蛋白质组学介绍

Proteomics

and

MassSpectroscopyProteomicsThedreamofhavinggenomescompletelysequencedisnowareality.Thecompletesequenceofmanygenomesincludingthehumanoneisknown.However,theunderstandingofprobablyhalfamillionhumanproteinsencodedbylessthan30,000genesisstillalongwayawayandthehardworktounravelthecomplexityofbiologicalsystemsisyettocome.Anewfundamentalconceptcalledproteome(PROTEincomplementtoagenOME)hasrecentlyemerged.Proteomicsshoulddrasticallyhelptounravelbiochemicalandphysiologicalmechanismsofcomplexmultivariatediseasesatthefunctionalmolecularlevel.Thedisciplineofproteomicshasbeeninitiatedtocomplementphysicalgenomicresearch.Theterm“proteome”wascoinedin1994byanAustraliangraduatestudent(MarkWilkins),ithascometobeusedanddefinedinavarietyofdifferentwaysProteomicsDefinition-Theidentification,characterizationandquantificationofallproteinsinvolvedinaparticularpathway,organelle,cell,tissue,organororganismthatcanbestudiedinconcerttoprovideaccurateandcomprehensivedataaboutthatsystem.Or-Acompletedescriptionofproteinsexpressedinanygivencellatanygiventime

ProteomicsAcellularproteomeisthecollectionofproteinsfoundinaparticularcelltypeunderaparticularsetofenvironmentalconditionssuchasexposuretohormonestimulation

Itcanalsobeusefultoconsideranorganism'scompleteproteome,whichcanbeconceptualizedasthecompletesetofproteinsfromallofthevariouscellularproteomes.Thisisveryroughlytheproteinequivalentofthegenome.Theterm"proteome"hasalsobeenusedtorefertothecollectionofproteinsincertainsub-cellularbiologicalsystems.Forexample,alloftheproteinsinaviruscanbecalledaviralproteome.ProteomicsSowhereareweinourunderstandingofthecell?31-60Ktotalgenesinthehumangenomewithlittledifferencebetweenthefruitflyandus!Wheredoesthediversitycomefrom?Answer:It’stheproteins!!!!!!!!!!!!

ProteomicsTheproteomeislargerthanthegenome,especiallyineukaryotes,inthesensethattherearemoreproteinsthangenes.Thisisdueto:

alternativesplicingofgenes

post-translationalmodificationslikeglycosylationorPhosphorylation.alternativesplicingofgenesAgivenpieceofpre-mRNAwhichhasbeentranscribedfromonegenecanbechoppedandreconnectedindifferentwaystoyieldvariousnewmRNAswhichthenexitthenucleustobetranslatedinthecytoplasm.Whenthepre-mRNAhasbeentranscribedfromtheDNA,itincludesseveralintronsandexons.

TheregulationandselectionofsplicesitesisdonebySerine/Arginine-residueproteins

alternativesplicingofgenesFourknownmodes

A-Alternativeselectionofpromoters:thisistheonlymethodofsplicingwhichcanproduceanalternativeN-terminusdomaininproteins.Inthiscase,differentsetsofpromoterscanbesplicedwithcertainsetsofotherexons.B-Alternativeselectionofcleavage/polyadenylationsites:thisistheonlymethodofsplicingwhichcanproduceanalternativeC-terminusdomaininproteins.Inthiscase,differentsetsofpolyadenylationsitescanbesplicedwiththeotherexons.

alternativesplicingofgenesFourknownmodes

C-Intronretainingmode:Insteadofsplicingoutanintron,theintronisretainedinthemRNAtranscript.However,theintronmustbeproperlyencodingforaminoacids.Theintron'scodemustbeproperlyexpressible,otherwiseastopcodonorashiftinthereadingframewillcausetheproteintobenon-functional.D-Exoncassettemode:Certainexonsaresplicedouttoalterthesequenceofaminoacidsintheexpressedprotein.post-translationalmodificationsPTMsinvolvingadditioninclude:Acetylation-theadditionofanacetylgroup,usuallyattheN-terminusoftheproteinAlkylation-theadditionofanalkylgroup(e.g.methyl,ethyl)Methylation-theadditionofamethylgroup,usuallyatlysineorarginineresidues.(Thisisatypeofalkylation.)Biotinylation-acylationofconservedlysineresidueswithabiotinappendageGlutamylation-covalentlinkageofglutamicacidresiduestotubulinandsomeotherproteins.

post-translationalmodificationsPTMsinvolvingadditioninclude:Glycylation-covalentlinkageofonetomorethan40glycineresiduestothetubulinC-terminaltailGlycosylation-theadditionofaglycosylgrouptoeitherasparagine,hydroxylysine,serine,orthreonine,resultinginaglycoproteinIsoprenylation-theadditionofanisoprenoidgroup(e.g.farnesolandgeranylgeraniol)Lipoylation-attachmentofalipoatefunctionality

post-translationalmodificationsPTMsinvolvingadditioninclude:Phosphopantetheinylation-theadditionofa4'-phosphopantetheinylmoietyfromcoenzymeA,asinfattyacid,polyketide,non-ribosomalpeptideandleucinebiosynthesisPhosphorylation-theadditionofaphosphategroup,usuallytoserine,tyrosine,threonineorhistidine

post-translationalmodificationsForinstance,thepeptidehormoneinsulinCuttwiceafterdisulfidebondsareformed,andapropeptideisremovedfromthemiddleofthechain;Theresultingproteinconsistsoftwopolypeptidechainsconnectedbydisulfidebonds.Proteomics-BridgingthegenometothefunctionsofthecellAreasofProteomicsProteinAnalysis/Chemistry-LookatPTmodifications,structureandfunction,enzymebehaviorExpression-whatwhyandwhen-2Dgels,MS,HPLC/proteinchips,CellMapping-protein-proteininteractions-affinitytags,twohybrid,antibodypulldownProteomicsAsurprisingfindingoftheHumanGenomeProjectisthattherearefarfewerprotein-codinggenesinthehumangenomethanproteinsinthehumanproteome20,000to25,000genescodingforproteins.about1,000,000proteins.

Thehumanbodymaycontainmorethan2millionproteins,eachhavingdifferentfunctions.

Thediscrepancyimpliesthatproteindiversitycannotbefullycharacterizedbygeneexpressionanalysis,thusproteomicsisusefulforcharacterizingcellsandtissues.

Sohowdoesitwork?Mostproteinsfunctionincollaborationwithotherproteins,andonegoalofproteomicsistoidentifywhichproteinsinteract.Thisoftengivesimportantcluesaboutthefunctionsofnewlydiscoveredproteins

Sohowdoesitwork?Proteinsareresolved,sometimesonamassivescale.Proteinseparationcanbeperformedusing2-Dgelelectrophoresis,`usuallyseparatesproteinsfirstbyisoelectricpointandthenbymolecularweight.

Onceproteinsareseparatedandquantified,theyareidentified

IndividualspotsarecutoutofthegelandcleavedintopeptideswithproteolyticenzymesSohowdoesitwork?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.ItisespeciallyusefulinproducingionsfrommacromoleculesbecauseitovercomesthepropensityofthesemoleculestofragmentwhenionizedSohowdoesitwork?Peptideselutingfromthecolumncanbeidentifiedbytandemmassspectrometry(MS/MS).

ThefirststageoftandemMS/MSisolatesindividualpeptideions,andthesecondbreaksthepeptidesintofragmentsandusesthefragmentationpatterntodeterminetheiraminoacidsequences.Labelingwithisotopetagscanbeusedtoquantitativelycompareproteinsconcentrationamongtwoormoreproteinsamples.Sohowdoesitwork?Finally,usedatabases.Computercomparessequencestoothersequencesstoredinaninternationallyaccessibledatabase.DeterminestheidentityoftheisolatedproteinAstheentirehumangenomeisknown,computersareabletodeterminenearlyeverypotentialprotein.Newproteinsare“discovered”whentheymatchsequencespredictedbythecomputerthathavenotpreviouslybeenfound.MedicalApplicationsAlzheimer’sdisease:Elevationsinbetasecretasecreatesamyloid/beta-protein,whichcausesplaquetobuildupinthepatient'sbrain,whichcausesdementia.Targetingthisenzymedecreasestheamyloid/beta-proteinandsoslowstheprogressionofthedisease.Aproceduretotestfortheincreaseinamyloid/beta-proteinisimmunohistochemicalstaining,inwhichantibodiesbindtospecificantigensorbiologicaltissueofamyloid/beta-protein.MedicalApplicationsHeartdisease:Commonlyassessedusingseveralkeyproteinbasedbiomarkers.StandardproteinbiomarkersforCVDincludeinterleukin-6,interleukin-8,serumamyloidAprotein,fibrinogen,andtroponins.cTnIcardiactroponinIincreasesinconcentrationwithin3to12hoursofinitialcardiacinjuryandcanbefoundelevateddaysafteranacutemyocardialinfarction.Anumberofcommercialantibodybasedassaysaswellasothermethodsareusedinhospitalsasprimarytestsforacutemyocardialinfarction.MedicalApplicationsRenalcellcarcinoma:Proteomicanalysisofkidneycellsandcancerouskidneycellsisproducingpromisingleadsforbiomarkersanddevelopingassaystotestforthisdisease.Inkidney-relateddiseases,urineisapotentialsourceforsuchbiomarkers.Recently,ithasbeenshownthattheidentificationofurinarypolypeptidesasbiomarkersofkidney-relateddiseasesallowstodiagnosetheseverityofthediseaseseveralmonthsbeforetheappearanceofthepathology.MedicalApplications.Phenylketonuria(PKU)Affectsinin5,000newbornsMostcommonnervoussystemdisorderAlleleisonchromosome12LacktheenzymeneededforthemetabolismoftheaminoacidphenylalanineAbuildupofabnormalbreakdownpathwayPhenylketoneAccumulatesinurine.Ifdietisnotchecked,canleadtoseverementalretardationOverviewofproteomicsNucleus DNA–RNACytoplasmProteinexpressionandmodificationProteinisolationMassspectroscopyProteinsequenceIdentificationOverviewofproteomicsProteomicsresearchishighlyinterdisciplinary,bringingtogether:

biologychemistryinstrumentationStatisticscomputerscienceOverviewofproteomicsSummarytime!Spend15-20minsgoingoverthisasagroupMAKENOTES!!!!!!!!!!!!/cfapps/research/data_admin/Site602/mainpageS602P0.htmlMassSpectroscopyMassSpectroscopyAnanalyticaltechniqueusedtomeasurethemass-to-chargeratioofions.Itismostgenerallyusedtofindthecompositionofaphysicalsamplebygeneratingamassspectrumrepresentingthemassesofsamplecomponents.Thetechniquehasseveralapplications,including:

1)identifyingunknowncompoundsbythemassofthecompoundmoleculesortheirfragmentsMassSpectroscopy2)determiningtheisotopiccompositionofelementsinacompound.

3)determiningthestructureofacompoundbyobservingitsfragmentation

4)quantifyingtheamountofacompoundinasampleusingcarefullydesignedmethods(massspectrometryisnotinherentlyquantitative)MassSpectroscopy5)studyingthefundamentalsofgasphaseionchemistry(thechemistryofionsandneutralsinvacuum)6)determiningotherphysical,chemical,orevenbiologicalpropertiesofcompoundswithavarietyofotherapproaches.

Amassspectrometerisadevicethatmeasuresthemass-to-chargeratioofions.Thisisachievedbyionizingthesampleandseparatingionsofdifferingmassesandrecordingtheirrelativeabundancebymeasuringintensitiesofionflux.MassSpectroscopyAllMassspecsconsistof:Ahighvacuumsystem10-6torrAsampleinletGC,HPLC,electronimpact,ordirectchemicalisolationAnionsourceConvertsmoleculestogas-phaseionsMALDI,fastatombombardmentMassSpectroscopyAllMassspecsconsistof:Amassfilter/analyzerTimeofflight,magneticsector,MALDI,oriontrapAdetectorArraydetector,conversiondynode,orelectronmultiplyerIonizationInmassspectrometry,asubstanceisbombardedwithanelectronbeamhavingsufficientenergytofragmentthemolecule

Thepositivefragmentswhichareproduced(cationsandradicalcations)areacceleratedinavacuumthroughamagneticfieldandaresortedonthebasisofmass-to-chargeratio.

IonizationSincethebulkoftheionsproducedinthemassspectrometercarryaunitpositivecharge,thevaluem/eisequivalenttothemolecularweightofthefragment.Theanalysisofmassspectroscopyinformationinvolvesthere-assemblingoffragments,workingbackwardstogeneratetheoriginalmolecule.IonizationElectronImpactionization(EI):Itcomprisesanelectrongun,atime-of-flightmassspectrometerwithposition-sensitivedetector(PSD).

Analytemustbeinavaporstate,limitingbiologicalmaterialbelow400DaUsefulformetabolites,pollutants,andpharmaceuticalcompoundsIonizationChemicalionization:EspeciallyusefultechniquefororganicchemistswhennomolecularionisobservedinEImassspectrumIonizationofsample(analyte)isachievedbyinteractionofitsmoleculeswithreagentssuchasCH4orNH3Verygoodfordeterminingmolecularmass

ashighintensitymolecularionsareproducedowingtolessfragmentation.Ionizationelectrosprayionization:Atechniqueusedinmassspectrometrytoproduceions.ItisespeciallyusefulinproducingionsfrommacromoleculesbecauseitovercomesthepropensityofthesemoleculestofragmentwhenionizedIstheprimaryionsourceusedinliquidchromatography-massspecbecauseit'saliquid-gasinterfacethatiscapableofcouplingliquidchomatographywithmassspectrometryMassAnalyzers

OnceionsarecreatedandleavetheionsourcetheypassintoamassanalyzerThisseparatestheionsandmeasurestheirmassesWhatisactuallymeasuredisthemasstochargeratio(m/z)ofeachionAtanygiventime,ionsofaparticularmasspassthroughandarecountedbythedetectorInthisway,theanalyzerscansthroughalargerangeofmassesMassAnalyzersQuadrupolemassspectrometry:Essentiallyamassfilterthatiscapableoftransmittingonlytheionofchoice.Amassspectrumisobtainedbyscanningthroughthemassrangeofinterestovertime.TwooppositerodshaveanoppositeappliedpotentialandaffectthetrajectoryofionstravelingdowntheflightpathcenteredbetweenthefourrodsMassAnalyzersQuadrupolemassspectrometry:ThemassrangeoftheoscillatingionsisscannedbychangingtheDCvoltageandthefrequency.TheresolutionofthespectrometercanbeincreasedbyeitheremployingeightpolesorbyconnectingtwoorthreeqaudupolesinseriesExcelatapplicationswhereparticularionsofinterestarebeingstudiedbecausetheycanstaytunedonasingleionforextendedperiodsMassAnalyzersIontrapmassspectrometry:Theiontrapconsistsofthreeelectrodeswithhyperbolicsurfaces,thecentralringelectrode,andtwoadjacentendcapelectrodesThedeviceisradiallysymmetrical

theelectrodesarealignedandisolatedusingceramicspacersandpostsMassAnalyzersIontrapmassspectrometry:Advantages:(i)highsensitivity(ii)compactnessandmechanicalsimplicityinadevicewhichisneverthelesscapableofhighperformance(iii)tandemmassspectrometryexperimentsareavailablebyperformingsequentialmassanalysismeasurements(iv)highresolution

MassAnalyzersMagneticsectoranalyzer:Generatedionsareacceleratedandarepassedaroundacurvedtrack(thesector)leadingtoadetector.Byincreasingthemagneticfieldappliedtotheions,heavierionswithhighermomentumcanbeinducedtofollowthecurvedtrack.MassAnalyzersMagneticsectoranalyzer:Onlyionsofmass-to-chargeratiothathaveequalcentripetalandcentrifugalforcespassthroughtheflighttubeTheionsthatreachthedetectorcanbevariedbychangingeitherthemagneticfieldortheappliedvoltageoftheionoptics.Sotheindividualionbeamsareseparatedspatiallyandeachhasauniqueradiusofcurvatureaccordingtoitsmass/chargeratioMassAnalyzersPlasmadesorptionionization:Firstavailabletoanalyzeproteinsandotherlargebiomolecules(well,lessthan35,000Mr).ThetechnologyisnowrelativelyobsoleteUsedradioactivecalifornium(252Cf)Requiredatimeofflight(TOF)massdetectorMassAnalyzersmatrix-assistedlaserdesorption-ionizationtime-of-flight

(MALDI-TOF)massspectrometry.

Relativelynoveltechniqueinwhichaco-precipitateofanUV-lightabsorbingmatrixandabiomoleculeisirradiatedbyananosecondlaserpulse,whichcausesthepeptidetoformcrystals.Mostofthelaserenergyisabsorbedbythematrix,whichpreventsunwantedfragmentationofthebiomolecule.

Theionizedbiomoleculesareacceleratedinanelctricfieldandentertheflighttube

MassAnalyzersmatrix-assistedlaserdesorption-ionizationtime-of-flight

(MALDI-TOF)massspectrometry.

Duringtheflightinthistube,differentmoleculesareseparatedaccordingtotheirmasstochargeratioandreachthedetectoratdifferenttimes.

Inthiswayeachmoleculeyieldsadistinctsignal.Itisaverysensitivemethod,whichallowsthedetectionoflow(10-15to10-18mole)quantitiesofsamplewithanaccuracyof0.1-0.01%.MassAnalyzers-MALDI-TOFProteinidentificationbythistechniquehastheadvantageofshortmeasuringtime(fewminutes)andnegligiblesampleconsumption(lessthan1pmol).Additionalinformationonpost-translationalmodificationsandpresenceofby-products!

MassAnalyzers-MALDI-TOFDrawbacksofMaldi-tofThesamplepreparationforMALDIisimportantfortheresult.Inorganicsaltswhicharealsopartofproteinextractsinterferewiththeionizationprocess.

Thematrixproteinmixtureisnothomogenousbecausethepolaritydifferenceleadstoaseparationofthetwosubstancesduringcrystallization

MassAnalyzers-MALDI-TOFThespotdiameterofthetargetismuchlargerthanthatofthelaser,whichmakesitnecessarytodoseverallasershotsatdifferentplacesofthetarget,togetthestatisticalaverageofthesubstanceconcentrationwithinthetargetspot.

Delaybetweenlaserpulses,delaytimeoftheaccelerationpower,laserwavelength,energydensityofthelaserandtheimpactangleofthelaseronthetargetareamongothersthecriticalvaluesforthequalityandreproducibilityofthemethod.DetectorTheelectronmultiplierisahighlysensitivedevicetodetectindividualenergeticparticlessuchaselectron,photons,orionsMultipliersarebasedontwoprinciples:(1)theparticle(s)tobedetectedhavetobeconvertedtoelectronsbeforetheamplificationcantakeplace(usingasocalledconversiondynode)Detector(2)theamplificationiscausedbyacascadeofaccelerationelectrodes(calleddynodes