《蛋白质组学介绍》PPT课件

ProteomicsandMassSpectroscopy,Proteomics,Thedreamofhavinggenomescompletelysequencedisnowareality.Thecompletesequenceofmanygenomesincludingthehumanoneisknown.However,theunderstandingofprobablyhalfamillionhumanproteinsencodedbylessthan30,000genesisstillalongwayawayandthehardworktounravelthecomplexityofbiologicalsystemsisyettocome.Anewfundamentalconceptcalledproteome(PROTEincomplementtoagenOME)hasrecentlyemerged.,Proteomics,shoulddrasticallyhelptounravelbiochemicalandphysiologicalmechanismsofcomplexmultivariatediseasesatthefunctionalmolecularlevel.Thedisciplineofproteomicshasbeeninitiatedtocomplementphysicalgenomicresearch.Theterm“proteome”wascoinedin1994byanAustraliangraduatestudent(MarkWilkins),ithascometobeusedanddefinedinavarietyofdifferentways,Proteomics,Definition-Theidentification,characterizationandquantificationofallproteinsinvolvedinaparticularpathway,organelle,cell,tissue,organororganismthatcanbestudiedinconcerttoprovideaccurateandcomprehensivedataaboutthatsystem.Or-Acompletedescriptionofproteinsexpressedinanygivencellatanygiventime,Proteomics,AcellularproteomeisthecollectionofproteinsfoundinaparticularcelltypeunderaparticularsetofenvironmentalconditionssuchasexposuretohormonestimulationItcanalsobeusefultoconsideranorganismscompleteproteome,whichcanbeconceptualizedasthecompletesetofproteinsfromallofthevariouscellularproteomes.Thisisveryroughlytheproteinequivalentofthegenome.Thetermproteomehasalsobeenusedtorefertothecollectionofproteinsincertainsub-cellularbiologicalsystems.Forexample,alloftheproteinsinaviruscanbecalledaviralproteome.,Proteomics,Sowhereareweinourunderstandingofthecell?31-60Ktotalgenesinthehumangenomewithlittledifferencebetweenthefruitflyandus!Wheredoesthediversitycomefrom?Answer:Itstheproteins!,Proteomics,Theproteomeislargerthanthegenome,especiallyineukaryotes,inthesensethattherearemoreproteinsthangenes.Thisisdueto:alternativesplicingofgenespost-translationalmodificationslikeglycosylationorPhosphorylation.,alternativesplicingofgenes,Agivenpieceofpre-mRNAwhichhasbeentranscribedfromonegenecanbechoppedandreconnectedindifferentwaystoyieldvariousnewmRNAswhichthenexitthenucleustobetranslatedinthecytoplasm.Whenthepre-mRNAhasbeentranscribedfromtheDNA,itincludesseveralintronsandexons.TheregulationandselectionofsplicesitesisdonebySerine/Arginine-residueproteins,alternativesplicingofgenes,FourknownmodesA-Alternativeselectionofpromoters:thisistheonlymethodofsplicingwhichcanproduceanalternativeN-terminusdomaininproteins.Inthiscase,differentsetsofpromoterscanbesplicedwithcertainsetsofotherexons.B-Alternativeselectionofcleavage/polyadenylationsites:thisistheonlymethodofsplicingwhichcanproduceanalternativeC-terminusdomaininproteins.Inthiscase,differentsetsofpolyadenylationsitescanbesplicedwiththeotherexons.,alternativesplicingofgenes,FourknownmodesC-Intronretainingmode:Insteadofsplicingoutanintron,theintronisretainedinthemRNAtranscript.However,theintronmustbeproperlyencodingforaminoacids.Theintronscodemustbeproperlyexpressible,otherwiseastopcodonorashiftinthereadingframewillcausetheproteintobenon-functional.D-Exoncassettemode:Certainexonsaresplicedouttoalterthesequenceofaminoacidsintheexpressedprotein.,post-translationalmodifications,PTMsinvolvingadditioninclude:Acetylation-theadditionofanacetylgroup,usuallyattheN-terminusoftheproteinAlkylation-theadditionofanalkylgroup(e.g.methyl,ethyl)Methylation-theadditionofamethylgroup,usuallyatlysineorarginineresidues.(Thisisatypeofalkylation.)Biotinylation-acylationofconservedlysineresidueswithabiotinappendageGlutamylation-covalentlinkageofglutamicacidresiduestotubulinandsomeotherproteins.,post-translationalmodifications,PTMsinvolvingadditioninclude:Glycylation-covalentlinkageofonetomorethan40glycineresiduestothetubulinC-terminaltailGlycosylation-theadditionofaglycosylgrouptoeitherasparagine,hydroxylysine,serine,orthreonine,resultinginaglycoproteinIsoprenylation-theadditionofanisoprenoidgroup(e.g.farnesolandgeranylgeraniol)Lipoylation-attachmentofalipoatefunctionality,post-translationalmodifications,PTMsinvolvingadditioninclude:Phosphopantetheinylation-theadditionofa4-phosphopantetheinylmoietyfromcoenzymeA,asinfattyacid,polyketide,non-ribosomalpeptideandleucinebiosynthesisPhosphorylation-theadditionofaphosphategroup,usuallytoserine,tyrosine,threonineorhistidine,post-translationalmodifications,Forinstance,thepeptidehormoneinsulinCuttwiceafterdisulfidebondsareformed,andapropeptideisremovedfromthemiddleofthechain;Theresultingproteinconsistsoftwopolypeptidechainsconnectedbydisulfidebonds.,Proteomics-Bridgingthegenometothefunctionsofthecell,AreasofProteomicsProteinAnalysis/Chemistry-LookatPTmodifications,structureandfunction,enzymebehaviorExpression-whatwhyandwhen-2Dgels,MS,HPLC/proteinchips,CellMapping-protein-proteininteractions-affinitytags,twohybrid,antibodypulldown,Proteomics,AsurprisingfindingoftheHumanGenomeProjectisthattherearefarfewerprotein-codinggenesinthehumangenomethanproteinsinthehumanproteome20,000to25,000genescodingforproteins.about1,000,000proteins.Thehumanbodymaycontainmorethan2millionproteins,eachhavingdifferentfunctions.Thediscrepancyimpliesthatproteindiversitycannotbefullycharacterizedbygeneexpressionanalysis,thusproteomicsisusefulforcharacterizingcellsandtissues.,Sohowdoesitwork?,Mostproteinsfunctionincollaborationwithotherproteins,andonegoalofproteomicsistoidentifywhichproteinsinteract.Thisoftengivesimportantcluesaboutthefunctionsofnewlydiscoveredproteins,Sohowdoesitwork?,Proteinsareresolved,sometimesonamassivescale.Proteinseparationcanbeperformedusing2-Dgelelectrophoresis,usuallyseparatesproteinsfirstbyisoelectricpointandthenbymolecularweight.Onceproteinsareseparatedandquantified,theyareidentifiedIndividualspotsarecutoutofthegelandcleavedintopeptideswithproteolyticenzymes,Sohowdoesitwork?,Thesepeptidescanthenbeidentifiedbymassspectrometry,Specifically:matrix-assistedlaserdesorption-ionizationtime-of-flight(MALDI-TOF)massspectrometry.Inthisprocedure,apeptideisplacedonamatrix,whichcausesthepeptidetoformcrystals.,Sohowdoesitwork?,Thenthepeptideonthematrixisionizedwithalaserbeamandanincreaseinvoltageatthematrixisusedtoshoottheionstowardadetectorinwhichthetimeittakesaniontoreachthedetectordependsonitsmass.Thehigherthemass,thelongerthetimeofflightoftheion.,Sohowdoesitwork?,InaMALDI-TOFmassspectrometer,theionscanalsobedeflectedwithanelectrostaticreflectorthatalsofocusestheionbeam.Thus,themassesoftheionsreachingtheseconddetectorcanbedeterminedwithhighprecisionandthesemassescanrevealtheexactchemicalcompositionsofthepeptides,andthereforetheiridentities!,Sohowdoesitwork?,Proteinmixturescanalsobeanalyzedwithoutpriorseparation.TheseproceduresbeginwithproteolyticdigestionoftheproteinsinacomplexmixtureTheresultingpeptidesareofteninjectedontoahighpressureliquidchromatographycolumn(HPLC)thatseparatespeptidesbasedonhydrophobicity.HPLCcanbecoupleddirectlytoatime-of-flightmassspectrometerusingelectrosprayionization,Sohowdoesitwork?,electrosprayionization:Atechniqueusedinmassspectrometrytoproduceions.Itisespeciallyusefulinproducingionsfrommacromoleculesbecauseitovercomesthepropensityofthesemoleculestofragmentwhenionized,Sohowdoesitwork?,Peptideselutingfromthecolumncanbeidentifiedbytandemmassspectrometry(MS/MS).ThefirststageoftandemMS/MSisolatesindividualpeptideions,andthesecondbreaksthepeptidesintofragmentsandusesthefragmentationpatterntodeterminetheiraminoacidsequences.Labelingwithisotopetagscanbeusedtoquantitativelycompareproteinsconcentrationamongtwoormoreproteinsamples.,Sohowdoesitwork?,Finally,usedatabases.Computercomparessequencestoothersequencesstoredinaninternationallyaccessibledatabase.DeterminestheidentityoftheisolatedproteinAstheentirehumangenomeisknown,computersareabletodeterminenearlyeverypotentialprotein.Newproteinsare“discovered”whentheymatchsequencespredictedbythecomputerthathavenotpreviouslybeenfound.,MedicalApplications,Alzheimersdisease:Elevationsinbetasecretasecreatesamyloid/beta-protein,whichcausesplaquetobuildupinthepatientsbrain,whichcausesdementia.Targetingthisenzymedecreasestheamyloid/beta-proteinandsoslowstheprogressionofthedisease.Aproceduretotestfortheincreaseinamyloid/beta-proteinisimmunohistochemicalstaining,inwhichantibodiesbindtospecificantigensorbiologicaltissueofamyloid/beta-protein.,MedicalApplications,Heartdisease:Commonlyassessedusingseveralkeyproteinbasedbiomarkers.StandardproteinbiomarkersforCVDincludeinterleukin-6,interleukin-8,serumamyloidAprotein,fibrinogen,andtroponins.cTnIcardiactroponinIincreasesinconcentrationwithin3to12hoursofinitialcardiacinjuryandcanbefoundelevateddaysafteranacutemyocardialinfarction.Anumberofcommercialantibodybasedassaysaswellasothermethodsareusedinhospitalsasprimarytestsforacutemyocardialinfarction.,MedicalApplications,Renalcellcarcinoma:Proteomicanalysisofkidneycellsandcancerouskidneycellsisproducingpromisingleadsforbiomarkersanddevelopingassaystotestforthisdisease.Inkidney-relateddiseases,urineisapotentialsourceforsuchbiomarkers.Recently,ithasbeenshownthattheidentificationofurinarypolypeptidesasbiomarkersofkidney-relateddiseasesallowstodiagnosetheseverityofthediseaseseveralmonthsbeforetheappearanceofthepathology.,MedicalApplications,.Phenylketonuria(PKU)Affectsinin5,000newbornsMostcommonnervoussystemdisorderAlleleisonchromosome12LacktheenzymeneededforthemetabolismoftheaminoacidphenylalanineAbuildupofabnormalbreakdownpathwayPhenylketoneAccumulatesinurine.Ifdietisnotchecked,canleadtoseverementalretardation,Overviewofproteomics,NucleusDNARNACytoplasmProteinexpressionandmodificationProteinisolationMassspectroscopyProteinsequenceIdentification,Overviewofproteomics,Proteomicsresearchishighlyinterdisciplinary,bringingtogether:biologychemistryinstrumentationStatisticscomputerscience,Overviewofproteomics,Summarytime!Spend15-20minsgoingoverthisasagroupMAKENOTES!/cfapps/research/data_admin/Site602/mainpageS602P0.html,MassSpectroscopy,MassSpectroscopy,Ananalyticaltechniqueusedtomeasurethemass-to-chargeratioofions.Itismostgenerallyusedtofindthecompositionofaphysicalsamplebygeneratingamassspectrumrepresentingthemassesofsamplecomponents.Thetechniquehasseveralapplications,including:1)identifyingunknowncompoundsbythemassofthecompoundmoleculesortheirfragments,MassSpectroscopy,2)determiningtheisotopiccompositionofelementsinacompound.3)determiningthestructureofacompoundbyobservingitsfragmentation4)quantifyingtheamountofacompoundinasampleusingcarefullydesignedmethods(massspectrometryisnotinherentlyquantitative),MassSpectroscopy,5)studyingthefundamentalsofgasphaseionchemistry(thechemistryofionsandneutralsinvacuum)6)determiningotherphysical,chemical,orevenbiologicalpropertiesofcompoundswithavarietyofotherapproaches.Amassspectrometerisadevicethatmeasuresthemass-to-chargeratioofions.Thisisachievedbyionizingthesampleandseparatingionsofdifferingmassesandrecordingtheirrelativeabundancebymeasuringintensitiesofionflux.,MassSpectroscopy,AllMassspecsconsistof:Ahighvacuumsystem10-6torrAsampleinletGC,HPLC,electronimpact,ordirectchemicalisolationAnionsourceConvertsmoleculestogas-phaseionsMALDI,fastatombombardment,MassSpectroscopy,AllMassspecsconsistof:Amassfilter/analyzerTimeofflight,magneticsector,MALDI,oriontrapAdetectorArraydetector,conversiondynode,orelectronmultiplyer,Ionization,Inmassspectrometry,asubstanceisbombardedwithanelectronbeamhavingsufficientenergytofragmentthemoleculeThepositivefragmentswhichareproduced(cationsandradicalcations)areacceleratedinavacuumthroughamagneticfieldandaresortedonthebasisofmass-to-chargeratio.,Ionization,Sincethebulkoftheionsproducedinthemassspectrometercarryaunitpositivecharge,thevaluem/eisequivalenttothemolecularweightofthefragment.Theanalysisofmassspectroscopyinformationinvolvesthere-assemblingoffragments,workingbackwardstogeneratetheoriginalmolecule.,Ionization,ElectronImpactionization(EI):Itcomprisesanelectrongun,atime-of-flightmassspectrometerwithposition-sensitivedetector(PSD).Analytemustbeinavaporstate,limitingbiologicalmaterialbelow400DaUsefulformetabolites,pollutants,andpharmaceuticalcompounds,Ionization,Chemicalionization:EspeciallyusefultechniquefororganicchemistswhennomolecularionisobservedinEImassspectrumIonizationofsample(analyte)isachievedbyinteractionofitsmoleculeswithreagentssuchasCH4orNH3Verygoodfordeterminingmolecularmassashighintensitymolecularionsareproducedowingtolessfragmentation.,Ionization,electrosprayionization:Atechniqueusedinmassspectrometrytoproduceions.ItisespeciallyusefulinproducingionsfrommacromoleculesbecauseitovercomesthepropensityofthesemoleculestofragmentwhenionizedIstheprimaryionsourceusedinliquidchromatography-massspecbecauseitsaliquid-gasinterfacethatiscapableofcouplingliquidchomatographywithmassspectrometry,MassAnalyzers,OnceionsarecreatedandleavetheionsourcetheypassintoamassanalyzerThisseparatestheionsandmeasurestheirmassesWhatisactuallymeasuredisthemasstochargeratio(m/z)ofeachionAtanygiventime,ionsofaparticularmasspassthroughandarecountedbythedetectorInthisway,theanalyzerscansthroughalargerangeofmasses,MassAnalyzers,Quadrupolemassspectrometry:Essentiallyamassfilterthatiscapableoftransmittingonlytheionofchoice.Amassspectrumisobtainedbyscanningthroughthemassrangeofinterestovertime.Twooppositerodshaveanoppositeappliedpotentialandaffectthetrajectoryofionstravelingdowntheflightpathcenteredbetweenthefourrods,MassAnalyzers,Quadrupolemassspectrometry:ThemassrangeoftheoscillatingionsisscannedbychangingtheDCvoltageandthefrequency.TheresolutionofthespectrometercanbeincreasedbyeitheremployingeightpolesorbyconnectingtwoorthreeqaudupolesinseriesExcelatapplicationswhereparticularionsofinterestarebeingstudiedbecausetheycanstaytunedonasingleionforextendedperiods,MassAnalyzers,Iontrapmassspectrometry:Theiontrapconsistsofthreeelectrodeswithhyperbolicsurfaces,thecentralringelectrode,andtwoadjacentendcapelectrodesThedeviceisradiallysymmetricaltheelectrodesarealignedandisolatedusingceramicspacersandposts,MassAnalyzers,Iontrapmassspectrometry:Advantages:(i)highsensitivity(ii)compactnessandmechanicalsimplicityinadevicewhichisneverthelesscapableofhighperformance(iii)tandemmassspectrometryexperimentsareavailablebyperformingsequentialmassanalysismeasurements(iv)highresolution,MassAnalyzers,Magneticsectoranalyzer:Generatedionsareacceleratedandarepassedaroundacurvedtrack(thesector)leadingtoadetector.Byincreasingthemagneticfieldappliedtotheions,heavierionswithhighermomentumcanbeinducedtofollowthecurvedtrack.,MassAnalyzers,Magneticsectoranalyzer:Onlyionsofmass-to-chargeratiothathaveequalcentripetalandcentrifugalforcespassthroughtheflighttubeTheionsthatreachthedetectorcanbevariedbychangingeitherthemagneticfieldortheappliedvoltageoftheionoptics.Sotheindividualionbeamsareseparatedspatiallyandeachhasauniqueradiusofcurvatureaccordingtoitsmass/chargeratio,MassAnalyzers,Plasmadesorptionionization:Firstavailabletoanalyzeproteinsandotherlargebiomolecules(well,lessthan35,000Mr).ThetechnologyisnowrelativelyobsoleteUsedradioactivecalifornium(252Cf)Requiredatimeofflight(TOF)massdetector,MassAnalyzers,matrix-assistedlaserdesorption-ionizationtime-of-flight(MALDI-TOF)massspectrometry.Relativelynoveltechniqueinwhichaco-precipitateofanUV-lightabsorbingmatrixandabiomoleculeisirradiatedbyananosecondlaserpulse,whichcausesthepeptidetoformcrystals.Mostofthelaserenergyisabsorbedbythematrix,whichpreventsunwantedfragmentationofthebiomolecule.Theionizedbiomoleculesareacceleratedinanelctricfieldandentertheflighttube,MassAnalyzers,matrix-assistedlaserdesorption-ionizationtime-of-flight(MALDI-TOF)massspectrometry.Duringtheflightinthistube,differentmoleculesareseparatedaccordingtotheirmasstochargeratioandreachthedetectoratdifferenttimes.Inthiswayeachmoleculeyieldsadistinctsignal.Itisaverysensitivemethod,whichallowsthedetectionoflow(10-15to10-18mole)quantitiesofsamplewithanaccuracyof0.1-0.01%.,MassAnalyzers-MALDI-TOF,Proteinidentificationbythistechniquehastheadvantageofshortmeasuringtime(fewminutes)andnegligiblesampleconsumption(lessthan1pmol).Additionalinformationonpost-translationalmodificationsandpresenceofby-products!,MassAnalyzers-MALDI-TOF,DrawbacksofMaldi-tofThesamplepreparationforMALDIisimportantfortheresult.Inorganicsaltswhicharealsopartofproteinextractsinterferewiththeionizationprocess.Thematrixproteinmixtureisnothomogenousbecausethepolaritydifferenceleadstoaseparationofthetwosubstancesduringcrystallization,MassAnalyzers-MALDI-TOF,Thespotdiameterofthetargetismuchlargerthanthatofthelaser,whichmakesitnecessaryto