Scott Nichollas E, Cordwell Stuart J
School of Molecular Bioscience, The University of Sydney, Sydney, NSW, 2006, Australia.
Methods Mol Biol. 2015;1295:355-68. doi: 10.1007/978-1-4939-2550-6_25.
Large-scale analysis of protein N- and O-linked glycosylation by mass spectrometry has traditionally been performed in eukaryotes by parallel approaches aimed at elucidating glycan structures (glycomics) and their formerly glycosylated peptides (glycoproteomics) without reference to their intact state. Such analyses depend heavily on commercial glycosidases (e.g. protein N-glycosidase F) that can remove glycans from the peptide backbone for separate analyses. Bacterial glycosylation has only recently been identified as a widespread phenomenon. In many cases however, unique bacterial sugars preclude enzymatic removal, therefore ultimately requiring a site-specific approach for intact glycopeptide analysis. Here, we describe protocols for the enrichment of bacterial glycopeptides using zwitterionic-hydrophilic interaction liquid chromatography (ZIC-HILIC) and their analysis using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) with collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) fragmentation for glycan structure elucidation and glycopeptide identification.
传统上，通过质谱对蛋白质N-糖基化和O-糖基化进行大规模分析，在真核生物中是通过并行方法进行的，旨在阐明聚糖结构（糖组学）及其以前糖基化的肽段（糖蛋白质组学），而不考虑其完整状态。此类分析严重依赖商业糖苷酶（如蛋白质N-糖苷酶F），这些酶可从肽主链上除去聚糖以进行单独分析。细菌糖基化直到最近才被确认为一种普遍现象。然而，在许多情况下，独特的细菌糖类无法通过酶法去除，因此最终需要一种位点特异性方法来进行完整糖肽分析。在这里，我们描述了使用两性离子亲水相互作用液相色谱（ZIC-HILIC）富集细菌糖肽的方法，以及使用液相色谱与电喷雾电离串联质谱（LC-MS/MS）联用，通过碰撞诱导解离（CID）和高能碰撞解离（HCD）碎裂来阐明聚糖结构和鉴定糖肽的分析方法。
