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
Id.
OK?
PMF
STOP 3% no
In-gel
digestion
ESI
yes
nanoESI
MS/MS
Id.
OK?
STOP
Split
extract
in half no
97%
de novo
sequencing
for homology
search or cloning
derivatize
sample
nanoESI
MS/MS
Shevchenko et al, PNAS USA, 1996, 93,

3. New workflows facilitated by MALDI MS/MS technologies10 20 30 40 50 60
Retention Time (Min)
1.7E+4 70 80 90
100
% Intensity
TIC
T28.4
T30.7
T33.2
T25.5
T31.5
T24.5
T40.0
T21.0
T21.4
T16.4
T35.6
T23.2
T19.7
T38.7
T32.1
T18.1
T56.8
T42.7
T24.2
T32.5
T47.9
T30.0
T11.4
T34.9
T22.0
T17.3
T25.3
T43.7
T49.9
T54.6
T37.7
SCX
nano-
HPLC
MALDI
(2-50,000 MS +
MS/MS spectra)
Protein #1
Protein #2
Protein #3
Protein #4

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.

6. H. ducreyi workflow 20 SCX fractions collected #2 #1 #3 #3 #4 50% 50%
~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

7. Flow of data processing
Fraction #1
Fraction #1
Fraction #2
Fraction #3
Fraction #4
Fraction #5
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

8. H. ducreyi proteins identified by 2D LC (requiring at least 1 significant peptide)
498
372
MALDI – AB 4700
Proteomics Analyzer
ESI - QSTAR®
Pulsar System
Successful
MS/MS
Spectra =
2498/7414
(34%)
Successful
MS/MS
Spectra =
1709/6222
(27%)
206
292 80
Graphical (Ven) diagram of the overlap in the proteins IDed by the 2 techniques
578 total unique proteins identified

9. Different ESI vs. MALDI characteristics on the peptide level
200
400
600
800
1000
1200
1400 1 2 3 4 5 6
Number of predicted charges ( = 1+SK+SH+SR)
MALDI peptides
ESI peptides
Number of identified peptides
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= MALDI
Length of peptides (AA) 50
100
150
200
250 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
MALDI peptides
ESI peptides
Number of identified peptides
Similar distribution was observed with the inclusion of all precursors (non-identified peptides)

10. How many peptides identify a protein? (h. ducreyi work)
MALDI
ESI 1 2 3 4 5 6 7 8 9 10
>10 2 3 4 5 6 7 8 9 10
>10 1
The distribution of h. ducreyi proteins identified by 1, 2, 3,..,etc. peptides

11. 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
This is a transition slide

12. Case Study 2: Fibroblast activation by TGF-b
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

14. Preliminary results from SCX fraction A795 60 45
MALDI – AB 4700
Proteomics Analyzer
155
ESI - QSTAR®
Pulsar System
140
200 unique proteins identified and quantified
Differentially
expressed
proteins
(>1STD) 21
Unique MALDI 26
Unique ESI 50
Common 60
Significant
proteins 45 95 68
Significant
peptides 61
104 20 40 60 80
100
120

15. A few examples of differentially expressed nuclear proteins identified by ESI or MALDI only

16. Comparison of quantification results
1800
1810
1820
1830
1840
1850
Mass (m/z)
1419.6 10 20 30 40 50 60 70 80 90
100
% Intensity
MALDI
ESI
heavy/light=2.28
heavy/light=2.49
gi| , enigma protein, AFYMEEGVPYC*ER (MH+= )
1150
1156
1162
1168
1174
1180
Mass (m/z)
1502.7 10 20 30 40 50 60 70 80 90
100
% Intensity
ESI
MALDI
heavy/light=2.17
heavy/light=2.12
gi|118090, peptidylprolyl isomerase B (cyclophilin B), DVIIADC*GK (MH+= )
MALDI ratio – ESI ratio
= /
(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