Protein glycosylation is one of the most common protein modifications. A major type of protein glycosylation is O-GalNAcylation, in which GalNAc-type glycans are attached to protein Ser or Thr residues via an O-linked glycosidic bond. O-GalNAcylation is thought to play roles in protein folding, stability, trafficking and protein interactions, and identification of the site-specific O-GalNAc glycoproteome is a crucial step toward understanding the biological significance of the modification. However, lack of suitable methodology, absence of consensus sequon of O-GalNAcylation sites and complex O-GalNAc glycan structures pose analytical challenges. We recently developed a mass spectrometry-based method called extraction of O-linked glycopeptides (EXoO) that enables large-scale mapping of site-specific mucin-type O-GalNAcylation sites. Here we provide a detailed protocol for EXoO, which includes seven stages of: (1) extraction and proteolytic digestion of proteins to peptides, (2) sequential guanidination and de-salting of peptides, (3) enrichment of glycopeptides, (4) solid-phase peptide conjugation and release of O-GalNAc glycopeptides using the OpeRATOR protease, (5) liquid chromatography with tandem mass spectrometry analysis of O-GalNAc glycopeptides, (6) identification of O-GalNAc glycopeptides by database search and (7) quantification of O-GalNAc glycopeptides. Using this protocol, thousands of O-GalNAcylation sites from hundreds of glycoproteins with information regarding site-specific O-GalNAc glycan can be identified and quantified from complex samples. The protocol can be performed by a researcher with basic proteomics skills and takes about 4 d to complete.

蛋白质糖基化是最常见的蛋白质修饰之一。蛋白质糖基化的一种主要类型是 O-GalNAcylation，其中 GalNAc 型聚糖通过 O-连接的糖苷键连接到蛋白质 Ser 或 Thr 残基上。O-GalNAcylation 被认为在蛋白质折叠、稳定性、运输和蛋白质相互作用中发挥作用，并且鉴定位点特异性 O-GalNAc 糖蛋白组是理解修饰的生物学意义的关键一步。然而，缺乏合适的方法、缺乏 O-GalNAcylation 位点的共有序列子和复杂的 O-GalNAc 聚糖结构带来了分析挑战。我们最近开发了一种基于质谱的方法，称为 O-连接糖肽 (EXoO) 的提取，可以对位点特异性粘蛋白型 O-GalNAcylation 位点进行大规模映射。在这里，我们提供了 EXoO 的详细协议，其中包括七个阶段：(1) 蛋白质提取和蛋白水解消化成肽，(2) 肽的顺序胍和脱盐，(3) 糖肽的富集，(4) 固体使用 OpeRATOR 蛋白酶的 O-GalNAc 糖肽的相肽缀合和释放，(5) 液相色谱与 O-GalNAc 糖肽的串联质谱分析，(6) 通过数据库搜索鉴定 O-GalNAc 糖肽和 (7) 定量O-GalNAc 糖肽。使用此协议，可以从复杂样品中识别和量化来自数百种糖蛋白的数千个 O-GalNAcylation 位点以及有关位点特异性 O-GalNAc 聚糖的信息。该协议可以由具有基本蛋白质组学技能的研究人员执行，大约需要 4 天才能完成。