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Application of Proteomics in Biological Research An introduction Jau-Song Yu Department of Cell and Molecular Biology Chang Gung University The central dogma of life science Gene (DNA) Transcription 1000 X Amplification mRNA Translation 100 X Amplification Protein Genomics: ---Identification and characterization of genes (gene expression) and their arrangement in chromosomes Proteomics (Functional Genomics): ---Functional analysis of gene products (proteins) Bioinformatics: ---Storage, analysis and manipulation of the information from genomics and proteomics Human Genome Project (HGP) --- 99% sequence of human genome published 16 February 2001 Volume 291 Number 5507 The Human Genome 15 February 2001, Volume 409, Number 6822 Global gene expression analysis --- cDNA microarray Breast cancer samples vs. normal tissues PNAS USA 98, 10869–10874 (2001) The extent of gene expression (i.e. the amount of mRNA) is only one of the many factors determining the protein function in cells mRNA level expressed protein level nor does it indicate the nature of the functional protein product mRNA stability, alternative splicing, etc. t-RNA t-RNA mRNA Ribosome t-RNA (....) Protein DNA t-RNA Post Translational Modifications X (....) X Active Protein CHO C 2H5 PO44 PO 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 Post-translational Modification of proteins (covalent modification, proteolytic cleavage, activator, inhibitor, etc) Genomics Proteomics genes characterization and identification functional analysis of gene products Bioimformatics Proteomics --Global analysis of hundreds to thousands of proteins in cells or tissues simultaneously (why we need?) How to analyze hundreds to thousands of proteins in cells or tissues simultaneously? ● Separation of proteins on one matrix --two-dimensional gel electrophoresis ● Identification of separated proteins in a high-throughput way --biomass spectrometry 2-Dimension Electrophoresis (2-DE) for Protein Separation One of the core technology of proteomics is 2-DE: At present, there is no other technique which is capable of resolving thousands of proteins in one separation procedure. Isoelectric point (pI): Isoelectric point is the pH of a solution at which the net charge of protein is zero. In electrophoresis there is no motion of the particles in an electric field at the isoelectric point. NH3+ NH3+ NH2 COOH COO- COO- NH3+ NH3+ NH2 COOH COO- COO- pH < pI Net positive charge pH = pI pH > pI Net negative charge 3 Isoelectric point Net charge 2 1 0 -1 -2 -3 2 3 4 5 6 7 pH 8 9 10 11 General principle and protocol of 2-dimension gel electrophoresis Ampholytes sample pH 9 - pH 3 + Isoelectric focusing (1st dimension) polyacrylamide MW 2nd dimension SDS-PAGE pH gradient Traditional Equipment for Isoelectric focusing (IEF): Ampholytes polyacrylamide Cathode (-) electrode solution Anode (+) electrode solution Immobilized pH Gradient (IPG) Acrylamide monomer Acidic buffering group: COO- CH2 - CH-C-NH-R Basic buffering group: NH3+ O Polyacrylamide gel Production of Immobilized pH Gradient (IPG) strip basic A acidic Gradient maker plastic support film D B E C F pH 3 pH 10 Equipment for Isoelectric focusing (IEF): IPGphor (IEF System) Protein IEF Cell Amersham Pharmacia Biotech Inc. Bio-Rad Laboratories Sample preparation Lysis solution: 8M 4% 40mM Urea NP-40 or CHAPS Tris base Cell line Lysis solution Sonication vacuum Lysis solution Centrifugation Measurement of [protein] 2-DE sample IPG strip rehydration and sample loading 2-DE sample Rehydration solution Rehydration solution: 8M 2% 2% 0.28% Trace IPG strip holder Position the IPG strip Urea NP-40 or CHAPS IPG buffer (Ampholyte) DTT Bromophenol blue IPG strip rehydration and sample loading Strip holder Anode (+) electrode Cathode (-) electrode 30 voltage 12hr First dimension: Isoelectric focusing Electrode pads 1. Place electrode pads (?) 2. 200 V step-n-hold 1.5hr 3. 500 V step-n-hold 1.5hr 4. 1000 V gradient 1500vhr Holder cover IPG strip Electrode Voltage 5. 8000 V gradient (?) Time 36000vhr Second dimension: SDS-PAGE • SDS equilibration • SDS-PAGE IPG strip SDS SDS equilibration buffer 50 mM 6M 30% 2% Trace Marker in paper SDS-PAGE SDS-PAGE Tris-HCl Urea Glycerol SDS Bromophenol 0.5% agarose in running buffer SDS-PAGE Detection of proteins separated on gels --Protocol of silver stain: 50% methanol 25% acetic acid 4hr ddH2O 30 sec ddH2O x 3 times 30min/time 3% Na2CO3 0.0185% formaldehyde 0.004% DTT solution 30min 2.3M citric acid 0.1% AgNO3 30min 5% acetic acid 25% methanol 2-DE separation of soluble E. coli proteins For cancer study ~ Clinical specimens Cryostat Laser-captured microdissector (LCM) Tumor cells SDS-PAGE Normal cells 2D gel electrophoresis Immage system isoelectrofocusing (?????) Identification of 2-DE-separated proteins in a high-throughput way using biomass spectrometry MALDI TOF/TOF MS LC/MSn What is a mass spectrometer and what does it do? Gary Siuzdak (1996) Mass Spectrometry for Biotechnology, Academic Press 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) Target plate Second detector Reflector Reflector Second Detector Time of Flight Target plate First detector First Detector Laser Laser 圖十一:在 MALDI-TOF MS中的反射器設計 M/Z 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 Mass Analyzer-Time of Flight (TOF) Kinetic Energy = ½ mv2 v = (2KE/m)1/2 m/z Sensitivity of MALDI-TOF MS ~10 fg 1347.7 g/mole x 5 x 10 -18 mole = 6.74 x 10 –15 g How to identify 2-DE-separated proteins by MALDI-TOF MS? Linking between genomics/bioinformatics/proteomics Clinical specimens Cryostat Laser-captured microdissector (LCM) Tumor cells SDS-PAGE Normal cells 2D gel electrophoresis Immage system isoelectrofocusing (?????) (?????) Digested by trypsin (Lys, Arg) (621, 754, 778, 835, 1204,, 1398, 1476, 1582) (664, 711, 735, 904, 1079, 1188, 1438) (602, 755, 974, 1166, 1244, 1374) (854, 931, 935, 1021, 1067, 1184, 1386, 1438) (Masses of tryptic peptides are predictable from gene sequence databases) MALDI-TOF MS analysis (621, 778, 835, 1204,, 1398, 1582) (735, 904, 1079, 1188, 1438) (755, 974, 1244, 1374) (854, 935, 1021, 1067, 1184, 1386, 1438) Database search/mapping Protein identified (100%?) (M/Z) An example ~ Identification of specific proteins purified from pig brain (A) (B) 10 pH 3 c 170 b 116.3 66.3 55.4 29 21.5 2 1 3 1 a MALDI-TOF analysis of tryptic fingerprint from the proteins purified from pig brain (a1) (b1) (b3) (d2) (c2) Data base search for the purified protein from pig brain (c2) 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 Proteomics solution IEF SDS-PAGE Direct identification of the amino acid sequence of peptides by tandem mass spectrometry Amino acid sequence analysis by MS - an example 2169 908 Press Release: The Nobel Prize in Chemistry 2002 9 October 2002 The Royal Swedish Academy of Sciences has decided to award the Nobel Prize in Chemistry for 2002 ”for the development of methods for identification and structure analyses of biological macromolecules” with one half jointly to John B. Fenn Virginia Commonwealth University, Richmond, USA, and Koichi Tanaka Shimadzu Corp., Kyoto, Japan ”for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules” and the other half to Kurt Wüthrich Swiss Federal Institute of Technology (ETH), Zürich, Switzerland and The Scripps Research Institute, La Jolla, USA ”for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution”. Revolutionary analytical methods for biomolecules This year’s Nobel Prize in Chemistry concerns powerful analytical methods for studying biological macromolecules, for example proteins. The possibility of analysing proteins in detail has led to increased understanding of the processes of life. Researchers can now rapidly and simply reveal what different proteins a sample contains. They can also determine threedimensional pictures showing what protein molecules look like in solution and can then understand their function in the cell. The methods have revolutionised the development of new pharmaceuticals. Promising applications are also being reported in other areas, for example foodstuff control and early diagnosis of breast cancer and prostate cancer. Mass spectrometry is a very important analytical method used in practically all chemistry laboratories the world over. Previously only fairly small molecules could be identified, but John B. Fenn and Koichi Tanaka have developed methods that make it possible to analyse biological macromolecules as well. In the method that John B. Fenn published in 1988, electrospray ionisation (ESI), charged droplets of protein solution are produced which shrink as the water evaporates. Eventually freely hovering protein ions remain. Their masses may be determined by setting them in motion and measuring their time of flight over a known distance. At the same time Koichi Tanaka introduced a different technique for causing the proteins to hover freely, soft laser desorption. A laserpulse hits the sample, which is “blasted” into small bits so that the molecules are released. The other half of the Prize rewards the further development of another favourite method among chemists, nuclear magnetic resonance, NMR. NMR gives information on the three-dimensional structure and dynamics of the molecules. Through his work at the beginning of the 1980s Kurt Wüthrich has made it possible to use NMR on proteins. He developed a general method of systematically assigning certain fixed points in the protein molecule, and also a principle for determining the distances between these. Using the distances, he was able to calculate the three-dimensional structure of the protein. The advantage of NMR is that proteins can be studied in solution, i.e. an environment similar to that in the living cell. The Nobel Prize in Chemistry for 2002 is to be shared between scientists working on two very important methods of chemical analysis applied to biological macromolecules: mass spectrometry (MS) and nuclear magnetic resonance (NMR). Laureates John B. Fenn, Koichi Tanaka (MS) and Kurt Wuthrich (NMR) have pioneered the successful application of their techniques to biological macromolecules. Biological macromolecules are the main actors in the makeup of life whether expressed in prospering diversity or in threatening disease. To understand biology and medicine at molecular level where the identity, functional characteristics, structural architecture and specific interactions of biomolecules are the basis of life, we need to visualize the activity and interplay of large macromolecules such as proteins. To study, or analyse, the protein molecules, principles for their separation and determination of their individual characteristics had to be developed. Two of the most important chemical techniques used today for the analysis of biomolecules are mass spectrometry (MS) and nuclear magnetic resonance (NMR), the subjects of this year’s Nobel Prize award. Bruker’s movie for MALDI-TOF Mass Spectrometry 長庚大學蛋白質體核心實驗室簡介