Department of Chemistry, Indiana University, Bloomington, Indiana, USA.
J Sep Sci. 2022 Sep;45(17):3348-3361. doi: 10.1002/jssc.202200223.
Structural isomers of sialylated N-glycans contribute to the diversity of the N-glycome and to a range of biological functions. Sialyl linkage isomers can be readily distinguished by mass spectrometry with mass differences between α2,3- and α2,6-linkages generated by a two-step sialic acid linkage-specific alkylamidation. To improve the identification of N-glycans from complex mixtures, we added a delactonization step after the first alkylamidation step, which regenerates negatively charged carboxylic acids on α2,3-sialic acids. N-glycan isomers with α2,3-sialic acids are then fractionated by ion-exchange chromatography prior to the second alkylamidation step. With this modified alkylamidation method, sialylated N-glycans were enriched and stabilized for structural characterization by capillary electrophoresis-mass spectrometry and tandem mass spectrometry. We identified 52 sialylated N-glycan structures, including 107 linkage isomers, in human serum and confirmed the presence of positional isomers of specific sialyl linkage isomers. Due to the reduced sample complexity after ion-exchange fractionation and CE separation, substructural features of N-glycans were rapidly evaluated and included core- and antenna-fucosylation and poly-lactosamine.
糖基化 N-聚糖的结构异构体有助于 N-聚糖组的多样性和一系列生物学功能。通过两步唾液酸连接特异性烷基酰胺化反应，可以在质谱中轻松区分α2,3-和α2,6-连接的唾液酸连接异构体，其质量差异产生。为了提高从复杂混合物中鉴定 N-聚糖的能力，我们在第一步烷基酰胺化反应后添加了去内酯化步骤，该步骤在α2,3-唾液酸上重新生成带负电荷的羧酸。具有α2,3-唾液酸的 N-聚糖异构体然后通过离子交换色谱在第二步烷基酰胺化之前进行分离。使用这种改良的烷基酰胺化方法，通过毛细管电泳-质谱和串联质谱对唾液酸化 N-聚糖进行了富集和稳定，以进行结构表征。我们在人血清中鉴定了 52 种唾液酸化 N-聚糖结构，包括 107 个连接异构体，并证实了特定唾液酸连接异构体的位置异构体的存在。由于离子交换分级和 CE 分离后样品复杂性降低，因此可以快速评估 N-聚糖的亚结构特征，包括核心和天线岩藻糖基化以及多乳糖胺。
