In sharp contrast with analysis of -glycan that can be prepared by PNGase F, -glycan analysis remains challenging due to a lack of versatile and simple procedures, especially those mediating cleavage of -glycans from proteins. Most -glycans and -glycans are modified with sialic acids at the non-reducing end and their glycosidic linkages are labile, making it difficult to measure glycans by mass spectrometric analysis. In addition, sialic acid residues present on glycan chains via α2,3-, α2,6-, and α2,8-linkages as structural isomers. In this study, we firstly established a direct and linkage-specific derivatization method for sialylated -glycans on proteins via linkage-specific lactone-opening aminolysis. In this procedure, labile sialylated glycans were not only stabilized, but also allowed distinguishing between sialyl linkages. Furthermore, we revealed that general reductive β-elimination was not useful for -glycan cleavages with undesirable degradations of resulting methyl amides. Using β-elimination in the presence of pyrazolone (PMP), with low pH despite alkali base concentration, SALSA-derivatized -glycans could be cleaved with minimal degradations. Cleaved and PMP-labeled -glycans could be efficiently prepared in an open reaction system at high temperature (evaporative BEP reaction) and detected by simple liquid-phase extraction. Moreover, in the evaporative BEP reaction by changing the alkali solution with LiOH, the lithiated -glycans could be observed and provided a lot of fragment information reflecting the complex structure of the -glycans. Direct sialic acid linkage-specific derivatization of -glycans on glycoproteins is simple protocol containing in-solution aminolysis-SALSA and acetonitrile precipitation for removal of excess reagents. Evaporative β-elimination with pyrazolone makes possible intact -linked glycan analysis just by liquid-phase extraction. These analytical methods established by the appropriate combination of direct-SALSA and evaporative β-elimination will facilitate -glycomic studies in various biological samples.

中文翻译：

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唾液酸的直接衍生化和温和的 β-消除用于键合特异性唾液酸 O-聚糖分析


与可通过 PNGase F 制备的 β 聚糖分析形成鲜明对比的是，由于缺乏通用且简单的程序，尤其是那些介导从蛋白质中裂解 β 聚糖的程序，β 聚糖分析仍然具有挑战性。大多数α-聚糖和β-聚糖在非还原端被唾液酸修饰，并且它们的糖苷键不稳定，使得通过质谱分析测量聚糖变得困难。此外，唾液酸残基通过 α2,3-、α2,6- 和 α2,8- 连接作为结构异构体存在于聚糖链上。在本研究中，我们首先通过连接特异性内酯打开氨解建立了蛋白质上唾液酸化β聚糖的直接和连接特异性衍生化方法。在此过程中，不稳定的唾液酸化聚糖不仅得到稳定，而且还可以区分唾液酸键。此外，我们发现一般的还原性β-消除对于β-聚糖裂解没有用，并且会产生不希望的甲基酰胺降解。在吡唑啉酮 (PMP) 存在下使用 β-消除，尽管碱浓度较低，但 pH 值较低，SALSA 衍生的 β-聚糖可以以最小的降解被裂解。裂解的和 PMP 标记的 β-聚糖可以在高温开放反应系统（蒸发 BEP 反应）中有效制备，并通过简单的液相萃取进行检测。此外，在用LiOH改变碱溶液的蒸发BEP反应中，可以观察到锂化β-聚糖，并提供大量反映β-聚糖复杂结构的片段信息。糖蛋白上 β-聚糖的直接唾液酸键特异性衍生化是一种简单的方案，包含溶液内氨解-SALSA 和乙腈沉淀以去除多余的试剂。 使用吡唑啉酮进行蒸发 β-消除使得仅通过液相萃取即可进行完整的连接聚糖分析。这些通过直接 SALSA 和蒸发 β-消除的适当组合建立的分析方法将促进各种生物样品中的 β-糖组学研究。