浙江大学蛋白质组学课件

„„„„„„„ProteomesProteomesOrganismtissuebiofluidcellsubcellularcompartmentProteincomplexesSignalpathwaysCellcycleApoptosisLocation:Function:Modification:PhosphorylationGlycosylationUbiquitationSumoylationDifferentiationMigrationMetabolism…………proteomeproteome19941994MacquarieMacquarieMarcWilkinsMarcWilkinsKeithWilliamsKeithWilliamsTheentireTheentirePROTEinPROTEinpopulationpopulation1995199577ElectrophoresisElectrophoresisPROTEOMEisthePROTEINSPROTEOMEisthePROTEINSexpressedbyagenomeoratissue.expressedbyagenomeoratissue.Proteomics()„(proteomics)„„„()(Proteomics)„Expressionproteomics)„(Functionalproteomics)WhyProteomics?!„,;„,,;„GenomicsProteomicsDNA(～30,000genes)mRNA(100,000)transcriptiontranslationPolypeptideChainsmodificationFunctionalProtein(1,000,000)GenomicsProteomics22,11875,000(4SNP)100,000()200,000()10,000,000DNA“Analysingtheentiresetofproteinsofanorganismisafarbiggerchallengethananythingingenomics.”Nature,1999mRNA„1cDNA„2„3„„„„„„„„„„„„„„„“”“”………„“”“”…………N„,,„,SDSPAGEConditionAConditionB34710pH+--+SAMPLEPREPARATIONFIRSTDIMENSIONSECONDDIMENSIONSTAININGIMAGINGANALYSIS&EXTRACTIONMASSSPECTROMETRICIDENTIFICATIONOFSPOTS2-DExpressionProteomicsMethodologySamplepreparationbasics•Duetothegreatdiversityofproteinsampletypesandorigins,thereisnouniversalsamplepreparationmethodapplicableforallproteins.•Unfortunately,anoptimalproceduremustbedeterminedempiricallyinmostcasesandtailoredforeachsampletype.Thiscanbeadauntingtask.•However,somegeneralsamplepreparationguidelinesshouldbekeptinmindtoavoidanumberofpitfallsduringsamplepreparationforproteomicstudies.„„„„„„-70oC„…1…2PBS…3110650l8mol/LUrea,4%CHAPS,40mmol/LTris,65mmol/LDTT…45s,10s,100-400W…54oC25000g1h…6-70oC5„„„„„„￭￭￭￭￭•Celldisruption/lysis•Proteinsolubilization•Sampleclean-up•SamplefractionationSamplepreparationworkflow„…………„……………„…„…„…„…„:„„Frenchpresspressurecell„,„Grinding„„„„„.1-3g„2-DE„…12-DE…22-DE…3„„„„CHAPSIPG2M5~7M„„„„„„ReductionofproteinsbyDTTIonisationofDTTCharacteristicsofDTTCys,.50mmol/L,.DTT,pKa8,pH.,DTTpH,,,DTT,,„„„ReagentAmount8Murea47mlof8.5stockor24gureain25mlH2O50mMDTTor2mMTBP385mgor500ulof200mMTBPstock4%CHAPS2g0.2%carrierampholytesSeenexttable0.0002%bromophenolblue100ulof0.1%stockddH2OTo50ml„„„„„„„PMSF(phenylmethylsulfonylfluoride)…,Y,,FactorXa,,,K,,„AEBSF(Pefabloc)…,,,,.„EDTA…,„Peptideproteaseinhibitors(leupeptinaprotinin)…,,,,,FactorXa„…Enzyme’sreactionvelocitydoubleswithtemperatureincreaseof10„…Protein’shalf-lifeextendedatlowtemperatures-4,-20,-80orinliquidnitrogen(-200)…Lyophilization,safest&mostconvenientmethod„Ammoniumsulfate„TCAprecipitation„Acetoneand/orethanol„TCAplusacetoneTCAacetone„Dialysis„Gelfiltration„Precipitation/resuspension„„„„pHpH2pHIPGpH„pHIPGpH3124-1210-12IPGpH10-12IPGpH3-124-12„8kD200kDIPG-2DTris/glycinedodelcylsulfateions„„(SDS)„,„„(NaCl):-,-(EEO),()„-:„10mM„40mM„-:,TCA/,„:-,-„:;„40mMTrisDNADNA„1ml150~300U20min„--:TCA„:-,-„-/„,„():„(SDS):--„:„SDS„(8:1;SDS0.25%).„„„„„„„Samplepreparationbasics„„„„„„„…/„:…A280…Bradford…BCAA280„280nmPheTrpTyr„280nm260nm„mg/mL=1.45A280nm-0.74A260nm„„PheTrpTyr„0.2mg/mL——2mg/mLBradford„G-250465nm595nm„:…25-200g/ml,…2…:…„…Bradford…TritonX-100SDS…Bicinchoninicacid(BCA)„Bicinchoninicacid(BCA)LoweryCu2+Cu2+Cu1+BCACu1+562nM„BCA„BradfordBCA„„…………„„syntheticcarrierampholyte,SCAPH„PHnon-equilibriumPHgradientelectrophoresis,NEPHGE„PH„’………•••„„„„„„…………„„The1stD:IsoelectricFocusing„„„„„„„„„µµµµµµµµµ„pH4-12pH3-10pH4-7pH5-8pH7-10pH6-11pH3-6pH3-4pH4-5pH5-6pH6-7pH7-8pH8-9pH9-10pH10-11pH11-12pH33„„„„„„„The2ndD:SDS-PAGESDS--PAGEMini-PROTEAN3High-throughput12gelcapacity„……………„„„„„0.1%R-250R-3500.5-1h.„10%5%1-3h2-3„0.1-1.0g„„„„„„„••••••„„„Extraction:ComparisonUreavsUrea/Thiourea8Murea7Murea/2MthioureaRatliverCHAPSvs.Amidosulfobetaine-C122%CHAPS2%ABSC-12ImprovedSolubilityUreaCHAPSDTTUreathioureaCHAPSSB3-10,TBPEffectofDNaseTreatmentDNaseDNaseE.colilysateprecipitatedwithTCA/acetoneandresuspendedCrudeE.colilysateNosalt30mMNaClEffectofdialysisPre-dialysissampleDialyzedsample