肝素/硫酸乙酰肝素四糖同分异构体的制备与表征

肝素/硫酸乙酰肝素（Hep/HS）寡糖结构多样，且与生物学、病理学功能息息相关，表征这些结构对解析它们的功能具有重要意义。本实验以市售猪肠肝素钠为原料，制备了9种肝素四糖，并用二糖组分分析方法结合反相离子对试剂色谱-串联离子阱飞行时间质谱(IPRP-LC/IT-TOF MS)进行结构解析。结果表明：9种肝素四糖中有两组同分异构体，且含己胺离子对试剂的RP-LC/MS能够成功分离肝素寡糖及其同分异构体；采用LC-MS/MS方法可以鉴定其中一组同分异构体的结构。该方法可为HS/Hep寡糖的研究提供标准品，并为解析HS/Hep同分异构体结构提供一种简单有效的方法，进而为更好地理解生物体内HS/Hep结构和生理生化功能的关系提供帮助。

Preparation and Characterization of Heparin/Heparan Sulfate Tetrasaccharide Isomers

Heparin(Hep)and heparan sulfate(HS)are complex,sulfated GAGs consist of repeating disaccharide units each composed of a uronic acid,either glucuronate or iduronatethe latter sometimes sulfated at C-2,i.e.IdoA2S],and a derivative of glucosamineeither 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 MS n 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.MS 2 analysis indicated that one dp4 was△HexA(2S)-GlcNS-HexA(2S)-GlcNS(6S)which had one glycosidic cleavage Y 1,two cross-ring cleavages 0,2 A 2 and 0,3 A 2 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 B 2 and Y 1,two cross-ring cleavages 1,4 A 2 and 0,3 A 2 in the first glucosamine residues,and a cross-ring cleavages 1,5 X 4 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.