Anal Chem. 2019 Dec 17;91(24):15993-16001. doi: 10.1021/acs.analchem.9b04727. Epub 2019 Dec 2.
Sialylated N-glycans play pivotal role in several important biological and pathological processes. Their sialyl-linkage isomers, mostly α-2,3- and α-2,6-linked, act differently during the cellular events and several diseases. While mass spectrometry (MS) technology is a powerful tool in N-glycome analysis, it still suffers from an inability to distinguish linkage isomers of native N-glycans. Herein, we described a sequential selective derivatization method, by which α-2,6- and α-2,3-linked sialic acids are sequentially labeled with methylamide incorporated with a different stable isotope. Isobaric labeling avoids inducing bias in ionization efficiency and chromatographic behavior. In optimized reaction conditions, high derivatization selectivity (∼99%) was achieved for both α-2,3- and α-2,6-linked sialic acid. High accuracy of quantitation within a dynamic range of 2 orders of magnitude and high reproducibility (CV < 20%, = 3) were demonstrated using standard glycans and multisialylated N-glycans. Finally, this method was applied in profiling the N-glycome of serum from CRC patients, where a level of six sialyl-linkage isomers were found to be altered significantly compared with that from healthy individuals.
唾液酸化 N-聚糖在许多重要的生物学和病理学过程中发挥着关键作用。它们的唾液酸连接异构体，主要是α-2,3-和α-2,6-连接的异构体，在细胞事件和几种疾病中表现出不同的作用。尽管质谱 (MS) 技术是 N-糖组分析的有力工具，但它仍然无法区分天然 N-聚糖的连接异构体。在此，我们描述了一种顺序选择性衍生化方法，通过该方法，α-2,6-和α-2,3-连接的唾液酸分别用带有不同稳定同位素的甲酰胺甲基酰胺标记。等重标记可避免诱导电离效率和色谱行为的偏差。在优化的反应条件下，α-2,3-和α-2,6-连接的唾液酸都实现了高的衍生化选择性（∼99%）。使用标准糖和多唾液酸化 N-聚糖，在 2 个数量级的动态范围内证明了定量的高精度（CV < 20%，n = 3）和高重现性。最后，该方法应用于结直肠癌患者血清 N-糖组的分析，与健康个体相比，发现六种唾液酸连接异构体的水平发生了显著改变。
