含N-乙酰化肝素寡糖的制备及序列分析  

Preparation and Sequencing of N-acetylated Heparin Oligosaccharides

作　　者：林江慧[1] 张建伟[1] 肖晓毛 高建平[1] 张惠芳[1] 魏峥[1] 

机构地区：[1]福州大学糖生物化学研究所,福州350002

出　　处：《分析化学》2015年第5期689-696,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.21343013);国家基础科学人才培养基金项目(No.1103303)资助~~

摘　　要：建立了含N-乙酰化肝素寡糖的分离提纯及其序列结构分析方法。首先应用肝素酶Ⅰ深度酶解低分子量肝素来富集含N-乙酰化结构寡糖,通过Bio-Gel P10凝胶色谱法分离制备了包括二糖至十四糖的系列肝素寡糖粗样品,Pro Pac PA-1强阴离子高效液相色谱(SAX-HPLC)等方法对粗样品进一步分离,提纯得到4种六糖和3种八糖片段。其次应用肝素酶Ⅰ,Ⅱ和Ⅲ复合酶解与HPLC法分析各纯化寡糖的二糖组分,并结合肝素酶Ⅰ底物特异性,初步推断4种六糖和3种八糖的序列结构。在寡糖的糖链两端均含有N-硫酸化二糖,而N-乙酰化二糖分布在糖链当中。应用电喷雾离子阱-飞行时间质谱(ESI-IT-TOF-MS)在负离子模式下进一步表征寡糖并分析其裂解规律。结果表明,各寡糖中均出现大量因SO2!3丢失形成的碎片离子峰,六糖中主要有双电荷和三电荷碎片离子峰;在八糖中出现了一系列从双电荷至五电荷的离子峰。各寡糖的双电荷离子峰质荷比进一步确定了上述寡糖的序列结构。六糖的裂解规律表明,裂解主要存在于糖苷键,N-乙酰葡糖胺和糖醛酸上的裂解方式分别为0,2X和0,2Z。本研究提供了切实有效的分离、分析未知结构肝素寡糖序列的新方法。A method for the preparation, purification and sequence analysis of heparin oligosaccharides with internal N-acetylated glucosamine residues was established. Firstly, low molecular weight heparin was completely digested by heparinase I , and separated by Bio-Gel P-10 gel filtration chromatography. A range of oligosaccharides from dp2 up to dpl4 was obtained. Four of dp6 and three of dp8 oligosaccharides with N-acetylated residues were purified by strong anion exchange （SAX） high performance liquid chromatography （HPLC）. Secondly, the disaccharide compositions of the purified oligosaccharides were analyzed by SAX-HPLC after combined digestion using heparinase Ⅰ , Ⅱ and Ⅲ. The sequence of oligosaccharides was deduced according to its disaccharide composition and the substrate specificity of heparinase Ⅰ. The sequencing results showed that the N-sulfated disaccharides were internal, with the terminal disaccharides being predominantly highly sulfated, as expected from the substrate specificity of heparinase Ⅰ. Finally, the purified oligosaccharides were characterized by electrospray ion TOF-MS） system in the negative ion model. The results showed source. The major ion fragments of dp6 oligosaccharides oligosaccharides the 0.2X fragmen trap time-of-flight mass spectrometry （ESI-IT- that many of the -SO32- groups were lost in ion- had 2 and 3 negative charges, while dp8 had 2 to 5 negative charges. The main fragmentation of dp6 appeared in glycosidic tation existed in strategy offers possibilities for the linkage, N-acetylgalactosamine while the 0.2Z one appeared in glucuronic acid. This sequencing of heparin oligosaccharides with rare structure.

关 键 词：肝素寡糖 飞行时间质谱 肝素酶 序列测定 

分 类 号：O629.1[理学—有机化学] O657.63[理学—化学]