Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824, USA.
Anal Chem. 2010 Mar 15;82(6):2421-5. doi: 10.1021/ac902734w.
Procedures are detailed for a quantitative release of O-linked glycans from peptides that now provide a shorter reaction time, a possible identification of O-linked sites, and a quantification of all reaction products. The release was initiated by a mild base, dimethylamine, and accelerated by microwave radiation. Differential analysis using standard glycoproteins has shown improved release efficiency concurrent with facile incorporation of dimethylamine into the former O-linked sites. In situ glycan reduction insures protection against peeling and is synchronous with subsequent studies by high performance MS(n) sequencing. The protocols were established with a synthetic O-GlcNAc peptide that would mimic the linkage chemistry and applied to a well characterized glycoprotein bovine fetuin with both N- and O-linked glycans and a highly glycosylated swine mucin.
详细介绍了一种从肽中定量释放 O-连接聚糖的方法，该方法反应时间更短，可能鉴定 O-连接位点，并定量所有反应产物。该释放由温和的碱二甲胺引发，并通过微波辐射加速。使用标准糖蛋白的差异分析表明，释放效率提高，同时二甲胺很容易掺入到以前的 O-连接位点中。原位糖还原可防止剥落，并与随后的高性能 MS(n)测序研究同步进行。该方案是在模拟连接化学的合成 O-GlcNAc 肽的基础上建立的，并应用于具有 N-和 O-连接聚糖的高度糖基化的牛胎球蛋白和高度糖基化的猪粘蛋白等特征明确的糖蛋白。
