Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing
Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing
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DOI：
10.1038/nchem.1817
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发表时间：
2014-01-01
期刊：
影响因子：
21.8
通讯作者：
Eyers, C. E.
中科院分区：
文献类型：
--
作者：
Both, P.;Green, A. P.;Eyers, C. E.
质谱是用于表征装饰细胞表面的复杂寡糖的主要分析技术。单糖结构单元通常是简单的差向异构体，当其组合时产生质谱法无法区分的非对映体糖缀合物。结构解析经常依赖于生物合成途径高度保守的假设。在这里，我们表明，生物合成酶可以显示出意想不到的混杂性，与人类糖基转移酶pp-a-GanT 2能够利用尿苷二磷酸N-乙酰葡糖胺和尿苷二磷酸N-乙酰半乳糖胺，导致在体外合成差向异构糖肽。使用离子迁移率质谱法（IM-MS）分离这些结构，并根据碰撞诱导解离后产生的单糖产物离子的漂移时间对连接的聚糖进行表征。最后，离子迁移率质谱碎片后被用来确定一系列差向异构体二糖和支链五糖Man 3聚糖的还原和非还原聚糖的性质，证明这种技术可能被证明是有用的复杂寡糖的测序。
Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-a-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry ( IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.