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.