mass spectrometry for protein quantification and identification of posttranslational modifications
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Mass Spectrometry for Protein Quantification and Identification of Posttranslational Modifications. Joseph A. Loo Department of Biological Chemistry David Geffen School of Medicine Department of Chemistry and Biochemistry University of California Los Angeles, CA USA. protein-ligand - PowerPoint PPT PresentationTRANSCRIPT
Mass Spectrometry for Protein Quantification and Identification of Posttranslational
ModificationsJoseph A. LooJoseph A. Loo
Department of Biological ChemistryDepartment of Biological ChemistryDavid Geffen School of MedicineDavid Geffen School of Medicine
Department of Chemistry and Department of Chemistry and BiochemistryBiochemistry
University of CaliforniaUniversity of CaliforniaLos Angeles, CA USALos Angeles, CA USA
Proteomics and posttranslational modifications
Patterson and Aebersold, Nature Genetics (supp.), 33, 311 (2003)
protein-ligandprotein-ligandinteractionsinteractions
protein-ligandprotein-ligandinteractionsinteractions
proteinproteincomplexescomplexes(machines)(machines)
proteinproteincomplexescomplexes(machines)(machines)
protein familiesprotein families(activity or structural)(activity or structural)
protein familiesprotein families(activity or structural)(activity or structural)
post-translationalpost-translationalmodified proteinsmodified proteins
post-translationalpost-translationalmodified proteinsmodified proteins
Eukaryotic cell.Examples of protein
properties are shown, including the interaction of proteins
and protein modifications.
Proteomic Analysis of Post-translational Modifications
Post-translational modifications (PTMs) Covalent processing events that change the properties
of a protein proteolytic cleavage addition of a modifying group to one or more amino
acids Determine its activity state, localization, turnover,
interactions with other proteins Mass spectrometry and other biophysical methods can
be used to determine and localize potential PTMs However, PTMs are still challenging aspects of
proteomics with current methodologies
Complexity of the ProteomeComplexity of the Proteome
Protein processing and modification comprise an important third dimension of information, beyond those of DNA sequence and protein sequence.
Complexity of the human proteome is far beyond the more than 30,000 human genes.
The thousands of component proteins of a cell and their post-translational modifications may change with the cell cycle, environmental conditions, developmental stage, and metabolic state.
Proteomic approaches that advance beyond identifying proteins to Proteomic approaches that advance beyond identifying proteins to elucidating their post-translational modifications are needed.elucidating their post-translational modifications are needed.
Use MS to determine PTM of isolated protein
Enzymatic or chemical degradation of modified protein
HPLC separation of peptides
MALDI and/or ESI used to identify PTM
MS/MS used to determine location of PTM(s)
Proteomic analysis of PTMs
Mann and Jensen, Nature Biotech. 21, 255 (2003)
Glycoprotein Gel StainGlycoprotein Gel Stain
CandyCane glycoprotein molecular weight standards containing alternating glycosylated and nonglycosylated proteins were electrophoresed through a 13% polyacrylamide gel. After separation, the gel was stained with SYPRO Ruby protein gel stain to detect all eight marker proteins (left). Subsequently, the gel was stained by the standard periodic acid–Schiff base (PAS) method in the Pro-Q Fuchsia Glycoprotein Gel Stain Kit to detect the glycoproteins alpha2-
macroglobulin, glucose oxidase, alpha1-glycoprotein and
avidin.
Pro-Q™ Glycoprotein Stain (DDAO phosphate)Molecular Formula: C15H18Cl2N3O5P (MW 422.20)
Detection of glycoproteins and total protein on an SDS-polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.
Nitro-Tyrosine Modification
Oxidative modification of amino acid side chains include methionine oxidation to the corresponding sulfone, S-nitrosation or S-nitrosoglutationylation of cysteine residues, and tyrosine modification to yield o,o’-dityrosine, 3-nitrotyrosine and 3-chlorotyrosine.
Nitric oxide (NO) synthases provide the biological precursor for nitrating agents that perform this modification in vivo. NO can form nitrating agents in a number of ways including reacting with superoxide to make peroxynitrite (HOONO) and through enzymatic oxidation of nitrite to generate NO·
2
Tyrosine nitration is a well-established protein modification that occurs in disease states associated with oxidative stress and increased nitric oxide synthase activity.
The combination of 2D-PAGE, western blotting, and mass spectrometry has been the more typical strategy to identify 3-nitrotyrosine-modified proteins.
Nitro-Tyrosine Modification
“Proteomic method identifies proteins nitrated in vivo during inflammatory challenge,” K. S. Aulak, M. Miyagi, L. Yan, K. A. West, D. Massillon, J. W. Crabb, and D. J. Stuehr, Proc. Natl. Acad. Sci. USA 2001; 98: 12056-12061.
Anti-nitrotyrosine immunopositive proteins in lung of rats induced with LPS.
116
98
55
37
30
20
kDa
3.5 9.54.5 5.1 5.5 6.0 7.0 8.4 3.5 9.54.5 5.1 5.5 6.0 7.0 8.4
MAPK phosphatase 2MAPK phosphatase 2E2
G1
enolaseenolasecasein kinase IIcasein kinase II
HSP70HSP70
Naf-1Naf-1
Diesel Exhaust Particle-Induced Nitro-Tyrosine ModificationsDiesel Exhaust Particle-Induced Nitro-Tyrosine Modifications
RAW 264.7 macrophage exposed to DEP (Xiao, Loo, and Nel - UCLA)
Sypro Rubyanti-nitro-tyrosine
trans. factor AP-2ßtrans. factor AP-2ß
MnSODMnSOD
Phosphorylation
Analysis of the entire complement of phosphorylated proteins in cells: “phosphoproteome”
Qualitative and quantitative information regarding protein phosphorylation important
Important in many cellular processes signal transduction, gene regulation, cell cycle, apoptosis
Most common sites of phosphorylation: Ser, Thr, Tyr MS can be used to detect and map
locations for phosphorylation MW increase from addition of
phosphate group treatment with phosphatase allows
determination of number of phosphate groups
digestion and tandem MS allows for determination of phosphorylation sites
MS/MS and Phosphorylation
Detection of phosphopeptides in complex mixtures can be facilitated by neutral loss and precurson ion scanning using tandem mass spectrometers
Allow selective visualization of peptides containing phosphorylated residues
Most commonly performed with triple quadrupole mass spectrometers
precursor ion transmission
collision cell (chamber)
mass analysis of product ions
MS/MS and Phosphorylation
Precursor ion scan Q1 is set to allow all the components of the mixture to enter the
collision cell and undergo CAD Q3 is fixed at a specific mass value, so that only analytes which
fragment to give a fragment ion of this specific mass will be detected
Phospho-peptide fragments by CAD to give an ion at m/z 79 (PO3) Set Q3 to m/z 79: only species which fragment to give a fragment
ion of 79 reach the detector and hence indicating phosphorylation
Q1 Q2collision cell
Q3
detector
MS/MS and Phosphorylation
Neutral loss scan Q1 and Q3 are scanned synchronously but with a
specific m/z offset The entire mixture is allowed to enter the collision cell,
but only those species which fragment to yield a fragment with the same mass as the offset will be observed at the detector
pSer and pThr peptides readily lose phosphoric acid during CAD (98 Da)
For 2+ ion set offset at 49 Any species which loses 49 from a doubly charged ion
would be observed at the detector and be indicative of phosphorylation
Enrichment strategies to analyze phosphoproteins/peptides
Phosphospecific antibodiesPhosphospecific antibodies Anti-pY quite successful Anti-pS and anti-pT not as successful, but may be used (M.
Grønborg, T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O. Ohara, M. Mann, O. N. Jensen, and A. Pandey, “Approach for Identification of Serine/Threonine-phosphorylated Proteins by Enrichment with Phospho-specific Antibodies.” Mol. Cell. Proteomics 2002, 1:517–527.
Immobilized metal affinity chromatography (IMAC)Immobilized metal affinity chromatography (IMAC) Negatively charged phosphate groups bind to postively charged
metal ions (e.g., Fe3+, Ga3+) immobilized to a chromatographic support
Limitation: non-specific binding to acidic side chains (D, E) Derivatize all peptides by methyl esterification to reduce non-
specific binding by carboxylate groups. Ficarro et al., Nature Biotech. (2002), 20, 301.
Direct MS of phosphopeptides bound to IMAC beads
Raska et al., Anal. Chem. 2002, 74, 3429
IMAC beads placed directly on MALDI target
Matrix solution spotted onto target
MALDI-MS of peptides bound to IMAC bead
MALDI-MS/MS (*) to identify phosphorylation site(s)
MALDI-MS spectrum obtained from peptide bound to IMAC beads applied directly to MALDI target
MALDI-MS/MS (Q-TOF) to locate phosphorylation site
Sample enrichment with minimal sample handling
contains phosphorylated
residue
Enrichment strategies to analyze phosphoproteins/peptides
Chemical derivatizationChemical derivatization Introduce affinity tag to enrich for
phosphorylated molecules e.g., biotin binding to immobilized
avidin/streptavidin
Enrichment strategies to analyze phosphoproteins/peptides
Oda et al., Nature Biotech. 2001, 19, 379 for analysis of pS and pT Remove Cys-reactivity by oxidation with performic acid Base hydrolysis induce ß-elimination of phosphate from pS/pT Addition of ethanedithiol allows coupling to biotin Avidin affinity chromatography to purify phosphoproteins
Enrichment strategies to analyze phosphoproteins/peptides
Zhou et al., Nature Biotech. 2001, 19, 375 Reduce and alkylate Cys-residues to eliminate their
reactivity Protect amino groups with t-butyl-dicarbonate (tBoc) Phosphoramidate adducts at
phosphorylated residues are formed by carbodiimide condensation with cystamine
Free sulfhydryls are covalently captured onto glass beads coupled to iodoacetic acid
Elute with trifluoroacetic acid
Chemical derivatization to Chemical derivatization to enrich for phosphoproteinsenrich for phosphoproteins
Developed because other methods based on affinity/adsorption (e.g., IMAC) displayed some non-specific binding
Chemical derivatization methods may be overly complex to be used routinely
Sensitivity may not be sufficient for some experiments (low pmol)
Phosphoprotein StainPhosphoprotein Stain
PeppermintStick phosphoprotein molecular weight standards separated on a 13% SDS polyacrylamide gel. The markers contain (from largest to smallest) beta-galactosidase, bovine serum albumin (BSA), ovalbumin, beta-casein, avidin and lysozyme. Ovalbumin and beta-casein are phosphorylated. The gel was stained with Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red). The digital images were pseudocolored.
Phospho
Phosphoprotein StainPhosphoprotein Stain
Visualization of total protein and phosphoproteins in a 2-D gel
Proteins from a Jurkat T-cell lymphoma line cell lysate were separated by 2-D gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain (blueblue) followed by SYPRO Ruby protein gel stain (redred). After each dye staining, the gel was imaged and the resulting composite image was digitally pseudocolored and overlaid.
T.H. Steinberg et al., Global quantitative phosphoprotein analysis using Multiplexed Proteomics technology, Proteomics 2003, 3, 1128-1144
RAW 264.7 exposed to DEP
Global Analysis of Protein PhosphorylationGlobal Analysis of Protein Phosphorylation
Pro-Q DiamondPro-Q Diamond Sypro RubySypro Ruby
Xiao, Loo, and Nel - UCLA
IEF
9.53.54.5 5.1 5.5 6.0 7.0 8.4
53
4
12
6 7
20
30
37
98
55
9.53.54.5 5.1 5.5 6.0 7.0 8.4
30
37
98
55
20
89
10
1112
13
14
TNFTNF convertase convertaseMAGUK p55MAGUK p55
PDIPDIProtein phosphatase 2AProtein phosphatase 2A
JNK-1JNK-1p38 MAPK alphap38 MAPK alpha
ERK-1ERK-1ERK-2ERK-2ErbB-2ErbB-2
TNFTNFHSP 27HSP 27
AA B
m/z
Re
l. A
bun
d.
QQH EEMass spectrometry is inherently not a quantitative technique. The intensity of a peptide ion signal does not accurately reflect the amount of peptide in the sample.
equimolar mixture equimolar mixture of 2 peptidesof 2 peptides
516.725 516.828m/z
(M+2H)2+ : [12C]-ion
[Val5]-Angiotensin II1031.5188 (monoisotopic)
Lys-des-Arg9-Bradykinin1031.5552 (monoisotopic)
= 0.036= 0.036equimolar mixture equimolar mixture of 2 peptidesof 2 peptides
Mass Spectrometry and Quantitative Measurements
AA B
m/z
Re
l. A
bun
d.
QQH EE
Two peptides of identical chemical structure that differ in mass because they differ in isotopic composition are expected to generate identical specific signals in a mass spectrometer.
equimolar mixture equimolar mixture of 2 peptidesof 2 peptides
Mass Spectrometry and Quantitative Measurements
QQH EE
13C13C13C
AA B
2D 2D
Methods coupling mass spectrometry and stable isotope tagging Methods coupling mass spectrometry and stable isotope tagging have been developed for quantitative proteomics.have been developed for quantitative proteomics.
ICAT: Isotope-Coded Affinity Tag
Alkylating group covalently attaches the reagent to reduces Cys-residues. A polyether mass-encoded linker contains 8 hydrogens (d0) or 8 deuteriums
(d8) that represents the isotope dilution. A biotin affinity tag is used to selectively isolate tagged peptides (by avidin
purification).
ICAT: Isotope-Coded Affinity Tag
The Cys-residues in sample 1 is labeled with d0-ICAT and sample 2 is labeled with d8-ICAT. The combined samples are digested, and the biotinylated ICAT-labeled peptides are enriched by avidin
affinity chromatography and analyzed by LC-MS/MS. Each Cys-peptide appears as a pair of signals differing by the mass differential encoded in the tag. The
ratio of the signal intensities indicates the abundance ratio of the protein from which the peptide originates.
MS/MS identifies the protein
Stable Isotope Amino Acid or 15N- in vivo Labeling
Metabolic stable isotope coding of proteomes
An equivalent number of cells from 2 distinct cultures are grown on media supplemented with either normal amino acids or 14N-minimal media, or stable isotope amino acids (2D/13C/15N) or 15N-enriched media.
These mass tags are incorporated into proteins during translation.
Enzymatic Stable Isotope Coding of Proteomes
Enzymatic digestion in the presence of 18O-water incorporates 18O at the carboxy-terminus of peptides
Proteins from 2 different samples are enzymatically digested in normal water or H2
18O.
RR33 RR44
NH2-CH-CO-NH-CH-COOH...NH-CH-CO-NH-CH-CO-...NH-CH-CO-NH-CH-CO-1818OOHH
RR11 RR22
...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH...NH-CH-CO-NH-CH-CO-NH-CH-CO-NH-CH-COOH
RR11 RR22 RR33 RR44
Trypsin /HTrypsin /H221818OO
(Arg, Lys)(Arg, Lys)
C-terminal peptide
Identification of Low Abundance Proteins
The identification of low abundance proteins in the presence of high abundance proteins is problematic (e.g., “needle in a haystack”)
Pre-fractionation of complex protein mixtures can alleviate some difficulties gel electrophoresis, chromatography,
etc Removal of known high abundance
proteins allows less abundant species to be visualized and detected
Identification of Low Abundance Proteins
GenWay Biotech
Additional Readings
R. Aebersold and M. Mann, Mass spectrometry-based proteomics, Nature (2003), 422, 198-207.
M. B. Goshe and R. D. Smith, “Stable isotope-coded proteomic mass spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 101-109.
W. A. Tao and R. Aebersold, “Advances in quantitative proteomics via stable isotope tagging and mass spectrometry.” Curr. Opin. Biotechnol. 2003; 14: 110-118.
S. D. Patterson and R. Aebersold, “Proteomics: the first decade and beyond.” Nature Genetics 2003; 33 (suppl.): 311-323.
M. Mann and O. N. Jensen, “Proteomic analysis of post-translational modification.” Nature Biotech. 2003; 21: 255-261.
D. T. McLachlin and B. T. Chait, “Analysis of phosphorylated proteins and peptides by MS.” Curr. Opin. Chem. Biol. 2001; 5: 591-602.
S. Gygi et al., “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.” Nature Biotech. 1999; 17: 994-999.
Proteomics in Practice: A Laboratory Manual of Proteome AnalysisReiner Westermeier, Tom NavenWiley-VCH, 2002
PART II: COURSE MANUAL Step 1: Sample Preparation Step 2: Isoelectric Focusing Step 3: SDS Polyacrylamide Gel Electrophoresis Step 4: Staining of the Gels Step 5: Scanning of Gels and Image Analysis Step 6: 2D DIGE Step 7: Spot Excision Step 8: Sample Destaining Step 9: In-gel Digestion Step 10: Microscale Purification Step 11: Chemical Derivatisation of the Peptide Digest Step 12: MS Analysis Step 13: Calibration of the MALDI-ToF MS Step 14: Preparing for a Database Search Step 15: PMF Database Search Unsuccessful
PART I: PROTEOMICS TECHNOLOGY Introduction Expression Proteomics Two-dimensional Electrophoresis Spot Handling Mass Spectrometry Protein Identification by Database Searching Methods of Proteomics
Proteins and Proteomics: A Laboratory ManualRichard J. SimpsonCold Spring Harbor Laboratory (2002)
Chapter 1. Introduction to Proteomics Chapter 2. One–dimensional Polyacrylamide Gel Electrophoresis Chapter 3. Preparing Cellular and Subcellular Extracts Chapter 4. Preparative Two–dimensional Gel Electrophoresis with
Immobilized pH Gradients Chapter 5. Reversed–phase High–performance Liquid Chromatography Chapter 6. Amino– and Carboxy– terminal Sequence Analysis Chapter 7. Peptide Mapping and Sequence Analysis of Gel–resolved Proteins Chapter 8. The Use of Mass Spectrometry in Proteomics Chapter 9. Proteomic Methods for Phosphorylation Site Mapping Chapter 10. Characterization of Protein Complexes Chapter 11. Making Sense of Proteomics: Using Bioinformatics to Discover a
Protein’s Structure, Functions, and Interactions