ESI and MALDI LC/MS-MS Approaches for Larger Scale Protein Identification and Quantification: Are They Equivalent?

1
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, MA
• 2University of Lund, Molecular Biology Dept.,
  Lund, Sweden

2
Introduction a conventional PMF MS/MS approach
for protein identification
MALDI
yes
PMF
3
no
ESI
yes
nanoESI MS/MS
Id. OK?
no
97
derivatize sample
nanoESI MS/MS
Shevchenko et al, PNAS USA, 1996, 93, 14440-14445
3
New workflows facilitated by MALDI MS/MS
technologies
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

5
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/
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H. ducreyi workflow
2
20 SCX fractions collected
1
200 mg cell lysate of h. ducreyi strain
35,000 reduced/alkylated and digested w. trypsin
50
50
3
3
45-min. gradient HPLC at 0.5 ml/min
90-min. gradient HPLC at 0.3 ml/min
matrix infusion at 1 ml/min
collection of 20-sec. fractions
Online MS-MS analysis on QStar Pulsar System
4
MS-MS analysis on AB 4700
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Flow of data processing
Fraction 1
Fraction 2
db search (Mascot)
db search (Mascot)
Fraction 3
Fraction 4
Fraction 5
Protein list 1 Protein list 2 Protein list
3 Protein list 4 ...
Protein list 1 Protein list 2 Protein list
3 Protein list 4 ...
non-redundant list of proteins
non-redundant list of proteins
non-significant proteins removed
non-significant proteins removed

• Compile in Oracle db
• generate quieries

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H. ducreyi proteins identified by 2D LC
(requiring at least 1 significant peptide)
498
372
ESI - QSTAR Pulsar System
MALDI AB 4700 Proteomics Analyzer
Successful MS/MS Spectra 2498/7414 (34)
Successful MS/MS Spectra 1709/6222 (27)
292
206
80
578 total unique proteins identified
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Different ESI vs. MALDI characteristics on the
peptide level
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How many peptides identify a protein?(h. ducreyi
work)
MALDI
ESI
The distribution of h. ducreyi proteins
identified by 1, 2, 3,..,etc. peptides
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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

12
Case Study 2 Fibroblast activation by TGF-b
Fibroblast
SMAD Pathway
TGF-b
SMAD complex
DNA binding partner
Transcription
DNA
Myofibroblast
13
Differential expression analysis of nuclear
proteins in human fibroblasts
30 SCX fractions collected
control
10 ng/ml TGF-b
2
1
3
Cys-containing peptides affinity purified/
cleaved with TFA
Protein preps. from 107 fibroblast nuclei are
labeled with acid cleavable ICATTM
reagent/trypsinized
4
4
45-min. gradient HPLC at 1 ml/min
90-min. gradient HPLC at 0.3 ml/min
matrix infusion at 2 ml/min
collection of 20-sec. fractions
Online MS-MS analysis on QStar Pulsar System
5
MS-MS analysis on AB 4700
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Preliminary results from SCX fraction A7
200 unique proteins identified and quantified
21
Unique MALDI
26
Unique ESI
50
Common
60
45
95
68
61
104
0
20
40
60
80
100
120
15
A few examples of differentially expressed
nuclear proteins identified by ESI or MALDI only
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Comparison of quantification results
MALDI
ESI
heavy/light2.28
heavy/light2.49
gi11416507, enigma protein, AFYMEEGVPYCER
(MH1828.826)
ESI
MALDI
heavy/light2.17
heavy/light2.12
gi118090, peptidylprolyl isomerase B
(cyclophilin B), DVIIADCGK (MH1168.625)
MALDI ratio ESI ratio
0.017 /- 0.122
(based on 75 common peptides)
avg. ratio
17
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