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

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, 14440-14445

New workflows facilitated by MALDI MS/MS technologies 10 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

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 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

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

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

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=0.92 - 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)

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

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

Case Study 2: Fibroblast activation by TGF-b SMAD Pathway TGF-b SMAD complex DNA binding partner Transcription DNA Myofibroblast

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

Preliminary results from SCX fraction A7 95 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

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

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 1828.84 1820.82 1844.82 MALDI ESI heavy/light=2.28 heavy/light=2.49 gi|11416507, enigma protein, AFYMEEGVPYC*ER (MH+=1828.826) 1150 1156 1162 1168 1174 1180 Mass (m/z) 1502.7 10 20 30 40 50 60 70 80 90 100 % Intensity 1168.622 1160.597 1154.632 ESI MALDI heavy/light=2.17 heavy/light=2.12 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