W. M. Keck FTMS Laboratory from Human Health Research, Department of Chemistry, North Carolina State University, Raleigh, NC, 27695, USA.
Anal Bioanal Chem. 2018 Feb;410(5):1409-1415. doi: 10.1007/s00216-017-0828-2. Epub 2017 Dec 26.
The INLIGHT™ strategy for N-linked glycan derivatization has been shown to overcome many of the challenges associated with glycan analysis. The hydrazide tag reacts efficiently with the glycans, increasing their non-polar surface area, allowing for reversed-phase separations and increased ionization efficiency. We have taken the INLIGHT™ strategy and adopted it for use with O-linked glycans. A central composite design was utilized to find optimized tagging conditions (45% acetic acid, 0.1 μg/μL tag concentration, 37 C, 1.75 h). Derivatization at optimized conditions was much quicker than any hydrazide derivatization strategy used previously. Human immunoglobulin A (IgA) and bovine submaxillary mucin (BSM) were then deglycosylated through hydrazinolysis and the removed glycans were tagged under optimum conditions. XIC of tagged glycans and MS2 data show successful hydrazide tagging of O-linked glycans for the first time. Graphical abstract The INLIGHT™ hydrazide tag was optimized using a central composite design for derivatization of O-linked glycans. Two glycoprotein standards were deglycosylated through hydrazinolysis and tagged at the optimized conditions. MS/MS data shows INLIGHT™ derivatization of glycans demonstrating successful hydrazide tagging of O-glycans for the first time.
INLIGHT™ 策略用于 N-连接糖基化衍生化，已被证明克服了许多与糖基分析相关的挑战。酰肼标签与糖基反应效率高，增加了它们的非极性表面积，允许进行反相分离和提高离子化效率。我们采用了 INLIGHT™ 策略，并将其应用于 O-连接糖基。利用中心复合设计找到了优化的标记条件（45%乙酸、0.1μg/μL 标记浓度、37°C、1.75 小时）。在优化条件下的衍生化比以前使用的任何酰肼衍生化策略都快得多。然后，通过肼解使人类免疫球蛋白 A（IgA）和牛颌下腺粘蛋白（BSM）去糖基化，并在最佳条件下标记去除的糖基。标记糖基的 XIC 和 MS2 数据首次显示成功地对 O-连接糖基进行了酰肼标记。
