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

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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

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% In

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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

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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

3

4

5

6

7

8

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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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