Developing peptide based approaches for systematic enzyme profiling

DEVELOPING PEPTIDE-BASED APPROACHES FOR SYSTEMATIC ENZYME PROFILING SUN HONGYAN (B.Sc., Wuhan University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 Acknowledgements I would like to express my deepest gratitude to my supervisor and mentor A/P Yao Shao Qin. He has brought me into the fascination of Chemical Biology and inspired me throughout my striving for scientific achievements. He has instilled with me unparalleled passion for scientific research, and empowered me to venture into unraveled territories in the Chemical Biology field. His indefatigable spirits, invaluable guidance and professionalism have been deeply engraved upon my mind, and they would be the gleams of light for me to shine through the gloom of scientific unknowns in the years ahead. My sincere thankfulness and appreciation also extend to my lab-mates in both DBS and Chemistry--Raja, Souvik, Mahesh, Laypheng, Grace, Eunice, Rina, Dawn, Hu Yi, Huang Xuan, Zhu Qing, Aparna, Resmi, Wang Gang, Elaine, Mingyu, Junqi, Wang Jun, Wei Lin, Su Ling, Candy, Liu Kai, Liqian, Farhana, Kitty, Mingyu, Pengyu, Wu Hao, Haibin, Jingyan, Kalesh. I would like to take this opportunity to thank each of you for invaluable assistances, fruitful discussions and happy memories over these years. Special thanks goes to my collaborators, Candy helped me to carry out the screening work for 14-3-3 projects as well as part of array work with Ser/Thr phosphatase project. Liqian and Haibing helped me with pTyr peptide library synthesis. Laypheng also helped out with the cloning work with pTyr peptide array project, Aparna has participated in the phage work of SrtA. Their timely assistance, superior team-works, and earnest friendship helped me to tide over the most demanding periods of my research pursuit. I would also like to thank Prof. Liou Yih-Cherng, Prof. Lu Yixin and Prof. Chang Young-Tae for writing the recommendation letters. I also appreciate the i support from Mdm Han Yanhui and Ms Peggy Ler from NMR lab and Mdm Wong Lai Kwan and Mdm Lai Hui Ngee from MS lab. A hearty “thank you” to my parents, my brother and Mr Du Xiaonan. I sincerely thank them for their understanding and supports over these years. This thesis will be dedicated to them. I also acknowledge kind support from NUS for providing me research scholarship. ii Table of Contents Page Chapter 1. Introduction 1.1 Summary 1.2 Conventional Enzyme Assay 1.3 Microarray Technology 1.3.1 1.3.2 1.3.3 Immobilization Strategies in Microarray Applications 1.3.1.1 Immobilization of Proteins 1.3.1.2 Immobilization of Peptides 1.3.1.3 Immobilization of Small Molecules Microarray-Based Screening Assays 10 1.3.2.1 Functional Annotation 10 1.3.2.2 Substrate Fingerprinting 12 1.3.2.3 Inhibitor Fingerprinting 18 Other Recent Developments 20 1.4 Bioimaging Technology 23 1.5 Objectives 24 Chapter 2. Activity Based Fingerprinting of Proteases Using FRET Peptides 26 2.1 Summary 26 2.2 Introduction 26 2.3 Results and Discussion 30 2.3.1 Protease Assays 30 2.3.2 FRET-Based Assay 32 2.3.3 Detection Limit 33 iii 2.3.4 Substrate Fingerprinting 2.4 Conclusion 35 37 Chapter 3. Quantitative Substrate Fingerprinting of Ser/Thr Phosphatases Using Peptide Microarrays 38 3.1 Summary 38 3.2 Introduction 39 3.3 Results and Discussion 41 3.3.1 Peptide Library Design 3.3.2 Determination of Peptide Immobilization Efficiency 41 and Pro-Q Detection Linear Range on Array 44 3.3.3 Time and Concentration Dependent Experiment 50 3.3.4 Substrate Fingerprints of Phosphatases 55 3.3.5 Pin1 Regulation Experiment and Cell Based Assay 57 3.4 Conclusion 60 Chapter 4. Rapid Affinity-Based Fingerprinting of 14-3-3 Isoforms Using Combinatorial Peptide Array 61 4.1 Summary 61 4.2 Introduction 61 4.3 Results and Discussion 64 4.3.1 Peptide Library Synthesis and Pin1 Fingerprinting 64 4.3.2 Substrate Fingerprinting of Seven 14-3-3 Isoforms 66 4.3.3 Identification of Isoform-Specific Binding Motif for 14-3-3 Sigma 4.4 Conclusion Chapter 5. Peptide Microarray for Studying PTPs 73 76 77 iv 5.1 Summary 77 5.2 Introduction 77 5.3 Results and Discussion 81 5.3.1 Peptide Library Synthesis 81 5.3.2 Substrate Fingerprinting of PTP mutants 83 5.3.3 Dual Labeling Assay 87 5.3.4 p(CAP) Peptide Array 92 5.4 Conclusion 96 Chapter 6. A Novel Strategy for SrtA-Mediated Site-Spcific Labeling of Proteins In vitro 97 6.1 Summary 97 6.2 Introduction 98 6.3 Results and Discussion 105 6.3.1 Cloning of Target Genes 105 6.3.2 Protein Expression and Purification 107 6.3.3 Site-Directed Mutagenesis of pTYB1-EGFP 108 6.3.4 Probe Synthesis 109 6.3.5 Protein Labeling 111 6.3.6 FRET Assay for SrtA and SrtA Mutants 115 6.4 Conclusion 118 Chapter 7. Experimental Procedures 120 7.1 General Procedures 120 7.1.1 Materials 120 7.1.2 Instrumentation 121 7.2 Solution Phase Synthesis 121 v 7.3 Peptide Synthesis 135 7.3.1 137 1,000-Member pSer/Thr Peptide Library Synthesis 7.3.2 p(CAP) Peptide Library Synthesis 138 7.4 Microplate Assay 138 7.4.1 FRET Assay against Proteases 138 7.4.2 p(CAP) Aassay against PTPs 139 7.4.3 FRET Assay with SrtA 139 7.5 Microarray-Based Enzyme Screening 140 7.5.1 Preparation of Avidin Slide 140 7.5.2 Microarray Preparation 141 7.5.3 Screening Peptide Array with pSer/Thr Phosphatases 141 7.5.3.1 Pro-Q Assay 141 7.5.3.2 Fingerprint Experiment 142 7.5.3.3 Time-Dependent Experiment 142 7.5.3.4 Solution Phase HPLC Assay 142 7.5.3.5 Phosphatase Inhibition Experiment 143 7.5.3.6 Pin1 Regulation Assay 143 Highthroughput Screening of Seven14-3-3 Isoforms 143 7.5.4.1 Protein Labeling and Screening on the Array 143 7.5.4.2 KD Analysis with Fluorescence Polarization 144 Screening Peptide Array with PTP Mutants 145 7.5.4 7.5.5 7.6 Gene Cloning and Protein Expression 145 7.6.1 Cloning SrtA Gene into pdest17 Vector 145 7.6.2 Cloning EGFP Gene into pdest17 Vector 146 vi 7.6.3 Site-Directed Mutagenesis of pTYB1-EGFP and PTP Mutants 147 7.6.3.1 SDM with pTYB1-EGFP 147 7.6.3.2 SDM with PTP Mutants 149 7.6.4 His-Tag Purification 150 7.6.5 CBD-Tag Purification 150 7.6.6 GST-Tag Purification 151 7.7 In vitro Protein Labeling with SrtA 151 7.8 Data Processing and Analysis 152 7.8.1 Dephosphorylation Ratio Analysis 152 7.8.2 Kobs Analysis 152 7.8.3 14-3-3 Fingerprinting Analysis 153 7.8.4 Quantiative 14-3-3 KD Analysis 154 7.8.5 PTP Substrate Fingerprinting Analysis 154 7.8.6 Quantiative PTP Mutant KD Analysis 155 Chapter 8. Concluding Remarks 156 Chapter 9. References 158 Chapter 10. Appendix 173 10.1 Supplemental Tables 173 vii Summary Enzymes are critical to the vital functioning of any living system and play an important role in regulation of cellular processes. There are around 18-29% of eukaryotic genomes which encode enzymes. However, little is still understood about the physiological role, substrate specificity and downstream targets of enzymes. There is a pressing need to develop platforms for high-throughput identification and characterization of enzyme activities. This research is now referred to as “Catalomics” which defines an emerging ‘-omics’ field where high-throughput studies of enzymes are carried out by using advanced chemical proteomics approaches. One key challenge in “Catalomics” is to develop robust tools for substrate fingerprinting of enzymes in high throughput. This thesis examines and addresses these challenges by integration of various emerging interdisciplinary and enabling platforms. Chapter describes a novel FRET assay for activity-based fingerprinting of proteases from different classes. Chapter presents a novel phosphorylated peptide array for substrate specificity studies of phosphotases. With this platform, new phosphatase biology was discovered. Chapter presents a combinatorial peptide microarray for large-scale studies of substrate specificity of 14-3-3 proteins. Chapter presents an affinity based microarray approach for mapping the substrate specificity of PTPs. Chapter demonstrates a site-specific protein labeling approach via SrtA mediation which could be potentially applied into bioimaging field to study the enzyme activity in vivo. These approaches should provide comprehensive insights into the catalytic mechanism of different clusters of enzymes. The substrate fingerprints obtained would not only provide the invaluable information for identification of physiological substrates but also aid in the design of selective and potent inhibitors. viii List of Publications (2003 – 2008) 1. Sun, H.; Tan, L.P.;Gao, L.; Yao, S.Q. High-throughput Screening of Catalytically Inactive Mutants of Protein Tyrosine Phosphatases (PTPs) in a Phosphopeptide Microarray. Chem. Commun., (2009), 677-679. 2. Sun, H.; Lu, C.H.S.; Uttamchandani, M.; Xia, Y.; Liou, Y.-C.; Yao, S.Q. Peptide Microarray for High-throughput Determination of Phosphatase Specificity and Biology. Angew. Chem. Intl. Ed., (2008), 47, 1698-1702. 3. Lu, C.H.S.; Sun, H.; Bakar, F.B.A.; Uttamchandani, M.; Zhou, W.; Liou, Y.-C.; Yao, S.Q. Deciphering the Substrate Preferences of 14-3-3 Isoforms Using Peptide Microarray, Angew. Chem. Intl. Ed., (2008), 47, 7438-7441. 4. Sun, H.; Lu, C.H.S.; Shi, H.; Gao, L.; Yao, S.Q. Peptide Microarrays for High-throughput Studies of Ser/Thr Phosphatases, Nature Protocols, (2008), 3, 1485-1493. 5. Uttamchandani, M; Li. J.; Sun, H.; Yao, S.Q Activity-Based Profiling: New Developments and Directions in Protein Fingerprinting, ChemBioChem, (2008), 9, 667-675. 6. Uttamchandani, M.; Wang, J.; Li, J.; Hu, M.; Sun, H.; Chen, K. Y.-T.; Liu, K.; Yao, S.Q. Inhibitor Fingerprinting of Matrix Metalloproteases Using a Combinatorial Peptide Hydroxamate Library. J. Am. Chem. Soc., (2007), 129, 7848-7858. 7. Lee, W.L.; Li, J.; Uttamchandani, M.; Sun, H.; Yao, S. Q. Inhibitor Fingerprinting of Metalloproteases Using Microplate and Microarray Platforms – An Enabling Technology in Catalomics Nat. Protocols, (2007), 2, 2126-2138. ix 87 a L8 SRLYRpSPSMPE 1741 Cdc25c (Xenopus) S285 b Y Due to patent issues, the protein source of these peptides cannot be revealed. ND =the correct peptide mass was detected by MS, but the peptide was not the major product. 175 Table 10.2 List of 53 peptide kobs values based on time-course experiment of lambda phosphatase, against the phospho-peptide library. They are selected based on stringent filtering criteria of accuracy curve fits (r > 0.85) and significant desphorylation signals of > 40% change from original Pro-Q readings. Absolute fluorescent intensity [Fluorescent Intensity – Background Intensity] of each peptide at designated time point is indicated in the table below. NO. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 ID Sequence B1 B3 C1 C5 C7 C8 D1 D2 D6 D8 E1 E2 E3 E6 E7 F1 F2 F3 F5 F6 F7 F8 G2 G3 G4 G6 G7 H1 H2 H7 I1 I2 J1 J2 J3 J4 J6 J7 J8 K1 K2 K4 K5 K6 K7 K8 L2 L3 L4 VDAAVpSPEERH APPAPpSPGPFA SQRFGpTPLSAS CHVEDpTPCPGE ANAFLpSPNCDW PQQTTpSPENAG SALSSpSPNNLS CTLTTpSPRLPV ELQVPpTPSLPS AMCMHpTPNTAP AVSLDpSPVSVG PVPPGpTPAPPG GAIGGpTPPAFN GTLGLpTPPTTE PSGLLpTPPQSG FELLPpTPPLSP LLGRSpSPSKNY AIVFRpTPPYAD PTPPLpSPSRRS VPSPPpSPPPLP KLPCDpTPGQGL LSTLSpSPGPKL AEMEMpSPAGQI GGGLGpTPLRLP TPCYIpSPELCE SRIILpSPCVQA YTVVGpTPCYIS EQAEGpSPGGPG SYDSGpTPLEDD CYENPpSPTAGI GMMGLpSPDAYR MHRQEpTVDC SDLLTpSPDVGL PVARTpSPLQTP LPEVApTPESEE PVVTCpTPSCTA AGPALpSPVPPV LEPLCpTPVVTC SDSLSpSPTLLA VDLACpTPTDVR ENNVLpSPLPSQ GSRSRpTPSLPT DDLMLpSPDDIE APAAPpTPAAPA VKMLGpSPVDSV VAVVRpTPPKSP SGSHCpTPPPPY PEQPLpTPVTDL MNILGpSPISAD Absolute fluorescence intensity of each peptide at designated time point. (min) 0’ 553 450 962 1674 905 468 813 1465 1022 1336 570 913 712 1065 1395 454 774 714 788 517 1957 689 957 901 1329 1192 1080 570 678 1629 1002 1416 1032 775 1191 1159 506 1539 597 1219 593 872 1114 651 781 531 1308 815 843 5’ 581 469 850 1574 690 471 794 1272 859 1161 496 602 729 823 963 396 802 644 664 545 1720 617 726 743 1317 1035 935 526 596 1387 900 1081 985 708 1085 1044 397 1561 511 1234 482 621 929 507 670 402 902 680 798 15’ 468 396 648 1590 678 477 823 1035 777 973 542 338 662 823 692 364 640 556 650 501 1639 532 700 607 1024 910 919 454 663 1555 999 921 819 699 987 756 368 1299 570 1113 305 567 955 362 699 409 727 559 826 30’ 361 295 603 1225 436 276 660 1142 748 696 468 347 447 611 688 305 630 388 561 479 1493 413 568 524 982 778 709 413 395 1167 774 761 568 658 971 763 308 1042 560 879 333 478 841 350 690 317 583 518 781 60’ 310 236 468 1149 411 239 514 844 580 692 411 289 407 586 550 308 486 399 457 339 1142 344 350 389 710 553 552 265 337 889 585 601 477 502 731 527 200 802 368 659 247 357 691 257 534 212 426 411 565 120’ 248 158 358 1001 443 192 434 727 418 666 335 202 373 541 408 265 390 361 395 288 1050 364 337 302 637 485 419 234 307 901 476 483 410 366 605 519 171 654 295 558 238 307 307 191 414 159 346 359 475 kobs 0.022 0.016 0.039 0.033 0.068 0.018 0.01 0.032 0.025 0.06 0.013 0.13 0.026 0.066 0.12 0.056 0.016 0.059 0.031 0.0048 0.028 0.048 0.04 0.042 0.026 0.032 0.024 0.023 0.026 0.026 0.012 0.057 0.033 0.008 0.019 0.046 0.036 0.022 0.005 0.017 0.09 0.064 0.0066 0.064 0.009 0.031 0.077 0.051 0.0045 R 0.95 0.96 0.97 0.99 0.92 0.86 0.92 0.88 0.93 0.98 0.9 0.97 0.9 0.92 0.94 0.96 0.95 0.93 0.95 0.93 0.97 0.99 0.94 0.98 0.97 0.99 0.98 0.98 0.86 0.87 0.92 0.96 0.88 0.98 0.97 0.95 0.96 0.98 0.85 0.97 0.95 0.92 0.94 0.95 0.89 0.93 0.96 0.97 0.92 Std. Dev. 0.008 0.006 0.007 0.006 0.022 0.013 0.009 0.015 0.008 0.01 0.008 0.03 0.012 0.021 0.034 0.013 0.005 0.018 0.008 0.007 0.006 0.006 0.011 0.007 0.007 0.004 0.005 0.004 0.014 0.013 0.008 0.012 0.012 0.004 0.005 0.012 0.009 0.005 0.01 0.06 0.024 0.02 0.006 0.016 0.008 0.009 0.018 0.01 0.007 176 50 51 52 53 L5 L6 L7 L8 HSSAApTPNLGP TFSGEpTPKRCL RLLCSpTPSFKK SRLYRpSPSMPE 534 1158 950 587 344 1086 865 507 372 967 874 515 208 904 665 461 212 673 474 285 180 591 460 232 0.077 0.022 0.023 0.016 0.87 0.99 0.95 0.94 0.033 0.004 0.008 0.007 177 Table 10.3 Dephosphorylation ratio of 87 phosphopeptide with each phosphatase dataset. Readings were obtained by treatment the peptide array with U of each enzyme and incubated at RT for h. NO. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 ID A1 B1 B3 B4 B5 B7 B8 C1 C2 C3 C4 C5 C6 C7 C8 D1 D2 D3 D4 D5 D6 D7 D8 E1 E2 E3 E4 E5 E6 E7 E8 F1 F2 F3 F4 F5 F6 F7 F8 G1 G2 G3 G4 G5 G6 G7 G8 H1 Peptide Sequence LPNAApSPGAEQ VDAAVpSPEERH APPAPpSPGPFA QQKKTpSPLNFK LTPPQpSGKKQS PLYVFpSPTEEL EGSGRpSPRYAL SQRFGpTPLSAS FFRIEpSPVKSF QVAPLpSPRSAN FFRVEpSPVKLF CHVEDpTPCPGE QLSCTpSPCYRE ANAFLpSPNCDW PQQTTpSPENAG SALSSpSPNNLS CTLTTpSPRLPV IYSSSpSPVSMK SNGEPpSPVAGP SPNNLpSPTGWS ELQVPpTPSLPS GMGAYpSPHSNG AMCMHpTPNTAP AVSLDpSPVSVG PVPPGpTPAPPG GAIGGpTPPAFN GSSCIpSPTIVQ VSVGSpSPPVKN GTLGLpTPPTTE PSGLLpTPPQSG HSESApSPSALS FELLPpTPPLSP LLGRSpSPSKNY AIVFRpTPPYAD VAGVHpSPMASS PTPPLpSPSRRS VPSPPpSPPPLP KLPCDpTPGQGL LSTLSpSPGPKL SLSSSpSPFVVL AEMEMpSPAGQI GGGLGpTPLRLP TPCYIpSPELCE LSHQPpSPFLNM SRIILpSPCVQA YTVVGpTPCYIS GGGLSpSPLRLP EQAEGpSPGGPG Dephosphorylation Ratio of Each Phosphotase Alkaline Lambda PP1 PP2B PP2A 0.36 0.49 0.11 0.53 0.17 0.41 0.24 0.01 0.32 0.09 0.21 0.34 0.14 0.54 0.00 0.18 0.08 0.05 0.60 0.00 0.00 0.08 0.04 0.57 0.00 0.12 0.09 0.00 0.06 0.07 0.17 0.09 0.00 0.20 0.00 0.26 0.40 0.40 0.59 0.00 0.08 0.08 0.00 0.38 0.08 0.14 0.29 0.02 0.44 0.00 0.13 0.00 0.00 0.20 0.27 0.04 0.46 0.43 0.21 0.00 0.03 0.32 0.40 0.00 0.01 0.15 0.49 0.30 0.04 0.30 0.27 0.43 0.01 0.01 0.00 0.29 0.32 0.05 0.41 0.15 0.10 0.38 0.17 0.45 0.15 0.14 0.02 0.00 0.29 0.00 0.24 0.00 0.00 0.00 0.00 0.05 0.08 0.00 0.08 0.07 0.06 0.40 0.34 0.46 0.03 0.14 0.16 0.08 0.19 0.03 0.12 0.52 0.53 0.27 0.11 0.39 0.19 0.04 0.18 0.00 0.21 0.64 0.50 0.53 0.20 0.16 0.49 0.33 0.24 0.00 0.21 0.60 0.43 0.38 0.23 0.29 0.18 0.05 0.07 0.10 0.04 0.42 0.21 0.18 0.00 0.03 0.56 0.11 0.04 0.21 0.22 0.11 0.09 0.24 0.08 0.14 0.45 0.17 0.29 0.10 0.25 0.09 0.09 0.44 0.00 0.20 0.41 0.16 0.29 0.12 0.15 0.32 0.08 0.07 0.00 0.17 0.20 0.14 0.43 0.01 0.10 0.34 0.00 0.13 0.19 0.10 0.40 0.42 0.20 0.00 0.21 0.42 0.28 0.45 0.00 0.40 0.17 0.16 0.53 0.32 0.14 0.37 0.05 0.35 0.06 0.16 0.39 0.37 0.61 0.18 0.13 0.00 0.00 0.19 0.00 0.05 0.00 0.01 0.48 0.04 0.11 0.45 0.41 0.25 0.00 0.07 0.48 0.57 0.28 0.11 0.02 0.33 0.18 0.31 0.05 0.29 0.08 0.02 0.25 0.30 178 49 H2 SYDSGpTPLEDD 0.00 0.44 0.29 0.32 0.30 50 H3 DPDVPpSPSNPK 0.62 0.02 0.00 0.29 0.00 51 H4 ASHPRpSPKGTV 0.40 0.13 0.01 0.43 0.00 52 H5 QQQPVpTPQTAA 0.09 0.29 0.09 0.39 0.00 53 H6 SLIIFpSPKGKL 0.55 0.22 0.21 0.40 0.00 54 H7 CYENPpSPTAGI 0.26 0.17 0.15 0.18 0.00 55 H8 TVTQLpSPMDRE 0.31 0.16 0.05 0.07 0.00 56 I1 GMMGLpSPDAYR 0.14 0.25 0.00 0.30 0.04 57 I2 MHRQEpTVDC 0.11 0.49 0.36 0.39 0.00 58 I3 RVSNGpSPSLER 0.20 0.01 0.00 0.31 0.15 59 I4 GSVEGpTPKKPG 0.18 0.00 0.00 0.46 0.11 60 I5 MEAQSpSPGLHM 0.26 0.01 0.00 0.36 0.02 61 I6 WFYpSPR 0.10 0.00 0.04 0.00 0.00 62 I7 NVSDGpSPNAGS 0.36 0.07 0.00 0.13 0.16 63 I8 RRPGTpSPGLLG 0.50 0.04 0.07 0.43 0.33 64 J1 SDLLTpSPDVGL 0.23 0.43 0.03 0.21 0.00 65 J2 PVARTpSPLQTP 0.12 0.36 0.01 0.26 0.00 66 J3 LPEVApTPESEE 0.36 0.46 0.08 0.13 0.06 67 J4 PVVTCpTPSCTA 0.07 0.56 0.59 0.24 0.10 68 J5 LLKLApSPELER 0.16 0.08 0.00 0.17 0.00 69 J6 AGPALpSPVPPV 0.17 0.48 0.00 0.49 0.04 70 J7 LEPLCpTPVVTC 0.00 0.54 0.45 0.21 0.05 71 J8 SDSLSpSPTLLA 0.33 0.00 0.00 0.19 0.13 72 K1 VDLACpTPTDVR 0.12 0.53 0.35 0.13 0.00 73 K2 ENNVLpSPLPSQ 0.22 0.49 0.04 0.36 0.07 74 K3 NNTSSpSPQPKK 0.16 0.17 0.03 0.40 0.22 75 K4 GSRSRpTPSLPT 0.09 0.29 0.32 0.56 0.25 76 K5 DDLMLpSPDDIE 0.31 0.29 0.00 0.00 0.00 77 K6 APAAPpTPAAPA 0.14 0.29 0.08 0.58 0.30 78 K7 VKMLGpSPVDSV 0.08 0.00 0.00 0.14 0.08 79 K8 VAVVRpTPPKSP 0.03 0.14 0.00 0.13 0.12 80 L1 YGPVLpSPMNKV 0.13 0.44 0.07 0.34 0.00 81 L2 SGSHCpTPPPPY 0.13 0.64 0.40 0.13 0.25 82 L3 PEQPLpTPVTDL 0.13 0.53 0.27 0.27 0.08 83 L4 MNILGpSPISAD 0.21 0.13 0.02 0.17 0.14 84 L5 HSSAApTPNLGP 0.23 0.45 0.34 0.51 0.20 85 L6 TFSGEpTPKRCL 0.15 0.28 0.38 0.25 0.07 86 L7 RLLCSpTPSFKK 0.10 0.41 0.50 0.53 0.10 87 L8 SRLYRpSPSMPE 0.07 0.26 0.03 0.17 0.15 Value indicates the dephosphorylation ratio which was calculated according to the following relationship. The intensity of each peptide is referred as Absolute fluorescent intensity = [Fluorescent Intensity – Background Intensity] Dephosphorylation ratio of x= 179 Table 10.4 Data from the Pin1-regulation experiments. The regulation scores of 76 mammalian peptide sequences are obtained based on Pin1-regulated dephosphorylation by PP2A. NO. 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 ID A01 A02 A03 A04 A05 A06 A07 A08 A09 A10 A11 A12 A13 A14 A15 A16 A17 B01 B02 B03 B04 B05 B06 B07 B09 B10 B12 B13 B14 B15 B16 C01 C02 C03 C04 C05 C06 C07 C08 C10 C11 C12 C13 C14 C15 C16 D01 Peptide Sequence YGPVLpSPMNKV RLLCSpTPSFKK ENNVLpSPLPSQ VPSPPpSPPPLP AVSLDpSPVSVG MEAQSpSPGLHM GSVEGpTPKKPG TPCYIpSPELCE EGSGRpSPRYAL NNTSSpSPQPKK LLKLApSPELER PVVTCpTPSCTA HSESApSPSALS IYSSSpSPVSMK SQRFGpTPLSAS ANAFLpSPNCDW PVARTpSPLQTP HSSAApTPNLGP MNILGpSPISAD APAAPpTPAAPA AIVFRpTPPYAD VSVGSpSPPVKN MHRQEpTVDC RRPGTpSPGLLG LPNAApSPGAEQ VKMLGpSPVDSV SDSLSpSPTLLA SALSSpSPNNLS GMGAYpSPHSNG CHVEDpTPCPGE FFRVEpSPVKLF SGSHCpTPPPPY SRLYRpSPSMPE FELLPpTPPLSP KLPCDpTPGQGL PVPPGpTPAPPG WFYpSPR EQAEGpSPGGPG APPAPpSPGPFA GSRSRpTPSLPT AGPALpSPVPPV GAIGGpTPPAFN SPNNLpSPTGWS SNGEPpSPVAGP FFRIEpSPVKSF PQQTTpSPENAG TFSGEpTPKRCL Dephosphorylation Source of protein Site P73 (human) S412 Cdc25c (Xenopus) T138 P53(human) S33 RAR-alpha(human) S77 SRC-3(human) S860 Sil(mice) S760 aH See footnote P27(human)T187 Nek8-3 IRF3-339 P53(human) S315 c-Jun S73 c-fos(human) T331 Raf-1(human) S289 Sil(mice) S643 a Hu-1 See footnote Hu-6 Bcl-70 P73 (human) T442 Cdc25c (Xenopus) S205 P53(human) T81 NF-kB p65(human) T254 SRC-3(human) S867 a See footnote Hu P21(human) S98 Daxx-1 Beta-catenin(mice) S246 c-fos(human) S374 Raf-1(human) S296 Sil(mice) S656 a See footnote Hu-2 Hu-7 P73 (human) T482 Cdc25c (Xenopus) S285 c-myc(human) T58 SRC-3(human) T24 P54nrb(mice) T412 a See footnote Hu P21(human) S120 Bim-EL Tau (human) T212 Bcl-2(human) S87 P54nrb(mice) T452 Raf-1(human) S301 Sil(mice) S664 a See footnote Hu-3 a See footnote Hu -8 Cdc25c (Xenopus) T67 Pin1 Regulation Score 0.07 0.07 0.07 -0.1 0 -0.09 0.06 0.08 -0.22 0.08 -0.05 0 -0.15 -0.01 0.02 -0.03 0.06 0.01 -0.01 0.08 -0.09 -0.08 -0.03 0 0.14 0.02 -0.02 -0.03 0.01 0.11 0.11 -0.03 0.06 -0.06 -0.07 180 48 D02 VDLACpTPTDVR cyclinD1 T286 0.15 49 D03 PTPPLpSPSRRS c-myc(human) S62 -0.01 50 D04 VAGVHpSPMASS SRC-3(human) S505 51 D05 GTLGLpTPPTTE P54nrb(mice) T430 a 52 D06 RVSNGpSPSLER See footnote Hu (human) S10 0.08 Nek8-1 53 D07 GGGLGpTPLRLP -0.02 54 D08 PLYVFpSPTEEL BTK-115 0.06 55 D10 VAVVRpTPPKSP Tau (human) T231 56 D11 LPEVApTPESEE c-fos(human) T232 57 D12 PSGLLpTPPQSG CyclinE(human) T380 0.31 58 D13 CTLTTpSPRLPV Raf-1(human) S642 -0.09 59 D14 AMCMHpTPNTAP Sil(mice) T686 0.04 a 60 D15 QLSCTpSPCYRE See footnote Hu -4 -0.01 61 D16 VDAAVpSPEERH APP668 0.03 62 E01 PEQPLpTPVTDL Cdc25c (Xenopus) T48 0.21 63 E02 DDLMLpSPDDIE P53(human) S46 0.07 64 E03 LLGRSpSPSKNY Tis/BTG2/pc3(human) S147 65 E04 LSTLSpSPGPKL SRC-3(human) S543 66 E05 GMMGLpSPDAYR Sil(mice) S699 0.12 a 67 E06 NVSDGpSPNAGS See footnote Hu P27(human) S178 -0.16 Nek8-2 68 E07 YTVVGpTPCYIS 0.12 69 E08 QQKKTpSPLNFK BTK-21 70 E10 SDLLTpSPDVGL c-Jun S63 71 E11 LEPLCpTPVVTC c-fos(human) T235 0.02 72 E12 GSSCIpSPTIVQ Raf-1(human) S29 -0.01 73 E13 ELQVPpTPSLPS Sil(mice) T574 0.15 a 74 E14 GGGLSpSPLRLP See footnote Hu Nek8-4 -0.05 Hu-5 75 E15 QVAPLpSPRSAN 76 E16 LTPPQpSGKKQS Cy-E Value indicates the Pin1 regulation score which was calculated according to the following relationship. The intensity of each peptide is referred as Absolute fluorescent intensity = [Fluorescent Intensity – Background Intensity] Pin1 regulation score of x= a Due to patent issues, the protein source of these peptides cannot be revealed. 181 Table 10.5 List of 144 peptides used in this study. The table below shows the identity of each peptide, its protein source, the corresponding PTP reported in the literature, as well as the peptide quality confirmed with LC-MS. NO. 384 well Peptide Sequence MW Plate1_A01 Plate1_A02 AEKPFYVNVEF AEMTGYVVTRW 1761.70339 1731.67106 10 11 12 13 14 15 16 17 18 19 20 21 Plate1_A03 Plate1_A04 Plate1_A05 Plate1_A06 Plate1_A07 Plate1_A08 Plate1_A09 Plate1_A10 Plate1_A11 Plate1_A12 Plate1_A13 Plate1_A14 Plate1_A15 Plate1_A16 Plate1_A17 Plate1_A18 Plate1_A19 Plate1_A20 Plate1_A21 AENAEYLRVAP DEELHY ASLNF DEGIHYSELIQ DEKVDYVQVDK DERVDYVVVDQ DESVD YVPMLD DKQVEYLDLDL DSGGFYITSRT DTETVYSEVRK EANSHYGHNDD EDEDYY KASVT EDGGVYSSSGL EDGISYTTLRF EDSTYYKASKG EDTLTYADLDM EGVATYAAAVL EGDNDYIIPLP ENGLNYIDLDL EPNVSYICSRY 1651.66259 1756.63644 1722.65209 1756.69395 1755.67355 1701.58638 1769.71435 1622.59967 1745.68921 1677.50756 1738.59939 1489.49928 1720.67283 1667.6099 1705.5813 1483.59786 1664.63537 1697.65684 1749.64525 22 23 Plate1_A22 Plate1_A23 ESDGSYQKPSY ESDGGYMDMSK 1679.57352 1638.4962 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Plate1_A24 Plate1_B01 Plate1_B02 Plate1_B03 Plate1_B04 Plate1_B05 Plate1_B06 Plate1_B07 Plate1_B08 Plate1_B09 Plate1_B10 Plate1_B11 Plate1_B12 Plate1_B13 ETDKEYYTVKD FEEDDYESPND FGMTRYVLDDE FLFNMYLTRER FMMTPYVVTRY GFLTEYVATRW GNNYVY IDPTQ GSAAPYLKTKF GWMEDYDYVHL GWMVHYTSKDT HNSALYSQVQK HRQLNYIQVDL IEDNEYTAREG IEDEDYYKASV 1809.67289 1778.52153 1764.6449 1908.79766 1826.71558 1761.71463 1702.62589 1601.68735 1846.62925 1743.63468 1693.68441 1817.78445 1715.60587 1750.63577 38 39 40 41 Plate1_B14 Plate1_B15 Plate1_B16 Plate1_B17 IEDNEYTARQG IESDIYAEIPD IESSNYMAPYD IRYHRYHGRSA 1714.62186 1683.62995 1708.57108 1834.77595 Corresponding Enzyme Protein Derived PTP1B Mitogen kinase BCR EGF SHP1 receptor SHP1 CD33 SHP1 SIGLEC2 SHP1, SHP2 Gab2 SHP2 Gab1 SHP2, TCPTP PDGF SHP2 GAB1 Src-1 SHP1 CD31 SHP1 CD31 Type 12 Photooncogene FER kinase SHP1 SIGLEC2 Fak chick kinase SHP2 SIRP SHP1 CATENIN TCPTP PDGF SHP2,PTP1B IRS1 Kinase gsk3 SELECTI SHP2 NE SHP2 PDGF JANUS TCPTP KINASE1 SHP1 SLP76 Tyrosine kinase TXK SHP1 HOAX1 JNK-2 kinase PTP1B ERK2 SHP1 KIT TCPTP STAT3 P130cas protein kinase c p62dok SHP2 FRS2 SHP1 LYN Type 12 SHP1,type 18,TCPTP C-SRC Type 12 PTP1B,TCPTP PDGF Protooncogene kinase, PIM pTyr Site 177 1197 340 822 643 689 751 657 713 663 762 470 654 1021 1179 603 740 1022 113 343 187 570 705 436 397 419 771 182 42 43 Plate1_B18 Plate1_B19 IYETDYYRKGG KAVDGYVKPQI 1783.68373 1636.72446 44 45 46 47 48 49 50 51 52 53 54 55 56 57 Plate1_B20 Plate1_B21 Plate1_B22 Plate1_B23 Plate1_B24 Plate1_C01 Plate1_C02 Plate1_C03 Plate1_C04 Plate1_C05 Plate1_C06 Plate1_C07 Plate1_C08 Plate1_C09 KDRMSYHVRSH KKRCPYTKHQT KVVALYDYMPM LISSDYELLSD LNSDGYTPEPA LNSKGYTKSID LPPEGYVVVVK MDTSVYESPYS MKDEEYEQMVK MTGDTYTAHAG NNHTEYASIQT NQSSGYRYGTD NSDVQYTEVQV NSKRDYTGCST 1834.73171 1808.7776 1748.69377 1673.64561 1582.55713 1644.67792 1618.73904 1697.5551 1848.66941 1543.50333 1696.61131 1666.56436 1700.63137 1650.57282 58 59 60 61 Plate1_C10 Plate1_C11 Plate1_C12 Plate1_C13 NVVPLYDLLLE PEGLNYACLTH PEGHEYYRVRE PEQDEYDIPRH 1706.75509 1636.59756 1933.71167 1817.66405 62 63 64 65 Plate1_C14 Plate1_C15 Plate1_C16 Plate1_C17 PGSLEYLCLPA PKGTGYIKTEL PLDKDYYVVRE PPDHQYYNDFP 1581.61689 1625.70848 1895.74632 1811.62113 66 67 68 69 70 71 Plate1_C18 Plate1_C19 Plate1_C20 Plate1_C21 Plate1_C22 Plate1_C23 PQDKEYYKVKE PSFSEYASVQV PSTRDYEIQRE PQDKEYYKVKE QGPVI YAQLDH QKQPIYIVMEL 1845.75689 1632.60917 1812.70626 1925.75689 1659.66768 1780.78536 72 73 74 75 76 77 78 79 80 81 82 83 Plate1_C24 Plate1_D01 Plate1_D02 Plate1_D03 Plate1_D04 Plate1_D05 Plate1_D06 Plate1_D07 Plate1_D08 Plate1_D09 Plate1_D10 Plate1_D11 QQQEVYGMMPR RNEGVYTAIAV REGLNYMVLAT SAEPQYQPGDQ SDDVRYVNAFK SDGHEYIYVDP SEEPIYIVTEY SESVVYADIRK SETDDYAEIID SPPALYAEPLD SSNPEYLSASD SSSSEYGSVSP 1785.65986 1611.66768 1685.68671 1638.5582 1732.68406 1713.59424 1761.67691 1685.70446 1689.56775 1591.61899 1588.53132 1505.49421 TCPTP INSULIN SHP2, PTP1B STAT5B Myc Zinc protein SHP1 HOAX10 BTK kinase TCPTP JAK3 LMW ZAP70 Erk kinase SHP2 PTK2B Tyrosine kinase Zap 70 type 13 type 12 SHP1, SHP2 SIRP Fyn kinase SHP1, SHP2 CD31 SHP2 MYELIN ESTROG EN RECEPT PTP1B OR SHP1 ROS1 PTP1B TYK2 P130cas INTERLU KIN RECEPT SHP1, SHP2 OR SHP2,TCPTP STAT1 TCPTP JAK3 TCPTP SHC JANUS SHP2,PTP1B KINASE2 SHP2 SIRP Fak kinase JAK kinase SHP2 MYELIN FES kinase SPECTRI LMW N Type 12 SHP1 ROS1 PHotooncogene FGR kinase SHP2 VEGF TCPTP PDGF SHP1 C-SRC SHP2 MYELIN Type 12 P62dok TCPTP INSULIN Mitogen kinase 1189 699 326 785 292 906 453 690 200 537 2334 1054 628 701 981 349 1007 496 241 1176 2274 1213 579 338 263 999 183 84 85 Plate1_D12 Plate1_D13 SSTVQYSTVVH STEPQYQPGEN 1626.63098 1668.56877 SHP2 SHP1,PTP1B 86 87 88 89 90 91 92 Plate1_D14 Plate1_D15 Plate1_D16 Plate1_D17 Plate1_D18 Plate1_D19 Plate1_D20 TDKEYYTVKDD TGGSVYTEDND TNDITYADLNL TSKLI YDFIED TSSVLYTAVQP VDADEYLIPQQ VDTPHYPRWIT 1795.65724 1576.49493 1671.6412 1762.70854 1584.64556 1709.65684 1803.73643 TCPTP Type SHP2 Type 12 SHP2 SHP2, PTP1B PKC Kinase 93 94 95 96 97 98 99 100 101 102 103 104 105 Plate1_D21 Plate1_D22 Plate1_D23 Plate1_D24 Plate1_E01 Plate1_E02 Plate1_E03 Plate1_E04 Plate1_E05 Plate1_E06 Plate1_E07 Plate1_E08 Plate1_E09 VNTTLYEKFTY VYESPYSDPEE YETDYYRKGGK YFMTEYVATRW AEGSAYEEVPT AENPEYLSEFS FDNLYYWDQDP AFGTVYKGIWI AFQFSYTAVFG AKIQDYHILTR APAEMYDIMKT ASEQGYEEMRA ATVGHYTAVQN 1797.72454 1733.57284 1798.69463 1885.71293 1571.54114 1704.59391 1894.64701 1673.72373 1656.62442 1776.79428 1688.621 1689.57247 1579.60509 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 Plate1_E10 Plate1_E11 Plate1_E12 Plate1_E13 Plate1_E14 Plate1_E15 Plate1_E16 Plate1_E17 Plate1_E18 Plate1_E19 Plate1_E20 Plate1_E21 Plate1_E22 Plate1_E23 Plate1_E24 Plate1_F01 Plate1_F02 Plate1_F03 Plate1_F04 Plate1_F05 AYRQLYLNPKG CPEKVYELMRA DINSLYDVSRM DRFIQYANPAF DVSRMYVDPSE EDIKSYYTVRQ EEIRFYQLGEE EKIQDYEKMPE ELGYEYMDVGS AELEFYMDYEA FGAKPYDGIPA GPQDIYDVPPV GQESEYGNITY GRETIYPNASL IGEGTYGTVFK IYIHRYENVSI KAEDEYVNEPL KPKQEYLNPVE LARDMYDKEYY LDSTFYRSLLE 1741.75717 1757.69008 1731.65581 1760.69423 1716.60852 1820.7365 1831.70485 1828.69733 1681.56017 1799.60203 1554.61384 1618.62989 1679.57352 1639.6626 1590.63498 1825.7783 1725.65175 1763.75141 1885.69767 1762.71978 126 127 Plate1_F06 Plate1_F07 LGQRIYQYIQS LLANAYIYVVQ 1787.76266 1685.74487 INTERLU KIN RECEPT OR C-SRC JANUS KINASE 759 530 1023 SIRP 429 PDGF EGFR 1009 1016 TEK SHP2 KINASE Tyrosine kinase zap-70 PTP1B,TCPTP INSULIN Mitogen kinase PLC-r Erb2 receptor Erb2 receptor Erb2 receptor CAAX Protease Jak2 Stem cell growth factor Erb3 Cyclin kinase activator Chromosomal associate protein Abl PLC-r Camp Phosphodiesterase PLC-r PTP1B Potassium gate IL-1 Erb-3 Alanine scanning Erb2 receptor P130cas PTN6 Carcinoembroyonic antigen Cell division kinase ATP cyclase Erb2 receptor Erb4 Met growth factor Erb2 receptor Dual specificity regulated kinase Neural cell adhesion 1108 1190 184 128 129 130 131 132 133 134 135 136 137 Plate1_F08 Plate1_F09 Plate1_F10 Plate1_F11 Plate1_F12 Plate1_F13 Plate1_F14 Plate1_F15 Plate1_F16 Plate1_F17 LMGHEYMEMKN LNKQGYKCRQC NKPTVYGVSPN PPDHQYYNDFP PEDTFYFDPEF PEPGPYAQPSV RHDSGYEVHHQ RNPGFYVEANP SADHLYVNVSE SSDPTYTSSLG 1801.62578 1759.69183 1594.64114 1811.62113 1825.6143 1560.58803 1783.64466 1682.64728 1652.61023 1533.52551 138 139 140 141 142 143 144 Plate1_F18 Plate1_F19 Plate1_F20 Plate1_F21 Plate1_F22 Plate1_F23 Plate1_F24 EEEPVYEAEPE STKYFYKQNGR TAEPDYGALYE VCAERYSQEVF VDSSLYNLPRS VSSTHYYLLPE VVIALYDYQTN 1727.68267 1810.74226 1647.57244 1749.64524 1669.67317 1727.68267 1717.69832 Cell division cycle protein 23 Protein kinase C Abl SHC transforming protein ISPK-1 Kinase Adaptor crk Alzheimer's disease PLC-r Tumor necrosis factor Tyrosine kinase receptor Hematopoietic lineage cell specific protein Erb4 PLC-r Apoptosis Protein cd27 Grb2 associated Activated cdc42 kinase T cell specific kinase 185 Table 10.6 The absolute fluorescent intensity of each PTP against pTyr peptide library NO. ID 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Plate1_A01 Plate1_A02 Plate1_A03 Plate1_A04 Plate1_A05 Plate1_A06 Plate1_A07 Plate1_A08 Plate1_A09 Plate1_A10 Plate1_A11 Plate1_A12 Plate1_A13 Plate1_A14 Plate1_A15 Plate1_A16 Plate1_A17 Plate1_A18 Plate1_A19 Plate1_A20 Plate1_A21 Plate1_A22 Plate1_A23 Plate1_A24 Plate1_B01 Plate1_B02 Plate1_B03 Plate1_B04 Plate1_B05 Plate1_B06 Plate1_B07 Plate1_B08 Plate1_B09 Plate1_B10 Plate1_B11 Plate1_B12 Plate1_B13 Plate1_B14 Plate1_B15 Plate1_B16 Plate1_B17 Plate1_B18 Plate1_B19 Plate1_B20 Plate1_B21 Plate1_B22 Plate1_B23 Plate1_B24 Plate1_C01 Plate1_C02 Plate1_C03 Plate1_C04 Plate1_C05 Plate1_C06 Plate1_C07 Plate1_C08 Sequence AEKPFYVNVEF AEMTGYVVTRW AENAEYLRVAP DEELHY ASLNF DEGIHYSELIQ DEKVDYVQVDK DERVDYVVVDQ DESVD YVPMLD DKQVEYLDLDL DSGGFYITSRT DTETVYSEVRK EANSHYGHNDD EDEDYY KASVT EDGGVYSSSGL EDGISYTTLRF EDSTYYKASKG EDTLTYADLDM EGVATYAAAVL EGDNDYIIPLP ENGLNYIDLDL EPNVSYICSRY ESDGSYQKPSY ESDGGYMDMSK ETDKEYYTVKD FEEDDYESPND FGMTRYVLDDE FLFNMYLTRER FMMTPYVVTRY GFLTEYVATRW GNNYVY IDPTQ GSAAPYLKTKF GWMEDYDYVHL GWMVHYTSKDT HNSALYSQVQK HRQLNYIQVDL IEDNEYTAREG IEDEDYYKASV IEDNEYTARQG IESDIYAEIPD IESSNYMAPYD IRYHRYHGRSA IYETDYYRKGG KAVDGYVKPQI KDRMSYHVRSH KKRCPYTKHQT KVVALYDYMPM LISSDYELLSD LNSDGYTPEPA LNSKGYTKSID LPPEGYVVVVK MDTSVYESPYS MKDEEYEQMVK MTGDTYTAHAG NNHTEYASIQT NQSSGYRYGTD NSDVQYTEVQV PTP1B 249 1171 435 428 51 587 56 1181 588 321 380 1248 468 251 2705 683 944 6722 781 585 0 43 3025 3664 2238 1538 3557 1362 90 1695 1565 834 524 1003 10095 1051 1559 1364 767 2355 1609 1235 39 0 1315 161 596 491 2480 9583 Absolute fluorescence intensity of each PTP TCPTP SHP1 SHP2 LMW 193 222 3462 126 864 1136 5527 5111 445 2352 169 4557 10457 1027 917 1271 9968 219 58 3009 612 22 472 4016 806 66 29 54 352 19 1974 8898 437 112 178 1233 1289 118 9680 9670 2350 0 48 834 140 305 1095 604 282 859 1956 586 6588 12343 1797 68 66 818 1593 355 3399 12782 229 3936 2849 3002 375 378 2252 5374 378 997 1392 12973 403 6488 2853 10213 5336 350 105 972 2942 304 1863 2278 0 154 62 0 0 291 135 814 165 1875 806 3317 5257 2531 4939 7381 9586 837 4328 3926 3147 524 1758 1433 325 950 4957 4457 12409 876 1044 719 663 178 1406 1045 707 1528 3328 4753 574 1501 10960 2762 0 1339 192 111 1077 1600 1946 119 1186 829 12914 15308 11860 288 793 600 2657 791 1723 3198 10879 833 1220 1155 3528 271 1099 5420 4579 558 1741 2722 6890 683 1105 3174 8123 852 1738 3630 4748 158 285 1196 0 268 0 128 830 1037 243 167 3076 11155 51 661 995 37 911 2910 137 916 1165 2102 5397 8582 1560 28 1286 1148 6459 1382 7188 2166 186 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 Plate1_C09 Plate1_C10 Plate1_C11 Plate1_C12 Plate1_C13 Plate1_C14 Plate1_C15 Plate1_C16 Plate1_C17 Plate1_C18 Plate1_C19 Plate1_C20 Plate1_C21 Plate1_C22 Plate1_C23 Plate1_C24 Plate1_D01 Plate1_D02 Plate1_D03 Plate1_D04 Plate1_D05 Plate1_D06 Plate1_D07 Plate1_D08 Plate1_D09 Plate1_D10 Plate1_D11 Plate1_D12 Plate1_D13 Plate1_D14 Plate1_D15 Plate1_D16 Plate1_D17 Plate1_D18 Plate1_D19 Plate1_D20 Plate1_D21 Plate1_D22 Plate1_D23 Plate1_D24 Plate1_E01 Plate1_E02 Plate1_E03 Plate1_E04 Plate1_E05 Plate1_E06 Plate1_E07 Plate1_E08 Plate1_E09 Plate1_E10 Plate1_E11 Plate1_E12 Plate1_E13 Plate1_E14 Plate1_E15 Plate1_E16 Plate1_E17 Plate1_E18 Plate1_E19 NSKRDYTGCST NVVPLYDLLLE PEGLNYACLTH PEGHEYYRVRE PEQDEYDIPRH PGSLEYLCLPA PKGTGYIKTEL PLDKDYYVVRE PPDHQYYNDFP PQDKEYYKVKE PSFSEYASVQV PSTRDYEIQRE PQDKEYYKVKE QGPVI YAQLDH QKQPIYIVMEL QQQEVYGMMPR RNEGVYTAIAV REGLNYMVLAT SAEPQYQPGDQ SDDVRYVNAFK SDGHEYIYVDP SEEPIYIVTEY SESVVYADIRK SETDDYAEIID SPPALYAEPLD SSNPEYLSASD SSSSEYGSVSP SSTVQYSTVVH STEPQYQPGEN TDKEYYTVKDD TGGSVYTEDND TNDITYADLNL TSKLI YDFIED TSSVLYTAVQP VDADEYLIPQQ VDTPHYPRWIT VNTTLYEKFTY VYESPYSDPEE YETDYYRKGGK YFMTEYVATRW AEGSAYEEVPT AENPEYLSEFS FDNLYYWDQDP AFGTVYKGIWI AFQFSYTAVFG AKIQDYHILTR APAEMYDIMKT ASEQGYEEMRA ATVGHYTAVQN AYRQLYLNPKG CPEKVYELMRA DINSLYDVSRM DRFIQYANPAF DVSRMYVDPSE EDIKSYYTVRQ EEIRFYQLGEE EKIQDYEKMPE ELGYEYMDVGS AELEFYMDYEA 602 688 2371 2158 153 2047 1006 1013 125 484 3564 1229 1095 6003 1902 1085 2574 1660 42 7119 1748 606 227 81 6184 446 358 482 484 12415 250 2735 10414 2208 5359 698 125 3281 1286 1535 783 696 1431 2329 2408 5096 1090 5048 1113 174 1285 19714 200 2290 1810 1697 86 241 50 3853 1909 676 9871 538 2040 2088 48 683 0 6577 2140 122 0 7606 0 75 10065 883 13706 314 3723 458 1319 1796 438 453 1162 2891 945 943 5805 1100 4868 1007 0 1034 17603 225 9095 704 589 709 123 5876 396 9279 1808 837 1106 5667 51 3147 531 310 17579 2122 0 382 9834 0 4384 346 19048 531 2926 1884 658 1651 6285 520 730 2258 1888 1314 347 3089 4011 1241 1946 3926 2951 0 5289 7643 730 576 17829 956 1323 5226 3183 932 751 444 5283 88 1963 19158 1234 1665 1188 11520 66 6727 2295 750 19127 4859 1028 424 1574 15367 757 513 10029 1613 20727 1875 4104 7611 2423 3795 1744 622 29 7640 4469 3274 896 436 5459 4092 2336 1484 8788 1847 4304 669 61 3714 7998 2425 445 2233 623 336 418 3217 571 48 1081 2771 1702 2309 1769 4117 1413 3622 164 3237 1144 5101 822 1000 5234 526 482 704 397 1270 4564 11381 284 6362 4279 2040 504 13900 1323 4850 1545 75 893 5038 555 3554 2062 1040 4370 610 3386 2470 187 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 Plate1_E20 Plate1_E21 Plate1_E22 Plate1_E23 Plate1_E24 Plate1_F01 Plate1_F02 Plate1_F03 Plate1_F04 Plate1_F05 Plate1_F06 Plate1_F07 Plate1_F08 Plate1_F09 Plate1_F10 Plate1_F11 Plate1_F12 Plate1_F13 Plate1_F14 Plate1_F15 Plate1_F16 Plate1_F17 Plate1_F18 Plate1_F19 Plate1_F20 Plate1_F21 Plate1_F22 Plate1_F23 Plate1_F24 FGAKPYDGIPA GPQDIYDVPPV GQESEYGNITY GRETIYPNASL IGEGTYGTVFK IYIHRYENVSI KAEDEYVNEPL KPKQEYLNPVE LARDMYDKEYY LDSTFYRSLLE LGQRIYQYIQS LLANAYIYVVQ LMGHEYMEMKN LNKQGYKCRQC NKPTVYGVSPN PPDHQYYNDFP PEDTFYFDPEF PEPGPYAQPSV RHDSGYEVHHQ RNPGFYVEANP SADHLYVNVSE SSDPTYTSSLG EEEPVYEAEPE STKYFYKQNGR TAEPDYGALYE VCAERYSQEVF VDSSLYNLPRS VSSTHYYLLPE VVIALYDYQTN 1186 10036 440 1180 1820 8592 662 903 1582 3196 510 1484 2677 486 599 538 738 611 3330 144 1424 860 1080 1719 360 0 18 8562 340 623 561 12658 0 1312 3509 346 470 1274 478 503 0 129 1878 0 503 361 1867 104 0 303 2960 773 190 3439 2995 231 438 882 2115 1025 3891 12417 608 958 216 605 215 407 5325 38 3461 40 4141 547 2234 377 1139 3041 1126 612 4191 6891 1842 1089 1546 3838 5032 1785 3457 6322 1945 1695 1881 987 689 2449 1486 1277 241 1018 1755 4766 1770 2733 351 2105 1288 2114 1298 5041 2449 0 339 117 3243 988 2220 1387 2149 267 362 1260 337 398 33 139 762 80 188 Table 10.7 The summary of KD against three PTP mutants including PTP1B, SHP1 and SHP2. Peptide NO. 384 well 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 PTP1B (µM) Plate1_A1 Plate1_A5 Plate1_A7 Plate1_A2 KD of each PTP mutant SHP1 (µM) SHP2 (µM) 1.27 2.7 0.73 0.56 0.74 Plate1_A4 Plate1_A6 Plate1_A9 4.7 0.35 0.65 0.59 Plate1_A11 Plate1_A13 Plate1_A15 19 1.83 Plate1_A16 Plate1_A17 Plate1_A19 7.9 2.63 Plate1_A21 Plate1_A18 Plate1_A20 4.8 1.58 0.43 Plate1_A22 Plate1_C2 Plate1_C6 Plate1_C8 Plate1_C9 Plate1_C11 Plate1_C15 Plate1_C12 Plate1_C14 1.9 0.56 1.1 0.18 1.6 1.86 0.47 3.14 6.9 0.27 1.9 Plate1_C22 Plate1_C24 Plate1_E5 0.79 4.7 5.4 0.69 0.93 14 0.62 1.48 0.62 Plate1_E11 Plate1_E13 Plate1_E15 Plate1_E19 Plate1_E21 Plate1_E18 0.9 0.96 0.77 5.5 0.29 Plate1_E10 Plate1_E12 Plate1_E14 0.37 1.6 Plate1_C19 Plate1_C21 Plate1_C23 Plate1_E4 Plate1_E6 Plate1_E9 0.96 0.88 1.47 1.3 0.18 1.57 2.6 2.7 0.33 0.86 1.22 5.2 1.8 2.2 0.46 0.18 1.8 1.12 1.9 0.54 0.54 0.41 1.1 0.84 0.49 0.94 0.5 0.31 0.79 0.91 Plate1_E22 Plate1_E24 Plate1_B3 1.7 5.2 Plate1_B5 Plate1_B7 Plate1_B4 2.3 1.9 3.1 4.4 2.8 1.39 Plate1_B8 1.3 189 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 Plate1_B11 Plate1_B15 Plate1_B10 0.61 1.96 Plate1_B16 Plate1_B17 Plate1_B19 Plate1_B21 Plate1_B18 Plate1_B20 1.6 2.6 Plate1_B22 Plate1_D1 Plate1_D5 Plate1_D7 Plate1_D2 Plate1_D4 0.29 Plate1_D16 Plate1_D19 Plate1_D21 0.55 Plate1_D23 Plate1_D18 Plate1_D20 0.43 0.97 1.52 7.7 1.83 0.93 0.41 10.5 2.1 2.8 0.82 0.38 2.2 2.14 0.15 0.45 0.56 0.69 0.13 3.1 0.03 1.93 0.92 0.16 0.29 0.17 0.55 1.2 0.28 0.35 0.52 1.2 12 Plate1_F16 Plate1_F21 Plate1_F23 Plate1_F22 1.6 0.46 0.91 0.11 Plate1_F7 Plate1_F6 Plate1_F8 Plate1_F9 Plate1_F15 Plate1_F10 0.27 1.92 3.1 Plate1_D8 Plate1_D11 Plate1_D12 Plate1_D24 Plate1_F1 Plate1_F5 1.4 0.76 1.28 1.2 19 190 [...]... al.;26 for the first time the array -based detection of kinase activity using peptide substrates was also demonstrated In essence, these pioneering studies set the precedent for development of various types of microarray -based enzyme assays capable of sensitive detection of different enzyme classes.5 At present, four key microarray -based platforms, namely protein-, peptide- , small-molecule- and cell -based. .. domain-selective peptides 91 5.3 The KD and selectivity ratio summary of SHP1-selective peptides 92 5.4 The KD and selectivity ratio summary of SHP2-selective peptides 92 5.5 List of 16 p(CAP) peptides used in the peptide microarray 94 5.6 The summary of microplate assay with p(CAP) peptide against 3 PTPs 95 10.1 List of 87 peptides used in the peptide microarray 173 10.2 List of 53 peptide kobs values based. .. Immobilization of Peptides 6 Peptides are natural substrates of many enzymes Correspondingly, peptide arrays have proved to be powerful tools for high-throughput enzyme screening and to generate substrate fingerprints of enzymes.39 Advances in peptide synthesis and combinatorial chemistry have made it possible to rapidly generate diverse libraries for such endeavours There are two main methodologies for fabrication... enzyme- catalysed chemical transformations visible are available in various formats for the different enzyme classes.6 Many different enzymes can be screened in parallel in 96-, 384- or even 1,536-well microtiter plates by using labelled substrates including fluorogenic or chromogenic substrates, FRET substrates or sensors for product information, for instance, pH indicators for ester hydrolyses.7 Such... N-terminal cysteine-peptides onto maleimide-derivatized SAM surfaces.42 Our group developed two novel approaches for peptide immobilization In the first strategy, N-terminal cysteine-containing peptides 7 were spotted onto the thioester-functionalized slide (Figure 1.2b).43 The strategy is advantageous as it allows facile synthesis of peptides, and the native peptide bond formed between the peptide and surface... plate enzyme assays have to be modified before they may be adapted for microarray -based screenings In the following sections, we shall examine the various microarray -based approaches in terms of three different aspects of highthroughput study of enzymes, namely functional annotation, substrate fingerprinting and inhibitor fingerprinting (Figure 1.3) 1.3.2.1 Functional Annotation In microtiter plate -based. .. substrate fingerprint analysis based on grouping pSer and pThr peptides separately 57 3.12 Pin1-regulated dephosphorylation by PP2A and cell -based experiments 59 4.1 Overall flow of the strategy 64 4.2 Images of the 1000-member peptide library screened against 7 different Cy3-labeled 14-3-3 isoforms 4.3 Scatter plot analysis of the data obtained form the 1000-member peptide library 4.4 70 Relative... traditional solution -based enzyme bioassays In addition, the salient features of the microarray technology, i.e miniaturization, parallelization and automation, make it very convenient to undertake thousands of enzyme assays simultaneously For a more comprehensive coverage of microarray field, please refer to several recent reports.5 1.2 Conventional Enzyme Assays Enzyme assays which render enzyme- catalysed... proteomics approaches, and its platform technologies and applications 1.3.1 Immobilization Strategies in Microarray Applications 1.3.1.1 Immobilization of Proteins Unlike DNA, proteins and especially enzymes are much more fragile Loss of enzyme activity often accompanies the immobilization of the enzyme onto the solid surface Consequently, one of the critical steps for the development of an enzyme array... unknown enzymes can be identified in high throughput Activity -based profiling (ABP), originally 10 developed by Cravatt et al in 1999 to study serine hydrolases,49 is mainly used for the identification and characterization of enzymes present in a mixture of unrelated proteins The method uses small-molecule probes capable of covalently reacting with a (or a class of) target enzyme( s) on the basis of the enzyme s . DEVELOPING PEPTIDE- BASED APPROACHES FOR SYSTEMATIC ENZYME PROFILING SUN HONGYAN (B.Sc., Wuhan University) A THESIS SUBMITTED FOR THE DEGREE. SHP2-selective peptides. List of 16 p(CAP) peptides used in the peptide microarray. The summary of microplate assay with p(CAP) peptide against 3 PTPs. List of 87 peptides used in the peptide microarray. enabling platforms. Chapter 2 describes a novel FRET assay for activity -based fingerprinting of proteases from different classes. Chapter 3 presents a novel phosphorylated peptide array for substrate