Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy , University of Gothenburg , Gothenburg , Sweden.
Oxford Glycobiology Institute, Department of Biochemistry , University of Oxford , Oxford OX1 3QU , United Kingdom.
Anal Chem. 2019 Aug 20;91(16):10604-10613. doi: 10.1021/acs.analchem.9b01772. Epub 2019 Aug 1.
Glycosylation is one of the most important post-translational modifications essential for modulating biological functions on cellular surfaces and within cells. Glycan structures are not predictable from the genome since their biosynthesis is nontemplate driven and subject to multiple sequential and competitive glycosyltransferases/glycosidases. From a structural viewpoint, their analysis presents a particular challenge in terms of sensitivity and structural characterization. Porous graphitized carbon liquid chromatography coupled mass spectrometry (LC-MS) is arguably the gold-standard for the structural characterization of glycoconjugates, especially complex mixtures typical in biological samples. This high performance is due in large part to chromatographic separation of isomers and the information delivered by collision induced fragmentation of each glycan in the mass spectrometer. More recently, ion mobility mass spectrometry (IM-MS) has emerged as an effective tool for gas-phase separation of isomeric oligosaccharides that has been demonstrated with small oligosaccharides and -glycans. Here, we present a direct comparison of the IM- and LC-separation of -glycans from porcine gastric and human salivary mucins. Our results identify structures, which are resolved by LC and/or IM, validating the combination of the two methods. Taken together, the incorporation of both techniques into a single platform would be powerful and undoubtedly valuable for determining the full glycome of unknown samples.
糖基化是最重要的翻译后修饰之一，对于调节细胞表面和细胞内的生物学功能至关重要。糖链结构不可从基因组中预测，因为它们的生物合成不是模板驱动的，并且受到多种连续的和竞争的糖基转移酶/糖苷酶的影响。从结构的角度来看，它们的分析在灵敏度和结构表征方面提出了一个特殊的挑战。多孔石墨化碳液相色谱-质谱联用（LC-MS）可以说是糖缀合物结构表征的金标准，尤其是在生物样品中典型的复杂混合物。这种高性能在很大程度上归因于异构体的色谱分离以及质谱仪中每个糖链的碰撞诱导碎裂所提供的信息。最近，离子淌度质谱（IM-MS）已成为对异构寡糖进行气相分离的有效工具，已经在小寡糖和β-聚糖上得到了证明。在这里，我们对来自猪胃和人唾液粘蛋白的β-聚糖的 IM 和 LC 分离进行了直接比较。我们的结果确定了通过 LC 和/或 IM 解析的结构，验证了这两种方法的结合。总之，将这两种技术结合到单个平台中对于确定未知样品的完整聚糖组将是强大的，无疑是有价值的。
