esi and maldi lc/ms-ms approaches for larger scale protein identification and quantification: are...
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ESI and MALDI LC/MS-MS Approaches for Larger Scale Protein Identification and Quantification: Are
They Equivalent?1P. Juhasz, 1A. Falick,1A. Graber, 1S. Hattan, 1N. Khainovski, 1J.
Marchese, 1S. Martin, 1D. Patterson, 1B. Williamson, 2J. Malmstrom, 2G. Westergren-Thorsson, 2G. Marko-Varga
1Applied Biosystems, Proteomics Research Center, Framingham, MA2University of Lund, Molecular Biology Dept., Lund, Sweden
Introduction: a conventional PMF + MS/MS approach for protein identification
In-geldigestion
3%
97%
PMFId.
OK?
Splitextract in half
nanoESI MS/MS
Id.OK?
STOP
derivatize sample
nanoESI MS/MS
de novo sequencing
for homology search or cloning
STOP
MALDI
ESI
yes
yes
no
no
Shevchenko et al, PNAS USA, 1996, 93, 14440-14445
New workflows facilitated by MALDI MS/MS technologies
10 20 30 40 50 60
Retention Time (Min)0
1.7E+4
0
10
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% In
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sity
TIC
T28.4
T30.7 T33.2
T25.5T31.5
T24.5 T40.0T21.0
T21.4T16.4 T35.6T23.2T19.7 T38.7
T32.1T18.1 T56.8T42.7T24.2 T32.5 T47.9T30.0T11.4 T34.9T22.0T17.3 T25.3 T43.7 T49.9 T54.6T37.710 20 30 40 50 60
Retention Time (Min)0
1.7E+4
0
10
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30
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% In
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sity
TIC
T28.4
T30.7 T33.2
T25.5T31.5
T24.5 T40.0T21.0
T21.4T16.4 T35.6T23.2T19.7 T38.7
T32.1T18.1 T56.8T42.7T24.2 T32.5 T47.9T30.0T11.4 T34.9T22.0T17.3 T25.3 T43.7 T49.9 T54.6T37.7
SCX
nano-HPLC
MALDI(2-50,000 MS + MS/MS spectra)
Protein #1 Protein #2 Protein #3 Protein #4
Objectives
• Characterize (dis)similarity of protein identification results from LC-ESI MS/MS and LC-MALDI MS/MS wokflows
• Interpret results based on the ESI vs. MALDI ionization preferences
• Compare performance (quantification and identification) in a protein differential expression study
Case Study 1: Haemophilus ducreyi
• H. ducreyi is a gram-negative bacterium that causes the sexually transmitted disease chancroid.
• Linked to the heterosexual transmission of HIV in developing countries.
• The sequencing of this genome (1.7Mb) has recently been completed and homology with H. influenzae is known.
• A proteomic study was undertaken to help with the sequence alignment and annotation. http://www.microbial-pathogenesis.org/H.ducreyi/
~200 g cell lysate of h. ducreyi strain 35,000reduced/alkylated anddigested w. trypsin
#1 20 SCX fractions collected#2
H. ducreyi workflow
90-min. gradient HPLCat 0.3 l/min
45-min. gradient HPLCat 0.5 l/min
matrix infusionat 1 l/min
MS-MS analysis on AB 4700
collection of 20-sec. fractionsOnline MS-MS
analysis on QStar®
Pulsar System
#3 #3
#4
50% 50%
Flow of data processing
Fraction #1Fraction #2
Fraction #3Fraction #4
Fraction #5
db search(Mascot)
Protein list #1Protein list #2Protein list #3Protein list #4...
Protein list #1Protein list #2Protein list #3Protein list #4...
Protein list #1Protein list #2Protein list #3Protein list #4...
db search(Mascot)
non-redundantlist of proteins
non-redundantlist of proteins
non-significantproteins removed
non-significantproteins removed
Fraction #1Fraction #2
Fraction #3Fraction #4
Fraction #5
•Compile in Oracle db•generate quieries•Compile in Oracle db•generate quieries
H. ducreyi proteins identified by 2D LC (requiring at least 1 significant peptide)
578 total unique proteins identified
ESI - QSTAR® Pulsar System
MALDI – AB 4700Proteomics Analyzer
292
372498
SuccessfulMS/MS
Spectra =2498/7414
(34%)
SuccessfulMS/MS
Spectra =1709/6222
(27%)
206 80
0
200
400
600
800
1000
1200
1400
1 2 3 4 5 6Number of predicted charges ( = 1+K+H+R)
MALDI peptides
ESI peptides
Nu
mb
er
of
ide
nti
fie
d p
ep
tid
es
• better success rates with ESI on doubly charged (small?) peptides
• better success rates with MALDI on more basic (bigger?) peptides
• K/R ratio=1.27 – ESI
• K/R ratio=0.92 - MALDI
Different ESI vs. MALDI characteristics on the peptide level
Length of peptides (AA)
0
50
100
150
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250
5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
MALDI peptides
ESI peptides
Nu
mb
er
of
ide
nti
fie
d p
ep
tid
es
Similar distribution was observed with the inclusion of all precursors (non-identified peptides)
How many peptides identify a protein?(h. ducreyi work)
1
2
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9
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>10
23
4
5
6
7
8
9
10>10
1
ESIESI MALDIMALDI
The distribution of h. ducreyi proteins identified by 1, 2, 3,..,etc. peptides
Conclusions from h. ducreyi work
• From very complex mixtures MALDI had better efficiency of identification (higher “MS/MS duty cycle”)
• Smaller peptides with K C-terminus identified more efficiently with ESI
• Larger/more basic peptides are more efficiently identified by MALDI
• A more complete sequence coverage of proteins is expected to “smooth out” differences
Fibroblast
Myofibroblast
TGF-
TGF-
SMADPathway
DNA
SMAD complex
Transcription
DNA binding partner
Case Study 2: Fibroblast activation by TGF-
Protein preps. from 107 fibroblast nuclei are labeled with acid cleavable ICATTM reagent/trypsinized
#1
Differential expression analysis of nuclear proteins in human fibroblasts
control 10 ng/ml TGF- 30 SCX fractions collected#2
Cys-containing peptidesaffinity purified/ cleaved with TFA
#3
90-min. gradient HPLCat 0.3 l/min
45-min. gradient HPLCat 1 l/min
matrix infusionat 2 l/min
MS-MS analysis on AB 4700
collection of 20-sec. fractions
Online MS-MSanalysis on QStar®
Pulsar System
#4 #4
#5
200 unique proteins identified and quantified
0 20 40 60 80 100 120
Significantpeptides
Significantproteins
Differentiallyexpressedproteins(>1STD)
Unique MALDIUnique ESICommon
Preliminary results from SCX fraction A7
ESI - QSTAR® Pulsar System
140MALDI – AB 4700Proteomics Analyzer
1559560 45
104
95
50
61
45
2621
60
68
Protein NameChange in rel.
expression level60S ribosomal protein L34 -300%(AF130077) PRO2619 [Homo sapiens] -250%ribosomal protein S12 -116%(X55525) type I collagen -84%ribosomal protein L5 [Homo sapiens] -83%KH-type splicing regulatory protein (FUSE binding protein 2); -63%splicing factor (CC1.3) [Homo sapiens] -60%U5 snRNP-specific protein, 116 kD [Homo sapiens] -60%(U36484) laminin-binding protein [Homo sapiens] -57%heterogeneous nuclear ribonucleoprotein L -56%alternative splicing factor ASF-2 - human -55%GUANINE NUCLEOTIDE-BINDING PROTEIN G(I) 56%25596 64%(Z85986) SRP20 (SR protein family member) 73%(S72370) pyruvate carboxylase, pyruvate:carbon dioxide ligase {EC 6.4.1.1} 85%(AF134838) endocytic receptor Endo180 92%elastin microfibril interface located protein 116%CLATHRIN LIGHT CHAIN B (BRAIN AND LYMPHOCYTE LCB) 268%
A few examples of differentially expressed nuclear proteins identified by ESI or MALDI only
1150 1156 1162 1168 1174 1180Mass (m/z)
1502.7
0
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1168
.622
1160
.597
1154
.632
1800 1810 1820 1830 1840 1850Mass (m/z)
1419.6
0
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% In
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sity
1828
.84
1820
.82
1844
.82
heavy/light=2.12heavy/light=2.17
heavy/light=2.49heavy/light=2.28
Comparison of quantification results
gi|11416507, enigma protein, AFYMEEGVPYC*ER (MH+=1828.826)
gi|118090, peptidylprolyl isomerase B (cyclophilin B), DVIIADC*GK (MH+=1168.625)
ESIESI
ESIESI MALDIMALDI
MALDIMALDI
MALDI ratio – ESI ratioavg. ratio
= 0.017 +/- 0.122 (based on 75 common peptides)
Conclusions from protein differential expression study
• ESI vs. MALDI are complementary: 52%(!) of proteins were identified with ESI or MALDI only when analysis is restricted to Cys-containing peptides.
• Protein quantification by isotope ratio measurements (using ICATTM reagent) yielded identical results with ESI and MALDI within the experimental errors