Through direct coupling of capillary electrophoresis (CE) to mass spectrometry (MS) with a sheathless interface, we have identified 77 potential N-glycan structures derived from human serum. We confirmed the presence of N-glycans previously identified by indirect methods, e.g., electrophoretic mobility standards, obtained 31 new N-glycan structures not identified in our prior work, differentiated co-migrating structures, and determined specific linkages on isomers featuring sialic acids. Serum N-glycans were cleaved from proteins, neutralized via methylamidation, and labeled with the fluorescent tag 8-aminopyrene-1,3,6-trisulfonic acid, which renders the glycan fluorescent and provides a −3 charge for electrophoresis and negative-mode MS detection. The neutralization reaction also stabilizes the labile sialic acids. In addition to methylamidation, native charges from sialic acids were neutralized through reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium to amidate α2,6-linked sialic acids in the presence of ammonium chloride and form lactones with α2,3-linked sialic acids. This neutralization effectively labels each type of sialic acid with a unique mass to determine specific linkages on sialylated N-glycans. For both neutralization schemes, we compared the results from microchip electrophoresis and CE.

通过直接将毛细管电泳（CE）耦合到具有无鞘接口的质谱（MS），我们已经鉴定出77种潜在的源自人血清的N-聚糖结构。我们确认了以前通过间接方法（例如电泳迁移率标准）鉴定的N-聚糖的存在，获得了我们先前工作中未鉴定的31个新的N-聚糖结构，不同的共迁移结构，并确定了具有唾液酸特征的异构体的特定键。从蛋白质上切下血清N-聚糖，通过甲基酰胺化进行中和，并用荧光标记8-aminopyrene-1,3,6-trisulfonic acid进行标记，该标记使聚糖具有荧光，并为电泳和负模式MS提供-3电荷检测。中和反应还稳定了不稳定的唾液酸。除了甲基酰胺化 在氯化铵存在下，通过与4-（4,6-二甲氧基-1,3,5-三嗪-2-基）-4-甲基吗啉反应中和唾液酸的天然电荷，以酰胺化α2,6-连接的唾液酸并与α2,3-连接的唾液酸形成内酯。这种中和作用以独特的质量有效地标记每种类型的唾液酸，以确定唾液酸化的N-聚糖上的特定键。对于这两种中和方案，我们比较了微芯片电泳和CE的结果。这种中和作用以独特的质量有效地标记每种类型的唾液酸，以确定唾液酸化的N-聚糖上的特定键。对于这两种中和方案，我们比较了微芯片电泳和CE的结果。这种中和作用以独特的质量有效地标记每种类型的唾液酸，以确定唾液酸化的N-聚糖上的特定键。对于这两种中和方案，我们比较了微芯片电泳和CE的结果。