Hanisch Franz-Georg
Institute of Biochemistry II, Medical Faulty, and Center for Molecular Medicine Cologne, University of Cologne, Köln, Germany.
Methods Mol Biol. 2012;842:179-89. doi: 10.1007/978-1-61779-513-8_10.
The sites of mucin-type O-glycosylation are difficult to predict, making structural analysis by mass spectrometry indispensible. This chapter refers to state-of-the-art techniques in the site localization of O-linked glycans and their structural characterization in situ using tandem ESI and MALDI mass spectrometry. Detailed protocols are provided that describe the application of nano-LC-ESI-MS/MS with alternative fragmentation modes (collision-induced dissociation vs. electron-transfer dissociation) for the analysis of O-glycopeptides. Moreover, a top-down sequencing approach by MALDI-MS is presented that is based on the in-source decay of intact glycoproteins or large glycopeptides and allows a ladder sequencing of up to 70 amino acid residues from both termini with unequivocal assignment of modified sites.
粘蛋白型O-糖基化位点难以预测，这使得质谱结构分析不可或缺。本章介绍了O-连接聚糖位点定位及其原位结构表征的最新技术，采用串联电喷雾电离（ESI）和基质辅助激光解吸电离（MALDI）质谱法。提供了详细的实验方案，描述了采用替代碎裂模式（碰撞诱导解离与电子转移解离）的纳升级液相色谱-电喷雾电离串联质谱（nano-LC-ESI-MS/MS）在O-糖肽分析中的应用。此外，还介绍了一种基于完整糖蛋白或大糖肽源内衰变的MALDI-MS自上而下测序方法，该方法可从两端对多达70个氨基酸残基进行阶梯式测序，并明确修饰位点。
