DEVELOPING SUB-PROTEOMIC METHODS FOR LARGE SCALE PROFILING TAN LAI PENG (B.Appl.Sci (Hons), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGMENTS I wish to thank my supervisor, Dr Yao Shao Qin for giving me the opportunity to research in his laboratory, for his unwavering patience and encouragement during these years, and all the advice and helps during the writing of the thesis To my lab mates for making the lab a warm, friendly and interesting place to work in To my net group members and friends, especially Cheryl for her prayers, Raymond for his company and Jonathan for going through this Accelerated Master Program with me To my family, especially my adorable siblings (David, Esther, John) for their support and encouragement To God i TABLE OF CONTENTS Acknowledgments .i Table of Contents ii Summary vi Publications …………….…………………………………………………viii List of Figures……………………………………………………………… ix List of Abbreviations…………………………………………………………xi Chapter 1: Introduction 1.1 Post-genomic era and its challenges 1.2 Addressing the proteomic challenges 1.2.1 Differential in-gel electrophoresis (DIGE) 1.2.2 Sub-proteomic expression profiling of proteins using activitybased probes (ABPs) 1.2.2.1 Reactive unit 1.2.2.2 Linker/recognition unit 10 1.2.2.3 Tag unit 11 1.2.2.4 Application of ABP in gel-based proteomics 12 1.3.1 Apoptosis 15 1.3.1.1 Caspases 16 1.3.1.2 Serine proteases in apoptosis 17 1.3 Aim of the project 19 1.3.2 Differential expression profiling of serine hydrolases in normal and apoptotic cells 20 ii 1.3.3 In vivo labeling of caspases in apoptotic HeLa cells 21 Chapter 2: Materials and Methods 24 2.1 Materials .24 2.2 GatewayTM Technology .24 2.2.1 PCR amplification of a known serine hydrolase gene 24 2.2.2 BP reaction 25 2.2.3 LR reaction 26 2.2.4 Transformation and induction of protein expression in BL21 (AI) 26 2.4.4 Sequencing confirmation of clones 27 2.3 Preparation of bacterial and yeast cell lysate 28 2.4 Estimation of protein concentration 28 2.5 In vitro labeling 28 2.6 Affinity purification .28 2.7 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) 29 2.8 Two-dimensional DIGE (2D DIGE) and imaging .29 2.9 Sypro ruby protein stain .30 2.10 Western blot 30 2.11 Elution of whole protein from SDS-PAGE gel 31 2.12 In-gel digestion of proteins 31 2.13 Matrix-assisted laser desorption ionization-time of flight (MALDITOF) mass spectrometry (MS) 32 iii 2.14 Preparation for apoptotic cells 32 2.14.1 Strains and culture condition 32 2.14.2 Apoptosis induction of HeLa cells by Ultraviolet (UV) irradiation 32 2.14.3 In vivo labeling 33 2.14.4 Evaluation of apoptosis 33 2.14.5 Preparation of HeLa cell extract 33 Chapter 3: Results 35 3.1 Labeling and identifying of a commercially available serine hydrolase 35 3.2 Optimization of sample preparation steps 37 3.3 Identification of serine hydrolases in yeast proteome 41 3.4 Differential profiling of serine hydrolases 44 3.4.1 Proof of concept 44 3.4.2 Differential profiling of serine hydrolases in normal and apoptotic HeLa cells 48 Chapter 4: Discussion 59 4.1 Labeling and identification of a commercially available serine hydrolase……………………………………………………………….59 4.2 Optimization of sample preparation step 60 4.3 Identification of serine hydrolases in a yeast proteome 62 4.4 Differential profiling of serine hydrolases 65 4.4.1 Proof of concept 65 iv 4.4.2 Differential profiling of serine hydrolases in normal and apoptotic cells 68 4.5 In vivo labeling of caspases 71 4.5.1 In vivo labeling of caspases 71 4.5.2 In vivo labeling of caspase-associated substrates 73 4.6 Activity-based profiling .77 Chapter 5: Conclusion 80 Chapter 6: References 81 Chapter 7: Appendix 113 v SUMMARY With the availability of complete genome sequence, emphasis has shifted towards the understanding of protein function Two-dimensional gel electrophoresis (2D-GE) is a state-of-the-art technique currently used for large-scale studies of proteomes from different organisms Due to its limited resolution of detection (detecting 1000-5000 proteins), this technique alone, however, is not sufficient to study complex proteomes, such as that of human (>40,000 proteins) Thus, there is an urgent need to develop a so-called subproteomic approach which is capable of analyzing, with higher-resolution, subsets of proteins in a proteome We have developed a functional proteomic methodology that makes use of a combination of an emerging technology, DIGE and fluorescent probes The probes are made up of fluorophosphonates (FP) which are mechanism-based suicide inhibitors of serine hydrolases In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1-propylindocarbocyanine halide (Cy3) FP probe and 1-(5- carboxypentyl)-1-methylindodi-carbocyanine halide (Cy5) FP probe, respectively The labeled proteins are mixed and separated in the same 2D gel, allowing quantification of differential serine hydrolases expression Using Gateway Technology by Invitrogen, a known serine hydolase was introduced into the bacterial expression host Differences vi in expression of the serine hydrolase between induced and uninduced condition were quantified by 2D DIGE This methodology was further extended into quantifying differences in the expression of serine hydrolases between normal and apoptotic cells In another part of the project, we report efficient labeling of caspases expressed inside apoptotic HeLa cells using fluorescently-labeled or biotinylated, fluoromethylketone (fmk)-containing probe Preliminary results with these probes indicated that they were highly cell-permeable, and caspase8 has been identified by biotinylated fmk-containing probe In conclusion, 2D DIGE, in combination with fluorescent probes and MS, will become a powerful tool for molecular characterization of cancer progression and identification of cancer-specific protein markers vii PUBLICATIONS Huang, X., Tan, E.L.P., Chen, G.Y.J., Yao, S.Q “Enzymetargeting small molecule probes for proteomics applications”, Appl Genomics Proteomics, 2003, in press Tan, E.L.P., Panicker, R.C., Tan, L.P., Chattopadhaya, S., Yao, S.Q “Developing chemical biology tools for the study of functional proteomics”, Proceedings of the 1st International Symposium on Biomolecular Chemistry, Japan Tan, E.L.P., Yao, S.Q “Activity-based, differential expression profiling of serine hydrolases in different proteomes”, manuscript in preparation Tan, E.L.P, Panicker, R.C., Yao, S.Q “In vivo activity-based profiling of caspases and their associated proteins”, manuscript in preparation viii LIST OF FIGURES Number Page Figure Strategy of 2D difference gel electrophoresis (DIGE) Figure General structure of an activity-based probe Figure Strategy of gel-based activity profiling using ABP 13 Figure Caspase activation cascade 17 Figure Structure of FP-Cy3 20 Figure Structure of FP-Cy5 21 Figure Structure of FP-Biotin 21 Figure Structure of caspase-biotin 22 Figure Structure of caspase-fluorescein 23 Figure 10 SDS-PAGE of FP-Cy5-labeled chymotrypsin 35 Figure 11 MALDI-TOF-MS spectrum of undigested chemotrypsin .36 Figure 12 MALDI-TOF-MS spectrum of digested chymotrypsin 36 Figure 13 Chymotrypsin matched peptide alignment sequences .37 Figure 14 Optimization of lysis and labeling condition 38 Figure 15 2D fluorescent gel of FP-Cy5 labeled yeast proteins 38 Figure 16 2D gel showing FP-Cy5 labeled yeast fluorescent spots 40 Figure 17 2D gel showing FP-biotin labeled proteins from yeast lysate .42 Figure 18 Protein sequence alignment of sec17 and sec11 using ClustalW 43 Figure 19 : The GatewayTM system 45 Figure 20 PCR analysis to verify the presence of the gene, YSP3, in bacteria expression host BL21 (AI) .45 Figure 21 YSP3 Basepair alignment of the sequencing results 46 Figure 22 Over-expression of YSP3 by arabinose induction in bacteria expression host BL21 (AI) .47 Figure 23 DIGE of induced and non-induced transformed bacterial lysates 47 ix Yuan, J and Yankner, B.A (2000) Apoptosis in the nervous system Nature 407: 802-809 Yuan, J., Shaham, S., Ledoux, S., Ellis, H.M and Horvitz, H.R (1993) The C elegans cell death gene Ced-3 encodes a protein similar to mammalian interleukin-1 betaconverting enzyme Cell 75: 641-652 Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., Flaig, M., Gillespie, J W., Hu, N., Taylor, P R., Emmert- Buck, M R., Liotta, L A., Petricoin III, E F., Zhao, Y., (2002) 2D Differential In-gel Electrophoresis for the Identification of Esophageal Scans Cell Cancer-speific Protein Markers Mol Cell Proteomics 1: 117–124 Zhu, Q., Huang, X., Chen, Y.J., Yao, S.Q (2003) Activity-based fluorescent probes that target phosphatases Tetrahedron Lett 44: 2669-72 90 CHAPTER APPENDIX PPENDIX MALDI-TOF mass spectra of tryptic peptides derived from yeast proteins ( Table 1) labeled by FP-biotin and their peptide alignment sequences 7.1.1 Subtilisin-like protease III Voyager Spec #1=>BC=>NR(2.00)[BP = 1066.0, 1070] 1066.0170 100 1070.1 90 1059.9894 80 70 1081.9883 % Intensity 60 1179.5420 50 933.4350 40 917.3788 1098.9688 936.4366 1707.7291 977.4572 1130.6207 1149.5005 1319.6769 938.8080 1343.6468 1142.5352 1792.7948 1437.5871 973.3506 20 1216.5788 1585.6697 1413.2121 1197.9640 1027.5213 1640.7180 30 10 899.0 1419.4 1939.8 2460.2 2980.6 3501.0 Mass (m/z) 51 101 151 201 251 301 351 401 451 MKFSTILPIL GNFHTEHQHV KLTEDDAFWR SQDPIIKFVE TEHEDFEGRA AVKVLRSNGE KSLAMEMAVN DDRAFFSNWG SYFLSLQPAP NGGGKKLDGF WANCCLCMII AHKTEFLPYR VISSSVSSKS QETTVKISNS EWGAVIPAND GTVSDVIKGI AAVDSGVHFA TCVDVFAPGI DSEFFNDAPS W PEFDGIVRFI YVIVFNEDIS QFGGIDNFFD SLQEEAPWGL EASDLNGHGT ELVTKEHIES IAAGNEDEDA NIMSTYIGSR PQELKEKVLK ENIDGTRSVR LQQIQSHMQV INGLFRGYTG HRVSHREKGV HCAGIIGSKH SKKKNKEFKG CLSSPAGAEK NATLSLSGTS FSTQGVLGDI AGEGLGQHDP VQKDHSTSVG YFTDEIIKII TSYVLDTGID FGVAKNTKIV STANLSLGSS SITVGASTFS MASPHVAGIL GDDTPNKLIY 91 7.1.2 Vesicular Transport Protein Sec17 Voyager Spec #1=>BC=>NR(2.00)[BP = 1963.9, 2242] 2241 100 1962.9114 90 80 1066.0043 70 % Intensity 60 1081.9821 50 40 933.4990 30 1320.5418 1179.5301 1493.7002 1707.7693 20 905.4055 1059.9962 1487.7273 912.1764 1234.6344 953.26231106.3759 1994.9498 1293.0111 1475.7542 10 994.4239 1977.8996 1124.3915 1657.9035 1839.9494 1470.5940 2071.1285 899.0 1419.4 1939.8 2370.1198 2651.4557 2460.2 2980.6 3501.0 Mass (m/z) 51 101 151 201 251 ISDPVELLKR KELNLAGDSF ENAIQIFTHR DQSVALSNKC YFLKKGLCQL GDSEQLSEHC AEKKGVPSSG LKAADYQKKA GQFRRGANFK FIKCADLKAL AATDAVAAAR KEFDNFMRLD FMKLFSGSDS GNEDEAGNTY FELGEILEND DGQYIEASDI TLQEGQSEDP KWKITILNKI YKFEEAADLC VEAYKCFKSG LHDYAKAIDC YSKLIKSSMG NFADSRESNF KESIQQQEDD VQAATIYRLR GNSVNAVDSL YELAGEWYAQ NRLSQWSLKD LKSLIDAVNE LL 92 7.1.3 Hypothetical 61.1 kDa protein in YPT52-DBP7 intergenic region Voyager Spec #1=>BC=>NR(2.00)[BP = 933.5, 1488] 100 933.4668 1487 90 1320.5230 80 70 977.4971 1065.9876 % Intensity 60 50 1707.7384 40 30 907.1786 1838.8874 1081.9754 1487.7000 1267.6347 912.3168 1116.3703 1475.7356 964.1767 2212.09162383.9219 20 1316.6530 2369.0715 988.1680 1157.4737 1567.6925 1008.2848 1206.76111387.5648 1759.9144 1525.6380 1745.7702 1014.9909 1274.6836 1810.92001979.0833 1514.8219 10 2138.0624 899.0 1419.4 1939.8 2706.2033 2460.2 2980.6 3501.0 Mass (m/z) 51 101 151 201 251 301 351 401 451 501 MMLRTTASRK AGGIIYSQKN AKSNDLLSGL GSLNDLINSL MIQRTSEVIT EEKLKANEEL EINSEVNDLS DKELSRLKLL KTNPPSLLSV SGILGQLTAK EVVSLKGWPH IVLRRGLASI DKFGDFFSNN TGSSQTRRSN NDSNLSIPES ELNTQYENSK LQAKHANEVG KSIDRSSKIL SNLLSTFNKK ALDELESTCS VFSLFLFTKT KVCESWIEDA NTGTTVASKK VPFAEDLLET RENIEVKKIL EFNSIKKSNQ REFEKNLQKN LLSITQVKEF SKNEALVQLT SCCDDGDCCS GKKILSNEQI GNPSNATDFD RRKLEVQRLV ASHKFRNTLW YEHYHDRPTL SLEPLNIETE NMLTNLSQLN LLQEVDEFKE NKIIKDKIEK FQVDEIKSRI CKKGNKNEGK YNRWNLLADD SVYARVGDNL EILDCEIRTL TIALSATAFY FLEDSWDGLK NSDPQLKEII ETLKEALSNY NLTKQKDKEL ERNGRLAHLE NNNNLPDVNI EGKISCKCKP FKTASLLPPN RVSNLNDAVE 93 MALDI-TOF mass spectra of tryptic peptides derived from yeast proteins (Table 2) labeled by caspase-biotin and their peptide alignment sequences 7.2.1 Glyceraldehyde 3-phosphate dehydrogenase Voyager Spec #1=>BC=>NR(2.00)[BP = 1765.1, 10239] 1.0E+4 100 1764.0808 90 80 70 % Intensity 60 50 40 30 20 1066.2319 10 908.4147 1614.1396 1082.2238 1141.3826 899.0 1531.0364 1779.1127 1419.4 2042.3915 2808.8067 1939.8 2460.2 3501.0 2980.6 Mass (m/z) Spot number:1 Voyager Spec #1=>BC=>NR(2.00)[BP = 1763.8, 11407] 1763.7676 100 1.1E+4 90 80 70 % Intensity 60 50 40 854.9932 30 20 1613.8637 808.2075 10 880.2215 846.2062 821.5518 1066.0310 1060.0257 1015.2792 800.0 Spot number:2 1530.7844 1264.4832 1623.1188 1778.7942 1261.7989 1428.1944 1340.2 2041.1343 2213.1920 2385.9398 1880.4 2420.6 2807.2931 2960.8 3080.3571 3501.0 Mass (m/z) 94 Voyager Spec #1=>BC=>NR(2.00)[BP = 1763.8, 5843] 1763.8237 100 5843.0 90 80 70 % Intensity 60 50 40 30 20 1613.9363 1766.1193 1066.0669 10 1006.2860 1167.3396 1320.5863 999.0 2042.1363 1707.8067 1499.4 2808.4127 1999.8 2500.2 3000.6 3501.0 Mass (m/z) Spot number:4 51 101 151 201 251 301 MGKVKVGVNG STHGKFHGTV VFTTMEKAGA SCTTNCLAPL GALQNIIPAS KPAKYDDIKK IALNDHFVKL FGRIGRLVTR KAENGKLVIN HLQGGAKRVI AKVIHDNFGI TGAAKAVGKV VVKQASEGPL ISWYDNEFGY AAFNSGKVDI GNPITIFQER ISAPSADAPM VEGLMTTVHA IPELNGKLTG KGILGYTEHQ SNRVVDLMAH VAINDPFIDL DPSKIKWGDA FVMGVNHEKY ITATQKTVDG MAFRVPTANV VVSSDFNSDT MASKE NYMVYMFQYD GAEYVVESTG DNSLKIISNA PSGKLWRDGR SVVDLTCRLE HSSTFDAGAG 95 7.2.2 Fructose-bisphosphate aldolase A Voyager Spec #1=>BC=>NR(2.00)[BP = 1675.8, 3978] 1675.8158 100 3977.8 90 80 70 1342.6855 1808.9365 % Intensity 60 50 2107.0927 40 2273.0959 854.9998 30 806.2121 870.9771 868.2297 909.2355 20 1093.5284 1434.7274 1059.2807 2088.1010 1631.0604 1791.8986 2061.2680 2228.0747 10 800.0 1340.2 1880.4 2420.6 2960.8 3501.0 Mass (m/z) 51 101 151 201 251 301 351 MPYQYPALTP NTEENRRFYR KGGVVGIKVD VLKIGEHTPS RCQYVTEKVL MATVTALRRT SYGRALQASA AAASESLFVS EQKKELSDIA QLLLTADDRV KGVVPLAGTN ALAIMENANV AAVYKALSDH VPPAVTGITF LKAWGGKKEN NHAY HRIVAPGKGI NPCIGGVILF GETTTQGLDG LARYASICQQ HIYLEGTLLK LSGGQSEEEA LKAAQEEYVK LAADESTGSI HETLYQKADD LSERCAQYKK NGIVPIVEPE PNMVTPGHAC SINLNAINKC RALANSLACQ AKRLQSIGTE GRPFPQVIKS DGADFAKWRC ILPDGDHDLK TQKFSHEEIA PLLKPWALTF GKYTPSGQAG 96 7.2.3 GTP-binding nuclear protein RAN Voyager Spec #1=>BC=>NR(2.00)[BP = 1785.0, 9298] 1784.9828 100 9.3E+ 90 80 70 % Intensity 60 50 2052.2188 40 30 20 2180.4688 10 1013.3594 999.0 1312.7101 2252.1462 1499.4 1999.8 2500.2 3000.6 3501.0 Mass (m/z) 51 101 151 201 GDASGRNAAM TLGVEVHPLV VTSRVTYKNV KNLQYYDISA ALAAQYEHDL AAQGEPQVQF FHTNRGPIKF PNWHRDLVRV KSNYNFEKPF EVAQTTALPD KLVLVGDGGT NVWDTAGQEK CENIPIVLCG LWLARKLIGD EDDDL GKTTFVKRHL FGGLRDGYYI NKVDIKDRKV PNLEFVAMPA TGEFEKKYVA QAQCAIIMFD KAKSIVFHRK LAPPEVVMDP 97 7.2.4 Peptidyl-prolyl cis-trans isomerase A Voyager Spec #1=>BC=>NR(2.00)[BP = 1947.0, 10406] 1.0E+ 100 1946.0493 90 80 70 % Intensity 60 50 1598.7820 40 30 20 1614.8116 10 1001.3161 1139.4189 999.0 1949.3310 1831.9807 1550.8031 1380.7684 1707.8497 1961.0250 1499.4 2286.9972 1999.8 2500.2 2580.8212 2793.5194 3000.6 3313.5478 3501.0 Mass (m/z) 51 101 151 MVNPTVFFDI SCFHRIIPGF ANAGPNTNGS KTSKKITIAD AVDGEPLGRV SFELFADKVP KTAENFRALS TGEKGFGYKG MCQGGDFTRH NGTGGKSIYG EKFEDENFIL KHTGPGILSM QFFICTAKTE WLDGKHVVFG KVKEGMNIVE AMERFGSRNG CGQLE 98 7.2.5 Alpha enolase Voyager Spec #1=>BC=>NR(2.00)[BP = 1909.0, 2452] 2452 100 1540.7703 90 80 70 1804.9298 % Intensity 60 50 40 30 20 10 1862.1631 1926.0118 1556.7395 2176.0744 1910.3297 2501.2474 1035.2904 1707.7654 2354.1523 1076.3655 1864.8237 3013.6418 3313.3681 1240.7694 1525.7715 2807.4371 1032.7092 2286.1183 1807.4499 1177.2292 1389.67361552.5767 2130.3029 2692.2068 2273.3088 2505.0698 1968.6410 1369.3776 1795.9649 2833.1217 1238.3901 2118.74042277.0749 1606.7210 3016.8807 2697.5799 2566.0591 2418.2697 1960.9884 2857.8353 3156.2375 3349.6934 3019.7749 3357.7058 999.0 1499.4 1999.8 2500.2 3000.6 3501.0 Mass (m/z) Spot number:11 Voyager Spec #1[BP = 1540.8, 13975] 1540.7504 100 1.4E+ 90 1907.9688 80 70 1805.9283 % Intensity 60 50 1526.7268 1923.9719 40 30 20 1543.7496 1910.9929 3013.6664 1425.6852 1557.7507 2354.16192511.0659 1926.9786 1029.6762 1495.0351 1708.73361860.9787 1010.2706 1179.5272 1808.9367 1994.9703 2178.0708 2502.2081 1529.7312 10 1015.5566 2357.0919 1962.9383 1320.5440 1168.7012 1464.7273 1628.0540 1813.0730 2101.0200 2273.00872432.99212585.1638 999.0 1499.4 1999.8 2500.2 2808.5598 3016.6557 3314.5981 2811.3819 2996.4772 3343.7228 2814.8257 2951.4018 3131.2746 3367.8082 3000.6 3501.0 Mass (m/z) Spot number:12 99 51 101 151 201 251 301 351 401 MSILKIHARE DNDKTRYMGK ENKSKFGANA NVINGGSHAG GKDATNVGDE FFRSGKYDLD WGAWQKFTAS TESLQACKLA SERLAKYNQL IFDSRGNPTV GVSKAVEHIN ILGVSLAVCK NKLAMQEFMI GGFAPNILEN FKSPDDPSRY AGIQVVGDDL QANGWGVMVS LRIEEELGSK EVDLFTSKGL KTIAPALVSK AGAVEKGVPL LPVGAANFRE KEGLELLKTA ISPDQLADLY TVTNPKRIAK HRSGETEDTF AKFAGRNFRN FRAAVPSGAS KLNVTEQEKI YRHIADLAGN AMRIGAEVYH IGKAGYTDKV KSFIKDYPVV AVNEKSCNCL IADLVVGLCT PLAK TGIYEALELR DKLMIEMDGT SEVILPVPAF NLKNVIKEKY VIGMDVAASE SIEDPFDQDD LLKVNQIGSV GQIKTGAPCR 100 7.2.6 Actin, beta Voyager Spec #1=>BC=>NR(2.00)[BP = 1516.8, 9859] 9.9E+ 100 1515.7803 90 80 70 % Intensity 60 1790.9501 50 40 30 2215.1192 20 10 1132.5128 1023.3268 1267.7241 1045.3422 999.0 3183.7567 1547.7709 1499.4 1960.9411 3137.7771 2169.3587 1999.8 2500.2 3000.6 3501.0 Mass (m/z) 51 101 151 201 251 301 351 LVVDNGSGMC AQSKRGILTL APLNPKANRE GVTHTVPIYE IVRDIKEKLC PEALFQPSFL GIADRMQKEI SKQEYDESGP KAGFAGDDAP KYPIEHGIVT KMTQIMFETF GYALPHAILR YVALDFEQEM GMESCGIHET TALAPSTMKI SIVHRKCF RAVFPSIVGR NWDDMEKIWH NTPAMYVAIQ LDLAGRDLTD ATAASSSSLE TFNSIMKCDV KIIAPPERKY PRHQGVMVGM HTFYNELRVA AVLSLYASGR YLMKILTERG KSYELPDGQV DIRKDLYANT SVWIGGSILA GQKDSYVGDE PEEHPVLLTE TTGIVMDSGD YSFTTTAERE ITIGNERFRC VLSGGTTMYP SLSTFQQMWI 101 7.2.7 Proteasome activator complex subunit Voyager Spec #1=>BC=>NR(2.00)[BP = 1337.7, 21698] 1337.7125 100 2.2E+ 90 80 70 % Intensity 60 50 40 30 20 10 1066.0811 999.0 1293.7304 1468.7678 1434.8280 1499.4 1706.3486 1883.9899 1973.2570 2179.5928 1999.8 2443.1910 2500.2 2920.8077 3000.6 3501.0 Mass (m/z) MASLLKVDQE VKLKVDSFRE RITSEAEDLV ANFFPKKLLE LDSFLKEPIL 51 NIHDLTQIHS DMNLPVPDPI LLTNSHDGLD GPTYKKRRLD ECEEAFQGTK 101 VFVMPNGMLK SNQQLVDIIE KVKPEIRLLI EKCNTPSGKG PHICFDLQVK 151 MWVQLLIPRI EDGNNFGVSI QEETVAELRT VESEAASYLD QISRYYITRA 201 KLVSKIAKYP HVEDYRRTVT EIDEKEYISL RLIISELRNQ YVTLHDMILK 251 NIEKIKRPRS SNAETLY 102 7.2.8 T-cell receptor beta chain C region Voyager Spec #1=>BC=>NR(2.00)[BP = 1267.7, 26096] 1267.7309 100 2.6E+ 90 80 70 % Intensity 60 1760.0037 50 40 30 1383.8388 20 10 999.0 1475.7809 1762.8195 1952.0524 1098.5821 1296.6940 1622.8797 1814.5209 1482.2236 1499.4 2186.2876 2333.2523 2202.2997 2336.2600 2515.9380 1999.8 2810.4214 2500.2 3501.0 3000.6 Mass (m/z) Spot number:24 Voyager Spec #1=>BC=>NR(2.00)[BP = 1267.7, 13427] 1267.7220 100 1.3E+ 90 80 70 % Intensity 60 50 40 30 1383.8101 20 10 1082.0318 1247.5754 1013.4651 1193.1093 999.0 2188.5037 1857.8026 1707.80511855.40771994.0622 1444.0995 2317.2825 1575.9028 2138.2774 1499.4 1999.8 2502.2854 2670.15002808.4508 2500.2 3082.1940 3000.6 3314.6219 3501.0 Mass (m/z) Spot number:25 NVPIDDSGMP EDRFSAKMPN ASFSTLKIQP SEPRDSAVYF CASSFDRGNS 51 YEQYFGPGTR LTVTEDL 103 104 ... molecule probes for proteomics applications”, Appl Genomics Proteomics, 2003, in press Tan, E.L.P., Panicker, R.C., Tan, L.P., Chattopadhaya, S., Yao, S.Q ? ?Developing chemical biology tools for the study... be analyze by 2D-GE, thereby rendering the technique insufficient for large- scale protein profiling in human 1.2 Addressing the proteomic challenges In this section, two strategies that have been... quantification Image overlay Image subtraction Figure Strategy of 2D DIGE 1.2.2 Sub- proteomic expression profiling of proteins using activity-based probes (ABPs) As mentioned before, more than 100 000 proteins