疾病蛋白质组学.ppt

第四讲疾病蛋白质组学（一）diseaseproteomics,一、基本概念和总体研究概况,疾病蛋白质组学diseaseproteomics,运用蛋白质组学研究手段，通过比较正常和病理情况下细胞、组织或体液中蛋白质在组成成分、表达水平、表达位置和修饰状态上的差异，寻找疾病诊断和预后的特异性蛋白质(群)，包括特异性抗原及相关抗原、受体、酶等，以及药物治疗的靶标等。通过深入了解这些疾病特异性蛋白质的结构和功能，揭示疾病过程中细胞内全部蛋白质的活动规律，为多种疾病发生、发展机制的阐明和早期诊断及治疗提供理论根据和解决途径。,研究进展,肿瘤蛋白质组：研究细胞的增殖、分化、异常转化、肿瘤形成白血病、乳腺癌、结肠癌、膀胱癌、前列腺癌、肺癌、肾癌、肝细胞癌和神经母细胞瘤等联合激光捕获微切割技术(Lasercapturemierodisseetion，LCM)，直接从肿瘤组织中提取纯肿瘤细胞，以克服组织内异质性的问题，为肿瘤蛋白质组研究提供了技术上的保障。鉴定了一批肿瘤相关蛋白，为肿瘤的早期诊断、药靶的发现、疗效和预后的判断提供了重要依据。在心脏、肺部、内分泌系统、神经系统疾病、药物成瘾性、环境毒理学、传染病、内耳相关疾病等方面，蛋白质组研究成果也为其提供了新的诊疗方向。国内：重点在肝病、恶性肿瘤、心血管、神经系统疾病和新发传染病等方面,存在问题和发展趋势,利用蛋白质组研究的人类疾病的范围虽然日趋扩大，但仍停留在初级比较阶段。进一步鉴定、验证，发展成应用于临床的生物标志物开展全方位的蛋白质组相互作用网络的分析进一步提高蛋白分离和鉴定的通量、灵敏度和规模；提高生物信息学应用范围与准确率，进行信息综合，准确地分析蛋白质的相互作用，界定相互作用连锁群；,二、心血管疾病蛋白质组学CardiovascularProteomics,thecardiovascular(CV)systemiscomposedofanumberofspecializedcelltypesincludingcardiacmyocytes,fibroblast,neurons,endothelialandsmoothmusclecellsandnewlydiscoveredstemandprogenitorcells.Todate,theproteomeofthesecellsarenotwellcharacterizednorhastheinterplaybetweenthecelltypesbeenestablishedinhealthordisease.ThisremainsasignificantchallengeasCVdiseaseisthenumberonekillerworldwide.,ResearchFocus,Themyofilamentproteome.Redoxmodificationsinthecardiacproteome.Cardiacbiomarkers.SecretorymicrovesiclesProteomicsofthesecretome,Themyofilamentproteome,Themyofilament（肌丝）proteinsareresponsibleforthecontractilenatureofthecardiacmyocytes.themyofilamentsubproteomeallowsthehearttoactasapump.Themyofilamentproteinsarehighlyregulatedbyanumberofspecificpost-translationalmodifications(PTMs)someofwhichhavebeendiscoveredthroughproteomicstudies.PTMsofmyofilamentproteinscandirectlyimpactonthecontractilityoftheheart.,Asimplifiedillustrationofthecardiacmyofilamentproteins.Thethickfilamentproteinsconsistofmyosinheavychain(MHC),myosin-bindingproteinC(MyBP-C),andtwomyosinlightchains(MLC1andMLC2).Thethinfilamentproteinsconsistofactin,tropomyosin(Tm),andthethreecomponentsoftroponin;troponinI(TnI),troponinC(TnC)andtroponinT(TnT).Phosphorylationsitesonthemyofilamentproteinsareindicatedwithasmalldiamond.Thelargescaffoldingprotein,titin,whichspansthesarcomere,isnotincludedinthisillustration.,肌球蛋白重链(MHC):myosinheavychain肌球蛋白轻链-1,2(MLC1,2):myosinlightchain-1,2肌动蛋白：Actin肌球蛋白结合蛋白C(MyBP-c):myosinbindingproteinC）肌钙蛋白(TnT,TnI,TnC):troponinT,I，C-原肌球蛋白(Tm)：-tropomyosin肌联蛋白:titin,Structureofaregionoftheoverlapregionofacardiacsarcomereindiastoleontheleftandduringsystoleontherightwithindicationsofmajorandfunctionallysignificantproteinphosphorylationsites.,Post-translationalmodificationsofmyofilamentproteins,Samplepreparation,Therearetwocommonlyusedmyofilamentprotein-enrichmentstrategies.Bothmethodsarecompatiblewith1-DEand2-DEanalysis：TFA(trifluoroaceticacid,三氟醋酸)extraction：cellsarelysedwithlowionicbuffer,andmyofilamentproteinsareextractedfromtheresultingpelletwith1%TFAv/v.appliedtoextractmyofilamentproteinsfromminuteamounts(20,50mg)ofbiopsysamples.（ref:Proteomics2002,2,978987.)Myofibrilisolation：intactmyofibrilscanbeisolatedformdetergent-skinned(detergentextraction)heartmuscleandstoredin50%glycerolat-20C.(ref:FASEBJ.2005,19,11371139.),DetectionMethodsforProteinmodification,phosphorylationchanges:1-D-IEF(phosphorylationsignificantlydecreasesproteinpIvalues)Westernblotswithphosphorylation-site-specificantibodiesMSanalysis:MALDI-TOFcoupledwithphosphatasetreatmentorPostsourcedecay(PSD)immobilizedmetalaffinitycolumn(IMAC)enrichmentandLCseparationfollowedbyMS/MSanalysis,Immobilizedmetalaffinitycolumn(IMAC),Schematicofaffinitybindingofphosphopeptidestoimmobilizedmetalionaffinitycolumns.,DetectionMethodsforProteinmodification,Proteindegradation:1-D-gelseparationfollowedbyWesternblot2-DE,2-DDIGEdirectsequencingfromtheNterminusorMS(exactsiteofdegradation)oxidationandnitrosylation:gelelectrophoresis(changeapparentMWandpIvalues)nano-ESILC/MS/MS(identifynitrotyrosineresidues)“top-down”MS(傅里叶转换离子回旋共振质谱）,文献阅读,ProteomicsClin.Appl.(2008)ChaoYuan,R.JohnSolaro.Myofilamentproteins:Fromcardiacdisorderstoproteomicchanges(p788-799)WenhaiJin,AnnaT.Brown,AnneM.Murphy.Cardiacmyofilaments:fromproteometopathophysiology(p800-810),2.Redoxmodificationsinthecardiacproteome,Myocardialischemiaresultsinoxidativestress,whichinvolvesthemitochondriaandmany/allaspectsofmyocytefunction.Duetothesusceptibilityofcardiacproteintooxidativedamage,proteomicscanhelptodiscover,quantify,andcharacterizetheredoxsignalingandoxidativePTMs.NitricoxideisakeymediatorofCVcellularresponseinacuteandchronicdiseasesettings.Newapproachesintheproteomicscanhelpidentifyanddefineimportantpathwayofnitricoxide-inducedPTMs.,Outlineofpotentialconsequencesofoxidativestressincellsystem,Oxidantscanreactwithproteinstocauseoneoftwobroadconsequences.Theycanoxidisecellularcomponentssuchasproteins,renderingthemdysfunctional,whichnegativelyaffectscellfunctionandpromotesdisease.Inthisscenario,antioxidantscanpreventthecellularproteinsfrombeingoxidisedandsoprovideprotection.Incontrast,oxidantscaninduceregulatorypost-translationaloxidativeproteinmodifications,whichareimportantforstressadaptation.Thus,antioxidantscaninterferewithhomeostaticcontrolandmightexplainwhyantioxidanttherapiescanbedetrimentalinsomecases.,MechanismsofROSgeneration.Sequentialreductionofmolecularoxygentogeneratesuperoxide,hydrogenperoxideandthenhydroxylradical.Listofaminoacidsparticularlysusceptibletomodification.,DiagramshowingtheproductionofNOandRNS,withtheireffectsonbiologicaltargets.Athighconcentrations,NOreactsmainlywithoxygensuperoxideformingperoxynitrite(ONOO)andperoxynitrousacid(ONOOH).Inthisway,NOisintimatelylinkedwithROS.Moreover,thereactionofNOwithO2leadstotheformationofthehighlypoisonousnitrogendioxide(NO2),dinitrogentetroxide(N2O4),orboth.Atlowconcentrations,thedirecteffectsofNOpredominate(dashedarrow)andhaemsandredoxmetalsatironsulphurcentresinproteinsarethemaintargets.Ni-NOR,nitrite:nitricoxidereductase;Ni,nitritereductase;NOS,nitricoxidesynthase;NR,nitratereductase;RSNOs,S-nitrosothiols.,Structureofcommonredoxmodificationsofaminoacidsidechains.ROSandRNScanchemicallymodifyaminoacids,particularlythesidechainsofthoseoutlinedhere.Clearly,cysteinethiolsaresubjecttoadiverserangeofalterations.,亚磺酸,磺酸,次磺酸,亚砜,亚硝基硫醇,羰基化,硝基化酪氨酸,Commonlyobservedoxidativemodificationsofproteinaminoacids(A)cysteine;(B)methionine;(C)tyrosine;(D)tryptophan.Alltheaminoacidsareschematicallyrepresentedaspartofapolypeptidechain.However,thenamesshownarethoseoffreeaminoacidsforconvenience.,Listofthemostutilizedmethodsinredoxproteomics,BiotinswitchmethodAhypotheticalproteinisindicatedwithcysteinesineitherthefreethiol,disulphide,ornitrosothiolconformations.Inthefirststep,freethiolsareblockedusingMMTS.Next,nitrosylatedcysteineresiduesareselectivelyreducedwithascorbateandthenewlygeneratedfreethiolsarefinallyS-biotinylatedwithbiotin-HPDP.,ThebiotinylatedproteinscanbedetecteddirectlybyWesternblottingwithantibodiesspecificforbiotinorusingavidinorstreptavidin.Antibodiescanberadiolabelled,fluorescentlyorenzymaticallylabelled,asisknownintheart.Additionally,taggedproteinscanalsobeisolatedfromaffinitycolumnsorbeads.PSH,proteinsulphhydrylgroups;PSNO,Snitrosatedproteins.,IsotopeCodedAffinityTagging(ICAT)(a).Thereagentconsistsofthreemoieties:anaffinitytagbiotin,alinkerthatcanincorporatesstableisotopes,andamaleimide(顺丁烯二酰亚胺)groupwhichreactsspecificallywiththethiolgroupofcysteine.Twolabelledformsofthereagentareused,theheavycontainingeightdeuteriums(氘)andthelightwithnone.,(b)ProteinsfromtwodifferentcellstatesarelabelledwiththelightorheavyICATreagents.Thesamplesarethencombinedanddigested.TheICAT-labelledpeptidesareisolatedbyaffinitychromatographyusinganavidincolumnandthenanalysedHPLC-MS(/MS)directlyorbyMALDIofthecollectedHPLCfractions.TheratioofthepeaksareasforspecificICAT-labelledpairsdefinestherelativeabundanceofitsparentproteinsbetweenthetwocellstates,quantificationofproteincysteineoxidation,Listofcardiacproteinsdemonstratedtoundergooxidativemodification,Ref:ProteomicsClin.Appl.2008,2,823836,3.Cardiacbiomarkers,DiagnosisofMIreliesonthedetectioninserumofacardiacspecificisoformofthemyofilamentprotein,troponinIwhichisreleasedintothebloodwhenthecardiacmyocytediesduesevereischemiaEarlierdetectionofMIordiagnosisofmyocardialischemiapriortocelldeathwillhelptoallowevenearlierinterventiontosave“potentiallyviable”teomicdiscoverypipelineforanalysisofhumanplasmasamplesforpatientswithinducedandcontrolMIhelpedtosetthestageforearlierdetectionofpatentsathighrisk.,CurrentgoldstandardmarkersofCVdistress,(i)electrophysiologicalandfunctionalchangesasmonitoredbyelectrocardiographyandechocardiographyrespectively(ii)elevatedserumlevelsofcardiacspecificproteins：myofilamentproteinsandcardiactroponin-Iand-T（myocardialinfarction）brainnatriureticpeptideandinflammation-relatedproteins,includingC-reactiveprotein(CRP),（heartfailure）.cardiacenzymeslactatedehydrogenaseandcreatinekinase(CK),Severalapproachescurrentlyusedtoquantitativelyproproteomicexpressionpatterns,fluorescence2-DDIGEcoupledtoMSanalysisProteinarraysinvitroandinvivostableisotopelabelLC-MStechniquesSignificantcostofusinglabeledreagentsinlarge-scalestudies.theapparentbiasofthesetechniquestowardslabelingtherelativelymostabundantspeciesinacomplexmixture,Morerecently,“label-free”differential(d)MS(无标记的质谱定量方法),Workflowforlabel-freedMSanalysisofplasmasamples.(A)Workflowchart.Thesixstagesoftheprocessarerepresentedwithinthisfigureincludingsamplepreparation,additionofinternalstandardsandMSanalysis.Eachstageplaysanimportantroleinleadingtoasuccessfulofdeterminationofmeaningfuldifferentials.,4.Secretorymicrovesicles,VascularsecretoryproteinandmembranevesiclescanaffecthomeostasisandcommunicationwithinentireCVsysteminresponsetoinjury.,Schematicfigureoftheuseofproteomicsforthecharacterizationofthenon-cellularproteinfractionsrelevantinatherosclerosis.Thefigurerepresentsanatheroscleroticplaqueanditscellularcomponents.Thecellsinvolvedinatheromaformationreleasesolubleproteinsandmembranebodiesthatmodifythevascularmicroenvironment.Proteomicscanbeappliedtothecharacterizationofthesenon-cellularcomponentsoftheatheroscleroticmicroenvironment.,Thelimitationsofplasmaproteomics,plasmaandserumareroutinelyusedforbiomarkerdiscoveryinproteomics.thehigh-abundanceproteins,notablyalbuminandimmunoglobulins,whichtogetherwithhaptoglobulin,antitrypsinandtransferrin,typicallyconstitutemorethan90%spectivebiomarkers:pgng/ml;albumin:3550mg/mlthelimitedabilityofproteomicstodetectlow-abundanceplasmaproteins,Proteomicsofextracellularsecretoryvesicles,(3)Matrixvesiclesareextracellularmembraneparticlesobservedintheinitialstagesofarterialcalcificationandcontainhighlevelsofcalcium-bindingacidicphospholipids.(4)Apoptoticbodiesarelargeparticlesreleasedfromcellsatthelaterstagesofprogrammedcelldeathandcharacterizedbylargediameter,nuclearcontent,andsurfaceligandsforphagocyticcellreceptors.(5)Heterogeneouspopulationorsecretorymicrovesicles.,(1)Microparticlesarereleasedfromtheplasmamembraneofstimulatedorapoptoticcells.Theirproteincompositionmayvaryinresponsetodifferentstimuli(highshearstress,apoptosis,etc.).(2)Exosomesarethesmallestofthesecretorymembraneparticlesandaresecretedasaconsequenceofthefusionoftheplasmamembranewiththemultivesicularbodies(MVB).MVBarelatecomponentsoftheendocyticpathway.,Thecriticalpatho-physiologicalroleofmicroparticles,Inthevascularcontext,microparticlesarereleasedbyendothelialcells,smoothmusclecells,lymphocytes,monocytes,erythrocytesandplatelets.Plasmalevelsofmicroparticlesaremarkedlyelevatedinpatientswithveinthrombosis,acutecoronarydisease,ischemicstroke,diabetes,myocardialinfarction,andhypertension.Microparticlesshowpro-coagulantactivity,pro-inflammatory,andpro-atheroscleroticactivities.modulatingtheendothelialsecretionofprostacyclinandnitricoxide;promotemonocyte-endotheliuminteractionbydirecttransferofarachidonicacidtotheplasmamembrane;physicallymediateleukocyte-leukocyteandleukocyte-endotheliuminteractionsviadirectbindingofcellsurfacereceptors,Proteomicsofmicroparticles,Proteomicanalysisofproteinexpressioninhumanplasmamicroparticles.MicroparticlesderivedfromtheperipheralbloodbycentrifugationwerelysedandlabelledwithCydyes(greenandredcolourinAandB,respectively).UsingDIGE,microparticleandmicroparticle-depletedplasmaproteinswereco-separatedinlargeformat2-Dgels.ImageswereacquiredonafluorescencescannerandproteinsidentifiedbyLC-MS/MS.Actinandhaemoglobinareenrichedinmicroparticles,comparedtomicroparticle-depletedplasma.,characterisationofmicroparticlesreleasedbyaparticularcelltypeinvitrobyproteomics,Besidestheinvestigationofthemixtureofmicroparticlescontainedinhumanplasma,proteomicscanbeappliedtothecharacterisationofmicroparticlesreleasedbyaparticularcelltypeinvitro.plateletmicroparticles(J.ProteomeRes.2005;4:15161521)surfaceproteinstypicalofplatelets,suchasintegrinaIIb,integrinb3andP-selectin,andchemokines,suchasCXCL4,CXCL7andCCL5,380proteinsnotpreviouslyidentifiedinplateletsEndothelialcellsinresponsetostimulationwith(TNFa).(Proteomics2005;5:44434455)cytoskeletonandcytoskeleton-bindingproteins(tubulin,actin,cofilin,vimentin,etc.)membrane-associatedproteinsthatcontroltransportandsignalling(caveolin,annexins,dynein,etc.)foldingchaperones(calnexin,calreticulin,etc.)Adhesionmolecules,suchasICAM-1andintegrinsb1,a5anda2,TheroleofExosomes,modulateimmuneresponseregulatehaemostaticbalancesupportthrombingenerationandinduceexpressionandsecretionofplasminogenactivatorinhibitor-1byendothelialcellsattenuatingfibrinolysisandpromotingpro-thromboticconditionsabilitytobeabsorbedtothecellsurfaceandmediatecell-cellinteractionsinthecardiovascularsystem,Proteomicsofexosomes,dendriticcell-derivedexosomes(J.Immunol.2001,166,73097318.)endocyticproteinswereabundantcomponentsoftheproteomeofexosomes.21newexosomalproteinswereidentified,includingcytoskeleton-relatedproteins,suchascofilin,profilinIorelongationfactor1a,andintracellularmembranetransportproteins,suchasannexins,rab7,11,rap1B,andsyntenin.aseriesofapoptosis-relatedproteins,includingthioredoxinperoxidaseII,Alix,14-3-3,andgalectin-3.mast-cellderivedexosomes(Arterioscler.Thromb.Vasc.Biol.2005,25,17441749)regulatethesecretionofplasminogena