Highly Efficient Release of Glycopeptides from Hydrazide Beads by Hydroxylamine Assisted PNGase F Deglycosylation for N-Glycoproteome Analysis

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• 作者：Junfeng Huang ; Hao Wan ; Yating Yao ; Jinan Li ; Kai Cheng ; Jiawei Mao ; Jin Chen ; Yan Wang ; Hongqiang Qin ; Weibing Zhang ; Mingliang Ye ; Hanfa Zou
• 刊名：Analytical Chemistry
• 出版年：2015
• 出版时间：October 20, 2015
• 年：2015
• 卷：87
• 期：20
• 页码：10199-10204
• 全文大小：380K
• ISSN：1520-6882

文摘

Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.