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简介：Insulinsecretorygranules(ISGs),agroupofdistinguishingorganellesinpancreaticβcells,areresponsibleforthestorageandsecretionofinsulintomaintainbloodglucosehomeostasis.ThemolecularmechanismsofISGbiogenesis,maturation,transportation,andexocytosisarestilllargelyunknownbecausetheproteinsinvolvedinthesedistinctstepshavenotbeenfullyidentified.Subcellularfractionationbydensitygradientcentrifugationhasbeensuccessfullyemployedtoanalyzetheproteomesofnumerousorganelles.However,useofthismethodtoelucidatetheISGproteomeislimitedbyco-fractionatedcontaminantsbecause1SGsareverydynamicandhaveabundantexchangesorcontactswithotherorganelles,suchastheGolgiapparatus,lysosomes,andendosomes.Inthisstudy,wedevelopedanewstrategyforidentifyingISGproteinsbyproteincorrelationprofiling(PCP)-basedproteomics,whichincludedISGpurificationbyOptiPrepdensitygradientcentrifugation,label-freequantitativeproteome,andidentificationofISGproteinsbycorrelatingfractionationprofilesbetweencandidatesandknownISGmarkers.Usingthisapproach,wewereabletoidentify81ISGproteins.Amongthem,TM9SF3,anine-transmembraneprotein,wasconsideredahighconfidenceISGcandidateproteinhighlightedinthePCPnetwork.FurtherbiochemicalandimmunofluorescenceassaysindicatedthatTMgSF3localizedinISGs,suggestingthatitisapotentialnewISGmarker.

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简介：Theplasmamembrane(PM)isacomplexenvironmentconsistingof>700speciesoflipidsandmanydifferenttypesofmembrane-associatingproteins.Theselipidsandmembraneproteinsaredistributedheterogeneouslyintonanometer-sizeddomains,callednanoclusters.ThelateralspatialsegregationinthePMgivesrisetodifferentcurvatureandlipidcomposition,whichdeterminestheefficiencyofeffectorbindingandsignaltransmission.Here,wedescribeanelectronmicroscopy(EM)-spatialmappingtechniquetoquantifytheextentofnanoclustersformationinthePM.Thenano-assembliesinthePMarequantifiedviaexpressingtheGFP-taggedproteinsorlipid-bindingdomainsinthecells,whicharethenimmunolabeledwiththegoldnanoparticlespre-coupledtotheanti-GFPantibody.ThegoldnanoparticlesarevisualizedviathetransmissionEMathighmagnification.ThestatisticalanalysisoftheRipley'sK-functioncalculatesthespatialdistributionofthegoldnanoparticles.Importantspatialparameters,suchastheextentofnanoclustering,theclusteredfraction,thenumberofproteinspercluster,theoptimalsizeofananocluster,andthenumberofproteinslocalizedtothePM,canbecalculated.Furtherdetailedaggregationpattern,suchasthepopulationsofmonomers,dimers,trimers,andhigherorderedoligomers,canalsobeextractedfromthespatialanalysis.TheEM-bivariateanalysisquantifiestheextentofco-localizationbetweentwodifferentcomponentsinthePMandprovideskeyinformationontheprotein-proteinandtheprotein-lipidinteractionsoveralong-distancescalefrom8to240nm.

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