1. Application of MALDI-TOF Mass Spectrometry in Post-Genomic Era

2. 99% sequence of human genome published Genomics: --Identification & characterization of genes & their arrangement in chromosomes Proteomics: --Functional analysis of gene products Bioinformatics: --Storage,analysis & manipulation of the information from genomics & proteomics

3. Genomics genes characterization and identification Proteomics functional analysis of gene products Bioimformatics

4. cDNA microarray: --measures changes in mRNA --interface between genomics and proteomics Two-dimensional gels & Mass spectrometry: --powerful tools for identifying proteins --post-translational modifications --protein-protein interaction Transgenic & knockout mouse: --functions of novel proteins in vivo

5. 99% sequence of human genome published Age of functional genomics cDNA microarray core lab Proteomics core lab Biological samples (C/E) ProteinscDNA Microarray2-D gels/MS Protein expression pattern mRNA expression pattern Bioinformatics core lab Function study in Tg/KO mouse core lab

6. Strategy for proteomic study in cancer biology Normal cells Tumor cells SDS-PAGE isoelectrofocusing MALDI-TOF MS analysis Laser-captured microdissector digested by trypsin Database search/mapping (?) Protein identified Clinical specimens

7. Mass Spectrometry for Biotechnology Gary Siuzdak The Department of Chemistry The Scripps Research Institute La Jolla, California Academic Press 1996 Reference Book

8. What is a mass spectrometer and what does it do?

9. Analogy between mass analysis and the dispersion of light

10. Components of a mass spectrometer

11. MALDI-TOF MS (Matrix-assisted laser desorption/ionization-Time of flight ) ( 基質輔助雷射脫附游離 - 飛行時間質譜儀 ) M/Z Time of Flight Solid probe

12. Ion Sources and Sample Introduction

13. Sample probes and matrix for MALDI

14. MALDI matrix # A nonvolatile solid material that absorbs the laser radiation resulting in the vaporization of the matrix and sample embedded in the matrix. #The matrix also serves to minimize sample damage from the laser radiation by absorbing most of the incident energy and the matrix is believed to facilitate the ionization process.

15. Matrix-assisted laser desorption/ionization source

16. Calibration for MALDI

17. Vacuum system in MS

18. Mass Analyzer-Time of Flight (TOF) Kinetic Energy = ½ mv 2 v = (2KE/m) 1/2 m/z

19. Ion Detector Electron multiplier Photomultiplier conversion dynode scintillation counting 1 10 6

20. Sensitivity of MALDI-TOF MS ~10 fg 1347.7 g/mole x 5 x 10 -18 mole = 6.74 x 10 –15 g

21. Strategy for proteomic study in cancer biology Normal cells Tumor cells SDS-PAGE isoelectrofocusing MALDI-TOF MS analysis Laser-captured microdissector digested by trypsin Database search/mapping (?) Protein identified Clinical specimens

22. The 2-D gel pattern of human kidney epithelial 293 cells expressed without (A) and with (B) the Epstein-Barr virus oncoprotein LMP1 (A)(B) Pick up spots of interest Digested by trypsin MALDI-TOF MS analysis IEF SDS-PAGE

23. 170 116.3 66.3 55.4 29 21.5 170 116.3 66.3 55.4 29 21.5 pH310 (A) pH310 a b c a' b' c' d (B) (A)(B) 1 1 2 13 The substrate of GSK-3  purified from pig brain

24. (b 1 ) (d 2 ) (a' 1 ) (b' 1 ) (b' 3 ) 472-480 526-532 533-552 558-565 472-480 526-532 533-552 558-565 472-480 526-532 533-552 558-565 533-552 558-565 MALDI-TOF analysis of tryptic fingerprint from the substrate for kinase F A /GSK-3  in pig brain

25. Data base search for the substrate of kinase F A /GSK-3  from pig brain MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG LIKQIGENLI VPGGVKTIEA HSRMVIPGGI DVHTRFQMPD QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRD IGAIAQVHAE NGDIIAEEQQ RILDLGITGP EGHVLSRPEE VEAEAVNRAI TIANQTNCPL YITKVMSKSS AEVIAQARKK GTVVYGEPIT ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTL IPEGTNGTEE RMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR IAVGSDADLV IWDPDSVKTI SAKTHNSSLE YNIFEGMECR GSPLVVISQG KIVLEDGTLH VTEGSGRYIP RKPFPDFVYK RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK TSPAKQQAPP VRNLHQSGFS LSGAQIDDNI PRRTTQRIVA PPGGRANITS LG *908.4 da --- 391-397 *2169.1da --- 533-552 *pI~5.95 Collapsin Response Mediator Protein-2 (CRMP-2, human) (d 2 )

26. (b 1 ) Amino acid sequence analysis by PSD technique in MALDI-TOF MS 2169 908

27. Tandem Mass Spectrometry

29. MS/MS with a TOF reflectron mass spectrometer

30. Peptide fragmentation postionization [Post Source Decay (PSD)]

31. (b 1 ) Amino acid sequence analysis by PSD technique in MALDI-TOF MS 2169 908

32. Amino acid sequencing

33. Determination of protein molecular weight

34. LC/MS

35. ESI Ion Trap LC/MS n ( 電噴霧離子井液相層析多重質譜 ) Posttranslational modification (?) Trypsin digest nano-LC (check minor difference) MS-MS(or MS n ) analysis for selected peptides Identification of modifications on protein NormalTumor MALDI-TOF =& ESI Ion trap LC/MS n MALDI-TOF

36. Two-dimensional gel electrophoresis system Sample purification/preparation (Laser-captured microdissector) 1 o -D Isoelectrophoresis 2 o -D SDS-PAGE 2D-gel image analysis system Quantitation of spots in 2D-gel Image comparison between 2D-gels Automatic spots pick-up Spot picker and Digester MALDI-TOF & ESI Ion trap LC/MS n MS spectrometric analysis system Automatic spots digestion/extraction MS spectrometry database Bioimformatics Aim: Set up proteomics core lab 貴儀中心

37. Nature Biotechnology 17, 994-999 (1999)

39. The End