膜技术8.docx
- 文档编号:5639253
- 上传时间:2022-12-29
- 格式:DOCX
- 页数:28
- 大小:1.40MB
膜技术8.docx
《膜技术8.docx》由会员分享,可在线阅读,更多相关《膜技术8.docx(28页珍藏版)》请在冰豆网上搜索。
膜技术8
JournalofChromatographyA,1217(2010)5328–5336
ContentslistsavailableatScienceDirect
JournalofChromatographyA
journalhomepage:
ExtractionmethodsofredbloodcellmembraneproteinsforMultidimensional
ProteinIdentificationTechnology(MudPIT)analysis*
AntonellaDePalmaa,1,AntonellaRoverib,1,MattiaZaccarinc,LouiseBenazzia,SimoneDaminellia,GiorgiaPantanod,MauroButtarelloe,FulvioUrsinib,MassimoGionf,PierLuigiMauria,∗
aProteomicsandMetabolomicsUnit,InstituteforBiomedicalTechnologies(ITB)–CNR,ViaFratelliCervi93,I-20090Segrate,Milan,Italy
bDepartmentofBiologicalChemistry,UniversityofPadua,I-35131Padua,Italy
cIstitutoOncologicoVeneto(IOV),IRCCS,I-35131Padua,Italy
dDepartmentofLaboratoryMedicine,AziendaOspedaliera,UniversityofPadua,I-35131Padua,Italy
eDepartmentofLaboratoryMedicine,AziendaOspedaliera,ULSS16,UniversityofPadua,I-35131Padua,Italy
fCentrefortheStudyofBiologicalMarkersofMalignancy,ConsortiumIstitutoOncologicoVenetoIRCCS,RegionalHospital,AULSS12,Venice,Italy
articleinfo
abstract
Articlehistory:
Received12May2010
Accepted16June2010
Availableonline25June2010
Keywords:
RedbloodcellsMembraneproteinsMudPIT
Massspectrometry
Sinceredbloodcells(RBCs)lacknucleiandorganelles,cellmembraneistheirmainload-bearingcom-ponentand,accordingtoadynamicinteractionwiththecytoskeletoncompartment,playsapivotalroleintheirfunctioning.Eveniferythrocytemembranesareavailableinlargequantities,thelowabun-danceandthehydrophobicnatureofcellmembraneproteinscomplicatetheirpurificationanddetectionbyconventional2Dgel-basedproteomicapproaches.So,inordertoincreasetheefficiencyofRBCmembraneproteomeidentification,herewetookadvantageofasimpleandreproduciblemembranesub-fractionationmethodcoupledtoMultidimensionalProteinIdentificationTechnology(MudPIT).Inaddition,theadoptionofastringentRBCfiltrationstrategyfromthewholeblood,permittedtoremoveexhaustivelycontaminants,suchasplateletsandwhitebloodcells,andtoidentifyatotalof275pro-teinsinthethreeRBCmembranefractionscollectedandanalysed.Finally,bymeansofsoftwarefortheelaborationofthegreatquantityofdataobtainedandprogramsforstatisticalanalysisandproteinclassi-fication,itwaspossibletodeterminethevalidityoftheentiresystemworkflowandtoassignthepropersub-cellularlocalizationandfunctionforthegreatestnumberoftheidentifiedproteins.
©2010ElsevierB.V.Allrightsreserved.
1.Introduction
Redbloodcell(RBC)isaveryuniquecelllackingnucleusandotherorganelles.Biconcaveinshape,itcirculatesmilliontimesinthebody,squeezinginthesmallestcapillariestoaccomplishitsmainfunction,thetransportandexchangeofgases.
Cellmembraneisthemainload-bearingcomponentofRBC,ensurestheosmoticbalance,allowsinteractionswiththeexternalenvironmentthroughreceptorsonitsoutersurfaceand,inasso-ciationwithitscytoskeletonnetworkaccountsforitselasticandmechanicalproperties[1].TheplasmamembraneofRBCconsists
Abbreviations:
2DC-MS/MS,two-dimensionalchromatographycoupledtotandemmassspectrometry;MudPIT,MultidimensionalProteinIdentificationTechnology;RBC,redbloodcell;WBC,whitebloodcell;2DE,bidimensionalelec-trophoresis;ROD,RepairOrDestroy.
*Financialsupport:
ThisworkwassupportedandfinancedbytheABOAssociation
(PLM).
∗Correspondingauthor.Tel.:
+390226422728;fax:
+390226422770.
E-mailaddress:
pierluigi.mauri@r.it(P.L.Mauri).
1Theseauthorscontributedequallytothework.
ofacomplex,orderedarrayoflipidsandproteinsstretchedovertheoutersurfaceofthecellintheformofalipidbilayerpunctuatedbypenetratingorattachedproteins[2].ThoughmuchisknownaboutRBCmembraneproteome,allisnotclearonlocalization,functionandinteractionsofmanyproteinswhichmainlydetermineRBCcytoskeletonelasticproperties[3].
Althoughthestudyofcellmembraneproteinsisofhighinterestsincetheyplaypivotalrolesinmanycellularandphysiologicalpro-cesses[4],theyarenotoriouslyunderrepresentedinconventional
2Dgel-basedproteomicapproaches[5].
Moreover,thestudiesonpreparationmethodstoenrichhydrophobicproteinsin2DEanalyses,asalternativetotheconven-tionalapproachbasedonosmoticlysiscoupledtocentrifugationsteps,didnotleadtosignificantimprovements[6].ProgressesinproteomeanalysisofRBCmembraneproteinsby2DEwereachievedbyBruschietal.[7],settingupadifferentialextractionprocedurecoupledtotheuseofdiluteimmobilinegels.Ofnote,therecenttechnologicalinnovationsinthefieldofproteomicappli-cationsofmassspectrometryseemtohavecontributedtotheincreaseofmembraneRBCproteinsidentifiedmorethantheopti-mizationstrategiesofextractionemployed[8].
0021-9673/$–seefrontmatter©2010ElsevierB.V.Allrightsreserved.
doi:
10.1016/j.chroma.2010.06.045
A.DePalmaetal./J.Chromatogr.A1217(2010)5328–53365329
However,atproteinand/orpeptidelevelsthecouplingofadvancedseparationtechniquestohighlysensitivemassspectrom-etersreducessamplecomplexityandsimultaneouslyincreasesthepossibilityofdetectingproteinsinawiderdynamicrange[9].Infact,therelativeabundanceofproteinswithinacellcanvaryfrom
7to10ordersofmagnitude.Inlarge-scaleproteomicsstudies,inordertoachieveabetterunderstandingoftheinnerwork-ingofacellandaccordinglytoavoidthatlessabundantproteinsaremaskedbythemoreabundantones,preliminaryfractionationstrategiestogetherwithpowerfulseparationtechniquesweresuc-cessfullyemployedpriortoMSanalysis[10,11].
Nevertheless,theoptimizationstrategiesemployedforRBCextractionproceduresmakeanimportantcontribution.Theyshouldlimitthecontaminationfromotherbloodcells,amongwhichthemostfrequentareduetoleukocytesandplatelets,becauseevenlowlevelsofthesecontaminantscouldbedetectedthankstothesensitivityofthemodernmassspectrometersand,therefore,maskionsignalsofthelessabundantRBCproteins.So,attemptstolimitthiscontaminationshouldbedevelopedprimarilywithaviewtoaddressingtheproteomicstudyofhematologicdis-orders,becauseinthesecasestheWBClevelsoftenincreasegreatly[12].Moreover,moststudiesaboutRBCsalsoregardretyculocytesaspossiblecontaminantsofRBCpopulation,butthiskindofcon-taminationismucheasiertoberemovedeitherbydensitygradientseparation[13],orbyinvitromaturation[14].
Basedontheseconsiderations,weherepresentasimpleandreproduciblefractionationmethodwhichallowstheconcurrentidentificationofbothmembraneandsolubleproteinsoftheRBCmembraneproteomebythetwodimensionalchromatographycoupledtotandemmassspectrometry(2DC-MS/MS,alsoreferredtoasshotgunproteomicsorMultidimensionalProteinIdentifi-cationTechnology,MudPIT)[15],whichpermitstheanalysisofthecorrespondingtrypticdigestedfractions.Briefly,theprocedureconsistsintheseparationofRBCmembraneproteomeintothreefractions.Thefirstfractionwascollectedaftertryptic“shaving”[16]ofintactRBC.Afterlysisanddetergent/ultracentrifugationsteps,asupernatantenrichedincytoskeletonproteinsandapelletenrichedinintegralmembraneproteinswereobtained,correspondingtothesecond(detergentsupernatant)andthird(residualpellet)fractionsrespectively.
Finally,employinganin-housesoftware(MAProMa)[17]andmultivariateanalysis,itwasperformedacomparisonofthedif-ferentproteinlistsobtainedfromthethreemembranefractionsinordertoevaluatethevalidityoftheentiresystemworkflowandtoassignthepropersub-cellularlocalizationandfunctionforthegreatestnumberoftheidentifiedproteins.
2.Materialsandmethods
2.1.Materials
LeukocyteandplateletdepletionfilterswerefromFreseniusKabiItalia,S.r.l,Cavezzo(Modena),Italy;Dulbecco’sphosphatebufferedsalinewithoutcalciumandmagnesium(PBS),trypsin,trypsinogeninhibitor,proteaseinhibitorsformammaliancells,RPMI1640,foetalcalfserum(FCS),TritonX-100,trifluoroaceticacid(TFA),acetonitrile(ACN),ethylenediaminetetraceticacid(EDTA),andTris(hydroxymethyl)aminomethane(Trisbase)werefromSigma–Aldrich,Inc.,StLouis,MO,USA;AlbuminOUTTMkitwasfromGenoTechnology,Inc.,St.Louis,MO,USA;monoclonalantibodiesantiCD45ECD(cloneJ33),antiCD41FITC(clonePZ),antiglycophorinPE(clone11E4B-7-6/KC16)andantiCD36
FITC(cloneFA6-152)werefromBeckman-Coulter,Milan,Italy;monoclonalantibodiesantiCD61PE(cloneVI-PL2)werefromBecton-Dickinson-Pharmingen,SanDiego,CA,USA;RapiGestTM
SFwasfromWatersCo,Milford,MA,USA;massspectrometry(MS)gradetrypsinwasfromPromega,Inc.,Madison,WI,USA;PepCleanTMC-18SpinColumnswerefromPierce,Rockford,IL,USA;waterwasdeionizedbypassingthroughaDirectQ(Millipore,Bed-ford,MA,USA).
2.2.Methods
2.2.1.Redbloodcells(RBCs)preparation
Bloodwaswithdrawnbyvenipuncturefromtwentyfasting,apparentlyhealthyvolunteers,whoprovidedtheirinformedcon-sentinaccordancewiththedeclarationofHelsinki,atthelocalbloodbank(ServizioImmunoematologiaeTrasfusionale,AziendaULSS12-Veneziana,Venice,Italy)andcollectedinB
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- 膜技术