疾病蛋白質(zhì)組學(xué)

疾病蛋白質(zhì)組學(xué)第1頁/共44頁一、基本概念和總體研究概況第2頁/共44頁疾病蛋白質(zhì)組學(xué)

diseaseproteomics運(yùn)用蛋白質(zhì)組學(xué)研究手段，通過比較正常和病理情況下細(xì)胞、組織或體液中蛋白質(zhì)在組成成分、表達(dá)水平、表達(dá)位置和修飾狀態(tài)上的差異，尋找疾病診斷和預(yù)后的特異性蛋白質(zhì)(群)，包括特異性抗原及相關(guān)抗原、受體、酶等，以及藥物治療的靶標(biāo)等。通過深入了解這些疾病特異性蛋白質(zhì)的結(jié)構(gòu)和功能，揭示疾病過程中細(xì)胞內(nèi)全部蛋白質(zhì)的活動(dòng)規(guī)律，為多種疾病發(fā)生、發(fā)展機(jī)制的闡明和早期診斷及治療提供理論根據(jù)和解決途徑。第3頁/共44頁研究進(jìn)展腫瘤蛋白質(zhì)組：研究細(xì)胞的增殖、分化、異常轉(zhuǎn)化、腫瘤形成白血病、乳腺癌、結(jié)腸癌、膀胱癌、前列腺癌、肺癌、腎癌、肝細(xì)胞癌和神經(jīng)母細(xì)胞瘤等聯(lián)合激光捕獲微切割技術(shù)(Lasercapturemierodisseetion，LCM)，直接從腫瘤組織中提取純腫瘤細(xì)胞，以克服組織內(nèi)異質(zhì)性的問題，為腫瘤蛋白質(zhì)組研究提供了技術(shù)上的保障。鑒定了一批腫瘤相關(guān)蛋白，為腫瘤的早期診斷、藥靶的發(fā)現(xiàn)、療效和預(yù)后的判斷提供了重要依據(jù)。在心臟、肺部、內(nèi)分泌系統(tǒng)、神經(jīng)系統(tǒng)疾病、藥物成癮性、環(huán)境毒理學(xué)、傳染病、內(nèi)耳相關(guān)疾病等方面，蛋白質(zhì)組研究成果也為其提供了新的診療方向。國內(nèi)：重點(diǎn)在肝病、惡性腫瘤、心血管、神經(jīng)系統(tǒng)疾病和新發(fā)傳染病等方面第4頁/共44頁存在問題和發(fā)展趨勢(shì)利用蛋白質(zhì)組研究的人類疾病的范圍雖然日趨擴(kuò)大，但仍停留在初級(jí)比較階段。進(jìn)一步鑒定、驗(yàn)證，發(fā)展成應(yīng)用于臨床的生物標(biāo)志物開展全方位的蛋白質(zhì)組相互作用網(wǎng)絡(luò)的分析進(jìn)一步提高蛋白分離和鑒定的通量、靈敏度和規(guī)模；提高生物信息學(xué)應(yīng)用范圍與準(zhǔn)確率，進(jìn)行信息綜合，準(zhǔn)確地分析蛋白質(zhì)的相互作用，界定相互作用連鎖群；第5頁/共44頁二、心血管疾病蛋白質(zhì)組學(xué)

CardiovascularProteomicsthecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.第6頁/共44頁ResearchFocusThemyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretome第7頁/共44頁ThemyofilamentproteomeThemyofilament（肌絲）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehearttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.第8頁/共44頁Asimplifiedillustrationofthecardiacmyofilamentproteins.Thethickfilamentproteinsconsistofmyosinheavychain(MHC),myosin-bindingproteinC(MyBP-C),andtwomyosinlightchains(MLC1andMLC2).Thethinfilamentproteinsconsistofactin,tropomyosin(Tm),andthethreecomponentsoftroponin;troponinI(TnI),troponinC(TnC)andtroponinT(TnT).Phosphorylationsitesonthemyofilamentproteinsareindicatedwithasmalldiamond.Thelargescaffoldingprotein,titin,whichspansthesarcomere,isnotincludedinthisillustration.肌球蛋白重鏈(MHC):myosinheavychain肌球蛋白輕鏈-1,2(MLC1,2):myosinlightchain-1,2肌動(dòng)蛋白：Actin肌球蛋白結(jié)合蛋白C(MyBP-c):myosinbindingproteinC）肌鈣蛋白(TnT,TnI,TnC):troponinT,I，C-原肌球蛋白(Tm)：-tropomyosin肌聯(lián)蛋白:titin第9頁/共44頁Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.第10頁/共44頁P(yáng)ost-translationalmodificationsofmyofilamentproteins第11頁/共44頁第12頁/共44頁SamplepreparationTherearetwocommonlyusedmyofilamentprotein-enrichmentstrategies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：TFA(trifluoroaceticacid,三氟醋酸)extraction：cellsarelysedwithlowionicbuffer,andmyofilamentproteinsareextractedfromtheresultingpelletwith1%TFAv/v.appliedtoextractmyofilamentproteinsfromminuteamounts(20,50mg)ofbiopsysamples.（ref:Proteomics2002,2,978–987.)Myofibrilisolation：intactmyofibrilscanbeisolatedformdetergent-skinned(detergentextraction)heartmuscleandstoredin50%glycerolat-20C.(ref:FASEBJ.2005,19,1137–1139.)第13頁/共44頁DetectionMethodsforProteinmodificationphosphorylationchanges:1-D-IEF(phosphorylationsignificantlydecreasesproteinpIvalues)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysis第14頁/共44頁Immobilizedmetalaffinitycolumn(IMAC)Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.第15頁/共44頁DetectionMethodsforProteinmodificationProteindegradation:1-D-gelseparationfollowedbyWesternblot2-DE,2-DDIGEdirectsequencingfromtheNterminusorMS(exactsiteofdegradation)oxidationandnitrosylation:gelelectrophoresis(changeapparentMWandpIvalues)nano-ESILC/MS/MS(identifynitrotyrosineresidues)“top-down”MS(傅里葉轉(zhuǎn)換離子回旋共振質(zhì)譜）第16頁/共44頁文獻(xiàn)閱讀ProteomicsClin.Appl.(2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathophysiology

(p800-810)

第17頁/共44頁2.RedoxmodificationsinthecardiacproteomeMyocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidativePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.第18頁/共44頁OutlineofpotentialconsequencesofoxidativestressincellsystemOxidantscanreactwithproteinstocauseoneoftwobroadconsequences.Theycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifications,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.第19頁/共44頁MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.Listofaminoacidsparticularlysusceptibletomodification.第20頁/共44頁DiagramshowingtheproductionofNOandRNS,withtheireffectsonbiologicaltargets.Athighconcentrations,NOreactsmainlywithoxygensuperoxideformingperoxynitrite(ONOO)andperoxynitrousacid(ONOOH).Inthisway,NOisintimatelylinkedwithROS.Moreover,thereactionofNOwithO2leadstotheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Atlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatiron–sulphurcentresinproteinsarethemaintargets.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthase;NR,nitratereductase;RSNOs,S-nitrosothiols.第21頁/共44頁Structureofcommonredoxmodificationsofaminoacidsidechains.ROSandRNScanchemicallymodifyaminoacids,particularlythesidechainsofthoseoutlinedhere.Clearly,cysteinethiolsaresubjecttoadiverserangeofalterations.亞磺酸磺酸次磺酸亞砜

亞硝基硫醇

羰基化

硝基化酪氨酸第22頁/共44頁Commonlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.However,thenamesshownarethoseoffreeaminoacidsforconvenience.第23頁/共44頁ListofthemostutilizedmethodsinredoxproteomicsFrom:JournalofExperimentalBotany,Vol.59,No.14,pp.3781–3801,2008第24頁/共44頁Biotinswitchmethod

Ahypotheticalproteinisindicatedwithcysteinesineitherthefreethiol,disulphide,ornitrosothiolconformations.Inthefirststep,freethiolsareblockedusingMMTS.Next,nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfreethiolsarefinallyS-biotinylatedwithbiotin-HPDP.ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin.Antibodiescanberadiolabelled,fluorescentlyorenzymaticallylabelled,asisknownintheart.Additionally,taggedproteinscanalsobeisolatedfromaffinitycolumnsorbeads.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.第25頁/共44頁IsotopeCodedAffinityTagging(ICAT)

(a).Thereagentconsistsofthreemoieties:anaffinitytagbiotin,alinkerthatcanincorporatesstableisotopes,andamaleimide(順丁烯二酰亞胺)groupwhichreactsspecificallywiththethiolgroupofcysteine.

Twolabelledformsofthereagentareused,theheavycontainingeightdeuteriums(氘)andthelightwithnone.

(b)ProteinsfromtwodifferentcellstatesarelabelledwiththelightorheavyICATreagents.

Thesamplesarethencombinedanddigested.

TheICAT-labelledpeptidesareisolatedbyaffinitychromatographyusinganavidincolumnandthenanalysedHPLC-MS(/MS)directlyorbyMALDIofthecollectedHPLCfractions.

TheratioofthepeaksareasforspecificICAT-labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstatesquantificationofproteincysteineoxidation第26頁/共44頁ListofcardiacproteinsdemonstratedtoundergooxidativemodificationRef:ProteomicsClin.Appl.2008,2,823–836第27頁/共44頁3.CardiacbiomarkersDiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein,troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierinterventiontosave“potentiallyviable”teomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk.第28頁/共44頁CurrentgoldstandardmarkersofCVdistress(i)electrophysiologicalandfunctionalchangesasmonitoredbyelectrocardiographyandechocardiographyrespectively(ii)elevatedserumlevelsofcardiacspecificproteins：myofilamentproteinsandcardiactroponin-Iand-T

（myocardialinfarction）brainnatriureticpeptideandinflammation-relatedproteins,includingC-reactiveprotein(CRP),（heartfailure）.cardiacenzymeslactatedehydrogenaseandcreatinekinase(CK)第29頁/共44頁Severalapproachescurrentlyusedtoquantitatively

profileglobalproteomicexpressionpatternsfluorescence2-DDIGEcoupledtoMSanalysisProteinarraysinvitroandinvivostableisotopelabelLC-MStechniquesSignificantcostofusinglabeledreagentsinlarge-scalestudies.theapparentbiasofthesetechniquestowardslabelingtherelativelymostabundantspeciesinacomplexmixture,Morerecently,“l(fā)abel-free”differential(d)MS(無標(biāo)記的質(zhì)譜定量方法)第30頁/共44頁第31頁/共44頁Workflowforlabel-freedMSanalysisofplasmasamples.(A)Workflowchart.Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation,additionofinternalstandardsandMSanalysis.Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials.第32頁/共44頁4.SecretorymicrovesiclesVascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury.Schematicfigureoftheuseofproteomicsforthecharacterizationofthenon-cellularproteinfractionsrelevantinatherosclerosis.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.第33頁/共44頁Thelimitationsofplasmaproteomicsplasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics.thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%spectivebiomarkers:pg~ng/ml;albumin:35–50mg/mlthelimitedabilityofproteomicstodetectlow-abundanceplasmaproteins第34頁/共44頁P(yáng)roteomicsofextracellularsecretory

vesicles(3)Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsofcalcium-bindingacidicphospholipids.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.(5)Heterogeneouspopulationorsecretorymicrovesicles.(1)Microparticlesarereleasedfromtheplasmamembraneofstimulatedorapoptoticcells.Theirproteincompositionmayvaryinresponsetodifferentstimuli(highshearstress,apoptosis,etc.).(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).MVBarelatecomponentsoftheendocyticpathway.第35頁/共44頁Thecriticalpatho-physiologicalroleofmicroparticlesInthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis,acutecoronarydisease,ischemicstroke,diabetes,myocardialinfarction,andhypertension.Microparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.modulatingtheendothelialsecretionofprostacyclinandnitricoxide;promotemonocyte-endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamembrane;physicallymediateleukocyte-leukocyteandleukocyte-endotheliuminteractionsviadirectbindingofcellsurfacereceptors第36頁/共44頁P(yáng)roteomicsofmicroparticlesProteomicanalysisofproteinexpressioninhumanplasmamicroparticles.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(greenandredcolourinAandB,respectively).UsingDIGE,microparticleandmicroparticle-depletedplasmaproteinswereco-separatedinlargeformat2-Dgels.ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC-MS/MS.Actinandhaemoglobinareenrichedinmicroparticles,comparedtomicroparticle-depletedplasma.第37頁/共44頁characterisationofmicroparticlesreleasedbyaparticularcelltypeinvitrobyproteomicsBesidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma,proteomicscanbeappliedtothecharacterisationofmicroparticlesreleasedbyaparticularcelltypeinvitro.plateletmicroparticles(J.ProteomeRes.2005;4:1516–1521)surfaceproteinstypicalofplatelets,suchasintegrinaIIb,integrinb3andP-selectin,andchemokines,suchasCXCL4,CXCL7andCCL5,380proteinsnotpreviouslyidentifiedinplateletsEndothelialcellsinresponsetostimulationwith(TNFa).(Proteomics2005;5:4443–4455)cytoskeletonandcytoskeleton-bindingproteins(tubulin,actin,cofilin,vimentin,etc.)membrane-associatedproteinsthatcontroltransportandsignalling(caveolin,annexins,dynein,etc.)foldingchaperones(calnexin,calreticulin,etc.)Adhesionmolecules,suchasICAM-1andintegrinsb1,a5anda2第38頁/共44頁TheroleofExosomesmodulateimmuneresponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor-1byendothelialcellsattenuatingfibrinolysisandpromotingpro-thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell-cellinteractionsinthecardiovascularsystem第39頁/共44頁P(yáng)roteomicsofexosomesdendriticcell-derivedexosomes

(J.Immunol.2001,166,7309–7318.)endocyticproteinswereabundantcomponentsoftheproteomeofexosomes.21newexosomalproteinswereidentified,includingcytoskeleton-relatedproteins,suchascofilin,profilinIorelongationfactor1a,andintracellularmembranetransportproteins,suchasannexins,rab7,11,rap1B,andsyntenin.aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,