A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase
A practical method of liberating O-linked glycans from glycoproteins using hydroxylamine and an organic superbase
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DOI：
10.1016/j.bbrc.2019.03.144
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发表时间：
2019-05-21
影响因子：
3.1
通讯作者：
Sakaguchi, Midori
中科院分区：
文献类型：
--
作者：
Kameyama, Akihiko;Tin, Wai Wai Thet;Sakaguchi, Midori
通过还原性β-消除和肼解从蛋白质中释放O-连接聚糖被广泛使用，但尚未满足最近在聚糖生物标志物研究和生物药物糖基化的微观异质性评价中对聚糖分析的需求。在此，我们介绍了一种替代方法，通过使用羟胺和有机超强碱，1，8-二氮杂双环[5.4.0]十一碳-7-烯（DBU），并优化了反应条件。所开发的方法提供了可比的肼解的结果，但较少的降解产物。此外，我们检查了优化的O-连接聚糖释放与变性剂和去污剂的相容性。优化的方法还释放了含有NeuGc的聚糖，而没有降解或脱酰作用。为了证明所开发的方法的可行性，我们分析了O-连接的聚糖分离的支持分子基质电泳（SMME），这是以前开发的粘蛋白。本文提出的O-聚糖释放和荧光标记方法简便、快速，对O-聚糖的分析具有实际意义。(C)2019作者爱思唯尔公司出版
O-Linked glycan liberation from proteins through reductive beta -elimination and hydrazinolysis is widely used, but have yet to satisfy the recent needs for glycan analysis in glycan biomarker research and microheterogeneity evaluation of biopharmaceutical glycosylation. Here, we introduce an alternative method by using hydroxylamine and an organic superbase, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and optimize the reaction conditions. The developed method afforded comparable results to those of hydrazinolysis, but with less degraded products. In addition, we examined the compatibility of the optimized O-Linked glycan liberation with denaturant and detergents. The optimized method also released glycans containing NeuGc without degradation or deacylation. To demonstrate the feasibility of the developed method, we analyzed O-Linked glycans of porcine submaxillary mucins separated by supported molecular matrix electrophoresis (SMME) which is previously developed to characterize mucins. The method for O-Linked glycan liberation and fluorescent labeling presented here was easy and rapid, and will be practically useful for O-Linked glycan analyses. (C) 2019 The Authors. Published by Elsevier Inc.