文献类型：期刊文献

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

作者：Huang, Junfeng[1,2];Wan, Hao[1,3];Yao, Yating[1,2];Li, Jinan[1,2];Cheng, Kai[1];Mao, Jiawei[1,2];Chen, Jin[1,2];Wang, Yan[1,2];Qin, Hongqiang[1];Zhang, Weibing[3];Ye, Mingliang[1];Zou, Hanfa[1]

机构：[1]Chinese Acad Sci, Dalian Inst Chem Phys, CAS Key Lab Separat Sci Analyt Chem, Natl Chromatog R&A Ctr, Dalian, Peoples R China;[2]Univ Chinese Acad Sci, Beijing 100049, Peoples R China;[3]E China Univ Sci & Technol, Shanghai Key Lab Funct Mat Chem, Shanghai 200237, Peoples R China

年份：2015

卷号：87

期号：20

起止页码：10199

外文期刊名：ANALYTICAL CHEMISTRY

收录：;EI(收录号：20154401469014);WOS:【SCI-EXPANDED(收录号:WOS:000363348300008)】；

基金：This work was supported by the National Key Basic Research Program of China (Grants 2013CB911202, 2012CB910101, and 2012CB910604), the Creative Research Group Project of NSFC (Grant 21321064), the National Natural Science Foundation of China (Grants 21235006, 81161120540, and 81361128015), and the Chinese National Key Project (Grant 2012ZX10002009-011).

语种：英文

外文关键词：Amines - Phase separation - Glycosylation

摘要：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 S-fold.