分子生物学分子生物学 (25).pdf

《分子生物学分子生物学 (25).pdf》由会员分享，可在线阅读，更多相关《分子生物学分子生物学 (25).pdf（16页珍藏版）》请在淘文阁 - 分享文档赚钱的网站上搜索。

1、Chapter 10Proteomics research methods 10.3 Research method of proteome expression pattern-Mass spectrometry10.3.1 Sample identification of proteome researchBasic principle:After sample ionization,the molecular weight of theprotein is separated and determined according to the difference in ion mass/c

2、harge ratio(m/z).Spot identificationSpot PickingDigestionMS Analysis and Database searchSpot cutterSpots of interestMass(m/z)01000020000300004000050000 1000 2000 3000 4000 Mass SpectrometryMolecularsIonsDetectorSuccessful ion pathQuadrupole Ion AnalyzerElectron Impact IonizerThe composition and stru

3、cture of the sample were analyzed by measuring the mass charge(m/z)of the sample ions.Characteristics of soft ionizationMoleculeintegritymaintainedwithoutproducing fragment ions;high sensitivity,fast speed,providingboth molecular weight and structureinformation;qualitative and quantitative;being eff

4、icient,due to the capability of connecting various chromatographies and thus processing complex system analysisMatrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS)Electrospray ionization mass spectrometry(ESI-MS)Main types of mass spectrometryIdentifying and ann

5、otating protein routes Search database for matching bypeptide mass spectrometry fingerprint(PMF)database;Search database for matching bymeasuring the secondary mass spectruminformation or amino acid sequence tagsof some peptides in the sample.Sample MS SpectrumTypical secondary mass spectrumReasons

6、for failing in PMF matchingsmall sample quantity leading to low signal-to-noise ratios of PMF;a protein spot on 2-DE glue is not a singleprotein;contamination of keratin or other proteins;many post-translational modifications of theproteins have not been recorded in the database;impure trypsin with

7、protease self-hydrolysingfragments;unknown new protein;a small database size.tissue section or imprint in situ mass spectrometryMALDI-TOF/TOFFourier transform cyclotron resonance mass spectrometry(FTMS)SELDI-TOF-MSCombined technology to accurately identify proteins10.3.2 Bioinformatics in proteomics

8、 researchBioinformatics is an indispensablepart of proteomics research.construction and analysis of 2-DEpatterns;search and construction of databases.Proteome databaseSWISS-PROT:http:/www.expasy.ch/sprot/(It has the largest and most diverse proteome database in the world.)NRDB:http:/www.nrdb.co.uk/d

9、bEST:http:/www.ncbi.nlm.nih.gov/NCBI:http:/www.ncbi.nih.gov/Genomes/EMBL:http:/www.embl.org/Prosite：http:/www.expasy.org/prosite/PDB：http:/www.rcsb.org/pdb/most commonly used onesQuestion:Besides the mass spectrometry mentionedin this lesson,what are the other massspectrometry techniques used to identifyproteins?