ESI and MALDI LC/MS-MS

Approaches for Larger Scale Protein

Identification and Quantification: Are
They Equivalent?
1
P. Juhasz, 1A. Falick,1A. Graber, 1S. Hattan, 1N. Khainovski, 1J.
Marchese, 1S. Martin, 1D. Patterson, 1B. Williamson, 2J. Malmstrom,
2
G. Westergren-Thorsson, 2G. Marko-Varga
1
Applied Biosystems, Proteomics Research Center, Framingham, MA
2
University of Lund, Molecular Biology Dept., Lund, Sweden
 Introduction: a conventional PMF + MS/MS
approach for protein identification

Id.
yes MALDI
PMF STOP
OK?

3% no

In-gel
digestion
ESI
nanoESI Id. yes
STOP
MS/MS OK?
97% Split no
extract
in half de novo
derivatize nanoESI sequencing
sample MS/MS for homology
search or cloning

Shevchenko et al, PNAS USA, 1996, 93, 14440-14445

 New workflows facilitated by MALDI
MS/MS technologies

SCX

TIC TIC

100 T28.4 1.7E+4 100 T28.4 1.7E+4

90 90

80 80
T30.7 T33.2 T30.7 T33.2
70 70
T25.5 T25.5

nano-
T31.5 T31.5
% Intensity

% Intensity
60 60

50
T24.5 T40.0 50
T24.5 T40.0
T21.0 T21.0
40 40

HPLC 30

20
T16.4

T18.1
T19.7
T21.4
T23.2

T24.2
T32.1
T35.6
T38.7
T42.7 T56.8
30

20
T16.4

T18.1
T19.7
T21.4
T23.2

T24.2
T32.1
T35.6
T38.7
T42.7 T56.8
10 T11.4 T30.0 T32.5 T47.9 10 T11.4 T30.0 T32.5 T47.9
T17.3 T22.0 T25.3 T34.9 T43.7 T49.9 T54.6 T17.3 T22.0 T25.3 T34.9 T43.7 T49.9 T54.6
T37.7 T37.7
0 0 0 0
10 20 30 40 50 60 10 20 30 40 50 60

Retention Time (Min) Retention Time (Min)

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/
 H. ducreyi workflow
#2 20 SCX fractions collected
#1

~200 g cell lysate of h.

ducreyi strain 35,000
reduced/alkylated and
digested w. trypsin
50% 50%

#3 #3 45-min. gradient HPLC

90-min. gradient HPLC at 0.5 l/min
at 0.3 l/min matrix infusion
at 1 l/min
collection of
20-sec. fractions
Online MS-MS
analysis on QStar®
Pulsar System
#4
MS-MS analysis
on AB 4700
 Flow of data processing
Fraction #1 Fraction #1
Fraction #2 db search db search Fraction #2
Fraction #3 Fraction #3
Fraction #4 (Mascot) (Mascot) Fraction #4
Fraction #5 Fraction #5
Protein list #1 Protein list #1
Protein list #2 Protein list #2
Protein list #3 Protein list #3
Protein list #4 Protein list #4
... ...

non-redundant non-redundant
list of proteins list of proteins

non-significant non-significant
proteins removed proteins removed

•Compile in Oracle db
•generate quieries
 H. ducreyi proteins identified by 2D LC
(requiring at least 1 significant peptide)

498 372
MALDI – AB 4700 ESI - QSTAR®
Proteomics Analyzer Pulsar System

Successful Successful
MS/MS MS/MS
Spectra = 206 292 80 Spectra =
2498/7414 1709/6222
(34%) (27%)

578 total unique proteins identified

 Different ESI vs. MALDI characteristics on the peptide level
1400
Number of identified peptides

1200
• better success rates with
MALDI peptides
ESI peptides
ESI on doubly charged
1000
(small?) peptides
800
• better success rates with
600 MALDI on more basic
400 (bigger?) peptides
200 • K/R ratio=1.27 – ESI
0 • K/R ratio=0.92 - MALDI
1 2 3 4 5 6
Number of predicted charges ( = 1+K+H+R)

250
Number of identified peptides

MALDI peptides
200 ESI peptides
Similar distribution was
150 observed with the
inclusion of all
100 precursors (non-
identified peptides)
50

0
5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
Length of peptides (AA)
 How many peptides identify a protein?
(h. ducreyi work)

ESI MALDI
>10
10
10
9 >10
9
8
8
7 1
7
6 1
6
5
5
4

4
3
3 2
2

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
Case Study 2: Fibroblast activation by TGF- 

Fibroblast TGF-

SMAD
Pathway

TGF- SMAD complex DNA binding

partner

Transcription DNA

Myofibroblast
 Differential expression analysis of nuclear proteins
in human fibroblasts
control 10 ng/ml TGF- #2 30 SCX fractions collected
#1 #3
Cys-containing
Protein preps. from 107 peptides
fibroblast nuclei are affinity purified/
labeled with acid cleaved with TFA
cleavable ICATTM
reagent/trypsinized

#4 #4 45-min. gradient HPLC

90-min. gradient HPLC at 1 l/min
at 0.3 l/min matrix infusion
at 2 l/min
collection of
20-sec. fractions
Online MS-MS
analysis on QStar®
Pulsar System
#5
MS-MS analysis
on AB 4700
 Preliminary results from SCX fraction A7

155 140
MALDI – AB 4700 60 95 45 ESI - QSTAR®
Proteomics Analyzer Pulsar System

200 unique proteins identified and quantified

Differentially 21 Unique MALDI

expressed Unique ESI
proteins
26
(>1STD) 50 Common

60
Significant
proteins 45
95

68
Significant 61
peptides
104

0 20 40 60 80 100 120
A few examples of differentially expressed nuclear proteins
identified by ESI or MALDI only

Change in rel.
Protein Name
expression level
60S ribosomal protein L34 -300%
(AF130077) PRO2619 [Homo sapiens] -250%
ribo s o mal pro te in S 12 -116%
(X55525) type I collagen -84%
ribosomal protein L5 [Homo sapiens] -83%
KH-type s plic ing re g ulato ry pro te in (FUS E binding pro te in 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%
he te ro g e ne o us nuc le ar ribo nuc le o pro te in L -56%
alternative splicing factor ASF-2 - human -55%
GUANINE NUCLEOTIDE-BINDING PROTEIN G(I) 56%
25596 64%
(Z85986) S RP20 (S R pro te in family me mbe r) 73%
(S 72370) pyruvate c arbo xylas e , pyruvate :c arbo n dio xide lig as e {EC 6.4.1.1} 85%
(AF134838) e ndo c ytic re c e pto r Endo 180 92%
e las tin mic ro fibril inte rfac e lo c ate d pro te in 116%
CLATHRIN LIGHT CHAIN B (BRAIN AND LYMPHOCYTE LCB) 268%
 Comparison of quantification results

1828.84
100 1419.6

ESI 90
MALDI
heavy/light=2.28 80

heavy/light=2.49
70

60

% Intensity

1820.82
50

40

30

20

1844.82
10

0
1800 1810 1820 1830 1840 1850
Mass (m/z)

gi|11416507, enigma protein, AFYMEEGVPYC*ER (MH+=1828.826)

1168.622
100 1502.7

ESI 90

heavy/light=2.17
MALDI
80

heavy/light=2.12 70

60

% Intensity

1160.597
50

40

30

20

1154.632
10

0
1150 1156 1162 1168 1174 1180
Mass (m/z)

gi|118090, peptidylprolyl isomerase B (cyclophilin B), DVIIADC*GK (MH+=1168.625)

MALDI ratio – ESI ratio

= 0.017 +/- 0.122 (based on 75 common peptides)
avg. ratio
 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