Heparin　(Hep)　and　heparan　sulfate　(HS)　are　complex,　sulfated　GAGs　consist　of　repeating　disaccharide　units　each　composed　of　a　uronic　acid,　either　glucuronate　or　iduronate　[the　latter　sometimes　sulfated　at　C-2,　i.e.　IdoA2S],　and　a　derivative　of　glucosamine　[either　N-acetylglucosamine,　N-sulfated　glucosamine,　or　unsubstituted　glucosamine]　that　can　be　variably　O-sulfated.　Hep/HS　has　important　biological　functions　in　developmental　processes,　angiogenesis,　blood　coagulation,　cell　adhesion,　and　tumour　metastasis.　These　involve　interactions　with　a　wide　variety　of　proteins,　i.e.　enzymes,　cytokines,　growth　factors　and　extracellular　matrix　proteins　through　their　specific　sequences,　patterns　or　densities　of　sulfation.　Mass　spectroscopy　is　now　becoming　a　much　more　useful　tool　for　the　structural　analysis　of　Hep/HS　oligosaccharides　because　of　its　high　detection　sensitivity　and　molecular　specificity.　MS/MS　of　heparin/HS　oligosaccharides　can　produce　information-rich　glycosidic　and　cross-ring　product　ions　which　can　be　used　to　determine　the　sites　of　acetylation　or　sulfation.　In　particular,　LC-MS　clearly　has　the　potential　now　to　sequence　most　oligosaccharides,　especially　with　the　recent　development　of　a　computational　framework　for　dealing　with　the　complex　data　generated.　In　this　study,　porcine　intestines　heparin　was　digested　by　heparinase　I.　The　obtained　crude　tetrasaccharides　(dp4s)　were　separated　and　further　purified　by　strong　anion-exchange　HPLC　and　size-exclusion　HPLC.　The　structures　of　dp4s　were　then　investigated　by　the　combination　of　disaccharide　analysis,　reversed-phase　liquid　chromatography/electrospray　ionization　ion　trap/time-of-flight　mass　spectrometry　(RP-LC/ESI-IT/TOF　MS)　and　a　computational　simulation　method.　The　results　showed　that　nine　tetrasaccharides　(dp4s)　were　obtained,　in　which　two　series　of　dp4s　isomer　were　existed.　The　dp4　isomers　were　successfully　separated　using　IPRP-LC/MS　system　with　hexylamine.　One　series　of　dp4s　contains　HexA(2S)-GlcNS　and　HexA(2S)-GlcNS(6S)　disaccharides,　and　other　ones　possess　HexA-GlcNS(6S)　and　HexA(2S)-GlcNS(6S).　Because　MSn　analysis　could　produce　abundant　structurally　useful　fragments,　the　former　dp4　isomers　were　selected　to　investigate　fragmentation　patterns　for　the　further　potential　structure　analysis　by　MS.　MS2　analysis　indicated　that　one　dp4　was　HexA(2S)-GlcNS-　HexA(2S)-GlcNS(6S)　which　had　one　glycosidic　cleavage　Y1,　two　cross-ring　cleavages　0,2A2　and　0,3A2　in　the　first　glucosamine　residues　(from　the　non-reducing　end)　of　oligosaccharide.　In　contrast,　the　other　dp4　was　HexA(2S)-GlcNS(6S)-HexA(2S)-GlcNS　which　contained　two　glycosidic　cleavages　B2　and　Y1,　two　cross-ring　cleavages　1,4A2　and　0,3A2　in　the　first　glucosamine　residues,　and　a　cross-ring　cleavages　1,5X4　in　the　first　glucuronic　acid　residues　of　the　oligosaccharide.　This　study　provides　a　simple　and　efficient　method　for　identifying　the　sequences　of　the　Hep/HS　oligosaccharide　isomers,　which　might　lead　to　a　greater　understanding　of　the　biological　roles　of　Hep/HS　oligosaccharides　in　organisms.　©　2020,　Editorial　Board　of　Journal　of　Chinese　Mass　Spectrometry　Society.　All　right　reserved.