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

Application of MALDI-TOF Mass Spectrometry in Post-Genomic Era. 99% sequence of human genome published. Genomics: --Identification & characterization of genes & their arrangement in chromosomes Proteomics: --Functional analysis of gene products Bioinformatics:

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

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  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 Biological samples (C/E) cDNA microarray core lab Proteomics core lab cDNA Proteins Microarray 2-D gels/MS mRNA expression pattern Protein expression pattern Bioinformatics core lab Function study in Tg/KO mouse core lab

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

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

  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) Solid probe Time of Flight M/Z

  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 mv2 v = (2KE/m)1/2 m/z

  19. Ion Detector Electron multiplier 1 106 Photomultiplier conversion dynode scintillation counting

  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 Clinical specimens Laser-captured microdissector Normal cells Tumor cells SDS-PAGE isoelectrofocusing (?) digested by trypsin MALDI-TOF MS analysis Database search/mapping Protein identified

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

  23. The substrate of GSK-3a purified from pig brain (A) (B) (A) 10 pH 3 c 170 b 116.3 1 a 66.3 55.4 29 21.5 (B) 10 pH 3 c' d 170 b' 116.3 2 1 3 a' 66.3 1 55.4 29 21.5

  24. MALDI-TOF analysis of tryptic fingerprint from the substrate for kinase FA/GSK-3a in pig brain (b1) 472-480 526-532 533-552 558-565 (d2) 472-480 526-532 533-552 558-565 (a'1) 472-480 (b'1) 472-480 526-532 533-552 558-565 (b'3) 533-552 558-565

  25. Data base search for the substrate of kinase FA/GSK-3a from pig brain (d2) Collapsin Response Mediator Protein-2 (CRMP-2, human) MSYQGKKNIP RITSDRLLIK GGKIVNDDQS FYADIYMEDG LIKQIGENLI VPGGVKTIEAHSRMVIPGGIDVHTRFQMPD QGMTSADDFF QGTKAALAGG TTMIIDHVVP EPGTSLLAAF DQWREWADSK SCCDYSLHVD ISEWHKGIQE EMEALVKDHG VNSFLVYMAF KDRFQLTDCQ IYEVLSVIRDIGAIAQVHAE NGDIIAEEQQRILDLGITGP EGHVLSRPEEVEAEAVNRAI TIANQTNCPL YITKVMSKSSAEVIAQARKK GTVVYGEPIT ASLGTDGSHY WSKNWAKAAA FVTSPPLSPD PTTPDFLNSL LSCGDLQVTG SAHCTFNTAQ KAVGKDNFTLIPEGTNGTEERMSVIWDKAV VTGKMDENQF VAVTSTNAAK VFNLYPRKGR IAVGSDADLVIWDPDSVKTI SAKTHNSSLE YNIFEGMECR GSPLVVISQG KIVLEDGTLHVTEGSGRYIP RKPFPDFVYK RIKARSRLAE LRGVPRGLYD GPVCEVSVTP KTVTPASSAK TSPAKQQAPPVRNLHQSGFSLSGAQIDDNIPRRTTQRIVAPPGGRANITS LG *908.4 da --- 391-397 *2169.1da --- 533-552 *pI~5.95

  26. Amino acid sequence analysis by PSD technique in MALDI-TOF MS (b1) 2169 908

  27. Tandem Mass Spectrometry

  28. MS/MS with a TOF reflectron mass spectrometer

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

  30. Amino acid sequence analysis by PSD technique in MALDI-TOF MS (b1) 2169 908

  31. Amino acid sequencing

  32. Determination of protein molecular weight

  33. LC/MS

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

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

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

  37. The End

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