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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: --Storage,analysis & manipulation of the information from genomics & proteomics mRNA level expressed protein level nor does it indicate the nature of the functional protein product t-RNA t-RNA mRNA Ribosome t-RNA (....) Protein DNA t-RNA Post Translational Modifications X (....) X CHO PO4 Active Protein 圖一:DNA序列是藍圖決定細胞的表現,蛋白質卻是實際上有功能 的工作者;分子階層的蛋白質及DNA分析是瞭解其功能的關鍵 Genomics Proteomics genes characterization and identification functional analysis of gene products Bioimformatics 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 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 Strategy for proteomic study in cancer biology Clinical specimens Laser-captured microdissector Tumor cells SDS-PAGE Normal cells isoelectrofocusing (?) digested by trypsin MALDI-TOF MS analysis Database search/mapping Protein identified Reference Book Mass Spectrometry for Biotechnology Gary Siuzdak The Department of Chemistry The Scripps Research Institute La Jolla, California Academic Press 1996 What is a mass spectrometer and what does it do? Analogy between mass analysis and the dispersion of light Components of a mass spectrometer 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 Reflector Second Detector Time of Flight First Detector Laser 圖十一:在 MALDI-TOF MS中的反射器設計 M/Z Ion Sources and Sample Introduction Sample probes and matrix for MALDI 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. Matrix-assisted laser desorption/ionization source Calibration for MALDI Vacuum system in MS Mass Analyzer-Time of Flight (TOF) Kinetic Energy = ½ mv2 v = (2KE/m)1/2 m/z Ion Detector Electron multiplier 1 Photomultiplier conversion dynode scintillation counting 106 Sensitivity of MALDI-TOF MS ~10 fg 1347.7 g/mole x 5 x 10 -18 mole = 6.74 x 10 –15 g Strategy for proteomic study in cancer biology Clinical specimens Laser-captured microdissector Tumor cells SDS-PAGE Normal cells isoelectrofocusing (?) digested by trypsin MALDI-TOF MS analysis Database search/mapping Protein identified The 2-D gel pattern of human kidney epithelial 293 cells expressed without (A) and with (B) the Epstein-Barr virus oncoprotein LMP1 IEF (B) SDS-PAGE (A) IEF Pick up spots of interest Digested by trypsin MALDI-TOF MS analysis The substrate of GSK-3a purified from pig brain (A) (B) 10 (A) pH 3 170 c b 116.3 1 a 66.3 55.4 29 21.5 (B) 10 pH 3 d 170 b' 116.3 66.3 55.4 29 21.5 2 1 3 1 a' c' 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 Data base search for the substrate of kinase FA/GSK-3a from pig brain (d2) Collapsin Response Mediator Protein-2 (CRMP-2, human) MSYQGKKNIP VPGGVKTIEA TTMIIDHVVP EMEALVKDHG NGDIIAEEQQ YITKVMSKSS FVTSPPLSPD IPEGTNGTEE IAVGSDADLV KIVLEDGTLH GPVCEVSVTP PRRTTQRIVA RITSDRLLIK HSRMVIPGGI EPGTSLLAAF VNSFLVYMAF RILDLGITGP AEVIAQARKK PTTPDFLNSL RMSVIWDKAV IWDPDSVKTI VTEGSGRYIP KTVTPASSAK PPGGRANITS *908.4 da --- 391-397 GGKIVNDDQS DVHTRFQMPD DQWREWADSK KDRFQLTDCQ EGHVLSRPEE GTVVYGEPIT LSCGDLQVTG VTGKMDENQF SAKTHNSSLE RKPFPDFVYK TSPAKQQAPP LG FYADIYMEDG QGMTSADDFF SCCDYSLHVD IYEVLSVIRD VEAEAVNRAI ASLGTDGSHY SAHCTFNTAQ VAVTSTNAAK YNIFEGMECR RIKARSRLAE VRNLHQSGFS *2169.1da --- 533-552 *pI~5.95 LIKQIGENLI QGTKAALAGG ISEWHKGIQE IGAIAQVHAE TIANQTNCPL WSKNWAKAAA KAVGKDNFTL VFNLYPRKGR GSPLVVISQG LRGVPRGLYD LSGAQIDDNI Amino acid sequence analysis by PSD technique in MALDI-TOF MS (b1) 2169 908 Tandem Mass Spectrometry MS/MS with a TOF reflectron mass spectrometer Peptide fragmentation postionization [Post Source Decay (PSD)] Amino acid sequence analysis by PSD technique in MALDI-TOF MS (b1) 2169 908 Amino acid sequencing Determination of protein molecular weight LC/MS 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 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 MS spectrometry database analysis system Bioimformatics 貴儀中心 Label & Quantify Isotope–Coded Affinity Tags ICAT™ • Breakthrough approach to quantitative protein expression analysis - 2D gel alternative heavy reagent: d8-ICAT (X=deuterium) light reagent: d0-ICAT (X=hydrogen) O NH HN S biotin tag O XX N H X XO O XX O N XX H linker (heavy or light) I S H cys reactive group Emerging Technology Nature Biotechnology 17, 994-999 (1999) The End Label & Quantify Isotope–Coded Affinity Tags ICAT™ • Breakthrough approach to quantitative protein expression analysis - 2D gel alternative heavy reagent: d8-ICAT (X=deuterium) light reagent: d0-ICAT (X=hydrogen) O NH HN S biotin tag O XX N H X XO O XX O N XX H linker (heavy or light) I reactive group Emerging Technology S H cys ICAT™ Protein ID and Quantification Process ProICAT provides Protein Identification and Quantitation information ICAT™ Features & Benefits • Overcomes some of the limitations of 2D Gels. • Ability to quantify membrane proteins. • ID and quantify low abundance proteins. • Broader range of protein MW or pI. • Allows for greater automated/higher throughput approach in the simultaneous quantification and identification of proteins. • Reduces complexity of analysis of protein digest -only cysteine containing peptides. • Applied Biosystems ‘Systems Approach’ for ICAT Technology including ICAT reagents, software and support.