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Sequencing the oligosaccharide pool in the low molecular weight heparin dalteparin with offline HPLC and ESI-MS/MS
Carbohydrate Polymers ( IF 11.2 ) Pub Date : 2017-11-13 , DOI: 10.1016/j.carbpol.2017.11.039
Zhangjie Wang , Tianji Zhang , Shaoshuai Xie , Xinyue Liu , Hongmei Li , Robert J. Linhardt , Lianli Chi

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI-MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI-MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC-MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.



中文翻译:

通过离线HPLC和ESI-MS / MS对低分子量肝素达肝素中的寡糖池进行测序

低分子量肝素(LMWHs)是广泛使用的抗凝药物。LMWH寡糖的组成和序列决定了它们的安全性和功效。与长链相比,LMWHs中的短寡糖池发生的解聚反应更多,并且是制造过程中最敏感的指标。电喷雾电离串联质谱(ESI-MS / MS)已被证明是对合成肝素寡糖进行测序的强大工具,但从未用于分析复杂混合物,如LMWHs。我们建立了离线强阴离子交换(SAX)-高效液相色谱(HPLC)和ESI-MS / MS方法来对达肝素钠的短寡糖进行测序。借助内部开发的MS / MS解释软件,获得了从四糖到八糖的18种代表性物种的序列。有趣的是,我们发现了一种新颖的2,3-二硫酸化的六糖醛酸结构,并通过互补肝素酶消化和LC-MS / MS分析对其进行了确认。这种方法为LMWHs的结构和肝素解聚反应机理提供了直接而深入的见解。

更新日期:2017-11-14
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