast complexity and large dynamic range of
the proteins Because disease identifying biomarkers are more likely to be low-abundance
proteins it is imperative to remove the high-abundance proteins or apply enrichment techniques
to allow detection and better coverage of the low-abundance proteins for MS analysis Several
strategies including depletion and protein equalizer approach have been used during sample
preparation to reduce sample complexity69 70
and the latest advances of these methods have been
reviewed by Selvaraju et al 71
Alternatively the complexity of biological samples can be
reduced by capturing a specific subproteome that may have the biological information of interest
The latter strategy is especially useful in the biomarker discovery where the changes in the
proteome are not solely reflected through the concentration level of specific proteins but also
through changes in the post-translational modifications (PTMs) Here we will mainly discuss
the enrichment of phosphoproteinpeptides glycoproteinpeptides and sample preparation for
peptidomics and membrane proteins
Phosphoproteomics
Phosphorylation can act as a molecular switch on a protein by turning it on or off within
the cell It is thought that up to 30 of the proteins can be phosphorylated72
and it plays
significant roles in such biological processes as the cell cycle and signal transduction73
Currently tens of thousands of phosphorylation sites can be proposed using analytical methods
available today74 75
The amino acids that are targeted for phosphorylation studies are serine
threonine and tyrosine with the abundance of detection decreasing typically in that order Other
25
amino acids have been reported to be phosphorylated but traditional phosphoproteomics
experiments ignore these rare events76
In a typical large-scale phosphoproteomics experiment the sample size is usually in
milligram amounts to account for the low stoichiometry of phosphorylated proteins The large
amount of protein is then digested typically with trypsin but alternatively experiments have
been performed with Lys-C digestion to produce large enzymatic peptides The larger peptides
produced from Lys-C render higher charged peptides during electrospray ionization (ESI) and
allow improved electron-based fragmentation to determine specific sites of phosphorylation77
From the pool of peptides phosphopeptides must be enriched otherwise they will be masked by
the vast number and higher ionization efficiency of non-phosphorylated peptides The two most
common enrichment techniques are immobilized metal ion affinity chromatography (IMAC) and
metal oxide affinity chromatography (MOAC) TiO2 being the most common oxide used for this
purpose A recent study reported that phosphorylation of neuronal intermediate filament proteins
in neurofibrillary tangles are involved in Alzheimerrsquos disease78
Glycoproteomics
Protein glycosylation is one of the most common and complicated forms of PTM Types
of protein glycosylation in eukaryotes are categorized as either N-linked where glycans are
attached to asparagine residues in a consensus sequence N-X-ST (X can be any amino acid
except proline) via an N-acetylglucosamine (N-GlcNAc) residue or the O-glycosylation where
the glycans are attached to serine or threonine Glycosylation plays a fundamental role in
numerous biological processes and aberrant alterations in protein glycosylation are associated
with neurodegenerative disease states such as Creutzfeld-Jakob Disease (CJD) and AD79 80
26
Due to the low abundance of glycosylated forms of proteins compared to non-glycosylated
proteins it is essential to enrich glycoproteins or glycopeptides in complex biological samples
prior to MS analysis Two of the most common enrichment methods used in glycoproteomics are
lectin affinity chromatography (LAC) and hydrazide chemistry The detailed methodologies of
LAC hydrazide chemistry and other enrichment methods in glycoproteomics have been
extensively reviewed in the past81 82
In particular LAC is of great interest in studies of
glycosylation alterations as markers of AD and other neurodegenerative diseases due to its recent
applications in brain glycoproteomics83
Our group has utilized multi-lectin affinity
chromatography containing concanavalin A (ConA) and wheat germ agglutinin (WGA) to enrich
N-linked glycoproteins in control and prion-infected mouse plasma84
This method enabled us to
identify a low-abundance glycoprotein serum amyloid P-component (SAP) PNGase F digestion
and Western blotting validation confirmed that the glycosylated form of SAP was significantly
elevated in mice with early prion infection and it could be potentially used as a diagnostic
biomarker for prion diseases
Membrane proteins
Membrane proteins play an indispensable role in maintaining cellular integrity of their
structure and perform many important functions including signaling transduction intercellular
communication vesicle trafficking ion transport and protein translocationintegration85
However due to being relatively insoluble in water and low abundance it is challenging to
analyze membrane proteins by traditional MS-based proteomics approaches Numerous efforts
have been made to improve the solubility and enrichment of membrane proteins during sample
preparation Several comprehensive studies recently covered the commonly used technologies in
27
membrane proteomics and different strategies that circumvent technical issues specific to the
membrane 86-90
Recently Sun et al reported using 1-butyl-3-methyl imidazolium
tetrafluoroborate (BMIM BF4) an ionic liquid (IL) as a sample preparation buffer for the
analysis of integral membrane proteins (IMPs) by microcolumn reversed phase liquid
chromatography (μRPLC)-electrospray ionization tandem mass spectrometry (ESI-MSMS)
The authors compared BMIM BF4 to the other commonly used solvents such as sodium dodecyl
sulfate methanol Rapigest and urea but they found that the number of identified IMPs from rat
brain extracted by ILs was significantly increased The improved identifications could be due to
the fact that BMIM BF4 has higher thermal stability and thus offered higher solubilizing ability
for IMPs which provided better compatibility for tryptic digestion than traditionally used solvent
systems38
In addition to characterization of membrane proteome the investigation of PTMs on
membrane proteins is equally important for characterization of disease markers and drug
treatment targets Phosphorylations and glycosylations are the two most important PTMs for
membrane proteins In many membrane protein receptors the cytoplasmic domains can be
phosphorylated reversibly and function as signal transducers whereas the receptor activities of
the extracellular domains are mediated via N-linked glycosylation Wiśniewski et al provides an
informative summary on recent advances in proteomic technology for the identification and
characterization of these modifications91
Our group has pioneered the development of detergent
assisted lectin affinity chromatography (DALAC) for the enrichment of hydrophobic
glycoproteins using mouse brain extract92
We compared the binding efficiency of lectin affinity
chromatography in the presence of four commonly used detergents and determined that under
certain concentrations detergents can minimize the nonspecific bindings and facilitate the
elution of hydrophobic glycoproteins In summary NP-40 was suggested as the most suitable
28
detergent for DALAC due to the higher membrane protein recovery glycoprotein recovery and
membranous glycoprotein identifications compared to other detergents tested In a different
study on mouse brain membrane proteome Zhang et al reported an optimized protocol using
electrostatic repulsion hydrophilic interaction chromatography (ERLIC) for the simultaneous
enrichment of glyco- and phosphopeptides from mouse brain membrane protein preparation93
Using this protocol they successfully identified 544 unique glycoproteins and 922 glycosylation
sites which were significantly higher than those using the hydrazide chemistry method
Additionally a total of 383 phosphoproteins and 915 phosphorylation sites were identified
suggesting that the ERLIC separation has the potential for simultaneous analysis of both glyco-
and phosphoproteomes
Peptidomics
Peptidomics can be loosely defined as the study of the low molecular weight fraction of
proteins encompassing biologically active endogenous peptides protein fragments from
endogenous protein degradation products or other small proteins such as cytokines and signaling
peptides Studies can involve endogenous peptides94
peptidomic profiling33
and de novo
sequencing of peptides95 96
Neuropeptidomics focuses on biologically active short segments of
peptides and have been investigated in numerous species including Rattus97 98
Mus musculus99
100 Bovine taurus
101 Japanese quail diencephalon
102 and invertebrates
103-106 The isolation of
peptides is typically performed through molecular weight cut-offs from either biofluids such as
CSF plasma or tissue extracts If the protein and peptide content is high such as for tissue or cell
lysates protein precipitation can be done via high organic solvents and the resulting supernatant
can be analyzed for extracted peptides where extraction solvent and conditions could have a
29
significant effect on what endogenous peptides are extracted from tissue107
A comparative
peptidomic study of human cell lines highlights the utility of finding peptide signatures as
potential biomarkers108
A thorough review of endogenous peptides and neuropeptides is beyond
the scope of this review and an excellent review on this topic is available elsewhere109
Fractionation and Separation
The mass spectrometer has a limited duty cycle and data dependent analysis can only
scan a limited number of mz peaks at any given time In addition significant ion suppression
can occur if there is a difference in concentration between co-eluting peptides or if too many
peptides co-elute Therefore one of the biggest challenges in biomarker discovery is the
complexity of the sample and the presence of high-abundance proteins in body fluids such as
CSF serum and plasma In addition to the removal of the most abundant proteins by
immunodepletion the reduction of the complexity of the sample by further fractionation is
indispensable to facilitate the characterization of unidentified biomarkers from the low
abundance proteins Traditionally used techniques for complex protein analysis include gel
based fractionation methods such as two-dimensional gel electrophoresis (2D-GE) and its
variation two-dimensional differential gel electrophoresis (2D-DIGE) or non-gel based such as
one- or multidimensional liquid chromatography (LC) and microscale separation techniques
such as capillary electrophoresis (CE)
2D-GE MS has been widely used as a powerful tool to separate proteins and identify
differentially expressed proteins ever since 2D gels were coupled to mass spectrometry In 2D-
GE MS thousands of proteins can be separated on a single gel according to pI and molecular
weight Individual protein spots that show differences in abundance between different samples
30
can then be excised from the gel digested into peptides and analyzed by MALDI MS or by
liquid chromatography tandem mass spectrometry (LC-MSMS) for protein identification The
introduction of 2D-DIGE adds a quantitative strategy to gel electrophoresis by enabling multiple
protein extracts to be separated on the same 2D gel thus providing comparative analysis of
proteomes in complex samples In 2D-DIGE protein extracts from two different conditions and
an internal standard can be labeled with fluorescent dyes for example Cy3 Cy5 and Cy2
respectively prior to two-dimensional gel electrophoresis Compared to traditional 2D-GE 2D-
DIGE provides the clear advantage of overcoming the inter-gel variation problem 110
Proteomic
profiling of CSF by 2D-GE and 2D-DIGE has led to the identification of putative biomarkers in
multiple neurological disorders For example Brechlin et al reported an optimized 2-DIGE
protocol profiled CSF from 36 CJD patients The applicability of their approach was proven by
the detection of known CJD biomarkers such as 14-3-3 protein neuron-specific enolase lactate
dehydrogenase and other proteins that are potentially relevant to CJD 111
In another study to
identify novel CSF biomarkers for multiple sclerosis CSF from 112 multiple sclerosis patients
and control individuals were analyzed by 2D-GE MS for comparative proteomics Ten potential
multiple sclerosis biomarkers were selected for validation by immunoassay 112
These
methodologies sample preparation techniques and applications of 2D-DIGE in
neuroproteomics were reviewed by Diez et al113
Although 2D gel provides excellent resolving
power and capability to visualize abundance changes there are some limitations to the method
For example gel based separation is not suitable for low abundance proteins extremely basic or
acidic proteins very small or large proteins and hydrophobic proteins114 115
Complementary to gel-based approaches shotgun proteomics coupled to LC have
become increasingly popular in proteomic research because they are reproducible highly
31
automated and capable of detecting low abundance proteins Furthermore another advantage of
LC-MS shotgun proteomics is the suitability for isotope labeling for protein quantification which
is reviewed in a later section In shotgun proteomics a protein mixture is digested and resulting
peptides are separated by LC prior to tandem MS fragmentation to identify different proteins by
peptide sequencing The most common separation for shotgun proteomics peptidomics or top-
down proteomics experiments use low-pH reversed phase (RP) C18 C8 or C4 columns RPLC
is well established which provides high resolution desalts the sample which can interfere with
ionization and the mobile phase is compatible with ESI Nanoscale C18 columns allow for
separation and introduction of sub microgram samples If larger amounts of sample are
available two dimensional separations are usually preferred to greatly enhance the coverage of
the investigated proteome which will be discussed in depth later It is preferable to have an
orthogonal separation method and since RP separates via hydrophobicity strong cation exchange
(SCX) was the original choice due to its separation by charge MudPIT (multidimensional
protein identification technology) usually refers to the use of SCX as the first phase of separation
and is a well-established platform116
SCX has the advantage over RP separation technologies to
effectively remove interfering detergents from the sample SCX separation is not based solely
off charge and hydrophobicity contributes to elution therefore a small amount of organic
modifier usually 10-15 is added to lessen the hydrophobicity effects117
The addition of
organic modifiers needs to be minimized otherwise binding to the C18 trap cartridge or C18
column will be reduced if performed on-line SCX can be used for PTMs and offers specific
applications for proteomic studies and an excellent current review is offered on this subject
elsewhere118
An alternative MudPIT separation scheme employing high pH RPLC as the first
phase of separation and low pH RPLC in the second dimension (RP-RP) has been successfully
32
applied to the proteomic analysis of complex biological samples119 120
The advantage of using
RP as the first dimension is the higher resolution for separation and better compatibility with
down-stream MS detection by eliminating salt Song et al reported a phosphoproteome analysis
based on this 2D RP-RP coupling scheme121
Hydrophilic interaction chromatography (HILIC) employs distinct separation modality
where the retention of peptides is increased with increasing polarity122
The loading of sample is
done by high organic and eluted by increasing the percentage of the aqueous phase or polarity of
the mobile phase opposite from RPLC thus establishing orthogonality of the two separation
modes123
HILIC has quickly become a very useful method and is actively used for proteomic
experiments124
for increased sensitivity125
phosphoproteomics126
glycoproteins127
and
quantification studies128
An alternative and modification to HILIC is ERLIC which adds an
additional mode of separation by electrostatic attraction An earlier study using ERLIC
demonstrated the ability to separate phosphopeptides from non-phosphorylated peptides at
pH=2129
A recent study looking into changes in the phosphoproteome of Marekrsquos Disease
applied ERLIC to chicken embryonic fibroblast lysate identifying only 13 phosphopeptides
out of all the identified peptides Due to the lack of isolation of phosphopeptides from ERLIC
the investigators performed immobilized metal affinity chromatography (IMAC) enrichment on
the fractions increasing identification of phosphopeptides over 50 fold130
A comparative study
of ERLIC to HILIC and SCX following TiO2 phospho-enrichment reported that
SCXgtERLICgtHILIC for phosphopeptide identifications126
Recent developments in instrumentation to combine LC with ion mobility spectrometry
(IMS) and MS (LC-IMS-MS) offered more advantages than conventional LC due to the rapid
high-resolution separations of analytes based on their charge mass and shape as reflected by
33
mobility in a given buffer gas The mobility of an ion in a buffer gas is determined by the ionrsquos
charge and its collision cross-section with the buffer gas The methodologies of IMS separations
and the application of LC-IMS-MS for the proteomics analysis of complex systems including
human plasma have been reviewed by Clemmerrsquos group131-133
They proposed a method that
employs intrinsic amino acid size parameters to obtain ion mobility predictions which can be
used to rank candidate peptide ion assignments and significantly improve peptide identification
134
Although 2D gel and LC are routinely used as separation techniques in MS-based
proteomics capillary electrophoresis (CE) has received increasing attention as a promising
alternative due to the fast and high-resolution separation it offers CE has a wide variety of
operation modes among which capillary zone electrophoresis (CZE) and capillary isoelectric
focusing (CIEF) have the greatest potential applications in MS-based proteomics thus will be
highlighted here CZE separates analytes by their charge-to-size ratios in buffers under a high
electrical field and is often used as the final dimension prior to MS analysis while the separation
feature of CIEF is based on isoelectric point and this technique is more suitable to be used as the
first dimension separation Detailed description of different CEndashMS interfaces sample
preconcentration and capillary coating to minimize analyte adsorption could be found in several
reviews135-141
CE technique is complementary to conventional LC in that it is suitable for the
analysis of polar and chargeable compounds Dovichirsquos group conducted proteomic analysis of
the secreted protein fraction of Mycobacterium marinum which has intermediate protein
complexity142
The tryptic digests were either analyzed by UPLC-ESI-MSMS in triplicates or
prefactionated by RPLC followed by CZE-ESI-MSMS It was demonstrated that the two
methods identified similar numbers of peptides and proteins within similar analysis times
34
However CZE-ESI-MSMS analysis of the prefractionated sample tended to identify more
peptides that are basic and have lower mz values than those identified by UPLC-ESI-MSMS
This analysis also presented the largest number of protein identifications by using CE-MSMS
suggesting the effectiveness of prefractionation of complex samples by LC method prior to CZE-
ESI-MSMS The use of CIEF as the first dimension of separation provides both sample
concentration and excellent resolving power The combination of CIEF and RPLC separation
has been applied to the proteomic analyses where the amount of protein sample is limited and
cannot meet the requirement of minimal load amount for 2D LC-MSMS143 144
So far CE-MS
has been widely applied to the proteomic analysis of various biological samples such as urine145
146 CSF
147 blood
148 frozen tissues
149 and the formalin-fixed and paraffin-embedded (FFPE)
tissue samples150
The recent CEndashMS applications to clinical proteomics have been summarized
in several reviews135 151 152
Protein Quantification
In 2D gel electrophoresis the quantitative analysis of protein mixtures is performed on
the gel by comparing the intensity of the protein stain The development of 2D-DIGE eliminated
the gel-to-gel variation and greatly improved the quantitative capability and reliability of 2D gel
methodology110
However the accuracy of 2D gel based protein quantification suffers from the
limitations that a seemingly single gel spot often contains multiple proteins and the difficulty of
detecting proteins with extreme molecular weights and pI values as well as highly hydrophobic
proteins such as membrane proteins Therefore non-gel based shotgun proteomics technology is
more suitable for accurate and large-scale protein identification and quantification in complex
samples Briefly the quantification in non-gel based shotgun proteomics can be categorized into
35
two major approaches stable isotope labeling-based and label-free methods The common
strategies for quantitative proteomic analysis are reviewed and summarized in Table 1
Isotope labeling methods
Because stable isotope-labeled peptides have the same chemical properties as their
unlabeled counterparts the two peptides within a mixture should exhibit identical behaviors in
MS ionization The mass difference introduced by isotope labeling enables the detection of a
pair of two distinct peptide masses by MS within the mixture and allowing for the measurement
of the relative abundance differences between two peptides Depending on how isotopes are
incorporated into the protein or peptide these labeling methods can be divided into two groups
In vitro chemical derivatization techniques which incorporate a label or tag into the peptide or
protein during sample preparation metabolic labeling techniques which introduce the isotope
label directly into the organism via isotope-enriched nutrients from food or media
1 In vitro derivatization techniques
There are multiple methods to introduce heavy isotopes into proteins or peptides in vitro
The commonly used strategies include 18
O 16
O enzymatic labeling Isotope-Coded Affinity Tag
(ICAT) Tandem Mass Tags (TMTs) and Isobaric Tags for Relative and Absolute Quantification
(iTRAQ) The 18
O labeling method enzymatically cleaves the peptide bond with trypsin in the
presence of 18
O-enriched H2O and introduces 4-Da mass shift in the tryptic peptides153
The
advantages of this method include 18
O-enriched water is extremely stable tryptic peptides will
be labeled with the same mass shift secondary reactions inherent to other chemical labeling can
be avoided Conversely widespread use of 18
O-labeling has been hindered due to the difficulty
of attaining complete 18
O incorporation and the lack of robustness154 155
Currently ICAT
36
TMTs and iTRAQ methods are extensively used in quantitative proteomics In ICAT cysteine
residues are specifically derivatized with a reagent containing either zero or eight deuterium
atoms as well as a biotin group for affinity purification of cysteine-containing peptides156 157
The advantage of ICAT is that the affinity purification via biotin moiety can facilitate the
detection of low-abundance cysteine-containing peptides In addition the mass difference
introduced by labeling increases mass spectral complexity with quantification from the different
precursor masses done by MS and peptide identification being achieved through tandem MS
(MSMS) This added complexity from different peptide masses was addressed by using isobaric
labeling methods such as TMTs and iTRAQ 158 159
where the same peptides in different samples
are isobaric after tagging and appear as single mz in MS scans thus enhancing the peptide limit
of detection and reducing the MS scan complexity Isobaric labeling reagents are composed of a
primary amine reactive group and an isotopic reporter group linked by an isotopic balancer group
for the normalization of the total mass of the tags The reporter group serves for quantification
purpose since it is cleaved during collision-induced dissociation (CID) to yield a characteristic
isotope-encoded fragment Moreover isobaric labeling methods allow the comparison of
multiple samples within a single experiment Recently a 6-plex version of TMTs was
reported160
and iTRAQ enables up to eight samples to be labeled and relatively quantified in a
single experiment161
8-plex iTRAQ reagents have been used for the comparison of complicated
biological samples such as CSF in the studies of neurodegenerative diseases 162
Recently our
group developed a novel N N-dimethyl leucine (DiLeu) 4-plex isobaric tandem mass (MS2)
tagging reagents with high quantitation efficacy DiLeu has the advantage of synthetic simplicity
and greatly reduced synthesis cost compared to TMTs and iTRAQ163
Xiang et al demonstrated
that DiLeu produced comparable iTRAQ ability for protein sequence coverage (~43) and
37
quantitation accuracy (lt15) for tryptically digested proteins More importantly DiLeu
reagents could promote enhanced fragmentation of labeled peptides thus allowing more
confident peptide and protein identifications
2 In Vivo Metabolic Labeling
Metabolic processes can also be employed for the incorporation of stable-isotope labels
into the proteins or organisms by enriching culture media or food with light or heavy versions of
isotope labels (2H
13C
15N) The advantage of in vivo labeling is that metabolic labeling does
not suffer from incomplete labeling which is an inherent drawback for in vitro derivatization
techniques In addition metabolic labeling occurs from the start of the experiment and proteins
with light or heavy labels are simultaneously extracted thus reducing the error and variability of
quantification introduced during sample preparation The most widely used strategy for
metabolic labeling is known as stable-isotope labeling of amino acids in cell culture (SILAC)
which was introduced by Mann and co-workers164 165
In SILAC one cell population is grown
in normal or light media while the other is grown in heavy media enriched with a heavy
isotope-encoded (typically 13
C or 15
N) amino acid such as arginine or leucine Cells from the
two populations are then combined proteins are extracted digested and analyzed by MS The
relative protein expression differences are then determined from the extracted ion
chromatograms from both the light and heavy peptide forms SILAC has been shown to be a
powerful tool for the study of intracellular signal transduction In addition this technique has
recently been applied to the quantitative analysis of phosphotyrosine (pTyr) proteomes to
characterize pTyr-dependent signaling pathways166 167
38
Labe-free quantification
Although various isotope labeling methods have provided powerful tools for quantitative
proteomics several limitations of these approaches are noted Labeling increases the cost and
complexity of sample preparation introduces potential errors during the labeling reaction It also
requires a higher sample concentration and complicates data processing and interpretation In
addition so far only TMTs and iTRAQ allow the comparison of multiple (up to eight) samples
simultaneously The comparison of more than eight samples in a single experiment cannot be
achieved by isotope labeling In order to address these concerns there has been significant
interest in the development of label-free quantitative approaches Current label-free
quantification methods for MS-based proteomics were developed based on the observation that
the chromatographic peak area of a peptide168 169
or frequency of MSMS spectra170
correlating
to the protein or peptide concentration Therefore the two most common label-free
quantification approaches are conducted by comparing (i) area under the curve (AUC) of any
given peptides171 172
or (ii) by frequency measurements of MSMS spectra assigned to a protein
commonly referred to as spectral counting173
Several recent reviews provided detailed and
comprehensive knowledge comparing label-free methods with labeling methods data processing
and commercially available software for label-free quantitative proteomics174-177
Dissociation Techniques
The vast majority of proteomic experiments have proteins or peptides being identified by
two critical pieces of data obtained from the mass spectrometer The first is the precursor ion
identified by its mz which is informative to the mass of the peptide being analyzed The second
is the use of tandem mass spectrometry to fragment or dissociate the precursor ion and record the
39
generated fragment ion pattern to discern the amino acid sequence The three most popular
dissociation or fragmentation techniques for peptides are CID electron-transfer dissociation
(ETD) and high-energy collision dissociation (HCD) A recent study on the human plasma
proteome demonstrated that combined fragmentation techniques enhance coverage by providing
complementary information for identifications CID enabled the greatest number of protein
identifications while HCD identified an additional 25 proteins and ETD contributed an
additional 13 protein identifications178
ETDECD
Electron capture dissociation (ECD) 179
preceded ETD but ECD was developed for use
in a Penning trap for Fourier transform ion cyclotron resonance (FTICR) mass spectrometers
ECD requires the ion of interest to be in contact with near-thermal electrons and for the electron
capture event to occur on the millisecond time scale but the time scale is inadequate for electron
trapping in Paul traps or quadrupoles in the majority of mass spectrometers180
ETD involves a
radical anion like fluoranthene with low electron affinity to be transferred to peptide cation
which results in more uniform cleavage along the peptide backbone The cation accepts an
electron and the newly formed odd-electron protonated peptide undergoes fragmentation by
cleavage of the N-Cα bond which results in fragmentation ions consisting of c- and zbull-type
product ions The uniformed cleavage results in reduced sequence discrimination to labile bonds
such as PTMs and also provides improved sequencing for larger peptides compared to CID181
The realization that larger peptides produced better MSMS quality spectra compared to CID led
to a decision tree analysis strategy where peptide charge states and size determined whether the
precursor peptide would be fragmented with CID or ETD182
One of the main benefits of
ETDECD is the ability to sequence peptides with labile PTMs such as phosphorylation77 183
40
sulfation184
glycosylation185
ubiquitination186
and histone modifications187
ETD also has the
benefit of providing better sequence information on larger neuropeptides when compared to
CID188
However a thorough analysis suggested that CID still yielded more peptideprotein
identifications than ETD in large scale proteoimcs189
HCD
High energy collision dissociation (HCD)190
is an emerging fragmentation technique that
offers improved detection of small reporter ions from iTRAQ-based studies191 192
HCD is
performed at a higher energy in a collision cell instead of an ion trap like CID thus HCD does
not suffer from the low-mass cutoff limitation Furthermore HCD offers enhanced
fragmentation efficiency assisting in MSMS spectra interpretation and protein identification193
A major drawback for HCD is that the spectral acquisition times are up to two-fold longer due to
increased ion requirement for Fourier transform detection in the orbitrap194
HCD has been
reported to increase phosphopeptide identifications over CID74
but in a different study CID was
reported to offer more phosphopeptide identifications over HCD194
Work has also been done to
transfer the decision tree analysis for HCD which basically switches CID with HCD claiming
better quality data determined by higher Mascot scores with more peptide identifications195
MSE
Data dependent acquisition (DDA) is the most commonly used ion selection process in
mass spectrometers for proteomic experiments An alternative process which does not have ion
selection nor switch between MS and MSMS modes is termed MSE MS
E is a data independent
mode and does not require precursor ions of a significant intensity to be selected for MSMS
analysis196
A data independent mode decouples the mass spectrometer choosing which
precursor ions to fragment and when the ions are fragmented MSE works by a low or high
41
energy scan and no ion isolation is occurring The low energy scan is where the precursor ion is
not fragmented and the high energy scan allows fragmentation The resulting mix of precursor
and fragmentation ions is then detected simultaneously197
The data will then need to be
deconvoluted using a proprietary time-aligned algorithm that is discussed elsewhere198
The
continuous data independent acquisition allows multiple MSMS spectra to be collected during
the natural analyte peak broadening observed in chromatography which provides more data
points for AUC label-free quantification In addition lower abundance peptides can be
sequenced as more MSMS spectra are collected throughout the elution of an LC peak allowing
better signal averaging for smaller analyte peak of interest during coelution and reducing
sampling bias in typical DDA experiments where only more abundant peaks can be selected for
fragmentation
A comparison of spiked internal protein standards into a complex protein digest provided
evidence that MSE was comparable to DDA analysis in LC-MS
199 MS
E has been used for label
free proteomics of immunodepleted serum in large scale proteomics samples200
In addition
MSE was performed for the characterization of human cerebellum and primary visual cortex
proteomes Hundreds of proteins were identified including many previously reported in
neurological disorders201
MSE is quickly becoming a versatile data acquisition method recently
used in such studies as cancer cells202
schizophrenia203
and pituitary proteome discovery204
The usefulness of MSE as an unbiased data acquisition method is being assimilated into multiple
proteomics studies including studies involving neurological disorders
Data Analysis
42
One of the major bottlenecks in non-targeted proteomic experiments is how to handle the
enormous amount of data obtained Database searches biostatistical analysis de novo
sequencing PTM validation all have their place and multiple available platforms are available
If the organism being studied has had its genome sequenced databases can be created
with a list of proteins in the FASTA format to be used in database searching There are
numerous database searching algorithms for sequence identification of MSMS data including
Mascot205
Sequest206
Xtandem207
OMSSA208
and PEAKS209
These searching algorithms are
performed by matching MSMS spectra and precursor mass to sequences found within proteins
How well the actual spectra match the theoretical spectra determines a score which is unique to
the searching algorithm and usually can be extrapolated to the probability of a random hit
Recently a database has been developed for PTM analysis by the use of the program SIMS210
Specifically for phosphopeptides Ascorersquos algorithm scans the MSMS data to determine the
likelihood of correct phosphosite identification from the presence of site identifying product
ions211
If the organism that is being analyzed has not had its genome sequenced and no (or very
limited) FASTA database is available a homology search can be performed using SPIDER212
available with PEAKS software Alternatively individual MSMS spectrum can be de novo
sequenced but software is available to perform automated de novo sequencing of numerous
spectra (PEAKS208
DeNovoX and PepSeq)
For large-scale protein identifications the false discovery rate (FDR) must be established
by the searching algorithm and that is accomplished by re-searching the data with a false
database created by reversing or scrambling the amino acid sequence of the original database
used for the protein search Any hits from the false database will contribute to the FDR and this
value can be adjusted usually around 1 An additional layer of confidence in the obtained data
43
can be achieved in shotgun proteomics experiments by removing all the proteins that are
identified by only one peptide
Once a set of confident proteins or peptides have been generated from database
searching bioinformatic analysis or biostatistical analysis is needed Numerous software
packages are available for different purposes FLEXIQuant is an example for absolute
quantitation of isotopically labeled protein or peptides of interest213
FDR analysis of
phosphopeptides or other specific PTMs can be adjusted with such software as Scaffold
providing data consisting only of a specific modification214
Bioinformatic tools such as
Scaffold or ProteoIQ also include gene ontology (GO) analysis which can classify identified
proteins by three categories cellular component molecular function or biological process
Custom bioinformatics programs can also be developed and are often useful in various proteomic
studies including biomarker discovery in neurological diseases215
More detailed review of
bioinformatics in peptidomics216
and proteomics217
can be found elsewhere
Validation of Biomarkers by Targeted Proteomics
The validation of putative biomarkers identified by MS-based proteomic analysis is often
required to provide orthogonal analysis to rule out a false positive by MS and providing
additional evidence for the biomarker candidate(s) from the study for future potential clinical
assays At present antibody-based assays such as Western blotting ELISA and
immunochemistry are the most widely used methods for biomarker validation Although accurate
and well established these methods rely on protein specific antibodies for the measurement of
the putative biomarker and could be difficult for large-scale validation of all or even a subset of a
long list of putative protein biomarkers typically obtained by MS-based comparative proteomic
44
analysis Large scale validation is impractical due to the cost for each antibody the labor to
develop a publishable Western blot or ELISA and the antibody availability for certain proteins
As an alternative strategy quantitative assays based on multiple-reaction monitoring (MRM) MS
using a triple quadrupole mass spectrometer have been employed in biomarker verification
MRM is the most common use of MSMS for absolute quantitation It is a hypothesis
driven experiment where the peptide of interest and its subsequent fragmentation pattern must be
known prior to the quantitative MRM experiments MRM involves selecting a specific mz (first
quadrupole) to be isolated for fragmentation (second quadrupole) followed by one or more of
the most intense fragment ions (third quadrupole) being monitored The ability to quantitate and
thus validate the proteins or peptides as potential biomarkers is achieved by performing MRM on
isotopically labeled reference peptide for targeted peptideprotein of interest The main obstacle
for quantification of peptides is interference and ion suppression effects from co-eluting
substances Since the isotopically labeled and native peptide will co-elute the same interference
and ion suppression will occur for both peptides and thus correcting these interfering effects
Peptides need to be systematically chosen for a highly sensitive and reproducible MRM
experiment to ensure proper validation of putative biomarkers Peptides require certain intrinsic
properties which include an mz within the practical mass detection range for the instrument and
high ionization efficiency If the desired peptide to be quantified is derived from a digestion
then peptides that have detectable incomplete digestion or missed cleavage site can be a major
source of variability Peptides with a methionine and to a lesser extent tryptophan are
traditionally removed from consideration from MRM quantitative experiments due to the
variable nature of the oxidation that can occur In addition if chromatographic separation is
performed the retention behavior of the peptide must be well behaved with little tailing effects
45
eluting late causing broadening of the peak and even irreversible binding to the column As an
example hydrophilic peptides being eluted off a C18 column may exhibit the previously
described concerns and a different chromatographic separation will need to be explored for
improved limits of detection quantitation and validation To determine consistent peptide
detection or usefulness of certain peptides databases such as Proteomics Database218
PRIDE219
PeptideAtlas220
have been developed to compile proteomic data repositories from initial
discovery experiments
After the peptide is selected for analysis the proper MRM transitions need to be selected
to optimize the sensitivity and selectivity of the experiment It is common for investigators to
select two or three of the most intense transitions for the proposed experiment It is imperative
that the same instrument is used for the determination of transition ions as different mass
spectrometers may have a bias towards different fragment ions
MRM experiments are still highly popular experiments for hypothesis directed
experiments221
biomarker analysis222
and validation223
Validation of putative biomarkers is
increasingly becoming a necessary step when performing large scale non-hypothesis driven
proteomics experiments The traditional validation techniques of ELISA Western blotting and
immunohistochemistry are still used but MRM experiments are becoming an attractive
alternative for validation of putative biomarkers due to its enhanced throughput and specificity
Current work is still being performed to both expand the linear dynamic range224
and
sensitivity225
of MRM A recent endeavor to increase the sensitivity for MRM experiments was
accomplished by ldquoPulsed MRMrdquo via the use of an ion funnel trap to enhance confinement and
accumulation of ions The authors claimed an increase by 5-fold for peak amplitude and a 2-3
fold reduction in chemical background225
46
Remaining Challenges and Emerging Technologies
Large sample numbers for mass spectrometry analysis
Multiple conventional studies in proteomics have been performed on a single or a few
biological samples As bio-variability can be exceedingly high the need for larger sample sizes
is currently being investigated Prentice et al used a starting point of 3200 patient samples
from the Womenrsquos Health Institute (WHI) to probe the plasma proteome using MS for
biomarkers The study did not test the 3200 patient samples by MS because even a simple one
hour one dimensional RP analysis on a mass spectrometer would take months of instrument time
for uninterrupted analysis Instead the authors pooled 100 samples together to bring the total
number of pooled samples to 32 To provide relevant plasma biomarkers the samples were then
subjected to immunodepletion 2-D protein separation (96 fractions total) and then 1-D RPLC of
tryptic peptide separation on-line interface to a mass spectrometer The large sample cohorts
help address bio-variability that can be a concern from small sample size proteomic experiments
and provide ample sample amounts to investigate the low abundance proteins226
Hemoglobin-derived neuropeptides and non-classical neuropeptides
Neuropeptides such as neuropeptide Y and enkephalin are short chains of amino acids
that are secreted from a range of neuronal cells that signal nearby cells In contrast non-classical
neuropeptides are termed as neuropeptides or ldquomicroproteinsrdquo which are derived from
intracellular protein fragments and synthesized from the cytosol227
MS was recently used to
determine that hemopressins which are hemoglobin-derived peptides are upregulated in Cpefatfat
mice brains Gelman et al designed an MS experiment to compare hemoglobin-derived
47
peptides comparing the brain blood and heart peptidome in mice The authors provided data
that specific hemoglobin peptides were produced in the brain and were not produced in the
blood Certain alpha and beta hemoglobin peptides were also up regulated in the brain for
Cpefatfat
mice and bind to CB1 cannabinoid receptors228
As discussed earlier in the review
peptidomics and specifically neuropeptidomics are popular fields of study utilizing MS and non-
classical neuropeptides is an exciting emerging area of research that could further expand the
diversity of cell-cell signaling molecules
Ultrasensitive mass spectrometry for single cell analysis
In addition to large scale analysis MS-based proteomics and peptidomics are making
progress into ultrasensitive single cell analysis The most successful MS-based techniques for
single cell analysis was performed with MALDI and studies that have been performed on
relatively large neurons are reviewed elsewhere229
The ultrasensitive MS analysis is currently
directed towards single cell analysis of smaller cells including cancer cells The first challenge
in single cell analysis is the isolation and further sample preparation to yield relevant data
Collection and isolation of a cell type can be accomplished using antibodies for fluorescence
activated cell sorting (FACS) and immune magnetic separation FACS works by flow cytometry
sorting cells by a laser that excites a fluorescent tag that is attached to an antibody Immune
magnetic separation allows separation by antibodies with magnetic properties such as
Dynabeads230
One exciting study combining FACS and MS termed mass cytometry This
technology works by infusing a droplet into an inductively coupled plasma mass spectrometer
(ICP-MS) containing a single cell bound to antibodies chelated to transition elements allowing a
quantifying response between single cells231
Clearly the future of single cell analysis for
48
biomarker analysis and proteomics is encouraging and has the potential to be an emerging field
in MS-based proteomics and peptidomics
Laserspray ionization (LSI)
Laserspray ionization (LSI) is an exciting new method to produce multiply charged mass
spectra from MALDI that is nearly identical to ESI232-234
Recently it has been reported that LSI
can be performed in lieu of matrix to produce a total solvent-free analysis234
The benefits of
being able to generate multiply charged peptides without any solvent may offer advantages
including MS analysis of insoluble membrane proteins or hydrophobic peptides avoidance of
chemical reactions while in solvents a reduction of sample loss due to liquid sample preparation
and ability to avoid diffusion effects from tissue imaging studies234
The multiply charged peptide and protein ions produced by LSI expand the mass range
for tissue imaging analysis More importantly the multiply-charged peptide ions are amenable
for electron-based fragmentation methods such as ETD or ECD which can be employed in
conjunction with tissue imaging experiments to yield in situ sequencing and identification of
peptides of interest235
Paper spray ionization
Paper spray (PS) is an ambient ionization method which was first reported using
chromatography paper allowing detection of metabolites from dried blood spots The original
method used a cut out piece of paper with a voltage clipped on the back while applying 10 microL of
methanolH2O236
Improvements have been made to this technology to enhance analysis
efficiency with a new solvent 91 dichloromethaneisopropanol (vv) and the use of silica paper
49
over chromatography paper237
Interesting applications or modifications have been made to PS
including direct analysis of biological tissue238
and leaf spray for direct analysis of plant
materials239
but both detect metabolites instead of proteins or peptides Paper spray ionization
was previously shown to enable detection of cytochrome c and bradykinin [2-9] standards in a
proof of principle study240
Clearly the utility of PS analysis in proteomics and peptidomics is
yet to be explored
niECD
New fragmentation techniques have been investigated for their utility in proteomics and
peptidomics including a recently reported negative-ion electron capture dissociation (niECD)
Acidic peptides which usually contain PTMs such as phosphorylation or sulfonation are often
difficult to be detected as multiply charged peptides in the positive ion mode As discussed
earlier multiply charged peptides are required for ECDETD fragmentation The fragmentation
of niECD is accomplished by a multiply negatively charged peptide adding an electron The
resulting fragmentation of multiply sulfated and phosphorylated peptide and protein standards
showed no sulfate loss and preserved phosphorylation site The resulting fragmentation pattern
from niECD was also improved in the peptide anions and provides a new strategy for de novo
sequencing with PTM localization241
Conclusions and Perspectives
Proteomics methodologies have produced large datasets of proteins involved in various
biological and disease progression processes Numerous mass spectrometry-based proteomics
and peptidomics tools have been developed and are continuously being improved in both
50
chromatographic or electrophoretic separation and MS hardware and software However several
important issues that remain to be addressed rely on further technical advances in proteomics
analysis When large proteomes consisting of thousands of proteins are analyzed and quantified
dynamic range is still limited with more abundant proteins being preferentially detected
Development and optimization of chemical tagging reagents that target specific protein classes
maybe necessary to help enrich important signaling proteins and assess cellular and molecular
heterogeneity of the proteome and peptidome Furthermore a significant bottleneck in
usefulness of proteomics research is the ability to validate the results and provide clear
significant biological relevance to the results The idea of P4 medicine242 243
is an attractive
concept where the four Prsquos stand for predictive preventive personalized and participatory
Proteomics is one of the critical ldquoomicsrdquo fields and has led to the development of enabling
innovative strategies to P4 medicine244
A goal of P4 medicine is to assess both early disease
detection and disease progression in a person A simplified example of how proteomics fits into
P4 medicine is that certain brain-specific proteins could be used for diagnosis with
presymptomatic prion disease244
The concept of proteomic experiments providing an individual
biomarker is becoming more obsolete with the revised vision being a biomolecular barcode that
could potentially be ldquoscannedrdquo or be a fingerprint for a specific disease or early onset to that
disease being closer to reality An excellent review on what biomarker analysis can do for true
patients is available245
Proteomics can also generate new hypothesis that can be tested by classical biochemical
approaches If a disease has an unknown pathogenesis proteomics is a good starting point to try
to assemble putative markers that can lead to further hypothesis for evaluation If a particular
protein or PTM is associated with a disease state either qualitatively or quantitatively potential
51
treatments could target that protein of interest or investigators could monitor that protein or
PTM during potential treatments of the disease Proteomics has expanded greatly over the last
few decades with the goal of providing revealing insights to some of the most complex
biological problems currently facing the scientific community
Acknowledgements
Preparation of this manuscript was supported in part by the University of Wisconsin Graduate
School Wisconsin Alzheimerrsquos Disease Research Center Pilot Grant and a Department of
Defense Pilot Award LL acknowledges an H I Romnes Faculty Fellowship
52
Figure 1 A summary of general workflows of biomarker discovery pipeline by MS-based
proteomic approaches
Biological sample (CSF blood urine saliva cell
lysate tissue homogenates secreted proteins etc)
Protein extraction Sample pretreatment
2D-GE2D-DIGE MS 1D or 2D LC-MSMS
MALDI-IMS
Identification of
differentially
expressed proteins
Protein identification
Potential biomarkers
Biomarker validation
- Antibody based immunoassays
- MRM
Quantitative analysis
- Isotope labeling
- Label free
Identification and
localization of
differentially expressed
biomolecules
Intact tissue
Sample preparation Slice frozen tissues
thaw-mounted on plate
Apply Matrix
53
Figure 2 Tissue expression and dynamic range of the human plasma proteome A pie chart
representing the tissue of origin for the high abundance proteins shows that the majority of
proteins come from the liver (A) Conversely the lower abundance plasma proteins have a much
more diverse tissue of origin (B and C) The large dynamic range of plasma proteins is presented
and the proteins can be grouped into three categories (classical plasma proteins tissue leakage
products interleukinscytokines) (D) Adapted from Zhang et al11
and Schiess et al246
with
permission
54
55
Table 1 A summary of the common strategies applied to MS-based quantitative proteomic
analysis
Gel based Stable isotope labeling Label free
2D-GE
2D-DIGE 110
In vitro derivatization
18O
16O
153
ICAT 156
TMT 159
iTRAQ 158
Formaldehyde 247
ICPL 248
In vivo metabolic labeling
14N
15N
249
SILAC 164
AUC measurement 169 172
Spectral counting 173
AUC Area Under Curve ICAT Isotope-Coded Affinity Tag TMT Tandem Mass Tags iTRAQ Isobaric Tags for
Relative and Absolute Quantification ICPL Isotope Coded Protein Labeling SILAC Stable Isotope Labeling by
Amino Acids in Cell Culture Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
56
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73
Chapter 3
Protein changes in immunodepleted cerebrospinal fluid from transgenic
mouse models of Alexander disease detected using mass spectrometry
Adapted from ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse
models of Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P
Messing A Li L Submitted
74
ABSTRACT
Cerebrospinal fluid (CSF) is a low protein content biological fluid with dynamic range
spanning at least nine orders of magnitude in protein content and is in direct contact with the
brain A modified IgY-14 immunodepletion treatment was performed to enhance analysis of the
low volumes of CSF that are obtainable from mice As a model system in which to test this
approach we utilized transgenic mice that over-express the intermediate filament glial fibrillary
acidic protein (GFAP) These mice are models for Alexander disease (AxD) a severe
leukodystrophy in humans From the CSF of control and transgenic mice we report the
identification of 266 proteins with relative quantification of 106 proteins Biological and
technical triplicates were performed to address animal variability as well as reproducibility in
mass spectrometric analysis Relative quantitation was performed using distributive normalized
spectral abundance factor (dNSAF) spectral counting analysis A panel of biomarker proteins
with significant changes in the CSF of GFAP transgenic mice has been identified with validation
from ELISA and microarray data demonstrating the utility of our methodology and providing
interesting targets for future investigations on the molecular and pathological aspects of AxD
75
INTRODUCTION
Alexander disease (AxD) is a fatal neurogenetic disorder caused by heterozygous point
mutations in the coding region of GFAP (encoding glial fibrillary acidic protein)1 The hallmark
diagnostic feature of this disease is the accumulation of astrocytic cytoplasmic inclusions known
as Rosenthal fibers containing GFAP Hsp27 αB-crystallin and other components2-5
Although
several potential treatment strategies6-8
are under investigation clinical trial design is hampered
by the absence of a standardized clinical scoring system or means to quantify lesions in MRI
that could serve to monitor severity and progression of disease One solution to this problem
would be the identification of biomarkers in readily sampled body fluids as indirect indicators of
disease
Cerebrospinal fluid (CSF) has a long history as a surrogate biopsy site for brain or spinal
cord in evaluating diseases of the central nervous system The protein composition of CSF is
well defined at least for the most abundant species of proteins and numerous studies exist that
characterize individual biomarkers or complex patterns of biomarkers in various diseases9 10
GFAP itself is present in CSF (albeit at much lower levels than in brain parenchyma) and in one
study of three Alexander disease patients its levels were markedly increased11
Whether an
increase in CSF GFAP will be a consistent finding in Alexander disease or whether other useful
biomarkers for this disease could be identified through an unbiased analysis of the CSF
proteome is not yet known
The rarity of Alexander disease makes analysis of human samples difficult However
mouse models exist that replicate key features of the disease such as formation of Rosenthal
fibers Unfortunately mouse CSF poses particular problems for proteomic studies and there is
76
an urgent need for technical improvements for dealing with this fluid For instance collection
from an adult mouse typically yields ~10 μL of CSF often with some contamination by blood12
To further complicate analysis CSF has an exceedingly low protein content (~04 μgμL) with
over 60 of the total protein content consisting of a single protein albumin13 14
A number of
techniques have been developed to remove albumin from biological samples including Cibacron
Blue15
IgG immunodepletion16
and IgY immunodepletion17-19
IgY which is avian in origin
offers reduced non-specific binding and increased avidity when compared to IgG antibodies from
rabbits goats and mice20-23
One widely used IgY cocktail is IgY-14 which contains fourteen
specific antibodies specific for albumin IgG transferrin fibrinogen α1-antitrypsin IgA IgM
α2-macroglobulin haptoglobin apolipoproteins A-I A-II and B complement C3 and α1-acid
glycoprotein Since existing protocols for IgY-14 depletion are optimized for use with large
volumes of serum new protocols must be developed to permit its use with the low volumes of a
low protein fluid represented by mouse CSF
Various improvements have also taken place in the field of proteomic analysis that could
facilitate analysis of mouse CSF Data dependent tandem mass spectrometry followed by
quantification of proteins is used in standard shotgun proteomics24-29
Several methods now exist
for introducing quantitation into mass spectrometry including stable isotope labeling30-32
isobaric tandem mass tags33 34
and spectral counting35 36
Spectral counting which is a
frequency measurement that uses MSMS counts of identified peptides as the metric to enable
protein quantitation is attractive because it is label-free and requires no additional sample
preparation Finally recent advances in spectral counting has produced a data refinement
strategy termed normalized spectral abundance factor (NSAF)37 38
and further developed into
distributive NSAF (dNSAF) to address issues with peptides shared by multiple proteins39
77
To identify potential biomarkers in AxD we report a novel scaled-down version of IgY
antibody depletion strategy to reduce the complexity and remove high abundance proteins in
mouse CSF samples The generated spectral counts data were then subjected to dNSAF natural
log data transformation and t-test analysis to determine which proteins differ in abundance when
comparing GFAP transgenics and controls with multiple biological and technical replicates
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) urea (gt99) sodium chloride (997) and ammonium
bicarbonate (certified) were purchased from Fisher Scientific (Fair Lawn NJ) Deionized water
(182 MΩcm) was prepared with a Milli-Q Millipore system (Billerica MA) Optima LCMS
grade acetonitrile and water were purchased from Fisher Scientific (Fair Lawn NJ) DL-
Dithiothreitol (DTT) and sequencing grade modified trypsin were purchased from Promega
(Madison WI) Formic acid (ge98) was obtained from Fluka (Buchs Switzerland)
Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris ge999 ) ammonium formate
(ge99995) glycine (ge985) and IgY-14 antibodies were purchased from Sigma-Aldrich
(Saint Louis MO)
Mice
Transgenic mice over-expressing the human GFAP gene (Tg737 line) were maintained
as hemizygotes in the FVBN background and genotyped via PCR on DNA prepared from tail
samples as described previously40
The mice were housed on a 14-10 light-dark cycle with ad
libitum access to food and water All procedures were conducted using protocols approved by
the UW-Madison IACUC
78
CSF collection
CSF was collected from mice as described previously12
Briefly mice were anesthetized
with avertin (400-600 mgkg ip) A midline sagittal incision was made over the dorsal aspect
of the hindbrain and three muscle layers carefully peeled back to expose the cisterna magna The
membrane covering the cisterna magna was pierced with a 30 gauge needle and CSF was
collected immediately using a flexible plastic pipette Approximately ten microliters of CSF was
collected per animal All samples used for MS analysis showed no visible contamination of
blood
Enzyme-linked immunosorbent assay (ELISA)
A sandwich ELISA was used to quantify GFAP8 Briefly a microtiter plate was coated
with a cocktail of monoclonal antibodies (Covance SMI-26R) Plates were blocked with 5
milk before addition of sample or standards diluted in PBS with Triton-X and BSA A rabbit
polyclonal antibody (DAKO Z334) was used to detect the GFAP followed by a peroxidase
conjugated anti-rabbit IgG antibody (Sigma A6154) secondary antibody The peroxidase activity
was detected with SuperSignal ELISA Pico Chemiluminescent Substrate (PIERCE) and
quantified with a GloRunner Microplate Luminometer Values below the biological limit of
detection (16ngL) were given the value 16ngL before multiplying by the dilution factor
Immunodepletion of abundant proteins
Currently there are no commercial immunodepletion products available for use with CSF
and to address the low protein content of this fluid (~04 microgmicroL) Therefore 100 microL of
purchased IgY-14 resin was coupled with a Pierce Spin Cup using a paper filter from Thermo
Scientific (Waltham MA) CSF samples from either transgenics or controls were first pooled to
100 microL and then added to 100 microL of 20 mM Tris-HCl 300 mM pH 74 (2x dilution buffer) and
79
allowed to mix with the custom IgY-14 depletion spin column on an end-over-end rotor for 30
minutes at 4oC The sample was then centrifuged at 04 rcf for 45 seconds with an Eppendorf
Centrifuge 5415D (Hamburg Germany) The antibodies were then washed with 50 microL 1x
dilution buffer vortexed for 5 minutes centrifuged at 04 rcf for 45 seconds and the flow through
was collected for tryptic digestion The antibodies were then stripped of the bound proteins with
four 025 mL washes of 01 M glycine pH 25 neutralized with two 025 mL washes of 01 M
Tris-HCl pH 80 and washed once with 025mL of 1x dilution buffer The immunodepletion
protocol was repeated for each transgenic or control pooled 100 microL sample (N=3)
Preparation of tryptic digests
The immunodepleted pooled mouse CSF samples (200 microL total volume) were
concentrated to 10 microL using a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA)
To each sample 8 microL of 133 M urea and 1 microL of 050 M DTT were added and allowed to
incubate at 37oC for one hour After incubation 27 microL of 055 M IAA was added for
carboxymethylation and the sample was allowed to incubate for 15 minutes in the dark To
quench the IAA 1 microL of 050 M DTT was added and allowed to react for 10 minutes To
perform trypsin digestion 70 microL of 50 mM NH4HCO3 was then added along with 025 microg
trypsin which had previously been dissolved in 50 mM acetic acid at a concentration of 05
microgmicroL Digestion was performed overnight at 37oC and quenched by addition of 25 microL of 10
formic acid The tryptic peptides were then subjected to solid phase extraction using a Varian
Omix Tip C18 100 microL (Palo Alto CA) Peptides were eluted with 50 ACN in 01 formic
acid concentrated and reconstituted in 30 microL H2O in 01 formic acid
RP nanoLC separation
80
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto an Agilent
Technologies Zorbax 300 SB-C18 5 microm 5x03 mm trap cartridge (Santa Clara CA) at a flow
rate of 5 microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm
Atlantis dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B
at 250 nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
81
range of 300-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot41
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt mus musculus
(house mouse) database (version 575) False positive analyses42
were calculated using an
automatic decoy option of Mascot Results from the Mascot results were reported using
Proteinscape 21 and technical replicates were combined and reported as a protein compilation
using ProteinExtractor (Bruker Daltonics Bremen Germany)
Mascot search parameters were as follows Allowed missed cleavages 2 enzyme
trypsin variable modifications carboxymethylation (C) and oxidation (M) peptide tolerance
plusmn12 Da maximum number of 13
C 1 MSMS tolerance plusmn05 Da instrument type ESI-Trap
Reported proteins required a minimum of one peptide with a Mascot score ge300 with bold red
characterization Spectral counts were determined from the number of MSMS spectra identified
from accepted proteins A bold red peptide combines a bold peptide which represents the first
query result from a submitted MSMS spectrum with the red peptide which indicates the top
peptide for the identified protein Requiring one bold red peptide assists in removal of
homologous redundant proteins and further improves protein results In addition requiring one
82
peptide to be identified by a score gt300 removes the ability for proteins to be identified by
multiple low Mascot scoring peptides
Each immunodepleted biological replicate had technical triplicates performed and the
technical triplicates were summed together by ProteinExtractor Peptide spectral counts were
then summed for each protein and subjected to dNSAF analysis Details for this method can be
found elsewhere37 39
but briefly peptide spectral counts are summed per protein (SpC) based on
unique peptides and a weighted distribution of any shared peptides with homologous proteins
ProteinScape removed 83 homologous proteins found in the current study to bring the total
number of proteins identified to 266 but some non-unique homologous peptides which are
shared by multiple proteins are still present in the resulting 266 remaining proteins To address
these non-unique homologous peptides distributive spectral counting was performed as
described elsewhere39
The dSpC is divided by the proteinrsquos length (L) and then divided by the
summation of the dSpCL from all proteins in the experiment (N) to produce each proteinrsquos
specific dNSAF value
N
i
i
kk
LdSpC
LdSpCdNSAF
1
)(
)()(
The resulting data were then transformed by taking the natural log of the dNSAF value The
means for each protein were calculated using the ln(dNSAF) from N=3 biological replicates and
the whole data set was subjected to the Shapiro-Wilk test for normalityGaussian distribution
performed on the software PAST (Version 198 University of Oslo Norway Osla) The
Shapiro-Wilk test was repeated multiple times to determine a non-zero value for zero spectral
83
counts A non-zero value is required to alleviate the errors of dividing by zero which was
experimentally determined to be 043 The Gaussian data were then subjected to the t-test to
identify statistically significant changes in protein expression
RESULTS AND DISCUSSION
General workflow
Individual CSF samples were manually inspected and samples were only selected that
showed no visual blood contamination Preliminary experiments showed that the maximum
degree of blood contamination estimated from counts of red blood cells in the CSF that was not
visible to the eye was less than 005 by total mass Approximately 10 individual mouse CSF
samples were pooled to achieve the desired 100 μL volume for a single biological replicate The
CSF samples were IgY immunodepleted followed by digestion with trypsin The resulting
digested samples were then de-salted concentrated reconstituted in 30 μL of 01 formic acid
and 5 μL was loaded onto a reversed phase nanoflow HPLC column subjected to a 90 minute
gradient and infused into the amaZon ETD ion trap mass spectrometer The workflow for
mouse CSF analysis is shown in Figure 1 The remainder of the desalted sample was saved for
technical replicates
Immunodepletion for CSF
Currently there are no immunodepletion techniques specifically designed for CSF
Nonetheless the protein profiles between CSF and serum are similar enough to use currently
available immunodepletion techniques designed for serum as a starting point The smallest
commercially available IgY-14 immunodepletion kit is for 15 μL of serum which is equivalent in
protein content to ~15 mL of CSF Therefore for 100 μL CSF one fifteenth of the total IgY-14
84
beads would be used for the equivalent immunodepletion which is 66 μL of proprietary bead
slurry The potential for irreversible binding of abundant proteins to their respective IgY
antibody even after an extra stripping wash and low amounts of total beads made using 66 μL
of IgY-14 bead slurry unrealistic In practice almost doubling the amount of IgY-14 beads (100
μL) used was not sufficient and resulted in albumin and transferrin peptides to be observed in
high abundance (data not shown) The most important protein to immunodeplete is albumin and
it has been reported to be a greater percentage of total CSF protein content (~60) than serum
(~49) in humans14
The difference in albumin percentage supports the results that proprietary
blends of immunodepletion beads for high abundance proteins such as albumin cannot be
scaled down on a strict protein scale and further modifications to the serum immunodepletion
protocol need to be made
Since IgY-14 beads were developed for use with serum all of its protocols need to be
taken into account to modify the protocol for CSF Serum samples should be diluted fifty times
before mixing with the IgY-14 beads but CSF protein concentration is at least one hundred times
lower than serum Therefore CSF is below half the recommended diluted protein concentration
for IgY immunodepletion Consequently multiple steps have been devised to address this
limitation First the binding time between the proteins targeted for removal from the CSF and
IgY-14 beads was doubled during the end-over-end rotor depletion step from the recommended
15 minutes to 30 minutes Second to prevent non-specific protein binding to the antibodies the
CSF samples were diluted with an equal amount (100 μL) of 2X dilution buffer The dilution
buffer of NaCl and Tris-HCl was needed to assist in reducing non-specific binding of proteins to
the 14 antibodies and ensuring the sample is held at physiological pH In addition to these
modifications a doubling of the starting IgY-14 bead slurry to 200 μL provided the desired
85
results Overall this modified protocol results in effective depletion of CSF abundant proteins
using only one-fifth of the antibodies provided by the smallest commercially available platform
Data Analysis
Spectral counting technique for relative quantitation provides numerous benefits for the
study of mouse CSF due to its label-free advantage In contrast isotopic labeling method often
involves additional sample processing that could cause sample loss which is highly undesirable
for low protein content and low volume samples Labeling methods also require a mixing of two
sets of isotopically labeled samples which would effectively increase the sample complexity and
reduce the amount of sample that can be loaded onto the nanoLC column by half In addition
more than two sets of samples can be compared by label-free methods The use of label-free
spectral counting method does not lead to an increase in sample complexity or interference in
quantitation from peptides in the mz window selected for tandem MS Using spectral counting
for relative quantitation however is dependent on reproducible HPLC separation and careful
mass spectrometry operation to minimize technical variability during the study To address
concerns of analytical reliability and run to run deviations base peak chromatograms from two
transgenic IgY-14 immunodepleted biological replicates including two technical replicates of
each were shown to be highly reproducible (Figure 2)
Each biological sample was analyzed in triplicate with the same protocols on the amaZon
ETD with three control and three transgenic samples From the three technical replicates for
each biological replicate the spectral counts of the peptides for the proteins identified were
summed The results from these mouse CSF biological triplicates are shown in Figure 3A for
GFAP overexpressor and Figure 3B for control The summation of spectral counts for each
biological replicate was performed to remove the inherent bias related to data dependent analysis
86
for protein identification One concern in grouping technical replicates is a potential loss of
information regarding analytical variability Figure 4 provides a graphical representation of
variability of technical replicates illustrating the standard deviation of technical replicates with
error bars Figure 4 shows that a statistically changed protein creatine kinase M (A) and an
unchanged protein kininogen-1 (B) have similar variability within (technical replicates) and
between samples (biological replicates) for each protein In addition Figure 4B illustrates that
even with the variability of kininogen-1 the resulting mean shown by the dashed line of control
and transgenic samples were almost equal whereas Figure 4A shows significantly different
expression level of creatine kinase M Performing replicate analysis of each biological sample
(n=3) helps correct for systematic errors deriving from sample handling and pooling of samples
helps reduce random error during the CSF sample collection process
Protein Identification and Spectral Counting Analysis
The data for dNSAF analysis like any mass spectrometry proteomics experiment
requires multiple layers of verification to ensure reliable data Our initial protein identifications
were subjected to a database search using a decoy database from Mascot which resulted in an
average false positive rate below 1 for all the experimental data collected Representative
MSMS spectra between control and GFAP transgenic samples are illustrated in Figure 5
Overall 266 proteins were identified in a combination of control and transgenic samples
(Supplemental Table 1) A total of 349 proteins were initially identified but 83 proteins were
isoforms of previously identified proteins and automatically excluded by ProteinExtractor The
next level of quality control was to only include ln(dNSAF) values from proteins identified by 2
or more unique peptides having a Mascot score of ge300 and observed in two out of three
biological replicates These selection parameters resulted in 106 proteins remaining after
87
dNSAF analysis (Supplemental Table 2) The resulting spectral counts were then subjected to
dSpC in order to account and correct for the systematic error of peptides shared by multiple
proteins (Supplemental Table 3)
It is inevitable in large scale and complex proteomics experiments that some proteins will
be seen in some samples and not others In addition when controls were compared to transgenic
samples as shown in Figure 3C 127 proteins were unique to either control or GFAP transgenic
mice and 139 proteins were seen in both samples From dNSAF equation if the spectral count
is zero the numerator is zero and the value will not be normalized between runs In order to
circumvent the zero spectral counts in dNSAF analysis zero spectral counts must be replaced by
an experimentally determined non-zero value determined to be 043 The 043 spectral counts
for zero spectral counts was calculated by serial Shapiro-Wilk tests to determine the lowest value
(043) for zero spectral counts and maintain a Gaussian distribution for all data sets The 043
value for zero spectral counts in the current study was higher than the 016 reported value for
zero spectral counts in the original NSAF spectral counting study37
Our study may have a
higher zero spectral count value than the previous study because the spectral counting data were
an addition of three technical replicates and three times 016 is close to 043 The normalized
Gaussian data were then subjected to t-test analysis and P-values below 005 are considered as
statistically significant and are presented in Table 1 The proteins with significant up or down
regulation from Table 1 can be further evaluated as how close significant proteins were to a p-
value of 005 such as ganglioside GM2 activator serine protease inhibitor A3N and collagen
alpha-2(I) chain having t-test scores of 0045 0047 and 0043 respectively Proteins exhibiting
a P-value close to 005 were more likely to be highly variable proteins or have smaller fold
changes between control and transgenic samples and thus provide less biological relevancy to
88
future studies The modest change in antithrobin-III which is reduced by 14-fold in transgenic
is included due a low pooled standard deviation in spectral counts
Spectral counting has been analyzed with fold changes derived directly from the average
spectral counts from the technical replicates and then the average of the three biological
replicates We decided to perform additional analysis using fold changes to dig deeper into
proteins that statistically support the null hypothesis from the dNSAF analysis To only pull out
highly confident protein identifications we used the same strict cut-off of two unique peptides
identified per protein as in dNSAF analysis We only accepted proteins with greater than three-
fold change in total spectral counts listed in Table 2 Two proteins contactin-1 (cntn1) and
cathepsin B (CB) illustrate the potential biomarkers missed by dNSAF analysis Cntn1 had zero
spectral count in the transgenic sample and had an average spectral count of 41 in control
samples The lack of any spectral counts in one biological control for cntn1 resulted in a large
standard deviation in the ln(dNSAF) means for the control which resulted in the t-test supporting
the null hypothesis Another example is CB which was detected by numerous spectral counts in
every GFAP transgenic biological replicate with an average spectral count of 97 (Table 2) The
presence of CB in one biological control sample (23 average spectral counts) resulted in a high
standard deviation in the mean of the control samples These examples exhibit a limitation of
dNSAF analysis which could cause a loss of potentially useful information
Previously Identified Proteins with Expression Changes
Previously three proteins have been described as increased in CSF from individual(s)
suffering from AxD In one study a single patientrsquos CSF was found to have elevated levels of
αβ-crystallin and HSP2744
In a second study three patients were reported to have elevated
levels of GFAP with concentrations from 4760 ngL to 30000 ngL (compared to lt175 ngL for
89
controls)11
GFAP was detected in our current study however the other two proteins were not
detected One possibility is that αB-crystallin and Hsp-27 levels in mouse CSF are too low for
detection by MS analysis In addition while the transgenic mice display the hallmark
pathological feature of AxD in the form of Rosenthal fibers they do not have any evident
leukodystrophy and thus may not display the full range of changes in CSF as might be found in
human patients
Creatine Kinase M
Creatine kinase M (M-CK) is a 43 kDa protein whose role is to reversibly catalyze
phosphate transfer between ATP and energy storage compounds M-CK has been primarily
found in muscle tissue and for humans creatine kinase M is diagnostically analyzed in the blood
for myocardial infarction (heart attack) In addition M-CK is also found in Purkinje neurons of
the cerebellum45 46
A related protein creatine kinase B (B-CK) also exhibited an apparent 21
fold increase in transgenic CSF over control but this difference was not statistically different
B-CK concentration is known to be elevated in CSF following head trauma47
or cerebral
infarction48
but decreased in astrocytes in individuals affected by multiple sclerosis49
Cathepsin
The data showed multiple cathepsins were up regulated in the CSF of transgenic mice
when compared to control mice The up regulated cathepsins were S L1 and B isoforms which
are all cysteine proteases Cathepsin S (CS) was never observed in control samples but
observed with an average of 73 spectral counts per analysis Cathepsin L1 (CL1) was up
regulated by 94 times in transgenic mice These two cathepsins exhibited significant changes
using dNSAF and t-test statistics as shown in Table 1 and Cathepsin B (CB) showed a 42 fold
increase in transgenic CSF as shown in Table 2
90
Cathepsins regulate apoptosis in cells50
which is the major mechanism for elimination of
cells deemed by the organism to be dangerous damaged or expendable CL and CB are
redundant in the system as thiol proteases and inhibition of CB by CA-047 causes a diminished
apoptosis response in multiple cell lines51
Intriguingly increased levels of CB or CL are
correlated with poor prognosis for cancer patients and shorter disease-free intervals It is
believed that these proteases degrade the extracellular membrane which allows tumor cells to
invade adjacent tissue and metastasize52
With regards to AxD the up regulation of these
cathepsins may be indicative of the bodyrsquos natural defense and response to Rosenthal fibers
Thus stimulation of these cathepsins may provide a further protective stress response but the
positive correlation between these proteases and cancer highlights the multiple roles of these
proteins in pathological response Alternatively it has been shown that increased CB is involved
with the tumor necrosis factor α (TNFα) induced apoptosis cascade53
The activation of the
TNFα could produce oligodendrocyte toxicity54
with the expression of TNFα being elevated in
tissue samples from mouse models and AxD patients55
The potential for a positive or a negative
effect in increasing or decreasing cathepsins warrant future research with cathepsins and AxD
Contactin-1
Contactin-1 (Cntn1) is a 133 kDa cell surface protein that is highly glycosylated and
belongs to a family of immunoglobulin domain-containing cell adhesion molecues56
Table 2
shows that Cntn1 is down regulated in transgenic mice since spectral counts were only observed
in control mice Cntn1 is expressed in neurons and oligodendrocytes and higher levels were
observed during brain development57
In addition Cntn1 leads to activation of Notch1 which
mediates differentiation and maturation of oligodendrocyte precursor cells (OPC) Although the
mechanism leading to a reduction of Cntn1 in CSF is not known it may reflect a change in
91
astrocyte interactions with either neurons or oligodendrocytes to alter their expression of this
protein
Validation of putative biomarkers and MS proteomics data using ELISA and RNA
microarray data
To further validate the relative protein expression data obtained via MS-based spectral
counting techniques orthogonal immunological and molecular biological approaches have been
examined As GFAP was shown to be elevated in the CSF of transgenic animals we used a
well-defined GFAP ELISA protocol to test its CSF concentration CSF from 8 week old male
mice was collected from both transgenic and control animals Five samples of transgenic CSF
was prepared by pooling four to eight animals for the GFAP ELISA In the case of controls
each sample represents a single animal GFAP concentrations observed by both the MS and
ELISA showed significant increases of GFAP in the CSF when comparing transgenic to control
animals
Another validation of MS spectral counts is observed in a microarray analysis performed
on transgenic mouse olfactory bulb tissue 55
In this paper nine of the proteins found by MS
showed similar gene expression in the microarray (Table 3) It is not surprising that all the genes
observed in the microarray are not the same as the proteins observed by MS analysis Gene
expression and protein synthesis and expression are not always correlated but the similarities
and overlapping trends observed with these two assays are encouraging As shown in Table 3
gene expression analysis also revealed the up regulation of several cathepsin proteins GFAP
and down regulation of contactin-1 in CSF collected from transgenic mice corroborating the
MS-based proteomics results
92
CONCLUSIONS
In this study we have produced a panel of proteins with significant up or down regulation
in the CSF of transgenic GFAP overexpressor mice This mouse model for AxD was consistent
with the previous studies showing elevation of GFAP in CSF The development of a modified
IgY-14 immunodepletion technique for low amounts of CSF was presented This improved
protocol is useful for future investigations to deal with the unique challenges of mouse CSF
analysis Modified proteomics protocols were employed to profile mouse CSF with biological
and technical triplicates addressing the variability and providing quantitation with dNSAF
spectral counting Validation of the MS-based proteomics data were performed using both
ELISA and RNA microarray data to provide further confidence in the changes in the putative
protein biomarkers This study presents three classes of interesting targets for future study in
AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
93
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6 Messing A Daniels C M Hagemann T L Strategies for treatment in alexander
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7 Tang G Yue Z Talloczy Z Hagemann T Cho W Messing A Sulzer D L
Goldman J E Autophagy induced by Alexander disease-mutant GFAP accumulation is
regulated by p38MAPK and mTOR signaling pathways Hum Mol Genet 2008 17 (11) 1540-
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8 Hagemann T L Boelens W C Wawrousek E F Messing A Suppression of GFAP
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9 Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C
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Res 2008 7 (1) 386-99
10 Sjodin M O Bergquist J Wetterhall M Mining ventricular cerebrospinal fluid from
patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma
biomarkers J Chromatogr B Analyt Technol Biomed Life Sci 878 (22) 2003-12
11 Kyllerman M Rosengren L Wiklund L M Holmberg E Increased levels of GFAP
in the cerebrospinal fluid in three subtypes of genetically confirmed Alexander disease
Neuropediatrics 2005 36 (5) 319-23
12 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M
Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta)
equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
13 Wong M Schlaggar B L Buller R S Storch G A Landt M Cerebrospinal fluid
protein concentration in pediatric patients defining clinically relevant reference values Arch
Pediatr Adolesc Med 2000 154 (8) 827-31
14 Roche S Gabelle A Lehmann S Clinical proteomics of the cerebrospinal fluid
Towards the discovery of new biomarkers PROTEOMICS ndash Clinical Applications 2008 2 (3)
428-436
15 Li C Lee K H Affinity depletion of albumin from human cerebrospinal fluid using
Cibacron-blue-3G-A-derivatized photopatterned copolymer in a microfluidic device Anal
Biochem 2004 333 (2) 381-8
94
16 Maccarrone G Milfay D Birg I Rosenhagen M Holsboer F Grimm R Bailey
J Zolotarjova N Turck C W Mining the human cerebrospinal fluid proteome by
immunodepletion and shotgun mass spectrometry ELECTROPHORESIS 2004 25 (14) 2402-
2412
17 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L
Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity
separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample
preparation and analysis Proteomics 2005 5 (13) 3314-28
18 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag
L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep
Biochem Biotechnol 2009 39 (3) 221-47
19 Huang L Fang X Immunoaffinity fractionation of plasma proteins by chicken IgY
antibodies Methods Mol Biol 2008 425 41-51
20 Greunke K Braren I Alpers I Blank S Sodenkamp J Bredehorst R Spillner E
Recombinant IgY for improvement of immunoglobulin-based analytical applications Clin
Biochem 2008 41 (14-15) 1237-44
21 Xiao Y Gao X Taratula O Treado S Urbas A Holbrook R D Cavicchi R E
Avedisian C T Mitra S Savla R Wagner P D Srivastava S He H Anti-HER2 IgY
antibody-functionalized single-walled carbon nanotubes for detection and selective destruction
of breast cancer cells BMC Cancer 2009 9 351
22 Liu T Qian W J Mottaz H M Gritsenko M A Norbeck A D Moore R J
Purvine S O Camp D G 2nd Smith R D Evaluation of multiprotein immunoaffinity
subtraction for plasma proteomics and candidate biomarker discovery using mass spectrometry
Mol Cell Proteomics 2006 5 (11) 2167-74
23 Hinerfeld D Innamorati D Pirro J Tam S W SerumPlasma depletion with
chicken immunoglobulin Y antibodies for proteomic analysis from multiple Mammalian species
J Biomol Tech 2004 15 (3) 184-90
24 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D
Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in
the discovery of candidate protein biomarkers in a diabetes autoantibody standardization
program sample subset J Proteome Res 2008 7 (2) 698-707
25 Ru Q C Zhu L A Silberman J Shriver C D Label-free semiquantitative peptide
feature profiling of human breast cancer and breast disease sera via two-dimensional liquid
chromatography-mass spectrometry Mol Cell Proteomics 2006 5 (6) 1095-104
26 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S
Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-
dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of
Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66
27 Rao P V Reddy A P Lu X Dasari S Krishnaprasad A Biggs E Roberts C T
Nagalla S R Proteomic identification of salivary biomarkers of type-2 diabetes J Proteome
Res 2009 8 (1) 239-45
28 Yu K H Barry C G Austin D Busch C M Sangar V Rustgi A K Blair I A
Stable isotope dilution multidimensional liquid chromatography-tandem mass spectrometry for
pancreatic cancer serum biomarker discovery J Proteome Res 2009 8 (3) 1565-76
29 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422
(6928) 198-207
95
30 Ong S E Blagoev B Kratchmarova I Kristensen D B Steen H Pandey A
Mann M Stable isotope labeling by amino acids in cell culture SILAC as a simple and
accurate approach to expression proteomics Mol Cell Proteomics 2002 1 (5) 376-86
31 Hsu J L Huang S Y Chow N H Chen S H Stable-isotope dimethyl labeling for
quantitative proteomics Anal Chem 2003 75 (24) 6843-52
32 Liu H Sadygov R G Yates J R 3rd A model for random sampling and estimation
of relative protein abundance in shotgun proteomics Anal Chem 2004 76 (14) 4193-201
33 Xiang F Ye H Chen R Fu Q Li L NN-dimethyl leucines as novel isobaric
tandem mass tags for quantitative proteomics and peptidomics Anal Chem 82 (7) 2817-25
34 Ross P L Huang Y N Marchese J N Williamson B Parker K Hattan S
Khainovski N Pillai S Dey S Daniels S Purkayastha S Juhasz P Martin S Bartlet-
Jones M He F Jacobson A Pappin D J Multiplexed protein quantitation in
Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents Mol Cell Proteomics
2004 3 (12) 1154-69
35 Zybailov B Coleman M K Florens L Washburn M P Correlation of relative
abundance ratios derived from peptide ion chromatograms and spectrum counting for
quantitative proteomic analysis using stable isotope labeling Anal Chem 2005 77 (19) 6218-
24
36 Old W M Meyer-Arendt K Aveline-Wolf L Pierce K G Mendoza A Sevinsky
J R Resing K A Ahn N G Comparison of label-free methods for quantifying human
proteins by shotgun proteomics Mol Cell Proteomics 2005 4 (10) 1487-502
37 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M
P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J
Proteome Res 2006 5 (9) 2339-47
38 Mosley A L Florens L Wen Z Washburn M P A label free quantitative
proteomic analysis of the Saccharomyces cerevisiae nucleus J Proteomics 2009 72 (1) 110-20
39 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome
quantitation how to deal with peptides shared by multiple proteins Anal Chem 82 (6) 2272-81
40 Messing A Head M W Galles K Galbreath E J Goldman J E Brenner M
Fatal encephalopathy with astrocyte inclusions in GFAP transgenic mice Am J Pathol 1998
152 (2) 391-8
41 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein
identification by searching sequence databases using mass spectrometry data Electrophoresis
1999 20 (18) 3551-67
42 Elias J E Gygi S P Target-decoy search strategy for increased confidence in large-
scale protein identifications by mass spectrometry Nat Methods 2007 4 (3) 207-14
43 You J S Gelfanova V Knierman M D Witzmann F A Wang M Hale J E The
impact of blood contamination on the proteome of cerebrospinal fluid Proteomics 2005 5 (1)
290-6
44 Takanashi J Sugita K Tanabe Y Niimi H Adolescent case of Alexander disease
MR imaging and MR spectroscopy Pediatr Neurol 1998 18 (1) 67-70
45 Hemmer W Wallimann T Functional Aspects of Creatine Kinase in Brain
Developmental Neuroscience 1993 15 (3-5) 249-260
46 Hemmer W Zanolla E Furter-Graves E M Eppenberger H M Wallimann T
Creatine kinase isoenzymes in chicken cerebellum specific localization of brain-type creatine
96
kinase in Bergmann glial cells and muscle-type creatine kinase in Purkinje neurons Eur J
Neurosci 1994 6 (4) 538-49
47 Nordby H K Tveit B Ruud I Creatine kinase and lactate dehydrogenase in the
cerebrospinal fluid in patients with head injuries Acta Neurochirurgica 1975 32 (3) 209-217
48 Bell R D Alexander G M Nguyen T Albin M S Quantification of cerebral
infarct size by creatine kinase BB isoenzyme Stroke 1986 17 (2) 254-60
49 Steen C Wilczak N Hoogduin J M Koch M De Keyser J Reduced Creatine
Kinase B Activity in Multiple Sclerosis Normal Appearing White Matter PLoS ONE 5 (5)
e10811
50 Chwieralski C Welte T Buumlhling F Cathepsin-regulated apoptosis Apoptosis 2006
11 (2) 143-149
51 Droga-Mazovec G Bojic L Petelin A Ivanova S Romih R Repnik U Salvesen
G S Stoka V Turk V Turk B Cysteine cathepsins trigger caspase-dependent cell death
through cleavage of bid and antiapoptotic Bcl-2 homologues J Biol Chem 2008 283 (27)
19140-50
52 Duffy M J Proteases as prognostic markers in cancer Clin Cancer Res 1996 2 (4)
613-8
53 Guicciardi M E Deussing J Miyoshi H Bronk S F Svingen P A Peters C
Kaufmann S H Gores G J Cathepsin B contributes to TNF-alpha-mediated hepatocyte
apoptosis by promoting mitochondrial release of cytochrome c J Clin Invest 2000 106 (9)
1127-37
54 Butt A M Jenkins H G Morphological changes in oligodendrocytes in the intact
mouse optic nerve following intravitreal injection of tumour necrosis factor J Neuroimmunol
1994 51 (1) 27-33
55 Hagemann T L Gaeta S A Smith M A Johnson D A Johnson J A Messing
A Gene expression analysis in mice with elevated glial fibrillary acidic protein and Rosenthal
fibers reveals a stress response followed by glial activation and neuronal dysfunction Hum Mol
Genet 2005 14 (16) 2443-58
56 Falk J Bonnon C Girault J A Faivre-Sarrailh C F3contactin a neuronal cell
adhesion molecule implicated in axogenesis and myelination Biol Cell 2002 94 (6) 327-34
57 Eckerich C Zapf S Ulbricht U Muumlller S Fillbrandt R Westphal M Lamszus
K Contactin is expressed in human astrocytic gliomas and mediates repulsive effects Glia
2006 53 (1) 1-12
97
Table 1 Statistically changed proteins between transgenic and control mouse CSF using
dNSAF analysis
Accession Protein Pa SC
b Fold
Changec
Control
dSpCd
Transgenic
dSpCd
KCRM_MOUSE Creatine kinase M-type 0034 425 30 182 541
HEXB_MOUSE Βeta-hexosaminidase subunit β 0012 183 uarr 0 59
CMGA_MOUSE Chromogranin-A 000078 153 darr 44 0
ANT3_MOUSE Antithrombin-III 0017 176 -14 67 47
SAP3_MOUSE Ganglioside GM2 activator 0045 415 darr 28 0
SPA3N_MOUSE Serine protease inhibitor A3N 0047 170 42 10 42
CO1A2_MOUSE Collagen alpha-2(I) chain 0043 56 darr 19 0
BLVRB_MOUSE Flavin reductase 00054 204 uarr 0 12
CATS_MOUSE Cathepsin S 00032 232 uarr 0 73
GFAP_MOUSE Glial fibrillary acidic protein 0021 107 uarr 0 21
RET4_MOUSE Retinol-binding protein 4 0040 189 darr 13 0
CBPE_MOUSE Carboxypeptidase E 0043 139 darr 11 0
CATL1_MOUSE Cathepsin L1 0015 87 94 02 19
The statistics are performed using the t-test from the ln(dNSAF) Gaussian data
a P p-value of the t-test where the null hypothesis states that there was no change in expression between
control and transgenic GFAP overexpressor b SC percentage of sequence coverage of the listed protein from
sequenced tryptic peptides c Fold Change positive values are indicative of a fold change for transgenic CSF
negative values are fold changes for control CSF (ie down-regulated in transgenic CSF) uarr indicates the protein
was only detected in transgenic CSF and darr indicates the protein was only detected in control CSF d dSpC
distributive spectral counts which represent the average spectral counts observed per run analysis on the mass
spectrometer and corrected using distributive analysis for peptides shared by more than one protein
98
Table 2 Proteins showing greater than three-fold changes with at least two unique
peptides identified for each protein
Accession Protein SC ()a Fold
Change b
Control
dSpC c
Transgenic
dSpC c
MUG1_MOUSE Murinoglobulin-1 precursor 147 42 155 37
CO4B_MOUSE Complement C4-B 113 54 22 118
PRDX6_MOUSE Peroxiredoxin-6 576 46 14 64
CNTN1_MOUSE Contactin-1 65 darr 41 0
CATB_MOUSE Cathepsin B 263 42 23 97
CAH3_MOUSE Carbonic anhydrase 3 396 76 11 84
APOH_MOUSE Beta-2-glycoprotein 1 128 44 44 1
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
PGK1_MOUSE Phosphoglycerate kinase 1 355 36 17 61
NDKB_MOUSE Nucleoside diphosphate kinase B 408 34 13 44
FHL1_MOUSE
Four and a half LIM domains
protein 1 243 39 13 51
NELL2_MOUSE
Protein kinase C-binding protein
NELL2 45 -43 13 03
MDHM_MOUSE
Malate dehydrogenase
mitochondrial 385 41 12 49
CSF1R_MOUSE Macrophage colony-stimulating
factor 1 receptor
80 44 14 62
a SC percentage of sequence coverage of the listed protein from sequenced tryptic peptides b Fold
Change positive values are indicative of a fold change for transgenic CSF negative values are fold changes for
control CSF and darr indicates the protein was only detected in control CSF c dSpC distributive spectral counts
which represent the average spectral counts observed per run analysis on the mass spectrometer and corrected using
distributive analysis for peptides shared by more than one protein
99
Table 3 Validation of changes in proteins revealed by MS-based spectral counting
consistent with previously published microarray data
Consistent changes in RNA and proteomic data
uarr regulated in transgenic darr regulated in transgenic
Cathepsin S Contactin-1
Cathepsin B Carboxypeptidase E
Cathepsin L1
Peroxiredoxin-6
Complement C4-B
Glial fibrillary acidic protein
Serine protease inhibitor A3N
Note Validation of putative biomarkers from the current proteomics dataset by previously
published RNA microarray data55
Both up and down regulated proteins were consistent with the
RNA microarray data
_
100
___________________________________________
SUPPLEMENTAL INFORMATION (Available upon request)
Table S1 Compilation list of proteins identified from all the control and transgenic biological
replicates
Table S2 Distributive spectral counting calculations performed for proteins observed to share
identified peptides
Table S3 Proteins with dNSAF spectral counting performed including t-test analysis for a
comparison between transgenic and control CSF
101
FIGURE LEGENDS
Figure 1 The general workflow indicating the major steps involved in sample collection sample
processing mass spectrometric data acquisition and analysis of mouse CSF samples
Figure 2 Assessment of run to run variability of the base peak chromatograms within and
between two biological and technical replicates The peak profile and intensity scale is
consistent between the four chromatograms The four panels show two biological replicates (Tg
4 and Tg5) with two technical replicates for each biological sample
Figure 3 A Venn-diagram of the biological triplicates (1 2 and 3) of transgenic mouse
CSF showing 78 proteins identified in all three experiments B Venn-diagram of the biological
triplicates (1 2 and 3) of control mouse CSF having 61 proteins identified by all three
replicates C The overlap between control and transgenic CSF proteomic analysis showing 139
proteins identified by both groups and 73 and 54 uniquely identified by respective groups
Figure 4 Assessment of technical replicate variability between biological replicates The error
bars in both A and B are the standard deviation derived from the technical triplicates for each
biological replicate Panel A shows creatine kinase M having more or equal variability in the
biological triplicates than each technical triplicate The means of the biological triplicates are
illustrated by the dashed line Panel B represents kininogen-1 which is unchanged between
control (Ctl) and transgenic (Tg) samples The biological variability coupled with the technical
replicates provides a barely noticeable difference in the pooled mean between control and
102
transgenic spectral counts The difference in means is contrasted with the three fold change
observed from creatine kinase M (A)
Figure 5 MSMS profile of the tryptic peptide LSVEALNSLTGEFK from creatine kinase M
(A) The top MSMS is from a control sample with an elution at 463 minutes (B) The bottom
MSMS is from the GFAP transgenic sample with an elution at 46 minutes These tandem MS
spectra show instrument reliability and consistent fragmentation patterns which are necessary for
spectral counting analysis
Figure 6 Validation of MS spectral counts using a GFAP ELISA GFAP content (ngL)
measured within mouse CSF from both transgenic and control animals The data represents the
average with standard deviation (n=5 each sample represents pooling of 1 to 8 animals) The
statistics are performed using a student t-test plt00001
103
Figure 1
104
Figure 2
105
Figure3
106
Figure 4
107
Figure 5
108
Figure 6
Ctl Tg
100
1000
10000
100000
Mouse CSF Sample
GF
AP
(n
gL
)
109
Table of Contents Summary
Cerebrospinal fluid (CSF) was obtained from transgenic mouse models of Alexander disease as
well as healthy controls We immunodepleted pooled CSF from mice by a modified IgY-14
protocol Shotgun proteomics utilizing trypsin digestion 1-D nanoRP separation CID tandem
mass spectrometry analysis Mascot database searching and relative quantitation via distributive
normalized spectral abundance factor resulted in the identification of 266 proteins and 27
putative biomarkers
110
Chapter 4
Genomic and proteomic profiling of rat adapted scrapie
Adapted from ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A
Cunningham R Wang J Wellner D Ma D Li L Aiken J In preparation
111
Abstract
A novel model of prion disease using rats called Rat Adapted Scrapie (RAS) was
developed with the genomics from brain and proteomics from cerebrospinal fluid (CSF) profiled
The CSF proteome from control and RAS (biological N=5 technical replicates N=3) were
digested and separated using one dimensional reversed-phase nanoLC coupled to data-
dependent tandem MS acquisition on an ion trap mass spectrometer In total 512 proteins 167
non-redundant protein groups and 1032 unique peptides were identified with a 1 false
discovery rate (FDR) Of the proteins identified 21 were statistically up-regulated (plt005) and
7 statistically down-regulated in the RAS CSF We also identified 1048 genes which were
differentially regulated in rat prion disease and upon mapping these changes to mouse gene
expression however only 22 of these genes were in common with mRNAs responding to
prion infection in mice suggesting that the molecular pathology observed in mice may not be
applicable to other species The proteins are compared to the differentially regulated genes as
well as to previously published proteins showing changes consistent with other prion animal
models
112
Introduction
Prion diseases are an unusual class of fatal transmissible neurodegenerative disorders
that affect the mammalian central nervous system They are caused by the accumulation of an
abnormal conformation of the normal host encoded cellular prion protein PrPC This
conformational rearrangement of PrPC is brought about by template directed misfolding wherein
seed molecules of the abnormal isoform PrPScrapie
PrPSc
convert PrPC into new PrP
Sc molecules
Scrapie in sheep and goats is the prototypic prion disease and is so named because clinically
affected animals scrape their wool off on pen and stall walls Laboratory investigation of prion
diseases typically relies upon rodents which can be infected with natural isolates of scrapie1
albeit with some difficulty as ovid scrapie isolates need time to adapt to rodents This adaptation
is characteristic of prion disease interspecies transmissions and properly reflects the molecular
adaptation that must occur to allow interaction between exogenous foreign PrPSc
and host PrPC
molecules selecting for conformers which exhibit template directed misfolding In some cases
no conformational solution is found reflecting a species barrier to disease transmission
In recent years advances in genomics and proteomics technologies have allowed
unprecedented examination of the biomolecules that are altered upon exposure to prion agents
These studies2 3
have relied upon analysis of gene and protein expression changes in response to
prion infection with the aim of trying to identify pathways that might underlie the mechanism of
prion-induced neurotoxicity A second important aim has been to identify signature molecules
that might act as surrogate biomarkers for these diseases as there are significant analytical
challenges associated with sensitively detecting and specifically distinguishing disease-induced
conformational changes (PrPSc
) of the prion protein from normal host conformations (PrPC)
113
Mass spectrometry (MS)-based proteomics has become a promising tool for biomarker
discovery from biological fluids such as CSF blood and urine4-6
Two-dimensional gel
electrophoresis MS (2D-GE MS) and two-dimensional differential gel electrophoresis (2D-DIGE
MS) are the most widely used methods for proteomic profiling of prion infected CSF so far due
to the advantage of ready separation and quantification of proteins in complex biological samples
Studies using 2D-GE MS or 2D-DIGE MS to profile proteins in CSF have led to the
identifications of currently accepted biomarker for sCJD 14-3-3 protein and other potential
biomarkers for prion diseases7-9
However the application of this method in biomarker
discovery is limited by insufficient sensitivity and potential bias against certain classes of
proteins as gel-based separation does not work well for the low abundance proteins very basic
or acidic proteins very small or large proteins and hydrophobic proteins 10 11
In contrast to 2D-
GE or 2D-DIGE shotgun proteomics employs enzymatic digestion of the protein samples
followed by chromatographic separation prior to introduction into a mass spectrometer for
tandem MS analyses Shotgun proteomics has become increasingly popular in proteomic
research because these methods are reproducible highly automated and have a greater
likelihood of detecting low abundance proteins12 13
Due to the sample complexity in CSF and
because albumin comprises over half of the protein content in CSF removal of high-abundance
proteins including albumin is necessary to improve proteomic coverage and identify low-
abundance proteins One method is IgY immunodepletion14 15
which is performed prior to LC-
MSMS and uses species specific antibodies from chicken yolk to remove abundant proteins in
biological samples such as CSF In the present work CSF from control and rat adapted scrapie
animal models (biological N=5 technical N=3 replicates) were analyzed by LC-MSMS and we
114
indentified 512 proteins and 167 non-redundant protein isoforms (referred to as protein groups)
with 21 proteins being statistically up-regulated (p lt 005) and 7 statistically down-regulated
By and large this work has been performed using laboratory mice for the gene
expression studies and human cerebrospinal fluid for proteomics mice do not provide sufficient
volumes of CSF for proteomic studies Both approaches are exceedingly valuable as the mouse
model allows cross-sectional time course experiments to be performed including the important
pre-clinical phase of disease Critically however the relevance and generalizability of mouse
prion responses to other prion diseases especially human disease is unknown Human proteomic
studies on CSF guarantee relevant protein identifications but are limited to the clinical phase of
the disease when apparent markers may reflect gross neurodegeneration covering up subtle but
more specific responses To address these issues we have adapted mouse RML prions into rats
with the aim of expanding the knowledge of prion disease responses addressing the limitations
of mice and opening up the possibility of preclinical proteomic profiling in a laboratory rodent
In the present work CSF samples from control and rat adapted scrapie were analyzed by system
biology approaches Here we show that Rat Adapted Scrapie (RAS) is a powerful tool for an -
omics based approach to decipher the molecular impact of prion disease in vivo with
applicability to the molecular mechanisms of disease and biomarker discovery We identified
1048 genes which were differentially regulated in rat prion disease Astonishingly on the whole
mouse orthologs of these genes did not change in mouse adapted scrapie and vice versa
questioning the universality of previous mouse gene expression profiles These RAS gene
expression changes were identified in the CSF proteome where we detected 512 proteins and 167
protein groups with 21 proteins being statistically up-regulated (plt005) and 7 statistically down-
115
regulated in the CSF of prion diseased rats Many of the proteins detected have previously been
observed in human CSF from CJD patients
Materials and Methods
Ethics Statement
This study was carried out in accordance with the recommendations in the NIH Guide for Care
and Use of Laboratory Animals and the guidelines of the Canadian Council on Animal Care The
protocols used were approved by the Institutional Animal Care and Use Committees at the
University of Wisconsin and University of Alberta
Chemicals
Acetonitrile (HPLC grade) and ammonium bicarbonate (certified) were purchased from
Fisher Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) Optima LCMS grade acetonitrile and water were purchased
from Fisher Scientific (Fair Lawn NJ) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) tris(hydroxymethyl)aminomethane (Tris
ge999 ) ammonium formate (ge99995) glycine (ge985) and IgY-7 antibodies were
purchased from Sigma-Aldrich (Saint Louis MO)
Rat Transmission and Adaptation
Prion agents used were the rocky mountain lab RML strain of mouse adapted scrapie
Stetsonville transmissible mink encephalopathy16
(TME) Hyper (Hy) strain of Hamster TME 17
1st passage Skunk adapted TME prepared as described and C from genetically defined
transmissions18
116
Brains from animals clinically affected with prion disease were aseptically removed and
prepared as 10 wv homogenates in sterile water 50 microL of 10 brain homogenate was
inoculated into weanling wistar rats intracranially After one year of incubation preclinical rats
from RML infections were euthanized by CO2 inhalation and the brain excised homogenized
and re-inoculated into naive animals Subsequent serial passages were from rats clinically
affected with rat adapted scrapie
Brains from rat passages were aseptically removed and bisected sagittally Brain halves
were reserved for immunohistochemistry (IHC) in formalin or frozen for immunoblotting RNA
isolation or subsequent passage For IHC tissues were first fixed in 10 buffered formalin
followed by antigen retrieval at 121 oC 21 bar in 10mM citrate buffer for 2 min After cooling
to room temperature sections were treated with 88 formic acid for 30 min and 4M guanidine
thiocyanate for 2 h Endogenous peroxidases were inactivated by 003 hydrogen peroxide and
tissue was blocked with 1 normal mouse serum Mouse anti-PrP mAB SAF83 (Cayman
Chemical) was biotinylated and applied at a 1250 dilution in blocking buffer overnight at 4 oC
Washes were performed in 10mM PBS with 005 tween-20 Streptavidin-peroxidase
(Invitrogen) was added for Diaminobenzidine (DAB) color reaction Anti-GFAP
immunohistochemistry was performed as above except that formic acid and guanidine treatment
steps were excluded Mouse anti-GFAP mab G-A-5 (Sigma) was used at a 1400 dilution
Brain samples for immunoblot were treated with or without Proteinase K (Roche) at a
ratio of 35 microg PK 100 ug protein at 50 microg mL for 30 minutes at 37 oC Phosphotungstenic acid
enrichments were performed as described14 19
Bis-Tris SDS-PAGE was performed on 12
polyacrylamide gels (Invitrogen) and transferred to PVDF Immunoblotting was performed using
117
mABs SAF83 (Cayman Chemical) 6H4 (Prionics) or 3F10 (a kind gift from Yong-Sun Kim) all
at a 120000 dilution
Gene Expression Profiling
RNA was extracted from frozen brain halves from clinically affected and control animals with
the QIAshredder and RNeasy mini kit (Qiagen Valencia CA) in accordance with the
manufacturerrsquos recommendations Due to the relatively large mass of rat brain an initial
homogenization was performed with a needle and syringe in 5mL of buffer RLT before further
diluting an aliquot in accordance with the manufacturers protocol Total RNA was amplified and
labeled in preparation for chemical fragmentation and hybridization with the MessageAmp
Premier RNA amplification and labeling kit (Life Technologies Grand Island NY) Amplified
and labeled cRNAs were hybridized on Affymetrix (Santa Clara CA) rat genome 230 20 high
density oligonucleotide arrays in accordance with the manufacturers recommendations
Differentially Expressed Genes were identified using Arraystar 50 (DNAStar Madison WI)
Robust multi-array normalization using the quantile approach was used to normalize all
microarray data A moderated T-test with a multiple comparison adjustment20
was used to reduce
the false discovery rate yet preserve a meaningful number of genes for pathway analysis
Pathway analysis was performed using the DAVID Bioinformatics database21
Comparative
analysis of genes induced by prions in mouse22
and rat disease was performed on genes
exhibiting at least a 2-fold change in expression Orthologs of rat and mouse genes were
identified using ENSEMBLE biomart release 6823
CSF Proteomic Profiling
118
CSF was collected from rats anaesthetized with isoflorane via puncture of the cisterna
magna A volume of 100-200 microL was routinely obtained CSF was touch spun for 30s at 2000xg
on a benchtop nano centrifuge to identify any blood contamination by the presence of a red
pellet CSF with blood contamination was not used for proteomic analysis CSF was prepared
for profiling by first depleting abundant proteins with an antibody based immunopartitioning
column IgY-R7 (Sigma-Aldrich Saint Louis MO) The manufacturers recommendations were
followed with a few deviations 40 microg of protein (~100 microL CSF) was bound to 100 microL of IgY
bead slurry in total volume of 600 microL formulated in 150mM ammonium bicarbonate The flow
through was collected and denatured by the addition of 10 microL of 8 M guanidine thiocyanate and
lyophilized in a speed-vac apparatus The sample was then reconstituted in 10 microL of water and 1
microL of 050 M DTT were added and allowed to incubate at 37oC for 30 minutes After incubation
27 microL of 055 M IAA was added for carboxymethylation and the sample was allowed to
incubate in the dark for 15 minutes After that 1 microL of 050 M DTT was added and allowed to
sit at room temperature for 10 minutes To perform trypsin digestion 70 microL of 50 mM
NH4HCO3 was then added along with 025 microg trypsin Digestion was performed overnight at
37oC and quenched by addition of 5 microL of 10 formic acid The tryptic peptides were then
subjected to solid phase extraction using an Agilent OMIX Tip C18 100 microL (Santa Clara CA)
Peptides were eluted with 50 ACN in 01 formic acid concentrated and reconstituted in 30
microL H2O with 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of a nanoAcquity (Milford MA) with a 10 microL injection
loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B consisted of
ACN Injections consisted of 5 microL of prepared CSF tryptic peptides onto a Waters 5 microm
119
Symmetry C18 180 microm x 20 mm trap cartridge (Milford MA) at a flow rate of 75 microLmin for 5
minutes at 95 A 5 B Separation was performed on a Waters 17 microm BEH130 C18 75 microm x
100 mm (Milford MA) with a gradient of 5 to 15 B over 10 minutes followed by 15 to
40 B over 80 minutes at room temperature
Mass spectrometry data acquisitions
An ion trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with a Bruker CaptiveSpray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Waters Acquity console software to perform MS acquisitions for all experiments Smart
parameter setting (SPS) was set to 700 mz compound stability and trap drive level was set at
100 Optimization of the CaptiveSpray source resulted in dry gas temperature of 160oC dry
gas flow of 30 Lmin capillary voltage of -1325 V end plate offset of -500 V MSMS
fragmentation amplitude of 10V and SmartFragmentation set at 30-300
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms one spectral average rolling average 2 acquisition
range of 350-1500 mz and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS
generation the ICC target was set to 300000 maximum accumulation time 5000 ms two
spectral averages acquisition range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
120
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required for spectral counting to prevent
loss of spectra Identification of peptides were performed using Mascot24
(Version 24 Matrix
Science London UK) Database searching was performed against a forward and reversed
concatenated SwissProt Rattus rattus Mascot search parameters were as follows Allowed
missed cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) peptide tolerance plusmn12 Da maximum number of 13
C 1 MSMS
tolerance plusmn05 Da instrument type ESI-Trap Data was then transformed into Dat file formats
and transferred to ProteoIQ (NuSep Bogart GA) False positive analyses were calculated using
ProteoIQ and set at 1
Results
Development of Rat Adapted Scrapie
To develop a rat adapted strain of prion disease we introduced 6 different prion agents RML
TME Hy Skunk Adapted TME and Chronic Wasting Disease (CWD) from wild type 96G and
96S deer16-18
into 6 rats (Fig 1) Of these primary transmissions only RML induced the
accumulation of Proteinase K resistant PrP after one year of incubation as determined by western
blotting on 10 brain homogenates and PrPSc
enriched phoshotungstenic acid precipitated brain
homogenates Second passage of rat adapted RML scrapie resulted in clinically affected rats at
565 days post inoculation Serial passages of TME or CWD agents were not performed Clinical
symptoms consisted of ataxia lethargy wasting kyphosis and myoclonus Prion affected rats
121
also showed low level porphyrin staining around their head Subsequent serial passage decreased
incubation time to 215 days
Proteinase K resistant prion protein was observed from all clinically affected animals both by
immunoblot (Fig 2) and by immunohistochemistry (Fig 3) Di- and mono-glycosylated bands
were the most abundant isoforms of PrPSc
PrPSc
was extensively deposited in the cerebral cortex
hippocampus thalamus inferior colliculus and granular layer of the cerebellum GFAP
expressing activated astrocytes were found throughout the brain particularly in the white matter
of the hippocampus thalamus and cerebellum Spongiform change was a dominant feature of
clinical rat
Gene expression Profiling
In total 1048 genes were differentially regulated within a 95 confidence interval
(Supplementary Table 1) and 305 genes were differentially expressed by greater than 2-fold (Fig
4) The 1048 genes that were statistically significant were used for pathway analysis using
DAVID Pathway analysis suggested that the gene expression profile was consistent with
immune activation and maturation as well as inflammation (Supplementary Table 2) a likely
interpretation given the observable astrocytosis (Fig 3) and gliosis associated with prion disease
Other pathways highlighted by the analysis included increases in transcription of genes involved
in lysosomes and endosomes
To further probe the gene expression data we compared genes which were differentially
expressed in rat adapted scrapie against their orthologs expression in mouse scrapie and vice
versa (Figs 5 and 6) We did not observe a high degree of correlation between expression fold
changes For example GFAP a gene whose up-regulation in prion disease is well known was
122
increased 25 fold in rat adapted scrapie but up-regulated 93 fold in mouse scrapie A
qualitative analysis of expression of orthologs in prion disease suggests that many genes
deregulated at least 2-fold in mouse or rat do not have orthologs that are differentially expressed
For example the gene RNAseT2 was up-regulated greater than threefold in rat adapted scrapie
but was not significantly up-regulated in mouse Similarly three genes important in metals
homeostatsis metallothionein 1a and 2a and ceruloplasmin were up-regulated in rat 46 43 and
3 fold respectively but were not differentially expressed in mouse prion disease
CSF Proteomics
Each immunodepleted biological replicate (N=5 for each control and RAS) had technical
triplicates performed The triplicates were summed together using ProteoIQ Peptide spectral
counts were then summed for each protein and subjected to dNSAF analysis using ProteoIQ
internal algorithms Details for this method can be found elsewhere25 26
but briefly peptide
spectral counts are summed per protein (SpC) based on unique peptides and a weighted
distribution of any shared peptides with homologous proteins T-tests were used to identify
significant changes in protein expression 1032 unique peptides which identify 512 proteins and
167 protein groups were found Of these 512 proteins 437 were identified in both RAS and
control CSF samples (Fig 7) The complete list of all 512 proteins can be viewed in
Supplemental Table 3 Using the ProteoIQ the most likely isoform was selected such as 14-3-3
protein gamma
From Table 1 we observe five proteins that agree with the genomic data for up
regulations in RAS granulin macrophage colony-stimulating factor 1 receptor cathepsin D
complement factor H and ribonuclease T2 In addition serine protease inhibitor A3N was not
123
detected as up regulated in the RAS genomic data but was found to be up-regulated in previous
genomic profiling of the mouse prion model22
One interesting trend from the data in Table 1 is
that the majority of proteins found to be up-regulated in the RAS model were not detected in the
control samples The absence of the detection of those proteins such as ribonuclease T2 in the
control CSF does not necessarily suggest the absence of the protein nonetheless it is below the
detection limits for this current proteomics protocol and instrumentation
Discussion
Mice have been the preferred laboratory rodent for prion diseases research because they
can be inexpensively housed and are amenable to transgenesis which allows for short incubation
periods as well as hypothesis testing upon targeted pathways Pursuant to the determination of
the mouse genome and the development of high density transcriptional arrays for measurements
of gene expression profiling mice have been used extensively to examine the molecular
pathology of prion disease probing the impact of disease and animal strain In order to expand
upon this foundation we adapted mouse prions to infect rats We reasoned that by taking a
comparative approach to the molecular pathology of prion disease inferences could be obtained
into the variability of the molecular response to prion diseases and that understanding this
variability might suggest whether human prion disease responses are more or less similar to
mouse responses A second rationale is the desire to identify surrogate markers of prion disease
While this approach has been taken before using gene expression profiling a more direct
approach has been to use proteomic approaches on cerebral spinal fluid (CSF) identifying
proteins that are increase in abundance with disease A rat prion disease is valuable for this
because the rat proteome is established and rats allow for the collection of relatively large
volumes of CSF (~100 microL per animal) which allows for robust analytical separations enhancing
124
detection of biomarkers Finally rats unlike humans can be used in a time course study of prion
disease This allows for the identification of early transcriptional and proteomic responses to
prion infection responses which are particularly valuable for the identification of surrogate
disease biomarkers
To initiate the development of a rat prion disease we attempted to adapt six different
prion disease agents PrPres
molecules to rat via intracranial inoculation of weanling animals
(Figure 1) Of these six agents only mouse RML prions were able to surmount the species
barrier to prion disease transmission Mouse PrP differs from rat by eight amino acid changes
six in the mature protein and four in the N-terminal octa-peptide repeat region (Supplementary
Fig 1) As rat shares the most prion protein sequence homology with mouse it is perhaps not
surprising that it transmitted whereas the other did not confirming that the primary prion protein
sequence is the most important determinant for interspecies transmission We conclude that there
is a large molecular species barrier preventing conversion of rat PrPc into PrP
res
The transmission of mouse RML into rats was characterized by a shortening of the
incubation period following each passage This is indicative of agent adaption to the new host
and increases in the titer present in end-stage brain Overall our adaptation of mouse prion
disease into rats resulted in a similar agent to that observed by Kimberlin27
The differences in
incubation period at second passage are largely due to our collecting the animals at 365 days post
inoculation upon first passage whereas Kimberlin and Walker allowed the first passage animals
to reach end-stage clinical rats
Rat adapted scrapie is in many ways similar to other prion diseases The clinical period of
disease is marked by a progressive neurological dysfunction with prominent ataxia kyphosis and
125
wasting The protease resistant PrP observed is typical as is the widespread deposition of PrPSc
in
the brain Spongiosis and reactive astrogliosis are as expected of a prion disease
Gene expression profiles from rats clinically affected with prion disease revealed a strong
neuronal inflammation associated with proliferated and activated astrocytes This is perhaps best
observed through the up-regulation of GFAP GFAP positive astrocytes were omnipresent
throughout the brain and GFAP mRNA was up-regulated 25 fold The up-regulation of GFAP is
a hallmark of the molecular response to prion infection and has been routinely observed Our
comparative analysis of gene expression changes in mouse RML versus rat adapted scrapie
suggest substantial differences in gene expression in response to prion disease despite the fact
that the overall response is neuro-inflammatory This suggests that the potential overlap between
mouse expression profiles and a putative human CJD expression profile could be quite different
at the level of individual transcripts that might be expected to be changed
CSF Proteomics
CSF proteomics can be exceedingly challenging due to the small sample available large
dynamic range in proteins and abundant proteins limiting sample loading onto nanoscale
columns Dynamic range reduction in the CSF sample was achieved using a custom amount of
IgY-7 antibodies to abundant proteins such as albumin After immunodepletion the total
protein content was reduced by ~90 limiting the proteomics analysis to one dimensional
separation Label free quantitation spectral counting was performed because it requires less
protein and does not increase sample complexity The proteins identified from the affected and
control N=5 are shown in Supplemental Table 2 Consistent detection of proteins in CSF from
both control and infected rats was observed (Fig 7C) Only two proteins were identified in
126
controls that were not observed in RAS and only 10 proteins were only observed in RAS Some
of these proteins that were only identified in RAS are significantly changed (Supplemental Table
3) One concern in proteomics data is the variability from run to run and the possibility that
certain proteins are identified from different unique peptides Figure 7A shows that the vast
majority of the unique peptides detected (783) were identified with a 1 FDR in both RAS and
control CSF samples highlighting the analytical reproducibility of our methodology
Proteomic analysis of the infected rat CSF provides a reasonable approach to cross
validate biomarkers detected by gene expression profiling For example RNAseT2 a secreted
ribonuclease was up-regulated 3-fold at the mRNA level and was also enriched in CSF from
infected rats (Table 1) Similarly coordinated increases in detection of colony stimulating factor
1 receptor complement factor H granulin and cathepsin D were also observed Conversely
proteomic analysis of CSF also allows for the observation of post-transcriptional responses to
prion disease that might be absent in gene expression profiling The 14-3-3 proteins and neuron
specific enolase both known markers for CJD are only detected by proteomic analysis Thus
gene expression profiling and proteomic detection serve to increase confidence in the
observation of up-regulation enhancing the likelihood that proteins detected by both
methodologies are specific and perhaps may be more sensitive at earlier time points
Comparison to human CSF prion disease proteome
In our comparative analysis 21 proteins were found to be up-regulated while 7 proteins
down-regulated in prion infected rats Included in the up-regulated proteins were the 14-3-3
proteins epsilon and gamma 14-3-3 zetadelta was also up-regulated but not statistically
significant Enolase 2 or neuron specific enolase (NSE) was also up-regulated in CSF of infected
127
rats These proteins are all in agreement with results from previous proteomic profiling of human
CSF from patients with CJD8 9
The detection of 14-3-3 protein is included in the diagnostic
criteria approved by World Health Organization for the pre-mortem diagnosis of clinically
suspected cases of sCJD28
although its application in large-scale screening of CJD is still
debated due to high false positive rate where elevated 14-3-3 protein levels were also reported in
other conditions associated with acute neuronal damage29 30
It was suggested that other brain-
derived proteins that are also specific to CJD can be used in conjunction with 14-3-3 protein to
increase diagnosis accuracy and specificity31
NSE is present in high concentration in neurons
and in central and peripheral neuroendocrine cells NSE serves as another valuable biomarker in
diagnosis of CJD as the elevated level of NSE in CSF was found in early and middle stages of
CJD 32
Other proteins detected in CSF included cystatin C and serpina3N although both of
these were not statistically changed These proteins were both previously identified as being
putative biomarkers for CJD33 34
Utility for biomarker discovery with emphasis on onset of biomarker appearance in CSF
The investigation of the protein changes in CSF from RAS compared to control rats
provides a solid foundation when investigating potential biomarkers with prion disease onset
The cross-validation of the genomic and proteomics data further emphasizes the targets for
consideration during disease onset Biomarker discovery provides the potential to determine if
animals such as cows in bovine spongiform encephalopathy (BSE) are affected instead of
having to be euthanized when BSE is expected In addition CSF collection in mice or hamsters
Prion models is extremely difficult and limited alternatively with the advent of the RAS model
CSF can be collected in sufficient amounts for CSF proteomics CSF collection for mice or
hamsters can be ~10 microL and could be contaminated with blood further complicating proteomic
128
analysis unlike rats which over 10 times more CSF can be collected per animal35
Due to the
amount of CSF available time course studies analyzing CSF proteomics is possible in RAS due
to animal numbers that are manageable and reasonable The RAS model further allows
investigators to bypass working with highly infections CJD CSF samples to investigate the CSF
proteome changes
Conclusion
In this study we have described the gene and protein expression changes in brain and
spinal fluid from a transmission of mouse prions into rats We find that while the overall gene
expression profile in rats is similar to that in mice the specific genes that make up that profile
are different suggesting that genes that change in response to prion disease in different species
may not be as conserved Analysis of CSF from rat adapted scrapie identified similar protein
changes as known in human CJD The rat will be a useful model to identify surrogate markers
that appear prior to the onset of clinical disease and thus may be of higher specificity and
sensitivity
Supplemental Information Available Upon Request
1 Chandler R L Encephalopathy in mice produced by inoculation with scrapie brain material Lancet 1961 1 (7191) 1378-9 2 Silva C J Onisko B C Dynin I Erickson M L Requena J R Carter J M Utility of Mass Spectrometry in the Diagnosis of Prion Diseases Anal Chem 2011 3 Wei X Herbst A Ma D Aiken J Li L A quantitative proteomic approach to prion disease biomarker research delving into the glycoproteome J Proteome Res 2011 10 (6) 2687-702 4 Zougman A Pilch B Podtelejnikov A Kiehntopf M Schnabel C Kumar C Mann M Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7 (1) 386-99 5 Sjodin M O Bergquist J Wetterhall M Mining ventricular cerebrospinal fluid from patients with traumatic brain injury using hexapeptide ligand libraries to search for trauma biomarkers J Chromatogr B Analyt Technol Biomed Life Sci 2003 878 (22) 2003-12 6 Cunningham R Ma D Li L Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery Frontiers in Biology 2012 7 (4) 313-335
129
7 Brechlin P Jahn O Steinacker P Cepek L Kratzin H Lehnert S Jesse S Mollenhauer B Kretzschmar H A Wiltfang J Otto M Cerebrospinal fluid-optimized two-dimensional difference gel electrophoresis (2-D DIGE) facilitates the differential diagnosis of Creutzfeldt-Jakob disease Proteomics 2008 8 (20) 4357-66 8 Harrington M G Merril C R Asher D M Gajdusek D C Abnormal proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease N Engl J Med 1986 315 (5) 279-83 9 Piubelli C Fiorini M Zanusso G Milli A Fasoli E Monaco S Righetti P G Searching for markers of Creutzfeldt-Jakob disease in cerebrospinal fluid by two-dimensional mapping Proteomics 2006 6 Suppl 1 S256-61 10 Lilley K S Razzaq A Dupree P Two-dimensional gel electrophoresis recent advances in sample preparation detection and quantitation Curr Opin Chem Biol 2002 6 (1) 46-50 11 Oh-Ishi M Maeda T Separation techniques for high-molecular-mass proteins J Chromatogr B Analyt Technol Biomed Life Sci 2002 771 (1-2) 49-66 12 Metz T O Qian W J Jacobs J M Gritsenko M A Moore R J Polpitiya A D Monroe M E Camp D G 2nd Mueller P W Smith R D Application of proteomics in the discovery of candidate protein biomarkers in a diabetes autoantibody standardization program sample subset J Proteome Res 2008 7 (2) 698-707 13 Aebersold R Mann M Mass spectrometry-based proteomics Nature 2003 422 (6928) 198-207 14 Huang L Harvie G Feitelson J S Gramatikoff K Herold D A Allen D L Amunngama R Hagler R A Pisano M R Zhang W W Fang X Immunoaffinity separation of plasma proteins by IgY microbeads meeting the needs of proteomic sample preparation and analysis Proteomics 2005 5 (13) 3314-28 15 Rajic A Stehmann C Autelitano D J Vrkic A K Hosking C G Rice G E Ilag L L Protein depletion using IgY from chickens immunised with human protein cocktails Prep Biochem Biotechnol 2009 39 (3) 221-47 16 Marsh R F Bessen R A Lehmann S Hartsough G R Epidemiological and experimental studies on a new incident of transmissible mink encephalopathy J Gen Virol 1991 72 ( Pt 3) 589-94 17 Bessen R A Marsh R F Biochemical and physical properties of the prion protein from two strains of the transmissible mink encephalopathy agent J Virol 1992 66 (4) 2096-101 18 Johnson C J Herbst A Duque-Velasquez C Vanderloo J P Bochsler P Chappell R McKenzie D Prion protein polymorphisms affect chronic wasting disease progression PLoS One 2011 6 (3) e17450 19 Safar J Wille H Itri V Groth D Serban H Torchia M Cohen F E Prusiner S B Eight prion strains have PrP(Sc) molecules with different conformations Nat Med 1998 4 (10) 1157-65 20 Benjamini Y Hochberg Y Controlling the False Discovery Rate A Practical and Powerful Approach to Multiple Testing Journal of the Royal Statistical Society Series B (Methodological) 1995 57 (1) 289-300 21 Huang da W Sherman B T Lempicki R A Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources Nat Protoc 2009 4 (1) 44-57 22 Moody L R Herbst A J Aiken J M Upregulation of interferon-gamma-induced genes during prion infection J Toxicol Environ Health A 2009 74 (2-4) 146-53 23 Flicek P Amode M R Barrell D Beal K Brent S Carvalho-Silva D Clapham P Coates G Fairley S Fitzgerald S Gil L Gordon L Hendrix M Hourlier T Johnson N Kahari A K Keefe D Keenan S Kinsella R Komorowska M Koscielny G Kulesha E Larsson P Longden I McLaren W Muffato M Overduin B Pignatelli M Pritchard B Riat H S Ritchie G R Ruffier M Schuster M Sobral D Tang Y A Taylor K Trevanion S Vandrovcova J White S Wilson M Wilder S P Aken B L Birney E Cunningham F Dunham I Durbin R Fernandez-Suarez X M Harrow J Herrero J
130
Hubbard T J Parker A Proctor G Spudich G Vogel J Yates A Zadissa A Searle S M Ensembl 2012 Nucleic Acids Res 2012 40 (Database issue) D84-90 24 Perkins D N Pappin D J Creasy D M Cottrell J S Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 1999 20 (18) 3551-67 25 Zybailov B Mosley A L Sardiu M E Coleman M K Florens L Washburn M P Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae J Proteome Res 2006 5 (9) 2339-47 26 Zhang Y Wen Z Washburn M P Florens L Refinements to label free proteome quantitation how to deal with peptides shared by multiple proteins Anal Chem 2010 82 (6) 2272-81 27 Kimberlin R H Cole S Walker C A Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters J Gen Virol 1987 68 ( Pt 7) 1875-81 28 World Health Organization Global surveillance diagnosis and therapy of human transmissible spongiform encephalopathies report from a WHO consultation 1998 World Health Organization Geneva Switzerland 9-11 February 1998 1-26 29 Bartosik-Psujek H Archelos J J Tau protein and 14-3-3 are elevated in the cerebrospinal fluid of patients with multiple sclerosis and correlate with intrathecal synthesis of IgG J Neurol 2004 251 (4) 414-20 30 Saiz A Graus F Dalmau J Pifarre A Marin C Tolosa E Detection of 14-3-3 brain protein in the cerebrospinal fluid of patients with paraneoplastic neurological disorders Ann Neurol 1999 46 (5) 774-7 31 Sanchez-Juan P Green A Ladogana A Cuadrado-Corrales N Saanchez-Valle R Mitrovaa E Stoeck K Sklaviadis T Kulczycki J Hess K Bodemer M Slivarichova D Saiz A Calero M Ingrosso L Knight R Janssens A C van Duijn C M Zerr I CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease Neurology 2006 67 (4) 637-43 32 Zerr I Bodemer M Racker S Grosche S Poser S Kretzschmar H A Weber T Cerebrospinal fluid concentration of neuron-specific enolase in diagnosis of Creutzfeldt-Jakob disease Lancet 1995 345 (8965) 1609-10 33 Sanchez J C Guillaume E Lescuyer P Allard L Carrette O Scherl A Burgess J Corthals G L Burkhard P R Hochstrasser D F Cystatin C as a potential cerebrospinal fluid marker for the diagnosis of Creutzfeldt-Jakob disease Proteomics 2004 4 (8) 2229-33 34 Miele G Seeger H Marino D Eberhard R Heikenwalder M Stoeck K Basagni M Knight R Green A Chianini F Wuthrich R P Hock C Zerr I Aguzzi A Urinary alpha1-antichymotrypsin a biomarker of prion infection PLoS One 2008 3 (12) e3870 35 DeMattos R B Bales K R Parsadanian M ODell M A Foss E M Paul S M Holtzman D M Plaque-associated disruption of CSF and plasma amyloid-beta (Abeta) equilibrium in a mouse model of Alzheimers disease J Neurochem 2002 81 (2) 229-36
131
Figure 1 Interspecies transmission of prion disease to rats End-stage clinical brain homogenates
were used to passage prion disease After one year of incubation animals were euthanized to
determine the extent of PrPres
accumulation Protease resistance PrP was only observed in those
animals infected with RML scrapie prions This material was serially passaged for two more
incubations before becoming rat-adapted as indicated by the shortening of the incubation period
132
Table 1 A list of selected proteins and mRNAs up-regulated in RAS CSF and brain tissue If
the protein is detected in the RAS CSF but not the control CSF the CSF expression is reported
with a infin If there is no change or data on certain genes related to an up regulated protein nd is
noted The mouse genomic data presented here was previously published22
Gene Protein Symbol Accession CSF
Expression
Rat
GEX
Mouse
GEX
14-3-3 protein zetadelta Ywhaz NP_037143 infin nd nd
14-3-3 protein epsilon Ywhae NP_113791 infin nd nd
14-3-3 protein gamma Ywhag NP_062249 infin nd nd
serine protease inhibitor A3N Serpin A3N NP_113719 54 nd 975
enolase 2 (gamma neuronal) Eno2 NP_647541 infin nd nd
granulin GRN NP_058809 62 364 184
macrophage colony-stimulating
factor 1 receptor
Csf1r NP_001025072 infin 293 205
cathepsin D CTSD NP_599161 infin 255 299
complement factor H Cfh NP_569093 376 234 nd
ribonuclease T2 RNAset2 NP_001099680 infin 302 nd
133
Figure 2 Accumulation of PrPSc
in rat adapted scrapie First second and third passage brain
homogenates were assayed for the presence of protease resistant PrP by immunoblot PrPSc
was
observed following phosphotungstenic acid enrichment at first passage By clinical disease in 2nd
and 3rd
passage rats PrPSc
had substantially accumulated
134
Figure 3 Histological appearance of rat adapted scrapie in the hippocampus at clinical disease
Infected animals showed intense immuno-staining for deposits of PrPSc
and GFAP expressing
astrocytes Spongiform change is an abundant feature in rat adapted scrapie
135
Figure 4 Scatter plot of gene expression profiling of rat adapted scrapie The expression of
individual genes from uninfected and infected animals were plotted to display up and down
regulation The dashed green line is no change Solid green lines are 2-fold changes in gene
expression
136
Figure 5 Quantitative relationship between orthologous pairs of two-fold up-regulated genes in
mouse and rat scrapie Genes up-regulated more than two fold were mapped to their orthologs
and the fold change was plotted Expression is log2 transformed
137
Figure 6 Comparison of genes upregulated in rat and mouse prion diseases Genes up-regulated
two fold in rodent scrapie were identified and the expression of their orthologs was determined
138
Figure 7 Venn diagram comparison of the CSF proteomics data between rat adapted scrapie
(RAS) model and control rats (A) Unique peptides from tryptic peptides produced for the
proteins identified (B) The total proteins identified including all isoforms within the protein
groups (C) The protein groups comparing only the top protein hit of the protein isoforms
showing very consistent protein identifications between RAS and control
139
Chapter 5
Investigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiae
Adapted from ldquoInvestigation of the differences in the phosphoproteome between starved vs
glucose fed Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M
Heideman W Li L In preparation
140
Abstract
This work explores comparative proteomics between starved and glucose fed
Saccharomyces cerevisiae (bakers yeast) Yeast depends on nutrients such as glucose to
survive and need to cycle between states of growth and quiescence Kinases such as protein
kinase A (PKA) and target of rapamycin kinase (TOR) are involved in the glucose response
Thus performing a large scale phosphoproteomic MS study can be highly beneficial to map the
signaling networks and cascades involved in glucose-dependent stimulated yeast growth Yeast
cell extract was digested and phosphopeptides were enriched by immobilized metal affinity
chromatography (IMAC) followed by two dimensional high pH-low pH reversed phase (RP)-RP
separation The low pH separation was infused directly into an ion trap mass spectrometer with
neutral loss electron-transfer dissociation (ETD)-triggered fragmentation to improve
phosphopeptide identifications In total 477 unique phosphopeptides are identified with 06
false discovery rate (FDR) and 669 unique phosphopeptides identified with 54 FDR This
study includes comparative phosphoproteins for one specific motif of PKA xxxKRKRxSxxxx
which is presented and differences between starved vs glucose fed are highlighted Phosphosite
validation is performed using a localization algorithm Ascore to provide more confident and
site-specific characterization of phosphopeptides
141
Introduction
Microorganisms such as Saccharomyces cerevisiae (yeast) cycle between growth when
nutrients are available and quiescence when nutrients are scarce When glucose is limited yeast
go into growth arrest state but when glucose is added growth quickly resumes Kinases such as
protein kinase A (PKA) and target of rapamycin (TOR) regulate growth in response to nutrient
conditions and have been well studied through transcriptional control1-4
Yeast execute large
transcriptome alterations in response to changing environmental growth conditions5 6
Gene
regulation by glucose introduction in yeast has been studied including genes used for growth on
alternative carbon sources and activation of genes coding for glucose transport and protein
synthesis7-10
Response to nutrients for survival is not limited to yeast biology and indeed all
living creatures both prokaryotes and multicellular eukaryotes like mammals require nutrient
responsiveness and coordinating cellular functions to survive
With regulation of certain genes well studied by glucose introduction the mechanism and
global protein modulation caused by glucose introduction remain unknown6 Large-scale
phosphorylation analysis has been previously performed in yeast using mass spectrometry11-14
Mapping the phosphoproteome in yeast transitioning from quiescence to growth conditions to
better understand the roles of phosphorylation in orchestrating growth is needed The
phosphorylation state during growth or starvation could be used to engineer cells to drive ectopic
activity (or inhibition) to promote growth and ethanol production on non-native sugars like
xylose
It has been reported that the phosphorylation state can be affected by the introduction of
glucose to carbon-starved yeast15
and phosphorylation plays a significant role in the cell cycle
and signal transduction16
Specifically O-Phosphorylation can function as a molecular switch by
142
changing the structure of a protein via alteration of the chemical nature of an amino acid for
serine threonine or tyrosine It has been reported that up to 30 of the proteome can undergo
phophorylation17
Mass spectrometry has evolved as a powerful tool to accomplish phosphosite
mapping using shotgun proteomics With available technology tens of thousands of
phosphorylation sites can be mapped to amino acids on thousands of proteins using shotgun
proteomics18-20
Mass spectrometry can offer sensitive automated non-targeted global analysis of
phosphorylation events in proteomic samples but in any large scale phosphoproteomic
investigation enrichment of phosphoproteinspeptides is required First phosphorylation is
usually a sub-stoichiometric process where only a percentage of all protein copies are
phosphorylated21
Various enrichment methods have been used for phosphopeptide enrichment
including metal oxide affinity chromatography (MOAC)22
such as TiO223
immobilized metal
affinity chromatography (IMAC)12 24 25
electrostatic repulsion-hydrophilic interaction
chromatography (ERLIC)26
and immunoaffinity of tyrosine phosphorylation27 28
After
enrichment MS analyses of phosphorylated peptides are still challenging due to ion suppression
from non-phosphorylated peptides
Even after phosphopeptide enrichment further sample preparation is needed for large
scale proteomic experiments Additional fractionation can increase protein coverage of a
sample by over ten fold such as MudPIT29
(multidimensional protein identification technology)
In online MudPIT a sample is injected onto a strong cation exchange (SCX) column coupled to
a RP column Successive salt bumps followed by low pH gradients provide the separation of
peptides in two dimensions Since the phosphate groups on phosphopeptides have a low pKa
value due to being more acidic then their unmodified counterparts they tend to elute earlier and
143
disproportionally in ion exchange chromatography like SCX Alternatively reversed-phase
reversed-phase (RP-RP) separation has been proposed as an alternative to MudPIT for offline
two dimensional (2D) separation30
One of the caveats of 2D separation is the potential for
wasted mass spectrometry time from early and late fractions having very few peptides present
all while having too much sample for middle fractions One simple method to reduce these
ldquowasted fractionsrdquo is to combine early or late fractions with the middle fractions to reduce MS
runs with little peptide content to analyze thus shortening the overall analysis time31
In addition the labile phosphorylation group has a large propensity to undergo cleavage
during collision induced dissociation (CID) producing a neutral loss The neutral loss can
produce insufficient backbone fragment ions for MSMS identification32
A solution to neutral
loss fragmentation in phosphopeptides is MS3 using CID to produce improved backbone
fragmentation13 14 33
An alternative fragmentation method to CID for fast sampling ion traps is
electron transfer dissociation (ETD)34-36
ETD produces a more uniform back-bone cleavage
where the cation peptide receives an electron from a low affinity radical anion37
The transferred
electron induces a dissociation at the N-Cα bond to produce c- and zbull-type product ions while
retaining the labile post-translational modifications (PTM) such as phosphorylation bound to the
product ions38
The Bruker amaZon ETD ion trap mass spectrometer has the potential to trigger
ETD fragmentation after a labile PTM produces a neutral loss from CID fragmentation This
method is termed neutral loss-triggered ETD fragmentation and provides a complementary
fragmentation pathway to labile poor fragmenting phosphorylated peptides
In this work we provide a qualitative comparative list of yeast phosphopeptides observed
in glucose fed vs glucose starved conditions
144
Experimental
EXPERIMENTAL DETAILS
Chemicals
Acetonitrile (HPLC grade) ammonium hydroxide (ACS plus certified) urea (gt99)
sodium chloride (997) ammonium bicarbonate (certified) Optima LCMS grade acetonitrile
Optima LCMS grade water and EDTA disodium salt (99) were purchased from Fisher
Scientific (Fair Lawn NJ) Deionized water (182 MΩcm) was prepared with a Milli-Q
Millipore system (Billerica MA) DL-Dithiothreitol (DTT) and sequencing grade modified
trypsin were purchased from Promega (Madison WI) Formic acid (ge98) was obtained from
Fluka (Buchs Switzerland) Iodoacetamide (IAA) ammonium formate (ge99995) Trizma
hydrochloride (Tris HCl ge990) Triton X-100 (laboratory grade) and iron (III) chloride
hexahydrate (97) were purchased from Sigma-Aldrich (Saint Louis MO) Sodium dodecyl
sulfate (SDS) (998) was purchased from US Biological (Marblehead MA) Nickel
nitrilotriacetic acid (Ni-NTA) magnetic agarose beads were purchased from Qiagen (Valencia
CA) Autoclaved yeast peptone dextrose (YPD) media was prepared in 1 L of deionized water
using 10 g yeast extract 20 g Peptone from Becton Dickinson and Company (Sparks MD) and
20 g D-(+)-Glucose (ge995) purchased from Sigma-Aldrich (Saint Louis MO)
Modified Mary Miller Yeast Protein Isolation
The yeast culture was prepared and protein extraction was performed using a modified
Mary Miller protocol39
Briefly yeast strain s288c was inoculated with YPD media and shook
for 72 hours to allow the yeast to be glucose starved After the 72 hours the yeast culture was
partitioned into two flasks To one flask glucose was added at 2 of the final concentration and
allowed to incubate and shake for 10 minutes Then 100 optical density units (ODU) of the yeast
145
culture were collected from each flask Each yeast sample was spun down in a Beckman-Coulter
J6B centrifuge (Indianapolis IN) at 2500xg for three minutes The media was aspirated and the
tubes were flash frozen in liquid nitrogen and stored at -80oC The yeast pellets were thawed on
ice and resuspended in 1 mL of lysis buffer (50 mM Tris HCl 01 triton x-100 05 SDS
pH=80) and 1X protease and phosphatase inhibitor cocktail from Thermo Scientific (Rockford
IL) and transferred to an Eppendorf tube To the yeast 200 microL of glass beads were added and
amalgamated for 25 minutes at 4oC The sample was inverted and the Eppendorf tube was
pierced with a hot 23 gauge needle through the bottom and the tube was placed atop a 5 mL
culture tube and centrifuge in the Beckman-Coulter J6B at 2500xg for three minutes at 4oC to
collect the liquid containing the yeast cells while the glass beads remain trapped in the
Eppendorf tubes The protein lysate was then centrifuged at 3000xg for five minutes at 4oC and
the supernatant was collected and stored at -80oC
Preparation of tryptic digests
The Saccharomyces cerevisiae protein cell extract for fed or starved were analyzed with a
BCA protein assay kit (Thermo Scientific Rockford IL) and 2 mg of protein was added to four
parts -20oC acetone and allowed to precipitate the protein for 1 hour at -20
oC The samples were
then centrifuged at 14000xg for 5 minutes and the supernatant was removed and the protein
pellet was reconstituted in 360 microl of 8 M urea To each sample 20 microL of 050 M DTT was
added and allowed to incubate at 37oC for 45 minutes After incubation 54 microL of 055 M IAA
was added for carbamidomethyl of cysteine and the sample was allowed to incubate for 15
minutes in the dark To quench the IAA 20 microL of 050 M DTT was added and allowed to react
for 10 minutes To perform trypsin digestion 1400 microL of 50 mM NH4HCO3 was then added
along with 20 microg trypsin to each sample Digestion was performed overnight at 37oC and
146
quenched by addition of 25 microL of 10 formic acid For each sample the tryptic peptides were
then subjected to solid phase extraction using a Hypersep C18 100 mg solid phase extraction
(SPE) column (Thermo Scientific Bellefonte PA) Peptides were eluted with 50 ACN in
01 formic acid concentrated and reconstituted in 500 microL of 80 ACN and 01 formic acid
Phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC)
One ml of Ni-NTA was washed three times with 800 microL of H2O and then the Ni was
removed with 800 microL of 100 mM EDTA in 50 mM ammonium bicarbonate and vortexed for 30
minutes The supernatant was removed and the NTA was washed with 800 microL of H2O three
times followed by addition of 800 microL 10 mM FeCl3 hexahydrate and vortexed for 30 minutes
The Fe-NTA was then washed three times with 800 microL of H2O and once with 80 ACN in 01
formic acid before being combined with the cell extract for phosphopeptide enrichment and
vortexed for 40 minutes The sample was washed three times with 200 microL of 80 ACN in 01
formic acid before elution with two aliquots of 200 microL of 5 ammonium hydroxide in 5050
ACNH2O with 15 minutes of vortexing The enriched phosphopeptides were then dried down
with a Thermo Scientific Savant SpeedVac (SVC100 Waltham MA) and reconstituted in 55 microL
25mM ammonium formate pH=75
First dimension neutral pH separation
Individually 50 microL of each yeast phosphopeptide enriched sample was injected onto a
Phenomenex Gemini-NX 3microm C18 110 Aring 150 x 2 mm column (Torrance CA) The Gemini
column was coupled to a Phenomenex SecurityGuard Gemini C18 2 mm guard cartridge
(Torrance CA) Mobile phase A consisted of 25 mM ammonium formate at pH=75 and mobile
phase B consisted of 25 mM ammonium formateACN with a 19 ratio respectively at pH=75
The column was operated at 50oC at a flow rate of 05 mLmin The flow profile was 2-30 B
147
over 65 minutes and then 5 min at 100 B A total of 22 fractions were collected every 3
minutes and the fractions were combined as follows 1 with 12 2 with 13 etc to 11 with 22
The combined fractions were dried down and reconstituted in 25 microL of 01 formic acid
RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN Injections consisted of 5 microL from each fraction onto an Agilent Technologies
Zorbax 300 SB-C18 5 microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5
microLmin for 5 minutes at 95 A 5 B Separation was performed on a Waters 3 microm Atlantis
dC18 75 microm x 150 mm (Milford MA) using a gradient from 5 to 45 mobile phase B at 250
nLmin over 90 minutes at room temperature Emitter tips were pulled from 75 microm inner
diameter 360 microm outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using
an in-house model P-2000 laser puller (Sutter Instrument Co Novato CA)
Mass spectrometry data acquisitions
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany)
equipped with an on-line nanospray source was used for mass spectrometry data acquisition
Hystar (Version 32 Bruker Daltonics Bremen Germany) was used to couple and control
Eksigent nanoLC software (Dublin CA Version 30 Beta Build 080715) for MS acquisition for
all experiments Smart parameter setting (SPS) was set to 700 mz compound stability and trap
drive level was set at 100 Optimization of the nanospray source resulted in dry gas
temperature of 125oC dry gas flow of 40 Lmin capillary voltage of -1300 V end plate offset of
-500 V MSMS fragmentation amplitude of 10V and SmartFragmentation set at 30-300
148
Data were generated in data dependent mode with strict active exclusion set after two
spectra and released after one minute MSMS spectra were obtained via collision induced
dissociation (CID) fragmentation for the six most abundant MS ions with a preference for doubly
charged ions An additional mode of MSMS fragmentation electron transfer dissociation
(ETD) was triggered on the precursor ion when a neutral loss was observed in CID
fragmentation from phosphoric acid H3PO4 (Δmz of 49 and 327 for 2+ and 3+ charge states
respectively) or for metaphosphoric acid (HPO3) (Δmz of 40 and 267 for 2+ and 3+ charge
states respectively) For MS generation the ion charge control (ICC) target was set to 200000
maximum accumulation time 5000 ms rolling average 2 acquisition range of 300-1500 mz
and scan speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target
was set to 300000 maximum accumulation time 5000 ms two spectral averages acquisition
range of 100-2000 mz and scan speed (Ultrascan) of 32000 mz
Data analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen
Germany) Deviations in parameters from the default Protein Analysis in DataAnalysis were as
follows intensity threshold 1000 maximum number of compounds 1E9 and retention time
window 0001 minutes These parameter changes were required to prevent artificial data
reduction Identification of peptides were performed using Mascot40
(Version 23 Matrix
Science London UK) Database searching was performed against SwissProt Saccharomyces
cerevisiae database (version 575) Mascot search parameters were as follows Allowed missed
cleavages 2 enzyme trypsin fixed modification carboxymethylation (C) variable
modifications oxidation (M) and phosphorylation (STY) peptide tolerance plusmn12 Da maximum
number of 13
C 1 MSMS tolerance peptide decoy (Mascot) checked plusmn05 Da instrument type
149
ESI-Trap Results from the Mascot search was exported as DAT files for analysis by Scaffold 3
and Scaffold PTM
Scaffold and Ascore data processing
Scaffold 3 (Proteome Software Portland OR)was used for MSMS proteomics data
comparison and false discovery rate analysis The DAT files were loaded into Scaffold 3 and
the fractions for the two dimensional fractionation were combined The resulting biological
triplicates for starved and glucose fed yeast were filtered with a 1 false discovery rate (FDR)
on the protein level (Supplemental Table 1) Due to the inherent poor fragmentation of
phosphopeptides a lenient FDR level may still yield biologically relevant data and thus a 54
FDR was generated for phosphopeptides (Supplemental Table 2) For a more conservative list of
phosphopeptides with a 06 FDR was generated from Scaffold 3 (Supplemental Table 3) FDR
analysis is sufficient at preventing poor data from being reported but does not prevent false
phosphosite identification in phosphopeptides To provide confidence in site identification
Scaffold PTM was used to perform Ascore41
analysis Ascore uses an algorithm to score the
probability of the phosphosite from a phosphopeptide identified by a database searching
algorithm such as Mascot Ascore can be freely accessed at httpascoremedharvardedu
Cell collection RNA isolation and microarray data analysis
All experiments were performed in biological duplicates Cell samples (10 ODU) were
taken at starved and fed states spun at 5000xg for 2 minutes at 30 oC the supernatant was
removed and the pellets snap frozen using liquid nitrogen RNA was isolated using the Epicentre
MasterPure Yeast Purification Kit (Epicentre Technologies) and quality was checked using gel
electrophoresis cRNA synthesis was carried out using the GeneChipreg Expression 3
Amplification One-Cycle Target Labeling and Control Reagents kit (Affymetrix) All
150
experiments followed the manufactures instructions cRNA samples were hybridized to
GeneChip Yeast Genome 20 Arrays for 16 hours Arrays were washed stained and scanned
according the manufactures recommendations Affymetrix CEL files were RMA normalized
with R and the Bioconductor Suite Data analysis was performed within TIGR Multiexperiment
Viewer v451 in-house Perl scripting R and Bioconductor
Results
Sample preparation for shotgun proteomics
As discussed in the introduction the purpose of this study is to provide an exploratory list
of qualitative differences in the phosphoproteome between starved vs glucose fed yeast After
yeast cell lysate production a substantial amount of sample preparation is performed to enhance
the depth of the phosphoproteome coverage A workflow of the steps following cell lysis is
outlined in Figure 1A The yeast proteins are isolated via acetone precipitation followed by
digestion with trypsin to produce peptides Phosphopeptides are intermixed with the entire
tryptic peptide sample and therefore Fe-NTA IMAC is used to enrich the phosphopeptides To
improve upon the number of phosphopeptides we then performed two dimensional separation
with high pH RP followed by low pH RP C18 and infused the eluent directly onto an ion trap
mass spectrometer Figure 1B show an improved technique for the first dimension of separation
to combine the early eluting and late eluting fractions from the first phase of separation to reduce
overall MS analysis time The combination of fractions 1 and 12 2 and 13 etc essentially
improves the duty cycle of the mass spectrometer by ensuring sufficient peptide content is
injected onto a low pH nanoLC RP C18 column
ETD-triggered mass spectrometry
151
In the present study labile phosphorylation can lead to non-informative neutral loss
During MS scanning mode the instrument will choose the 6 most abundant peaks with correct
isotopic distribution and select them for MSMS CID fragmentation During CID fragmentation
it is common for labile PTMs such as phosphorylation to undergo neutral loss and thus limited
informative b and y-type ions are formed Alternatively ETD fragmentation can be used on
specific MSMS scans which produce a neutral loss indicative of phosphorylation (98Daz or
80Daz) The subsequent ETD fragmentation on these putative phosphopeptides will lead to
uniform backbone cleavage resulting in confident identification of phosphopeptides with site-
specific localization during MSMS It is important to note that CID fragmentation still produces
very informative fragmentation for phosphorylation but ETD provides an orthogonal
fragmentation pathway to further increase the phosphoproteome coverage Additionally the
duty cycle for ETD fragmentation is roughly half that of CID therefore about half as many
potential peptides would be fragmented and sequenced if the instrument was using ETD
fragmentation exclusively
Protein Data
Even with phosphopeptide enrichment it is inevitable that non-phosphopeptides will also
be identified All data were searched with Mascot and in total over 1000 proteins were identified
with a 1 FDR rate and are listed in Supplemental Table 1 The proteins listed in Supplemental
Table 1 include both phosphoproteins and non-phosphorylated proteins In addition to the
proteins identified in the fed and starved states the unique peptides and spectral counts are also
listed for reference The phosphopeptides are specifically listed with a 54 FDR rate in
Supplemental Table 2 and comparisons between starved and fed with Ascore analysis are listed
for every phosphopeptide identified A higher confidence of phosphopeptide identification is
152
sometimes required before investing in time consuming biological experiments so a list of
phosphopeptides identified in starved and fed was taken from a setting directing Scaffold to
produce a 05 FDR which resulted in the generated FDR of 06 FDR and listed in
Supplemental Table 3
A summary of specific phosphorylation sites are listed in Table 1 with a 06 FDR and
Table 2 with a 54 FDR Each table gives totals for unique phosphopeptides identified having
an Ascore localization score ge80 without Ascore and phosphorylation events on each unique
peptides As expected the majority of phosphorylation events (over 50) occurred on serine
whereas fewer phosphorylation events (~10) occurred on tyrosine In addition the vast
majority of phosphorylation events were single phosphorylation (ge65) with very few
identifications having more than two phosphosites per peptide For specific phosphopeptide
identification and subsequent phosphoprotein identification refer to Supplemental Table 2 and 3
Discussion
Transcriptional response to glucose feeding
Yeast responds to the repletion of glucose after glucose-depletion by broad
transcriptional changes to induce growth Roughly one-third of the yeast genome is altered by at
least two fold when glucose is introduced as shown in Figure 2 Figure 2 is a heat map from a
microarray analysis showing 1601 genes up regulated and 1457 genes down regulated after
addition of glucose compared to the starved state The arbitrary cut-offs for these values were as
follows two fold induction after nutrient repletion and a false discovery rate (q-value) of 001
Red color in the heat map indicates a particular gene is up-regulated in the fed state compared to
the starved state Alternatively genes coded in green are less expressed in the fed state
compared to the starved condition The intensity of the green or red colors is indicative of the
153
intensity of the fold change in gene expression These large transcriptional changes after glucose
repletion drive and complement the current phosphoproteomic study
PKA motif analysis
One benefit of a large scale phosphoproteomics experiment is to examine the different
phosphopeptides from known motifs for specific kinases PKA and TOR are responsible for the
majority of the transcriptional response and thus PKA is a good target for motif analysis Figure
3 shows the PKA motif sequence xxxRKRKxSxxxxxx with the phosphorylation occurring on
the serine Other than F16P (fructose-16-bisphosphatase) all the proteins listed are unique to the
starved or fed samples A motif sequence will inevitably show up by random chance in any
analysis For this study the control for motif analysis uses the swissprot protein list for the
entire yeast proteome for the background Compared to background this specific PKA kinase
from Figure 3 is up-regulated by 264 fold when compared to the background One interesting
protein emerged from this motif analysis in the fed sample but not the starved sample is
Ssd1which is implicated in the control of the cell cycle in G1 phase42
Ssd1 also is
phosphorylated by Cbk1 and Cbk1 has been proposed to inhibit Ssd143
and provides an
intriguing target for future studies on starved vs glucose fed yeast growth
Localization of the phosphorylation sites
When a phosphopeptide contains any number of serine threonine or tyrosine amino
acids the localization of the phosphosite can sometimes be ambiguous Database searches used
to identify peptides like Mascot do not provide any probability for localization of correct
phosphorylation sites Ascore is an algorithm that uses the same MSMS spectra as Mascot but
instead of comparing theoretical peptide MSMS to experimental spectra Ascore searches for
informative a b or y-type peptide fragments giving evidence for phosphosite The Scaffold
154
program adds a localization probability to the Ascore values and the values are listed in
Supplemental Table 2 and 3 Figure 4 has a representative Ascore mass spectra assigning the
peaks identified and providing evidence that the phosphorylation site occurs at the threonine
instead of the serine Incorporating Ascore into this study provides a layer of validation for
putative phosphosite identification
Plasma Membrane 2-ATPase
A previous study identified and localized phosphorylation sites on plasma membrane 1-
ATPase after glucose was introduced to starved yeast15
In the current study PMA2 (plasma
membrane ATPase 2) was identified in glucose fed and not starved samples The doubly
threonine phosphorylated peptide identified in fed yeast is PMA2 with amino acid sequence
IKMLTGDAVGIAKETCR (583-599) whereas the homologous peptide in PMA1 has the exact
same amino acid sequence except for the first isoleucine substituted for valine
VKMLTGDAVGIAKETCR (554-570 not observed in data) PMA2 was observed in the 06
FDR list of phosphopeptides and has an Ascore localization probably of 100 A plant study
showed that PMA2 phosphorylation level was higher in early growth phase than when in
stationary phase44
In addition PMA2 expression in yeast permits the growth of yeast and
threonine phosphorylation has been reported on Thr-95545
The identification of PMA2 in the
fed glucose cell extract provides an interesting target for future study on the molecular
mechanisms involved in regulation growth in starved vs glucose fed yeast
Conclusion
In conclusion this work provides a qualitative comparison in the phosphoproteome
between starved and glucose fed yeast A large scale IMAC enrichment of yeast cell lysate
followed by 2-D separation provided a rich source of phosphopeptides for neutral loss triggered
155
ETD analysis using mass spectrometry A specific motif for PKA is highlighted to show the
differences in proteins identified between starved vs fed conditions In total 477 unique
phosphopeptides are identified with 06 FDR and 669 unique phosphopeptides identified with
54 FDR Phosphosite validation is performed using a localization algorithm Ascore to
provide further confidence on the site-specific characterization of these phosphopeptides The
proteins Ssd1 and PMA2 emerged as potential intriguing targets for more in-depth studies on
protein phosphorylation involved in glucose response
Supplemental Tables 1 2 and 3 are available upon request
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159
Figure 1 The general workflow indicating the major steps involved in sample collection
sample processing mass spectrometric data acquisition and analysis of comparative
phosphoproteomics for starved vs glucose fed yeast (A) The high pH RP separation
procedure for combining fractions to reduce low peptide containing fractions from the
UV-VIS trace is also shown (B)
160
Figure 2 Heat map for the global transcriptional response to glucose fed vs starved samples
S288C cells starved for glucose until growth was arrested and subsequently glucose was added
161
Samples for microarray analysis were taken at the starved state and 60 minutes after glucose was
added The heat map shows the fed log2 fold change for each gene relative to the starved state
across the genome performed in biological replicate (A) Black indicates no change in
expression level while red indicates higher expression for the fed relative to the starved state
Green indicates higher expression for the starved state compared to the fed state (Adapted from
Dr Michael Conways Thesis)
162
Figure 3 The motif for protein kinase A (PKA) that was observed for fed vs starved which is
xxxRKRKxSxxxxxx with the phosphorylation occurring on the serine The motif occurred at a
rate 264 fold higher than the yeast proteome used for background In addition one protein was
observed in both starved and fed with accession identification of F16P (Fructose-16-
bisphosphatase)
163
06 FDR phosphopeptide analysis
Table 1 The number of phosphopeptides identified with a 06 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
Starved Fed All
Ascore ge80 score
unique
STY 164 153 317
S 87 (530) 82 (536) 169 (533)
T 60 (366) 55 (359) 115 (363)
Y 17 (104) 16 (105) 33 (104)
Unique no Ascore
STY 242 235 477
S 131 (541) 133 (566) 264 (553)
T 86 (355) 78 (332) 164 (344)
Y 25 (103) 24 (102) 49 (103)
Phosphorylation events
on each unique peptide
1 102 113 187
2 36 40 68
3 12 11 22
4 or more 8 3 11
164
54 FDR phosphopeptide analysis
Starved Fed All
Ascore ge80 score
unique
STY 217 217 434
S 115 (530) 113 (521) 228 (525)
T 78 (359) 78 (359) 156 (359)
Y 24 (111) 26 (120) 50 (115)
Unique no Ascore
STY 337 332 669
S 193 (573) 180 (542) 373 (558)
T 111 (329) 116 (349) 227 (339)
Y
Phosphorylation events
on each unique peptide
1
2
3
4 or more
33 (98)
135
56
16
11
36 (108)
169
55
14
3
69 (103)
280
100
27
13
Table 2 The number of phosphopeptides identified with a 54 FDR rate for starved and
glucose fed yeast Only unique phosphopeptides are included in the numbers listed Ascore
localization probability ge80 and no Ascore value which totals all phosphopeptides regardless
of Ascore value even if the Ascore localization value was 0 are shown for starved or fed
samples The number of phosphorylation events on each unique peptide is also included The
vast majority of the phosphopeptides identified have a single phosphosite
165
Figure 4 Ascore validation of the phosphopeptide LSLAtKK from the protein SGM1 (slow
growth on galactose and mannose protein 1) with 100 localization probability observed
in only fed but not starved yeast samples Ascorersquos algorithm identifies a b and y-type
ions and looks to identify peaks that provide evidence for a specific phosphorylation site
For this example Ascore assigns this phosphosite to threonine (T5) instead of the serine
(S2) The intense peaks at 4024 mz and 5250 mz are not identified by any a b or y-
type ions From the ladder sequence of the peptide shown numerous ions indicate the
threonine is phosphorylated while the serine is not Among these ions used for
localization are b2 y2 y5+H2O y3 y4 and y5
166
Chapter 6
Use of electron transfer dissociation for neuropeptide sequencing and
identification
Adapted from ldquoDiscovery and Characterization of the Crustacean Hyperglycemic Hormone
Precursor Related Peptides (CPRP) and Orcokinin Neuropeptides in the Sinus Glands of the Blue
Crab Callinectes sapidus Using Multiple Tandem Mass Spectrometry Techniquesrdquo Hui L
Cunningham R Zhang Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
167
Abstract
The crustacean Callinectes sapidus sinus gland (SG) is a well-defined neuroendocrine site that
produces numerous hemolymph-borne agents including the most complex class of endocrine
signaling moleculesmdashneuropeptides One such peptide is the crustacean hyperglycemic hormone
(CHH) precursor-related peptide (CPRPs) which was not previously sequenced Electron
transfer dissociation (ETD) was used for sequencing of intact CPRPs due to their large size and
high charge state In addition ETD was used to sequence the sulfated peptide Cholecystokinin
CCK-like Homarus americanus using a salt adduct Collectively these two examples
demonstrated the utility of ETD in sequencing neuropeptides with large molecular weight or
with labile modifications
168
Introduction
Neuropeptides are the largest and most diverse group of endocrine signaling molecules in
the nervous system They are necessary and critical for initiation and regulation of numerous
physiological processes such as feeding reproduction and development1 2
Mass spectrometry
(MS) with unique advantages such as high sensitivity high throughput chemical specificity and
the capability of de novo sequencing with limited genomic information is well suited for the
detection and sequencing of neuropeptides in endocrine glands and simultaneously provides the
potential for information on post-translational modifications such as sulfonation3-6
The sinus glands (SG) are located between the medulla interna and medulla externa of the
eyestalk in Callinectes sapidus It is a well-known neuroendocrine site that synthesizes and
secretes peptide hormones regulating various physiological activities such as molting
hemolymph glucose levels integument color changes eye pigment movements and
hydro-mineral balance7 Our labrsquos previous neuropeptidomic studies of SG in several
crustacean species including Cancer borealis8-11
Carcinus maenas12
and Homarus americanus13
14 used multiple MS strategies combining MALDI-based high resolution accurate mass profiling
biochemical derivatization and nanoscale separation coupled to tandem MS for de novo
sequencing In the current study we explore the neuropeptidome of the SG in the blue crab
Callinectes sapidus a vital species of economic importance on the seafood market worldwide In
total 70 neuropeptides are identified including 27 novel neuropeptides and among them the
crustacean hyperglycemic hormone precursor-related peptides (CPRPs) and orcokinins represent
major neuropeptide families known in the SG
The crustacean hyperglycemic hormone (CHH) and its precursor-related peptide (CPRP) are
produced concurrently during the cleavage of CHH from the CHH preprohormone protein15
The
169
CPRP peptide is located between the signal peptide and the CHH sequence and is separated from
the CHH by a dibasic cleavage site The functions of CPRPs are currently unknown16
However
the complete characterization of CPRPs provides a foundation for future experiments elucidating
their functional roles in crustaceans The cDNA of CHH preprohormone in SG of Callinectes
sapidus has been characterized17
but the direct detection of CPRP has not been reported due to
its relatively large size and possible post-translational modifications While small fragments of
CPRPs can be identified using nanoLC-ESI-Q-TOF tandem mass spectrometry the intact
peptide is difficult to detect due to the large molecular weight of CPRPs
Peptidomics traditionally uses collision induced dissociation (CID) for tandem MS
experiments for de novo sequencing Recently an alternative peptide fragmentation method has
been developed using the transfer of an electron termed electron transfer dissociation (ETD)18 19
ETD involves a radical anion with low electron affinity to be transferred to peptide cation which
results in reduced sequence discrimination and thus provides improved sequencing for larger
peptides compared to CID20
Specifically for an ion trap ETD the fluoranthene radical anion is
allowed to react with peptide cations in the three dimensional trap The resulting dissociation
cleaves at the N-Cα bond to produce c and zbull type product ions The peptidome of model
organisms like Callinectes sapidus can be further explored and expanded utilizing ETD as a
complementary fragmentation technique to CID Previous peptidomic analysis has been
completed using ETD as an additional fragmentation method21
It was observed that
enzymatically produced peptides with a higher mz produced improved sequence coverage using
ETD This observation termed decision tree analysis determined that a charge state of ge6 all
peptides endogenous or enzymatic should be fragmented via ETD22
In the present study the
highly charged peptide CPRP with an intact molecular weight of 3837 readily produces plus six
170
charge states The higher charge state of CPRP is highly amenable to fragmentation by ETD
which produces remarkably improved fragmentation and thus increased sequence coverage when
compared to CID
Sulfonation of tyrosine is a post-translational modification (PTM) that is thought to occur on
trans-membrane spanning and secreted proteins23
Cholecystokinin-8 (CCK-8) is a sulfated
peptide which has been linked to satiety24-26
and a CCK-like peptide has been observed in
lobster27
Sulfonation is an extremely labile modification and is often lost during soft
ionization such as ESI or MALDI but can be avoided by optimizing the ionization process28
One potential strategy to identify sulfopeptides is to scan for a neutral loss of 80 Da after CID
but this method does not allow for identification of site of sulfonation and has the risk to be
mistaken for phosphorylation The sulfonation PTM is a negatively charged modification on
the peptide which allows for negative ion scanning in the mass spectrometer but provides
minimal MSMS sequence coverage during CID due to preferential loss of the sulfate group
Alternatively electron-based dissociation technique enables more rapid radical driven
fragmentation where the cleavage pattern is more uniform along the peptide backbone without
initially cleaving the labile sulfated group thus preserving the site of modification These types
of dissociation techniques only work for multiply-charged ions thus a method to install a
multiply-charged cation on the target peptide is desirable It has been shown that the electron
capture dissociation (ECD) can be used to sequence sulfated peptides when a multi-charged
cation is added to the solution29
We use a similar multi-charge cation solution technique to
sequence a sulfated peptide from Homarus americanus via ETD on an ion trap mass
spectrometer Here we presented the use of the ETD fragmentation technique for the analysis
of large peptides and peptides containing labile post-translational modification
171
Experimental Section
Chemical and materials
Acetonitrile methanol magnesium chloride (ACS grade) potassium chloride (ACS grade) and
calcium chloride (ACS grade) were purchased from Fisher Scientific (Pittsburgh PA) Formic
acid was from Sigma-Aldrich (St Louis MO) Human sulfated CCK-8 and CCK-like peptide
(E(pyro)FDEY(Sulfo)GHMRFamide) were purchased from American Peptide (Sunnyvale CA)
Fused-silica capillary with 75 μm id and 360 μm od was purchased from Polymicro
Technologies (Phoenix AZ) Millipore C18 Ziptip column was used for sample cleaning and all
water used in this study was deionized water (182 MΩcm) prepared from a Milli-Q Millipore
system (Billerica MA) The physiological saline consisted of 440 mM NaCl 11mM KCl 26
mM MgCl2 13 mM CaCl2 11 mM Trizma base and 5 mM maleic acid in pH 745
Animals and dissection
Callinectes sapidus (blue crab) were obtained from commercial food market and maintained
without food in artificial sea water at 10-12 ordmC Animals were cold-anesthetized by packing on
ice for 15-30min before dissection The optic ganglia with the SG attached were dissected in
chilled (~4ordmC) physiological saline and individual SGs were isolated from the optic ganglia by
micro-dissection and immediately placed in acidified methanol (90 methanol 9 glacial acetic
acid and 1 water) and stored at -80ordmC until tissue extraction
Tissue homogenization
Acidified methanol was used during the homogenization of SGs The homogenized SGs were
immediately centrifuged at 16100 g using an Eppendorf 5415D tabletop centrifuge (Eppendorf
172
AG) The pellet was washed using acidified methanol and combined with the supernatant and
further dried in a Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation) The
resulting dried sample was re-suspended with a minimum amount (20 microL) of 01 formic acid
Fractionation of homogenates using reversed phase (RP)-HPLC
The re-suspended extracts were then vortexed and briefly centrifuged The resulting supernatants
were subsequently fractionated via high performance liquid chromatography (HPLC) HPLC
separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax
UV-D II absorbance detector (Rainin Instrument Inc Woburn MA) The mobile phases included
Solution A (deionized water containing 01 formic acid) and Solution B (acetonitrile containing
01 formic acid) About 20 μl of extract was injected onto a Macrosphere C18 column (21 mm
id x 250 mm length 5 microm particle size Alltech Assoc Inc Deerfield IL) The separation
consisted of a 120 minute gradient of 5-95 Solution B Fractions were automatically collected
every two minutes using a Rainin Dynamax FC-4 fraction collector (Rainin Instrument Inc
Woburn MA) The fractions were then concentrated using in Savant SC 110 SpeedVac
concentrator (Thermo Electron Corporation) and re-suspended with minimum amount of 01
formic acid
Nano-LC-ESI-Q-TOF MSMS
Nanoscale LC-ESI-Q-TOF MSMS was performed using a Waters nanoAcquity UPLC system
coupled to a Q-TOF Micro mass spectrometer (Waters Corp Milford MA)
Chromatographic separations were performed on a homemade C18 reversed phase capillary
column (75 microm internal diameter x100 mm length 3 microm particle size 100 Aring) The mobile phases
173
used were 01 formic acid in deionized water (A) 01 formic acid in acetonitrile (B) An
aliquot of 50 microl of a tissue extract or HPLC fraction was injected and loaded onto the trap
column (Zorbax 300SB-C18 Nano trapping column Agilent Technologies Santa Clara CA)
using 95 mobile phase A and 5 mobile phase Bat a flow rate of 10 microlmin for 10 minutes
Following this the stream select module was switched to a position at which the trap column
came in line with the analytical capillary column and a linear gradient of mobile phases A and B
was initiated For neuropeptides the linear gradient was from 5 buffer B to 45 buffer B over
90 min The nanoflow ESI source conditions were set as follows capillary voltage 3200 V
sample cone voltage 35 V extraction cone voltage 1 V source temperature 100ordmC A data
dependent acquisition was employed for the MS survey scan and the selection of three precursor
ions and subsequent MSMS of the selected parent ions The MS scan range was from mz
400-1800 and the MSMS scan was from mz 50-1800
Peptide Prediction De Novo Sequencing and Database Searching
De novo sequencing was performed using a combination of MassLynxTM
41 PepSeq software
(Waters) and manual sequencing Tandem mass spectra acquired from the Q-TOF were first
deconvoluted using MaxEnt 3 software (Waters) to convert multiply charged ions into their
singly charged forms The resulting spectra were pasted into the PepSeq window for sequencing
analysis The candidate sequences generated by the PepSeq software were compared and
evaluated for homology with previous known peptides The online program blastp (National
Center for Biotechnology Information Bethesda MD httpwwwncbinlmnihgovBLAST)
was used to search the existing NCBI crustacean protein database using the candidate peptide
sequences as queries For all searches the blastp database was set to non-redundant protein
174
sequences (ie nr) and restricted to crustacean sequences (ie taxid 6657) For each of the
proteins putatively identified via blastp the BLAST score and BLAST-generated E-value for
significant alignment are provided in the appropriate subsection of the results Peptides with
partial sequence homology were selected for further examination by comparing theoretical
MSMS fragmentation spectra generated by PepSeq with the raw MSMS spectra If the
fragmentation patterns did not match well manual sequencing was performed
NanoLC Coupled to MSMS by CID and ETD
Setup for RP nanoLC separation
The setup for nanoLC consisted of an Eksigent nanoLC Ultra (Dublin CA) with a 10 microL
injection loop Mobile phase A consisted of H2O in 01 formic acid and mobile phase B
consisted of ACN (Fisher Scientific Optima LCMS grade Fair Lawn NJ) Injections
consisted of 5 microL of prepared tissue extract onto an Agilent Technologies Zorbax 300 SB-C18 5
microm 5 x 03 mm trap cartridge (Santa Clara CA) at a flow rate of 5 microLmin for 5 minutes at 95
A5 B Separation was performed on a Waters HPLC column with 3 microm Atlantis dC18 75 microm
x 150 mm (Milford MA) on a gradient from 5 to 45 mobile phase B at 250 nLmin over 90
minutes at room temperature Emitter tips were pulled from 75 microm inner diameter 360 microm
outer diameter capillary tubing (Polymicro Technologies Phoenix AZ) using a commercial
laser puller model P-2000 (Sutter Instrument Co Novato CA)
Mass Spectrometry Data Acquisitions for Collision Induced Dissociation (CID)
An ion-trap mass spectrometer (amaZon ETD Bruker Daltonics Bremen Germany) equipped
with an on-line nanospray source was used for mass spectrometry data acquisition Hystar
(Version 32 Bruker Daltonics Bremen Germany) was used to couple and control Eksigent
175
nanoLC software (Dublin CA Version 30 Beta Build 080715) to MS acquisition for all
experiments Smart parameter setting (SPS) was set to mz 700 compound stability and trap
drive level were set at 100 Optimization of the nanospray source resulted in dry gas
temperature 125oC dry gas 40 Lmin capillary voltage -1300 V end plate offset -500 V
MSMS fragmentation amplitude 10V and SmartFragmentation set at 30-300
Data was generated in data dependent mode with strict active exclusion set after two spectra and
released after one minute MSMS was obtained via CID fragmentation for the six most
abundant MS peaks with a preference for doubly charged ions and excluded singly charged ions
For MS generation the ion charge control (ICC) target was set to 200000 maximum
accumulation time 5000 ms four spectral average acquisition range of mz 300-1500 and scan
speed (enhanced resolution) of 8100 mz s-1
For MSMS generation the ICC target was set to
200000 maximum accumulation time 5000 ms three spectral averages acquisition range of
mz 100-2000 and scan speed (Ultrascan) of 32000 mz s-1
Mass Spectrometry Data Acquisitions for Electron Transfer Dissociation (ETD)
The amaZon ETD ion-trap mass spectrometer was operated in electron transfer dissociation for
MSMS fragmentation with the same optimized settings as reported for CID unless otherwise
stated Smart parameter setting (SPS) was set to 500 mz compound stability and trap drive
level were set at 100 MSMS was obtained via ETD fragmentation for the four most
abundant MS peaks with no preference for specifically charged ions except to exclude singly
charged ions The ETD reagent parameters were set to 400000 target ICC for the fluoranthene
radical anion The fluoranthene radical anion has an mz of 210 and therefore the 210 mz value
was removed from the resulting MSMS spectra and 160 mz was set for the MSMS low mz
cut-off
176
Direct Infusion of Sulfated Peptide Standards into the Mass Spectrometer using ETD and
CID Fragmentation
The sulfated peptide standards were infused into the mass spectrometer at a flow rate of 300
nlmin using a fused-silica capillary with 75 μm id and 360 μm od coupled to a custom pulled
tip The CCK-8 human peptide (20 microM) was infused in 5050 ACNH2O and detected in
negative ionization mode with an ICC of 70000 and fragmented with CID using the same
settings as the previous CID experiments Alternatively the CCK-like lobster sulfated peptide
(2 microM) was mixed with 30 microM of different salts (MgCl2 KCl or CaCl2) and 01 formic acid in
5050 ACNH2O The plus three charge of the CCK-like lobster peptide was selected for ETD
fragmentation in positive mode with the same setting as the previous ETD experiments The
data were then de novo sequenced for coverage and localization of the sulfation site
Data Analysis
MS data were processed with DataAnalysis (Version 40 Bruker Daltonics Bremen Germany)
Deviations in parameters from the default Protein Analysis in DataAnalysis were as follows
intensity threshold 1000 maximum number of compounds 1E7 and retention time window 05
minutes These parameter changes assisted in providing better quality spectra for sequencing
Identification of peptides was performed using Mascot (Version 23 Matrix Science London
UK) Searches were performed against a custom crustacean database none enzyme allow up to
1 missed cleavage amidated C-terminal as variable modifications peptide tolerance mass error
12 Da MSMS mass error tolerance is 06 Da
Results and Discussion
177
Identification and Characterization of Intact CPRPs Using ETD
Using ESI-Q-TOF 9 truncated CPRPs fragments were sequenced to cover the amino acid
sequence of the intact CPRP peptide RSAEGLGRMGRLLASLKSDTVTPLRGFEGETGHPLE
A representative CID MSMS spectrum of ASLKSDTVTPLR is shown in Figure 1 (a) CID
using ESI-Q-TOF mainly produces fragmentation for doubly and triply charge molecules which
is adequate to sequence peptides with relatively small molecular weight (less than 2000 Da)
However CID fragmentation is insufficient to sequence the larger intact CPRP in a complex
sample whose molecular mass is 3837 Da and therefore it is extremely difficult to directly
sequence by traditional CID ESI-Q-TOF ETD is an attractive fragmentation alternative to
sequence the highly charged CPRP peptide In ETD a protonated peptide reacts with an anion
(fluoranthene radical) where the anion transfers an electron to the peptide cation and the resulting
fragmentation occurs along the N-Cα bond by free-radical-driven-cleavage The large size of
CPRP offers multiple basic amino acid sites to sequester charge and reduce the sequence
coverage from collision induced dissociate by preventing random backbone cleavage whereas
ETD does not cleave the weakest bond and provides the alternative fragmentation pathway to
obtain superior sequence coverage to larger peptides As shown in Figure 1 (cd) the
fragmentation of ETD is highly amenable to ETD compared to the CID fragmentation in Figure
1 (b) As evident from comparison ETD offers more extensive fragmentation than CID thus
providing enhanced coverage for large intact peptides such as CPRPs Specifically we observe
125 of the b- and y- cleavages for CID as compared to an average of 535 of c- and zbull-
fragments More than a four-fold increase in fragments using ETD suggests the utility of this
relatively new tandem MS fragmentation method as complementary techniques for de novo
sequencing of large intact endogenous peptides and novel neuropeptide discovery endeavors
178
Negative Mode Sulfated Peptide Identification
An accepted method for identification and quantification for sulfated peptides is negative
ionization30
CCK-8 sulfated peptide standards show intense signal in negative ionization mode
without needing to have additives added such as salts Figure 2 shows the CID MSMS
spectrum from the negative ionization and produces an intense b6 ion at 430 mz The transition
from the doubly negatively charged MS ion of 570 mz to the b6 ion provides a selected reaction
monitoring transition for quantification studies but the sequence information is limited and the
presence of the methionine produces variable oxidation In addition Figure 2 shows the
MSMS product ions with the loss of the sulfate group thus making site-specific location of
sulfation more difficult
Investigation of Cation Salt Adducts to Produce Multiply Charged Sulfated Peptides
Sulfated peptides can be isolated in positive scanning mode but usually only in a plus one
state with low signal intensity If ETD is performed on the singly charged peptide cation a
neutral is formed and is lost in the mass spectrometer and thus no sequence information can be
obtained In order to remedy this situation a technique that adding metal salts to peptides to
increase charge state for ECD used in Fourier transform ion cyclotron resonance mass
spectrometry (FTICR-MS)29
inspired the investigation of increasing charge state of targeted
peptide via metal salt adduction followed by ETD analysis in a Bruker amaZon ion trap
Various salts were investigated including MgCl2 CaCl2 and KCl each used at a concentration of
30 microM and mixed with a sulfated peptide standard at 2 microM The addition of KCl produced
mostly doubly charged ion of the lobster CCK-like peptide standard K adduct which produced
insufficient sequence information from ETD fragmentation (data not shown) In comparison
the MgCl2 and CaCl2 produced intense triply charged peptide ions but CaCl2 produced lower
179
signal intensity compared to MgCl2 (data not shown)
Cation Assisted ETD Fragmentation of CCK-like Lobster Sulfated Peptide and Future
Directions
The addition of MgCl2 to the lobster CCK-like sulfated peptide is shown in Figure 3
Except for z1 the complete z-series is obtained including the z7 ion with and without the
sulfation PTM The spectrum is complicated by the additional Mg adducts but several peaks
are shown without any Mg adduct such as z2 z3 z4 etc Clearly the method of cation
assisted ETD fragmentation can be useful using ion trap instruments and can properly sequence
sulfated peptides that are prone to neutral loss from the labile PTM One concern about future
direction in sulfopeptide isolation in non-genome sequenced species is isolation of sulfopeptides
Currently antibodies to specific sulfopeptides is the only commonly used enrichment technique
for sulfopeptides Also since metal cations are needed for this method direct infusion into an
ESI mass spectrometer is preferred to prevent running large amounts of adduct forming salts
through the LC system With direct infusion the lack of separation confounds the problem in
sulfopeptide detection because any co-eluting peptides could have ionization efficiency and thus
reduce the signal of the sulfopeptide Once discovered and sequenced a selected reaction
monitoring (SRM) method could be developed using LC retention coupled with negative
ionization mode followed by CID MSMS or possibly MSMSMS to perform quantitative
studies for sulfopeptides
Conclusions
In this study ETD was performed to improve the sequence coverage of large endogenous
neuropeptides with higher charge and bigger size The intact CPRP from Callinectes sapidus was
identified and characterized with 68 sequence coverage by MS for the first time Cation
180
assisted ETD fragmentation was also shown to be a powerful tool in de novo sequencing of
sulfated neuropeptides These endeavors into using ETD for certain neuropeptides will assist in
future analysis of large neuropeptides and PTM containing neuropeptides
181
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E Li L J Mass spectral characterization of peptide transmittershormones in the nervous system and
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183
Figure 1 Comparison of tandem MS spectra of truncated CPRP (ASLKSDTVTPL mz 128766)
by ESI-Q-TOF (a) and intact CPRP (MW 3837) by the amaZon ETD for both CID and ETD
fragmentation (a) MSn of precursor ion with mz 640 with charge state +6 by CID represent
loss of NH3 ordm represent loss of H2O (b) MS+6
of precursor ion with mz 640 with charge state +6
by ETD at z represent z+1 z represent z+2 (c) MS+5
of precursor ion with mz 768 with charge
state +5 by ETD z represent z+1 z represent z+2 For all labels in Figure 1 charge state +1 is
not specified
184
185
Figure 2 Negative CID spectrum of CCK-8 peptide standard (20 microM) via direct infusion show
the doubly charged b6 ion provides the most intense MSMS transition
186
Figure 3 ETD spectrum of E(pyro)FDEY(Sulfo)GHMRF-amide (2 microM) obtained on the
amaZon ETD via direct infusion with 30 microM MgCl2 The sulfated peptide can be identified
with a visible z-series of z2 to z9 and identified sulfate loss
187
Chapter 7
Investigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysis
Adapted from ldquoInvestigation and reduction of sub-microgram peptide loss using molecular
weight cut-off fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J
Wellner D Li L Journal of Mass Spectrometry In Press
188
ABSTRACT
This work investigates the introduction of methanol and a salt modifier to molecular
weight cut-off membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide
quantities Using a neuropeptide standard bradykinin sample loss was reduced over two orders
of magnitude with and without undigested protein present Additionally a bovine serum
albumin (BSA) tryptic digestion was investigated Twenty-seven tryptic peptides were identified
from MALDI mass spectra after enriching with methanol while only two tryptic peptides were
identified after the standard MWCO protocol The strategy presented here enhances recovery
from MWCO separation for sub-microg peptide samples
INTRODUCTION
Molecular weight cut-off membrane-based centrifugal filter devices (MWCO) are
commonly used to desalt and concentrate large molecular weight proteins [1] Greeing and
Simpson recently investigated various MWCO membranes for large amounts of starting material
(~6 mg) focusing on optimal conditions for the sub 25 kDa protein fraction [2] The authors
recovered 200 μg to 29 mg of protein from multiple MWCO experiments and demonstrated that
a 1090 acetonitrile (ACN)H2O elution solvent produced optimal results [3] In addition Manza
et al provided an alternative approach to isolate proteins with a 5 kDa MWCO by using
NH4HCO3 and recovering the retained proteins [4] Alternatively the elution from MWCOs can
be collected to recover only low molecular weight peptides Multiple peptidomic studies have
utilized MWCOs for peptide isolation during the first few steps of sample preparation [5 6]
When sample amount is limited or peptide content is below 1 microg sample loss is a significant
concern when using MWCOs to isolate endogenous peptides Optimized protocols have been
189
investigated using ACN [3 7] salt [4 8] SDS [5] or native sample [6 9 10] but these
experiments primarily focused on large sample amounts rather than sub-microgram peptide
quantities
MWCOs separate large molecules from small molecules The small molecule fraction
may be rich with signaling peptides (SP) cytokines and other small molecules involved in cell-
cell signaling Signaling peptides perform various functions in the body including cell growth
cell survival and hormonal signaling between organs [11] Individual SP contribute to different
aspects of behavior such as pain (enkephalins) [12] feeding (neuropeptide Y) [13] and blood
pressure (bradykinin) [14] MWCO separations can be used to enrich biologically important SP
and explore the peptide content from biological fluids with relatively low peptide content like
blood or cerebrospinal fluid (CSF) In a recent investigation the detection of neuropeptides and
standards in crustacean hemolymph was improved when methanol and protease inhibitors were
present before performing MWCO neuropeptide isolation The impact of methanol on MWCO
sample loss was not investigated in the study [15] In another study a large-scale mass
fingerprinting protocol of endogenous peptides from CSF used a combination of salts before
MWCO fractionation but the impact of adding salts was not discussed [16] The most
commonly used brand of MWCO in the publications and in peptidomic studies is Millipore
Therefore Millipore MWCOs (using regenerated cellulose as the membrane) were used in the
present study The purpose of this work is to provide an optimized sample preparation technique
for MWCO filtering to reduce sample loss and allow sub-microg detection of peptides using MALDI
mass spectrometry
MATERIALS AND METHODS
190
Materials and Chemicals
Water acetonitrile methanol (optima LCMS grade) and sodium chloride (995) were
purchased from Fisher Scientific (Fair Lawn NJ) The α-cyano-4-hydroxy-cinnamic acid (99)
formic acid (FA) (ge98) and bovine serum albumin (ge96) were purchased from Sigma-
Aldrich (St Louis MO) Amicon Ultra 05 mL 10000 MWCO centrifugal filters and ZipTips
packed with C18 reversed-phase resin were purchased from Millipore (Billerica MA) Trypsin-
digested bovine serum albumin (BSA) was purchased from Waters (Milford MA) Bradykinin
was purchased from American Peptide Company (Sunnyvale CA)
MALDI MS Instrumentation
An AutoFLEX III MALDI TOFTOF mass spectrometer (Bruker Daltonics Billerica
MA) was operated in positive ion reflectron mode The MALDI MS instrument is equipped with
a proprietary smart beam (Bruker Daltonics Billerica MA) laser operating at 200 Hz The
instrument was internally calibrated over the mass range of mz 500minus2500 using a standard
peptide mix Two thousand laser shots were collected per sample spot at an accelerating voltage
of 19 kV and a constant laser power using random shot selection The acquired data were
analyzed using FlexAnalysis software (Bruker Daltonics Billerica MA) Mass spectrometry
data acquisition was obtained by averaging 2000 laser shots
Molecular weight cut off separation procedure
The MWCO separations were performed using Amicon Ultra 05 mL 10000 MWCO
centrifugal filters (Billerica MA) Before MWCO separation three washing steps were
performed to remove contaminants from the filter The three washes were 500 μL of 5050
H2OMeOH 500 μL of H2O and 400 μL of the solution used for MWCO separation For the
191
100 H2O solution 1 microg of BSA or bradykinin was used for separation All the other MWCO
separation experiments used 500 ng of BSA or 100 ng or less of bradykinin The MWCO filter
was then centrifuged at 14000 rcf for 5 min at room temperature in an Eppendorf 5415 D
microcentrifuge (Brinkmann Instruments Inc Westbury NY) The filtrate was concentrated in a
Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation West Palm Beach FL)
and acidified The resulting sample was desalted according to the manufacturer using C18
ZipTips from Millipore (Billerica MA) by washing the ZipTip with three times 100 ACN
three aqueous washes of 01 FA binding the peptides from the solution and one aqueous wash
of 01 FA Peptides were then eluted from the ZipTips using 15 microL of 50 ACN in 01 FA
Matrix deposition
Equal volumes of 05 μL from the 15 μL sample solution (including standards not subject
to MWCO filtering) and α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix solution in 50
ACN50 H2O were mixed using a dried-droplet method and spotted on a MALDI target The
resulting droplets were allowed to air dry prior to mass spectrometry acquisition
RESULTS AND DISCUSSION
Analysis of two orders of magnitude increase for bradykinin standard
Bradykinin was selected to assess the potential peptide loss in the flow-through after
performing MWCO separation As shown in Figure 1 1 microg of bradykinin standard does not
produce a detectable signal by MALDI mass spectrometry analysis after a 10 kDa MWCO
separation in water (performed in triplicate) For comparison 1 ng of bradykinin standard
diluted to 15 microL produced an intense signal on the MALDI mass spectrometer suggesting
192
significant sample loss occurs when the target analyte is low in quantity (data not shown
performed in triplicate) Figure 1 shows that the addition of a salt in this case NaCl improves
the limits of detection and decreases sample loss when 7030 watermethanol was compared to
7030 aqueous 1 M NaClmethanol The reproducible results gave a relative standard deviation
(RSD) of 6 for peak intensity Figure 1 shows that even when starting with 1 μg of bradykinin
too much sample is lost during the MWCO separation in water to detect the remainder
However an intense signal is observed for 10 ng of bradykinin after the MWCO separation when
7030 aqueous 1 M NaClmethanol is used as an elution solvent Sample loss from zip-tipping
was estimated in triplicate by calculating the decreases in peak intensity When 10 ng of
bradykinin was used sample loss was ~41 In comparison the calculated yield for 10 ng of
bradykinin after the 7030 aqueous 1 M NaClmethanol MWCO separation and zip-tip desalting
showed an estimated sample loss of 63 meaning more loss can be attributed to sample clean-
up than MWCO filtration
A series of experiments were performed to determine if 7030 aqueous 1 M
NaClmethanol is an optimal solution to recover peptides during a MWCO separation (data not
shown) A 5050 aqueous 1 M NaClmethanol and a 5050 watermethanol elution were
performed in duplicate but signal intensity of the resulting bradykinin was poor and numerous
polymer peaks were detected in the flow through A lower salt concentration 01 M NaCl was
used but also produced greatly reduced signal when compared to aqueous 1 M NaCl To assess
the optimized methodrsquos compatibility with lower sample amounts the 7030 aqueous 1 M
NaClmethanol solution was added to 1 ng of bradykinin for MWCO separation but no signal
was obtained (data not shown) Using a neuropeptide standard the addition of methanol and
NaCl salt significantly improved the sample recovery in sub-microg amounts
193
BSA tryptic peptide mixture analysis
After demonstrating the importance of using an optimized solution for MWCO
separations with an individual peptide the new method was applied to 500 ng of BSA tryptic
digest to investigate its utility with more complex peptide mixtures Table 1 lists the BSA
tryptic peptides identified in the MALDI MS analysis from different solution conditions
processed by MWCO separation As shown in Table 1 a directly spotted BSA tryptic peptide
standard in the absence of any MWCO filtration enabled identification of 39 tryptic peptides by
accurate peptide mass measurements Once again when using 100 H2O for MWCO
separations the starting amount was doubled to 1 microg (also done with 500 ng data not shown)
However many tryptic peptides were not detected due to low signal intensities and non-optimal
elution conditions Instead of H2O a 1 M NaCl solution was used for the MWCO elution but
only two tryptic peptides were identified (Table 1) The addition of 30 methanol into the
sample before MWCO filtration produced the first increase in identified BSA tryptic peptides
The remaining data from Table 1 shows improved BSA tryptic peptide identifications as the
sample (elution) conditions were further optimized Figure 2 shows the actual mass spectra
associated with the three most promising elution solutions along with 100 H2O
The BSA tryptic peptide intensities are shown in Figure 2A and the most intense tryptic
peptide YLYEIAR mz 92749 was observed in the four different solutions shown in Figure 2B
but not in the 100 H2O or 100 1 M NaCl solutions (data not shown) In Figure 2A all mass
spectra are normalized to an intensity of 7 x 104 to illustrate two points First the MWCO
filtering step still produced sample loss regardless of the solvent conditions chosen Second
there is a noticeable increase in peptide peak intensity using the optimized solvent 6040
194
aqueous 1 M NaClmethanol (Figure 2A) Figure 2C displays a zoomed-in view of a BSA
tryptic peptide signal LKECC
DKPLLEK mz 153266 (
carbamidomethyl) observed only in
the optimized solvent The detection of the mz 153266 peptide in Figure 2C highlights the
potential gain in sample and detectable peptides by using an optimized saltMeOH combination
A non-optimized saltMeOH combination will still reduce sample loss but further minimizing
sample loss during sample preparation will always be desirable in any analytical protocol
MWCO composition
The purpose of this application note is to provide evidence of sub-microg sample loss during
MWCO separations of peptide samples and a solution to overcome this limitation The
explanation of why adding MeOH and NaCl to the sample solution provides a significant
reduction in sample loss is beyond the scope of this application note Regardless Supplemental
Table S1 is an expanded version of Table 1 showing the amino acid sequence hydrophobicity
calculated using GRAVY scores and pI of the identified peptides in this study No discernible
trend was obtained from the data The membrane of commonly used MWCO in peptidomics and
for this study is comprised of chemically treated (regenerated) cellulose which is a
polysaccharide containing β (1rarr4) linked D-glucose Glucose has numerous free hydroxyl
groups which could non-specifically adsorb peptides flowing through the MWCO The addition
of MeOH has the most significant effect on signal which could be due to disrupting the
interaction between peptides and hydroxyl groups from glucose NaCl has a less significant
effect on sample recovery compared to MeOH but a detectable reduction in sample loss is noted
This improvement in sample recovery could be analogous to the use of NaCl in
195
immunodepletion protocols to reduce non-specific binding which is accomplished by adding
150 mM NaCl [17]
Analysis of bradykinin in the presence of undigested BSA
When using MWCO for peptide isolation proteins are typically present in the samples
usually in larger amounts Figure 3 shows the effect that adding BSA protein to a 10 ng
bradykinin solution before MWCO fractionation has on the resulting recovery of bradykinin
Adding 10 μg of BSA to the optimized 7030 aqueous 1 M NaClmethanol solution slightly
decreased bradykininrsquos signal with a RSD of 10 More severe signal reduction occurred after
adding 100 microg BSA with a RSD of 2 (N=2) It is not unexpected that more signal reduction
due to sample loss would occur especially in the 100 microg BSA sample because the BSA protein
has a molar ratio of 160 BSA protein molecules to one bradykinin peptide Figure 3 shows the
usefulness of the MWCO method with samples containing large amounts of proteins
RecommendationConclusion
The present work provides solutions to reduce sample loss from the use of MWCO for
sub-microg peptide isolation with or without non-digested proteins in the sample Despite its
widespread utility significant sample loss often occurs during the MWCO fractionation step
which is particularly problematic when analyzing low-abundance peptides from limited starting
material This application note aims to reduce sample loss during MWCO separations
specifically for sub-microg peptide isolation If complex samples are processed with MWCO
separation the authors recommend eluting the sample with 6040 aqueous 1 M NaClmethanol
solution as a starting point to minimize sample loss This application note provides a viable
196
alternative for sub-microg peptide MWCO separation circumventing the need to increase the starting
material by minimizing sample loss from using a MWCO membrane-based centrifugal filter
device
References
[1] HM Georgiou GE Rice MS Baker Proteomic analysis of human plasma failure of
centrifugal ultrafiltration to remove albumin and other high molecular weight proteins
Proteomics 2001 1 1503
[2] DW Greening RJ Simpson Low-molecular weight plasma proteome analysis using
centrifugal ultrafiltration Methods Mol Biol 2011 278 109
[3] DW Greening RJ Simpson A centrifugal ultrafiltration strategy for isolating the low-
molecular weight (ltor=25K) component of human plasma proteome J Proteomics 2010 73
637
[4] LL Manza SL Stamer AJ Ham SG Codreanu DC Liebler Sample preparation and
digestion for proteomic analyses using spin filters Proteomics 2005 5 1742
[5] D Theodorescu D Fliser S Wittke H Mischak R Krebs M Walden M Ross E Eltze O
Bettendorf C Wulfing A Semjonow Pilot study of capillary electrophoresis coupled to mass
spectrometry as a tool to define potential prostate cancer biomarkers in urine Electrophoresis
2005 26 2797
[6] K Antwi G Hostetter MJ Demeure BA Katchman GA Decker Y Ruiz TD Sielaff LJ
Koep DF Lake Analysis of the plasma peptidome from pancreas cancer patients connects a
peptide in plasma to overexpression of the parent protein in tumors J Proteome Res 2009 8
4722
[7] LP Aristoteli MP Molloy MS Baker Evaluation of endogenous plasma peptide extraction
methods for mass spectrometric biomarker discovery J Proteome Res 2007 6 571
[8] A Zougman B Pilch A Podtelejnikov M Kiehntopf C Schnabel C Kumar M Mann
Integrated analysis of the cerebrospinal fluid peptidome and proteome J Proteome Res 2008 7
386
[9] X Yuan DM Desiderio Human cerebrospinal fluid peptidomics J Mass Spectrom 2005 40
176
[10] X Zheng H Baker WS Hancock Analysis of the low molecular weight serum peptidome
using ultrafiltration and a hybrid ion trap-Fourier transform mass spectrometer J Chromatogr A
2006 1120 173
[11] L Li JV Sweedler Peptides in the brain mass spectrometry-based measurement approaches
and challenges Annu Rev Anal Chem 2008 1 451
[12] GB Stefano G Fricchione Y Goumon T Esch Pain immunity opiate and opioid
compounds and health Med Sci Monit 2005 11 MS47
[13] J Jensen Regulatory peptides and control of food intake in non-mammalian vertebrates Comp
Biochem Physiol A Mol Integr Physiol 2001 128 471
197
[14] A Kuoppala KA Lindstedt J Saarinen PT Kovanen JO Kokkonen Inactivation of
bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma Am
J Physiol Heart Circ Physiol 2000 278 H1069
[15] R Chen M Ma L Hui J Zhang L Li Measurement of neuropeptides in crustacean
hemolymph via MALDI mass spectrometry J Am Soc Mass Spectrom 2009 20 708
[16] H Jahn S Wittke P Zurbig TJ Raedler S Arlt M Kellmann W Mullen M Eichenlaub H
Mischak K Wiedemann Peptide fingerprinting of Alzheimers disease in cerebrospinal fluid
identification and prospective evaluation of new synaptic biomarkers PLoS One 2011 6
e26540
[17] NA Cellar AS Karnoup DR Albers ML Langhorst SA Young Immunodepletion of high
abundance proteins coupled on-line with reversed-phase liquid chromatography a two-
dimensional LC sample enrichment and fractionation technique for mammalian proteomics J
Chromatogr B Analyt Technol Biomed Life Sci 2009 877 79
198
Table 1 Identified BSA tryptic peptides from various MWCO separation conditions
BSA tryptic
peptide (MH+)
100
H2O 1microg
100
1 M NaCl
70
H2O
80
1 M NaCl
70
1 M NaCl
60
H2O
60
1 M NaCl
5083
5453
6894
7124
8985
9275
10345
10725
11385
11636
12496
12837
13057
13997
14157
14197
14398
14636
14798
15026
15118
15328
15547
15677
15768
16399
16678
16738
17248
17408
17477
17497
18809
18890
19019
19079
20450
21139
22479
Total 39 2 2 6 8 15 15 27
199
Figure 1 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard showing improvement over two orders of magnitude in detection limits Each MWCO
separation was performed at minimum in triplicate with representative spectrum selected for
each with a calculated RSD from the peak heights Three different amounts of bradykinin were
tested to assess the magnitude of sample loss under different MWCO solvent conditions The
top panel shows 1 microg of bradykinin standard after MWCO separation with 100 H2O elution
produced no signal The addition of 40 or 30 MeOH produced very low bradykinin signals
for both 100 ng (RSD of 28) and 10 ng (SNlt3 no RSD calculated) respectively In the
bottom two spectra each showed very large intensity but the 7030 aqueous 1 M NaClmethanol
10 ng bradykinin was processed with a 10kDa MWCO and zip-tipped and was reproducible with
200
a RSD of 6 The last spectrum was from 10 ng bradykinin (RSD of 3) which was diluted to
an equivalent volume as all the other experiments and directly spotted onto the MALDI plate
201
Figure 2 Representative MALDI mass spectra from MWCO separation of a BSA tryptic
peptide standard showing sample loss Stacked mass spectra from mz range 875-2150
normalized to 7 x 104 intensity representing the detection difference from a BSA tryptic peptide
standard from different MWCO separation conditions (A) It should be noted that when the
solvent for MWCO elution was 100 H2O 1 microg of BSA tryptic peptides was processed instead
of 500 ng A zoomed in view of the most abundant BSA tryptic peptide detected YLYEIAR
mz 92749 (B) Various percentages of MeOH produced significant signal but addition of a salt
(1 M NaCl) increases the signal which is closest to a directly spotted BSA tryptic peptide
standard A zoomed in view of a representative low intensity BSA tryptic peptide detected
LKECC
DKPLLEK mz 153266 (C) The optimized solution to be used for MWCO filtration
202
6040 aqueous 1 M NaClmethanol was the only procedure that enabled the detection of the
tryptic peptide in Figure 2C which was also detected in the directly spotted BSA tryptic peptide
standard All experiments were performed a minimum of two times with nearly identical results
) Carbamidomethyl amino acid modification
ordm) Tryptic peptide identified in three of the spectra in Figure 2A
dagger) Tryptic peptide identified in two of the spectra in Figure 2A
) Tryptic peptide identified in a single spectrum in Figure 2A
203
Figure 3 Representative MALDI mass spectra after MWCO separation of a bradykinin
standard with a BSA protein present showing optimized solvent conditions minimized samples
losses Each experiment was performed in duplicate Two different amounts of BSA protein
were tested to assess the magnitude of sample loss caused by the presence of a protein The top
panel shows 10 microg of BSA protein and the second panel shows 100 microg of BSA protein added
while only 10 ng of bradykinin was added Detectable sample loss was observed when the BSA
protein was added but panel two shows that the amount of BSA protein was 1 x 104 greater
(equivalent to 160 fold molar excess) than bradykinin The last two spectra were controls using
a MWCO with the optimized solution in panel 3 and panel 4 using 10 ng bradykinin which was
diluted to an equivalent volume as all the other experiments and directly spotted onto the
MALDI plate
204
Supplemental Table 1 Expanded Table 1 including grand average of hydropathicity (GRAVY)
score theoretical pI and the sequence from the underlying amino acid sequence for the peptides
identified in the BSA digest The GRAVY and pI scores were obtained from ExPASy
Bioinformatics and modifications were not taken into consideration
(httpwebexpasyorgprotparam) Peptide assignments to the recorded peaks were done by
BSA
tryptic
peptide
(MH+)
GRAVY
score
Theoretical
pI
Sequence 100
H2O
1microg
100
1 M
NaCl
70
H2O
80
1 M
NaCl
70
1 M
NaCl
60
H2O
60
1 M
NaCl
5083 NA NA FGER
5453 0900 972 VASLR
6894 0267 979 AWSVAR
7124 -0950 647 SEIAHR
8985 0529 674 LcVLHEK
9275 -0071 600 YLYEIAR
10345 -0725 674 NEcFLSHK
10725 -0211 538 SHcIAEVEK
11385 0 599 ccTESLVNR
11636 0130 453 LVNELTEFAK
12496 -1250 545 FKDLGEEHFK
12837 0264 675 HPEYAVSVLLR
13057 -0582 532 HLVDEPQNLIK
13997 0567 437 TVMENFVAFVDK
14157 0567 437 TVmENFVAFVDK
14197 0058 530 SLHTLFGDELcK
14398 -0133 875 RHPEYAVSVLLR
14636 -0515 465 TcVADESHAGcEK
14798 0292 600 LGEYGFQNALIVR
15026 -0625 409 EYEATLEEccAK
15118 0207 597 VPQVSTPTLVEVSR
15328 -0617 617 LKEccDKPLLEK
15547 -0823 441 DDPHAcYSTVFDK
15677 -0085 437 DAFLGSFLYEYSR
15768 -0985 456 LKPDPNTLcDEFK
16399 -0067 875 KVPQVSTPTLVEVSR
16678 0064 437 MPCTEDYLSLILNR
16738 -1723 550 QEPERNEcFLSHK
17248 0064 437 MPcTEDYLSLILNR
17408 0064 437 mPcTEDYLSLILNR
17477 -0914 414 YNGVFQEccQAEDK
17497 -0621 410 EccHGDLLEcADDR
18809 -0537 606 RPcFSALTPDETYVPK
18890 -0567 674 HPYFYAPELLYYANK
19019 -1275 466 NEcFLSHKDDSPDLPK
19079 0044 454 LFTFHADIcTLPDTEK
20450 -0812 839 RHPYFYAPELLYYANK
21139 -0682 480 VHKEccHGDLLEcADDR
22479 -0458 423 EccHGDLLEcADDRADLAK
Total 39 2 2 6 8 15 15 27
205
mass matching with no tandem mass spectrometry performed Lower case amino acids indicate
a modification present in the peptide of carbamidomethyl (c) or oxidation (m)
206
Chapter 8
Conclusions and Future Directions
207
Summary
Comparative shotgun proteomics investigating numerous biological changes in various
species and sample media and peptidomic method development have been reported The
developed comparative shotgun proteomics based on label-free spectral counting with nanoLC
MSMS platform have been employed in mouse CSF rat CSF yeast culture and other biological
specimens Mass spectrometry (MS)-based peptidomic enhancements using ETD and improved
sample preparation methods for molecular weight cut-offs have been reported Together these
studies add to the Li Labs capabilities in comparative shotgun proteomics and expand available
methods for peptidomic research
Immunodepletion of CSF for comparative proteomics
Chapters 3 and 4 used similar methods to generate a list of differentially expressed
proteins providing insight into how Alexander disease (AxD) perturbs the proteome and how the
new prion model rat adapted scrapie (RAS) proteome varies from control rats In the GFAP
overexpressor mouse CSF study in Chapter 3 we have produced a panel of proteins with
significant up or down regulation in the CSF of transgenic GFAP overexpressor mice MS-based
proteomic study of this mouse model for AxD was consistent with the previous studies showing
elevation of GFAP in CSF The development of a modified IgY-14 immunodepletion technique
for low amounts of CSF with recommendations for future antibody depletion techniques to deal
with the unique challenges of mouse CSF was presented Modified proteomics protocols were
employed to profile mouse CSF with biological and technical triplicates addressing the
variability and providing quantitation with dNSAF spectral counting Validation of the data was
performed using both ELISA and RNA microarray data to provide corroboration with the
208
changes in the putative biomarkers This work presents numerous interesting targets for future
study in AxD including CK-MCK-B cathepsin S L1 and B isoforms and contactin-1
Using the protocol developed in Chapter 3 we performed a similar study in RAS CSF
compared to control rat CSF Two differences in sample preparation for the rat CSF compared
to the mouse CSF are noted (1) rat IgY-7 immunodepletion was performed and (2) each rat
CSF sample was collected from one animal due to sufficient volume instead of pooling from
multiple animals for the mouse CSF sample After immunodepletion the CSF samples from
control and RAS (biological N=5 technical replicates N=3) were digested and separated using
one dimensional RP nanoLC separation We were able to identify 167 proteins with redundant
isoforms removed which was derived from total 512 proteins with a 1 FDR from all the CSF
samples Comparative analysis was performed using dNSAF spectral counting and 21 proteins
were significantly changed Our data were consistent with previous prion CSF studies showing
14-3-3 protein increased in CSF of prion infected animals Genomic analysis was also
performed and was used to cross-validate our proteomic data and numerous proteins were found
to be consistent including a novel marker related to prion disease RNAset2 (ribonuclease T2)
In summary this work provides a foundation for investigation of the perturbed proteome of a
new prion model RAS
Yeast Phosphoproteomic Comparison between Starved and Glucose Fed Conditions
This work presented a qualitative comparison of the phosphoproteome between starved
and glucose fed Saccharomyces cerevisiae (bakers yeast) A large scale IMAC enrichment of
yeast cell lysate followed by 2-D separations provided a rich source of phosphopeptides for CID
MSMS and neutral loss triggered ETD analysis using mass spectrometry A specific motif for
PKA was highlighted to show the differences in proteins identified between starved and glucose
209
fed conditions In total 477 unique phosphopeptides were identified with 06 FDR and 669
unique phosphopeptides identified with 54 FDR Phosphosite validation was performed using
a localization algorithm Ascore to provide further confidence on the site-specific
characterization of these phosphopeptides Two proteins Ssd1 and PMA2 emerged as potential
intriguing targets for more in-depth studies on protein phosphorylation involved in glucose
response
Methods for Peptide Sample Preparation and Sequencing
In this study ETD was performed to improve the sequence coverage of endogenous large
neuropeptides and labile tyrosine sulfation CCK-like neuropeptides found in the blue crab
Callinectes sapidus The intact CPRP from Callinectes sapidus was identified and characterized
with 68 sequence coverage by MS for the first time Cation assisted ETD fragmentation using
MgCl2 was also shown to be a powerful tool in de novo sequencing of sulfated neuropeptides
These endeavors into using ETD for certain neuropeptides will assist in future analysis of large
neuropeptides and PTM containing neuropeptides
In addition to ETD sequencing I presented a protocol on improving recovery of minute
quantities of peptides by adding methanol and a salt modifier (NaCl) to molecular weight cut-off
membrane-based centrifugal filters (MWCO) to enrich sub-microgram peptide quantities
Despite its widespread utility significant sample loss often occurs during the MWCO
fractionation step which is particularly problematic when analyzing low-abundance peptides
from limited starting material This work presented a method to reduce sample loss during
MWCO separations specifically for sub-microg peptide isolation Using a neuropeptide standard
bradykinin sample loss was reduced by over two orders of magnitude with and without
210
undigested protein present The presence of bovine serum albumin (BSA) undigested protein
and the bradykinin standard lends evidence that sample loss occurs because of the MWCO and
not the presence of the protein Additionally a BSA tryptic digestion is presented where twenty-
seven tryptic peptides are identified from MALDI mass spectra after enriching with methanol
while only two tryptic peptides are identified after the standard MWCO protocol
Ongoing Projects and Future Directions
CSF Projects
Rat Adapted Scrapie and Time Course Study of Rat CSF
In ongoing experiments from the work described in Chapter 4 related to rat adapted
scrapie (RAS) we are working towards more complete proteome coverage of CSF and a time
course study of RAS After the promising results of the 1-D proteomic comparison between
RAS and control CSF we want to perform IgY immunodepletion on 1 mL of rat CSF followed
by 2-D high pH-low pH RP-RP LC-MSMS IgY immunodepletion is still required and
afterwards approximately 40 microg of low abundance protein would remain Following traditional
urea tryptic digestion (see Appendix 1) and solid phase extraction (SPE) clean-up the sample
would be dried down and reconstituted in mobile phase A (25 mM ammonium formate basic
pH) for high pH RP separation Fractions would be dried and subjected to MS analysis similar to
the work described in Chapter 4 The purpose of this work would be to increase the proteome
coverage from a few hundred to a thousand CSF proteins identified A time course study of RAS
is also desirable to gain insight into disease progression Rats at different stages will be
sacrificed and CSF will be collected The goal is to perform isobaric labeling between the time
courses with spectral counting being an alternative for relative protein expression We will use
the targets identified in Chapter 4 to track certain proteins for time course analysis Overall
211
these future projects will dig deeper into the proteome and how this novel prion model RAS
perturbs the proteins expressed in rats over time
Glycoproteomics and Peptidomic Analysis of CSF Samples from Individuals with
Alzheimerrsquos Disease
Alzheimerrsquos disease is an incurable progressive neurodegenerative disorder which results
in cognitive impairment Our aim is to provide additional biomarkers andor targets for drug
treatment Currently we used 500 microL of human CSF for non-membrane glycoprotein
enrichment (see Appendix 1) followed by 2-D high-pH-low-pH RP-RP amaZon ion trap LC-
MSMS analysis The initial work was a failure due to low amount of signal and significant
sample loss (data not shown) Through a comparison with Dr Xin Weirsquos PhD thesis we
estimated roughly 20 microg of proteins was obtained after glycoprotein enrichment and 1-D analysis
produced over 100 protein identifications (data not shown) but the additional off-line 2-D
separation and sample clean up resulted in low number of protein identifications for each fraction
analyzed by MS (data not shown) We have recently obtained 1 mL of human CSF samples
from healthy controls and individuals suffering from Alzheimerrsquos disease and plan to perform
the same experiments with double the starting amount and reduce the fractions collected from 2-
D separation from 11 to 6 In addition to the glycoproteomics anything not enriched will be
subjected to MWCO separation using the method presented in Chapter 7 The obtained peptide
sample will be subjected to Q-TOF nanoLC MSE data acquisition followed by de novo
sequencing using various programs including PEAKS and Mascot Collectively we feel this
project has great potential to lead to interesting targets and further expand the proteomic
knowledge of Alzheimerrsquos disease
GFAP Knock-in Mouse CSF
212
In a direct follow-up to Chapter 3 we aim to perform comparative proteomics on control
vs glial fibrillary acidic protein (GFAP) knock-in mouse CSF samples The sample preparation
protocol as presented in Chapter 3 will be used For quantitative proteomics we plan on
performing isobaric labeling followed by performing high energy collision induced dissociation
(HCD) on the Orbitrap Elite The MS acquisition will also perform a top ten scan where the top
ten MS peaks will be selected for tandem MS analysis In addition targeted quantitation of
specific proteins using multiple reaction monitoring (MRM) can be performed on potential
biomarkers and identified in this follow-up study and the work presented in Chapter 3 Also any
CSF samples with noticeable blood content cannot be used for the exploratory proteomics
experiments but can potentially be used for the MRM analysis and should be kept for such
experiments in the future
Large Scale Proteomics
Proteomics of Mouse Amniotic Fluid for Lung Maturation
The overall goal of this project is to determine what proteins are present in amniotic fluid
when the embryo is 175 weeks compared to amniotic fluid at 155 weeks The rationale behind
why these two time points matter was investigated through a lung explant culture where amniotic
fluid was added at 155 weeks and 175 weeks Visual maturation of lungs occurred with the
175 week amniotic fluid and lung maturation genes were up regulated in the mRNA in the lung
explant culture when compared to the 155 week amniotic fluid The compound which is
causing the maturation of the lungs is unknown and search for a secreted protein might provide a
clue to this process In order to test this hypothesis we carried out discovery proteomics
experiments The workflow involves tryptic digestion SPE and 1-D low pH nanoLC separation
coupled to an amaZon ETD ion trap for tandem MS analysis The resulting mass spectrometric
213
acquisition identified less than 100 proteins with a 1 FDR (data not published) To address the
lack of depth in the proteome coverage we purchased an IgY immunodepletion column to
remove the most abundant proteins in amniotic fluid similarly to Chapter 3 and 4 but on a larger
scale One abundant protein alpha-fetoprotein is present in amniotic fluid but absent or present
in very low concentration in CSF serum or plasma Alpha-fetoprotein is similar to albumin and
thus we ran amniotic fluid on an IgY immunodepletion column and observed significant
reduction in alpha-fetoprotein amount (data not published) After immunodepletion 2-D high
pH-low pH RP-RP will be performed followed by nanoLCMSMS on the amaZon ETD ion trap
We will perform mass spectrometry experiments using the 155 week amniotic fluid and 175
week amniotic fluid which have average protein contents ~2 mgmL We anticipate a minimum
of tenfold increase in the proteome coverage of amniotic fluid and provide meaningful
hypothesis driven biological experiments from this work
Phosphoproteomics of JNK Activation
c-Jun N-terminal kinase (JNK) is an important cellular mediator of stress activated
signaling Under conditions of oxidative stress JNK is activated resulting in the downstream
phosphorylation of a large number of proteins including c-Jun However the cellular response
to JNK activation is extremely complex and JNK activation can result in extremely different
physiological outcomes For example JNK is at the crossroads of cellular death and survival
and exhibits both pro-death and pro-survival activities These highly disparate outcomes of JNK
activation are highly contextual and depend on the type of stressor and duration of stress In the
brain JNK has been shown to be activated in models of Alzheimerrsquos disease Parkinsonrsquos
disease amyotrophic lateral sclerosis and stroke It is thought that JNK is activated in these
diseases as a result of oxidative stress and it is unknown whether this activation is pro-survival or
214
pro-death To elucidate JNK signaling in the brain we chose to analyze primary cortical
astrocytes under conditions of oxidative stress Since hydrogen peroxide is a physiologically
relevant oxidant in the brain and known to activate JNK we chose to use this to treat astrocytes
and then analyze changes to the phosphoproteome by mass spectrometry By doing this we
hope to elucidate important JNK-dependent targets involved in stress signaling in the brain and
that identifying these targets could lead to the identification of novel disease mechanisms and
potentially new therapeutic targets for neurodegeneration Specifically we plan on performing
stable isotope labeling by amino acids (SILAC) of astrocytes for control hydrogen peroxide
treated and hydrogen peroxide treated with JNK inhibitor The SILAC labeled astrocyte cell
lysate will be digested enriched using Fe-NTA IMAC and separated using 2-D high pH-low pH
RP-RP and infused into the amaZon ETD ion trap The protocol is similar to the yeast
comparative phosphoproteomics from Chapter 5 After analysis we plan on processing the data
using ProteoIQ to identify phosphoproteins with significant changes
Immunoprecipitation Followed by Mass Spectrometry
Stb3 Mass Spectrometry Analysis
Stb3 (Sin3-binding protein) has previously been shown to change location depending on
the presence or absence of glucose (Heideman Lab Dr Michael Conways Thesis) An
immunoprecipitation of Stb3 was performed in parallel to the work described in Chapter 5 Two
separate experiments were performed one with wild type Stb3 and another tagged with myc for
improved protein immunoprecipitation Myc tagging is a polypeptide tag which can be
recognized by an antibody and provide a better enrichment compared to using a Stb3 antibody
alone The myc tagging was done because of the low abundance of Stb3 and the limited amount
of protein that was pulled down by the antibody for Stb3 Immunopreciptation experiments were
215
performed for both starved and glucose fed samples All samples were tryptically digested
followed by low pH RP nanoLCMSMS Both CID and ETD were used for fragmentation
analysis of each sample (n=2) The Stb3 wild type had two peptide hits in one MS run which is
not surprising due to low Stb3 antibody affinity In contrast a significant amount of Stb3 was
pulled down from Myc tagged starved and glucose fed samples For the glucose starved
samples 22 unique peptides from Stb3 were identified whereas glucose fed samples yielded 21
unique Stb3 peptides identified (unpublished data) The identified Stb3 in the myc samples
allowed us to investigate what other proteins were pulled down that are not present in the wild
type samples
From previous work by our collaborator Dr Heideman it had been suggested that Stb3
forms a complex with Sin3 and Rpd3 Our proteomic data shows multiple significant peptide
hits for Sin3 and Rpd3 in myc tagged but not in wild type where Stb3 is only observed once
with a low Mascot score When looking at the unique proteins identified in myc tagged glucose
fed sample but not included in the wild type samples the myc fed sample contained eight unique
ribosomal proteins with numerous unique peptides for each The ribosomal proteins identified in
myc fed sample supports the novel hypothesis that in the glucose fed state the complex of Stb3
Rpd3 and Sin3 interact with ribosomal proteins Other proteins unique to myc tagged glucose
starved samples are glyceraldehyde-3-phosphate dehydrogenase 2 and transcriptional regulatory
protein UME6 Also three proteins were observed in both myc fed and starved but not in the
wild type samples including transposon Ty1-DR1 Gag-Pol polyprotein(YD11B) SWIRM
domain-containing protein (FUN19) and RNA polymerase II degradation factor 1 (DEF1) Our
proteomics data for the Stb3 immunoprecipitation experiments with starved vs glucose fed
216
samples provide exciting evidence to support previous observation made by the Heideman group
and highlight the utility of MS-based approach to deciphering protein-protein interactions
Conclusions
The majority of the work described in this dissertation revolves around sample
preparation for proteomics and peptidomics with a focus on generating biologically testable
hypotheses In the area of neuropeptides CPRP was fully sequenced analytical methods were
transformed for use in an ETD ion trap and sub-microg peptides can now be analyzed by mass
spectrometry after MWCO separation In the field of comparative proteomics comparisons
between mouse CSF in GFAP overexpressors and control or rat adapted scrapie model and
control CSF yeast fed vs glucose starved cell extract have all been presented Collectively this
thesis has developed new techniques for neuropeptide sample preparation and presented
numerous comparative proteomic analyses of various biological samples and how the proteomes
are dynamically perturbed by various treatments and disease conditions
217
Appendix 1
Protocols for sample preparation for mass spectrometry based
proteomics and peptidomics
218
Small Scale Urea Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 8 μL of urea solution
(400mg05mL) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Allow to digest overnight in 37degC water bath
10 Acidify with 10μL 10 formic acid
11 Perform solid phase extraction using tips dependent of sample amount
a Sub-5μg amounts ndash Millipore Ziptips
b 5-75μg amounts ndash Bond-Elut tips from Agilent (OMIX tips)
12 Dry down in Speedvac as needed for experiment
219
Small Scale ProteaseMAX Tryptic Digestion (below 20 microg)
1 Concentratedilute sample to 10 μL starting volume
2 All solutions are in a 50mM NH4HCO3 buffer (395mgmL)
3 Add 1 μL of 05M Dithiothreitol (DTT) (77mg100μL) and 2 μL of 1 of
ProtesaeMAX (Promega) to the sample
4 Place sample in 37degC water bath for 45 minutes
5 Add 27μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
6 Quench reaction with 1μL of DTT solution and let sit for 10 minutes
7 Dilute with 70μL of NH4HCO3 solution
8 Use 1μL of trypsin (05μgμL)
9 Add 1 μL ProteaseMAX and let sit for 3-4 hours
10 Acidify with 2μL 10 formic acid
11 Dry down in Speedvac as needed for experiment
220
Large Scale Urea Tryptic Digestion (mg of proteins)
1 All solutions are in a 50 mM NH4HCO3 buffer (395 mgmL)
2 Add 20μL of 05M Dithiothreitol (DTT) (77mg100μL) and 160μL of urea solution
(400mg05mL) to sample
3 Allow sample to denature 45-60 minutes in a 37degC water bath
4 Add 54μL of Iodoacetamide (IAA) (10mg100μL) and allow to react (in the dark) for
15 minutes
5 Quench reaction with 20μL of DTT solution
6 Dilute with 14mL of NH4HCO3 solution
7 Add 100μg of trypsin
8 Allow to digest overnight in 37degC water bath
9 Acidify sample with 100μL of 10 formic acid
10 Perform solid phase extraction with Hypersep C18 Cartridges with 100 mg of C18
bead volume (Thermo)
11 Dry down with the Speedvac as needed for experiment
221
Fe-NTA Preparation from Ni-NTA Beads
1 Centrifuge (5 min at 140 rcf) and use a magnet to keep beads in place as supernatant
is removed
2 Wash 3 times with 800μL of H2O (using same technique of centrifuging and using
magnet to keep beads in places as supernatant is removed)
3 Add 800μL of 100mM EDTA (372mgmL) in a 50mM NH4HCO3 (395mgmL)
buffered solution (pH around 8) and vortex for 30 minutes to chelate the Ni
centrifuge and remove supernatant
4 Wash 3 times with 800μL of H2O
5 Add 800μL of 10mM FeCl3 hexahydrate (27mgmL) and vortex for 30 minutes to
bind Fe to beads centrifuge and remove supernatant
6 Wash 3 times with 800μL H2O
7 Resuspend in a storage solution of 111 ACNMeOH001 Acetic Acid (1mL total)
222
Fe-NTA IMAC Phospho-enrichment
1 Use wash solution (80ACN 01Formic Acid) to rinse beads vortex 1 minute
centrifuge and remove supernatant
2 Add sample (around 500μL in 80ACN 01Formic Acid) and vortex 40 minutes to
allow sample to bind dispose of supernatant after centrifuging
3 Wash 3 times with 200μL of wash solution discard supernatant
4 Add 200μL of elution solution (5050 ACN5Ammonium Hydroxide) vortex 15
minutes and save supernatant
5 Add 200μL of elution solution vortex 10 minutes and save supernatant
6 Wash 2 time with wash solution (collect supernatant of first wash)
7 If reusing store in 1mL solution of 111 ACNMeOH001 Acetic Acid
223
High pH Off-line Separation
1) In general a minimum of 20 microg of peptides are needed to gain any benefit
from off-line 2D fractionation It is better to inject 100 microg of peptides on
column
2) Use a Gemini column or a similar column that can handle pH=10 and for this
protocol a 21 mm x 150 mm column was used
3) Prepare ldquobuffer Ardquo 25 mM NH4 formate pH=10 Also prepare ldquobuffer Brdquo
4) Dry down desired sample and reconstitute in buffer A
5) Inject based off sample loop (current Alliance HPLC has a 50 microL sample
loop)
6) Run gradient at bottom of the page collecting fractions every 3 minutes except
for the 1st minute which is the void volume
7) Optional If you want to reduce instrument time you can combine fractions 1
with 12 2 with 13 etc until 11 with 22
Time Mobile phase A Mobile phase B Flow Rate
05mlmin
0 98 2 05 mLmin
65rsquo 70 30 05 mLmin
65rsquo1rdquo 5 95 05 mLmin
70 5 95 05 mLmin
224
Non Membrane Glycoprotein Enrichment
1 For protocols involving membrane glycoprotein enrichment see Dr Xin Weirsquos
thesis
2 Add 75 microL of Wheat germ agglutinin (WGA) bound to agarose beads and 150 microL
of Concanavalin A (ConA) to a Handee spin cup (Thermo) Wash 2 times with
lectin affinity chromatography (LAC) binding buffer (015 M NaCl 002 M Tris-
HCl 1 mM CaCl2 pH=74) centrifuging at 400 g for 30 seconds
3 Place stopper on bottom of spin cup and add 200 microg of sample (500 microL for CSF)
Bring up to 300 microL using lectin LAC binding buffer
4 Incubate for 1 hour with continuous mixing at room temperature
5 Centrifuge at 400 g for 30 seconds
6 Perform two more 300 microL LAC binding washes followed by centrifugation
7 Add 300 microL LAC eluting buffer (0075 M NaCl 001 M Tris-HCl 02 M alpha-
methyl mannoside 02 M alpha-methyl glucoside and 05 M acetyl-D-
glucosamine) vortex for 10 minutes (have stopper in place while vortexing)
centrifuge and collect
7 Add another 300 microL LAC eluting buffer centrifuge and collect
225
MWCO separation for Sub-microg peptide concentrations
1 Wash molecular weight cut-off (MWCO) with 5050 waterMeOH centrifuge at
14000 g for 5 minutes for 10 kDa filter (time will vary Millipore Amicon Ultra
filters)
2 Wash with 100 water centrifuge at 14000 g for 5 minutes
3 Add methanol to the sample to get the concentration to 30 methanol and add
salt to achieve a roughly 05 M NaCl before adding the sample to the MWCO
4 Centrifuge at 14000 for 10 minutes collect flow through
226
Immunoprecipitation
Modified from Thermo Fisher Scientific Classic IP Kit
1 10 microL Polyclonal antibody added to 1 microg protein standard in a Handee spin cup
(Thermo) diluted with 1x TBS buffer to 400 microL and incubated overnight at on
shakerend-over-end rotator
2 Wash Protein GA beads with 2x 200microL TBS buffer and added to the
antibodysample for 15 hours at 4oC
3 Centrifuge at 400 g for 30 seconds and discard flow through
4 Wash 3x with 100microL TBS buffer centrifuge at 400 g for 30 seconds and discard
flow through
5 Wash with 1x conditioning solution (neutral pH buffer) centrifuge at 400 g for 30
seconds and discard flow through
6 Eluted with elution buffer (2x100microL) centrifuge at 400 g for 30 seconds and
collect flow through
227
C18 Solid Phase Extraction (SPE)
1 Determine amount of peptides for SPE ge5 microg use Ziptips from Millipore If
between 5-75 microg use OMIX from Agilent 100 microL version and if ge75 microg use SPE
cartridges such as 100 mg Hypersep from Thermo
2 Ensure no detergents are in the sample and it is acidified
3 The three SPE procedures all use the same sets of solutions only volumes vary
4 3x Wetting solution (100 ACN) for 60 microL OMIX (20 microL ziptip and 15 mL for
100 mg cartridge)
5 3x Wash solution (01 formic acid in 100 H2O) same volumes as 4
6 Draw up the sample about 10 times minimum (generally 30-40 is preferred)
without letting the bead volume dry out
7 1X Wash solution same volumes as 4
8 Back load the eluting solution 01 formic acid in 5050 H2OACN into the
Ziptips (15 microL) or OMIX (50 microL) tips For the SPE cartridge add 700 microL of
eluting solution
9 Dry down and prepare for next step in sample preparation
228
Laser Puller Programs and Protocols
1) Obtain about two feet of Fused-silica capillary with 75 μm id and 360 μm od
2) Wash with methanol and then air dry using the bomb
3) Cut into one foot or desired length
4) Use a lighter to burn the middle portion (about an inch in length) of the tubing
5) Remove the ash by rinsing with methanol and rubbing with a Kimwipe
6) Make sure the laser puller has been on for at least 30 minutes before use to allow
for the instrument to warm up
7) Place capillary in instrument with the burnedexposed portion in the center
making sure that the length of the capillary is pulled taut
8) Enter desired program (next page) and press pull
229
Laser Puller Programs
Program 99 (default lab program)
Heat Filament Velocity Delay Pull
250 0 25 150 15
240 0 25 150 15
235 0 25 150 15
245 0 25 150 15
Program 97 (developed for larger inner diameter tips)
Heat Filament Velocity Delay Pull
230 - 25 150 -
220 - 25 150 -
215 - 25 150 8
230
On column Immunodepletion (serum plasma CSF amniotic fluid)
1) Prepare buffer A ldquoDilution Bufferrdquo 10 mM Tris-HCl pH=74 150 mM NaCl
2) Prepare buffer B ldquoStripping Bufferrdquo 01 M Glycine-HCl pH=25
3) Prepare buffer C ldquoNeutralization Bufferrdquo 100 mM Tris-HCl pH=80
4) Determine loading amount (LC1=~1000 ug of serum or plasma less for CSF due
to the increased amount of albumin percentage in CSF)
5) Add Dilution buffer to sample before injection and ensure the sample is proper
pH (~7)
6) Use gradient below
Time A B C Flow Rate
(mLmin)
0rsquo 100 0 0 02
4rsquo59rdquo 100 0 0 02
5rsquo 100 0 0 05
8rsquo59rdquo 100 0 0 05
9rsquo 0 100 0 05
22rsquo 0 100 0 05
22rsquo1rdquo 0 0 100 05
39rsquo 0 0 100 05
7) Store the column in 1x dilution buffer until next use
231
Small Scale Immunodepletion (100 microL of CSF)
1) Use 75 microg (per 100 microL CSF) of Sigma Seppro IgY-14 or IgY-7 bead slurry
2) Add an equal volume of 2x dilution buffer (20 mM Tris-HCl pH=74 300 mM
NaCl) to the starting amount of CSF
3) Add to a spin cup with a filter and allow to mix for 30 minutes
4) Centrifuge at 400 g for 30 seconds and collect the flow through
5) Add 50 microL of 1x dilution buffer mix for 5 minutes centrifuge at 400 g for 30
seconds and collect the flow through
6) Use 1x stripping washes (01 M Glycine-HCl pH=25) centrifuge as before and
discard Repeat four times
7) Use 1x neutralization buffer (100 mM Tris-HCl pH=80) centrifuge as before
and discard Repeat two times
8) Store the beads in the spin column in 1x dilution buffer until next use
232
Alliance Maintenance Protocol
Seal Wash
10 methanol no acetonitrile This wash cleans behind the pump-head seals to
ensure proper lubrication Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime SealWsh gt Start
2 Press Stop after 5 minutes
Prime Injector
10 methanol for maintenance high organic solvent for dirty runs (eg 95
acetonitrile) Done before injecting any real samples to ensure no bubbles are
present in the injector Minimum once per week
1 On instrument interface navigate MenuStatus gt Diag gt Prime NdlWsh gt Start
2 After completion press Close
Purge Injector
Solvent is dependent on run Run this protocol at beginning of experiments each day
Minimum once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Navigate Direct Function gt 4 Purge Injector gt OK
3 Set Sample loop volumes 60 leave Compression Check unchecked gt OK
Prime Solvent Pumps
Solvent is dependent on run If solvents are changed run this protocol Minimum
once per week for maintenance
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys choose composition A type 100 Enter x4
3 Navigate Direct Function gt 3 Wet Prime gt OK
4 Set Flow Rate 7000 mLmin Time 100 min gt OK
5 Repeat for all changedactive solvent pumps
Condition Column
Dependent on user Use starting conditions for run
1 On instrument interface navigate MenuStatus gt Status screen
2 Using arrow keys type starting solvent compositions for run
3 Navigate Direct Function gt 6 Condition Column gt OK
4 Set Time as desired
233
Appendix 2
List of Publications and Presentations
234
PUBLICATIONS
ldquoDiscovery and characterization of the crustacean hyperglycemic hormone precursor related
peptides (CPRP) and orcokinin neuropeptides in the sinus glands of the blue crab Callinectes
sapidus using multiple tandem mass spectrometry techniquesrdquo Hui L Cunningham R Zhang
Z Cao W Jia C Li L J Proteome Res 2011 10(9) 4219-29
ldquoInvestigation and reduction of sub-microgram peptide loss using molecular weight cut-off
fractionation prior to mass spectrometric analysisrdquo Cunningham R Wang J Wellner D Li L
Journal of Mass Spectrometry In Press
ldquoMass spectrometry-based proteomics and peptidomics for systems biology and biomarker
discoveryrdquo Cunningham R Ma D Li L Frontiers in Biology 2012 7(4) 313-35
ldquoProtein changes in immunodepleted cerebrospinal fluid from transgenic mouse models of
Alexander disease detected using mass spectrometryrdquo Cunningham R Jany P Messing A Li
L Journal of Proteome Research Submitted
ldquoInvestigation of the differences in the phosphoproteome between starved vs glucose fed
Saccharomyces cerevisiaerdquo Cunningham R Grunwald D Wellner D Conway M Heideman
W Li L In preparation
ldquoIdentification of astrocytic JNK targets by phosphoproteomics using mass spectrometryrdquo
Cunningham R Dowell J Wang J Wellner D Li L Milhelm M In preparation
ldquoGenomic and proteomic profiling of rat adapted scrapierdquo Herbst A Cunningham R Wellner
D Wang J Ma D Li L Aiken J In preparation
235
INVITED SEMINARS AND CONFERENCE PRESENTATIONS
Robert Cunningham Davenne Mayour and Shoshanna Coon ldquoMorphology and Thermal
Stability of Monolayers on Porous Siliconrdquo The 231th
ACS National Meeting 2006 Atlanta
GA
Robert Cunningham Xin Wei Paige Jany Albee Messing and Lingjun Li ldquoMass
Spectrometry-Based Analysis of Cerebrospinal Fluid Peptidome and Proteome for Biomarker
Discovery in Alexander Diseaserdquo The 57th
ASMS Conference 2009 Philadelphia PA
Robert Cunningham ldquoWhat to Expect in a PhD Graduate Schoolrdquo Invited seminar University
of Northern Iowa 2010 Cedar Falls IA
Robert Cunningham Dustin Frost Albee Messing and Lingjun Li ldquoInvestigation of an
Optimized ProteaseMAX Assisted Trypsin Digestion of Human CSF for Pseudo-SRM
Quantification of GFAP and Identification of Biomarkersrdquo The 58th
ASMS Conference 2010
Salt Lake City UT
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo Oral presentation at Pittcon Conference and Expo 2011 2011 Atlanta
GA
Robert Cunningham Michael Conway Daniel Wellner Douglas Grunwald Warren
Heideman and Lingjun Li ldquoDevelopment of optimized phosphopeptide enrichment methods for
comparison of starved and glucose fed yeast Saccharomyces cerevisiaerdquo The 59th
ASMS
Conference 2011 Denver CO
Robert Cunningham Paige Jany Albee Messing and Lingjun Li ldquoMass Spectrometry-Based
Analysis of GFAP Overexpressor Micersquos Cerebrospinal Fluid for Protein Biomarker Discovery
in Alexander Diseaserdquo 2011 Wisconsin Human Proteomics Symposium 2011 Madison WI
