Abstract Bisecting N-glycan structures, which are commonly observed in glycoproteins, regulate many important functions in organisms. Tandem mass spectrometry is the most frequently utilized approach for the identification of bisecting N-glycan structures. However, it can be difficult to obtain fragments to recognize bisecting N-glycans based on the analysis performed, i.e., by using hydrophilic interaction liquid chromatography equipped with a fluorescence detector and a quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). The misinterpretation of bisecting N-glycans for other types of N-glycans is possible. Therefore, a method is needed to specifically recognize bisecting N-glycan structures. This report introduces a facile strategy based on tandem mass spectrometry to identify bisecting N-glycans by using a procainamide labeling approach that increases both the mass spectrometric and the fluorescence detection sensitivity of the N-glycans. In this strategy, the precursor ions belonging to bisecting N-glycans were used by extracting the detected diagnostic fragment ions, including proc-H1N3 (m/z 1009.481+) and proc-H1N3F1 (m/z 1155.539+), in the corresponding tandem mass spectra. Subsequently, the structures of the bisecting N-glycans were confirmed. The presented strategy was applied to human IgG glycoprotein and human plasma glycoproteome. Finally, stepping energy-collision induced dissociation (SE-CID) was applied to validate the diagnostic fragments. This approach enables bisecting N-glycans to be verified and can be used for further mass spectrometry-based glycan analysis of biological samples.

中文翻译：

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使用普鲁卡因酰胺标记方法对串联质谱中的二等分 N-聚糖进行鉴定，以对生物样品进行深入的 N-聚糖分析

摘要 在糖蛋白中常见的二等分 N-聚糖结构调节生物体中的许多重要功能。串联质谱法是鉴定二等分 N-聚糖结构最常用的方法。然而，基于所进行的分析，即通过使用配备有荧光检测器和四极杆飞行时间串联质谱 (HILIC-FLD- QTOF-MS/MS)。将 N-聚糖对分为其他类型的 N-聚糖的误解是可能的。因此，需要一种方法来特异性识别二等分 N-聚糖结构。本报告介绍了一种基于串联质谱的简便策略，通过使用普鲁卡因酰胺标记方法来识别二等分的 N-聚糖，该方法增加了 N-聚糖的质谱和荧光检测灵敏度。在该策略中，通过提取检测到的诊断碎片离子，包括 proc-H1N3 (m/z 1009.481+) 和 proc-H1N3F1 (m/z 1155.539+)，在相应的串联中使用属于二等分 N-聚糖的母离子质谱。随后，证实了二等分N-聚糖的结构。所提出的策略应用于人 IgG 糖蛋白和人血浆糖蛋白组。最后，应用步进能量碰撞诱导解离 (SE-CID) 来验证诊断片段。