简介：Venomousanimalsontheearthhavebeenfoundtobevaluableresourcesforthedevelopmentoftherapeutics.Enzymaticandnon-enzymaticproteinsandpeptidesarethemajorcomponentsofanimalvenoms,manyofwhichcantargetvariousionchannels,receptors,andmembranetransporters.Comparedtotraditionalsmallmoleculedrugs,naturalproteinsandpeptidesexhibithigherspecificityandpotencytotheirtargets.Inthisreview,wesummarizethevarietiesandcharacteristicsoftoxinsfromafewrepresentativevenomousanimals,anddescribethecomponentsandapplicationsofanimaltoxinsaspotentialdrugcandidatesinthetreatmentofhumandiseases,includingcancer,neurodegenerativediseases,cardiovasculardiseases,neuropathicpain,aswellasautoimmunediseases.Inthemeantime,therearemanyobstaclestotranslatenewtoxindiscoverytotheirclinicalapplications.Thechallenges,strategies,andperspectivesinthedevelopmentoftheproteintoxin-baseddrugsarediscussedaswell.

简介：Thereisaccumulatingevidencethatcysteinesulfenation（cys-SOH）inproteinsplaysanimportantroleincellularresponsetooxidativestress.Thepurposeofthepresentstudywastoidentifymitochondrialproteinsthatundergochangesincys-SOHduringaging.Studieswereconductedinratswhentheywere5or30monthsofage.FollowingblockingoffreeproteinthiolswithN-ethylmaleimide,proteinsulfenicacidswerereducedbyarsenitetofreethiolgroupsthatweresubsequentlylabeledwithbiotin-maleimide.Sampleswerethencomparativelyanalyzedbytwo-dimensionalWesternblots,andproteinsshowingchangesinsulfenationwereselectivelyidentifiedbymassspectrometrypeptidesequencing.Asaresult,fiveproteinswereidentified.Proteinsshowinganage-relateddecreaseinsulfenationincludepyruvatecarboxylaseandpyruvatedehydrogenase;whilethoseshowinganage-relatedincreaseinsulfenationincludeaconitase,mitofilin,andtubulin（α-1）.Resultsofthepresentstudyprovideageneralpictureofmitochondrialproteinsulfenationinbrainoxidativestressandimplicatetheinvolvementofproteinsulfenationinoveralldeclineofmitochondrialfunctionduringbrainaging.

简介：Numerouscellularfunctionsoccurinspatiallyandtemporallyconfinedregions.Recentstudieshaveshownthatmembrane-lessorganellesandcompartmentsinthecellareassembledvialiquid-liquidphaseseparation(LLPS).InvitroLLPSassaysusingrecombinantexpressedandpurifiedproteinsarenecessaryforustofurtherunderstandhowtheassemblyofphase-separatedcompartmentsisregulatedincells.However;uniformstandardsandprotocolsarelackingfortheseinvitrostudies.Here,wedescribeastep-by-stepprotocolcommonlyusedtoinvestigateinvitroLLPSusingpurifiedproteins.Thisprotocolincludesexpressionandpurificationofthestudiedproteins,inductionofLLPSofthepurifiedproteins,andstudiesofthebiophysicalpropertiesoftheliquiddropletsformedbyLLPS.TheseprotocolscanbeeasilyfollowedbyresearcherstoinvestigatetheLLPSbehaviorsofproteinsofinterest.

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简介：Micro-electrondiffraction(MicroED)isanemergingtechniquetousecryo-electronmicroscopetostudythecrystalstructuresofmacromoleculefromitsmicro-/nano-crystals,whicharenotsuitableforconventionalX-raycrystallography.However,thistechniquehasbeenpreventedforitswideapplicationbythelimitedavailabilityofproducinggoodmicro-/nano-crystalsandtheinappropriatetransferofcrystals.Here,wedevelopedacompleteworkflowtopreparesuitablecrystalsefficientlyforMicroEDexperiment.Thisworkflowincludesinsituon-gridcrystallization,single-sideblotting,cryo-focusionbeam(cryo-FIB)fabrication,andcryo-electrondiffractionofcrystalcryo-lamella.ThisworkflowenablesustoapplyMicroEDtostudymanysmallmacromolecularcrystalswiththesizeof2-10μm,whichistoolargeforMicroEDbutquitesmallforconventionalX-raycrystallography.Wehaveappliedthismethodtosolve2.5Acrystalstructureoflysozymefromitsmicro-crystalwithinthesizeof10×10×10μm^3.OurworkwillgreatlyexpandtheavailabilityspaceofcrystalssuitableforMicroEDandfillupthegapbetweenMicroEDandX-raycrystallography.

简介：Amolecularmodelofpancreaticzymogengranule（ZG）iscriticalforunderstandingitsfunctions.WehaveextensivelycharacterizedthecompositionandmembranetopologyofratZGproteins.Inthisstudy,wereportthedevelopmentoftargetedproteomicsapproachestoquantifyrepresentativemouseandhumanZGproteinsusingLC-SRMandheavyisotope-labeledsyntheticpeptides.TheabsolutequantitiesofmouseRab3DandVAMP8weredeterminedas1242±218and2039±151（mean±SEM）copiesperZG.ThesizedistributionandtheaverageddiameterofZGs750±23nm（mean±SEM）weredeterminedbyatomicforcemicroscopy.TheabsolutequantificationofRab3Dwasthenvalidatedusingsemi-quantitativeWesternblottingwithpurifiedGST-Rab3Dproteinsasaninternalstandard.Toextendourproteomicsanalysistohumanpancreas,ZGswerepurifiedusinghumanaciniobtainedfrompancreaticislettrans-plantationcenter.OnehundredandeightyhumanZGproteinswereidentifiedforthefirsttimeincludingboththemembraneandthecontentproteins.Furthermore,thecopynumberperZGofhumanRab3DandVAMP8weredeterminedtobe1182±45and485±15（mean±SEM）.ThecomprehensiveproteomicanalysesofmouseandhumanpancreaticZGshavethepotentialtoidentifyspecies-specificZGproteins.ThedeterminationofproteincopynumbersonpancreaticZGsrepresentsasignificantadvancetowardsbuildingaquantitativemolecularmodelofaprototypicalsecretoryvesicleusingtargetedproteomicsapproaches.TheidentificationofhumanZGproteinslaysafoundationforsubsequentstudiesofalteredZGcompositionsandsecretioninpancreaticdiseases.

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：Disulfidebondsarevitalforproteinfunctions,butlocatingthelinkagesiteshasbeenachallengeinproteinchemistry,especiallywhenthequantityofasampleissmallorthecomplexityishigh.In2015,ourlaboratorydevelopedasensitiveandefficientmethodformappingproteindisulfidebondsfromsimpleorcomplexsamples（LuetaLinNatMethods12：329,2015）.Thismethodisbasedonliquidchromatography-massspectrometry（LC-MS）andapowerfuldataanalysissoftwaretoolnamedpLink.Tofacilitateapplicationofthismethod,wepresentstep-by-stepdisulfidemappingprotocolsforthreetypesofsamples--purifiedproteinsinsolution,proteinsinSDS-PAGEgels,andcomplexproteinmix-turesinsolution.Theminimumamountofproteinrequiredforthismethodcanbeaslowasseveralhundrednanogramsforpurifiedproteins,ortensofmicrogramsforamixtureofhundredsofproteins.Theentireworkflow--fromsamplepreparationtoLC-MSanddataanalysis--isdescribedingreatdetail.Webelievethatthisprotocolcanbeeasilyimplementedinanylaboratorywithaccesstoafast-scanning,high-resolution,andaccurate-massLC-MSsystem.

简介：Fluorescentproteins(FPs)withemissionwavelengthsinthefar-redandinfraredregionsofthespectrumprovidepowerfultoolsfordeep-tissueandsuper-resolutionimaging.Thedevelopmentofred-shiftedFPshasevokedwidespreadinterestandcontinuousengineeringefforts.Inthisarticle,basedonacomputationaldesignandgeneticcodeexpansion,wereportarationalapproachtosignificantlyexpandandred-shiftthechromophoreofgreenfluorescentprotein(GFP).WeappliedcomputationalcalculationstopredicttheexcitationandemissionwavelengthsofaFPchromophoreharboringunnaturalaminoacids(UAA)andidentifyinsilicoanappropriateUAA,2-amino-3-(6-hydroxynaphthalen-2-yl)propanoicacid(naphthol-Ala).OurmethodologyallowedustoformulateaGFPvariant(cpsfGFP-66-Naphthol-Ala)withred-shiftedabsorbanceandemissionspectralmaximaexceeding60and130nm,respectively,comparedtothoseofGFP.TheGFPchromophoreisformedthroughautocatalyticpost-translationalmodificationtogenerateaplanar4-(p-hydroxybenzylidene)-5-imidazolinonechromophore.WesolvedthecrystalstructureofcpsfGFP-66-naphthol-Alaat1.3■resolutionanddemonstratedtheformationofamuchlargerconjugatedn-systemwhenthephenolgroupisreplacedbynaphthol.Theseresultsexplainthesignificantred-shiftingoftheexcitationandemissionspectraofcpsfGFP-66-naphthol-Ala.

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