Max Planck Unit for the Science of Pathogens, Berlin, Germany.
Institute for Biology, Humboldt University, Berlin, Germany.
Methods Mol Biol. 2023;2718:151-165. doi: 10.1007/978-1-0716-3457-8_9.
Glycosylation is one of the most common and complex post-translation modifications that influence the structural and functional properties of proteins. Glycoproteins are highly heterogeneous and exhibit site- and protein-specific expression differences. Mass spectrometry in combination with liquid chromatography has emerged as the most powerful tool for the comprehensive characterization of glycosylation. The analysis of intact glycopeptides has emerged as a promising strategy to analyze glycoproteins for their glycan heterogeneity at both protein- and site-specific levels. Nevertheless, intact glycopeptide characterization is challenging as elucidation of the glycan and peptide moieties requires specific sample preparation workflows that, combined with the tandem mass spectrometry approach, enable the identification of single glycopeptide species. In this chapter, we provide a detailed description of the methods that include procedures for (i) proteolytic digestion using specific proteases, (ii) optional glycopeptide enrichment using hydrophilic interaction liquid chromatography, (iii) nano-LC-MS/MS analysis of glycopeptides, and (iv) data analysis for identification of glycopeptides. Together, our workflow provides a framework for the system-wide site-specific analysis of N- and O-glycopeptides derived from complex biological or clinical samples.
糖基化是最常见和最复杂的翻译后修饰之一，影响蛋白质的结构和功能特性。糖蛋白高度不均一，并表现出位点和蛋白质特异性表达差异。质谱联用液相色谱已成为全面表征糖基化的最有力工具。完整糖肽分析已成为分析糖蛋白在蛋白质和位点特异性水平上聚糖异质性的一种很有前途的策略。然而，完整糖肽的表征具有挑战性，因为糖基和肽部分的阐明需要特定的样品制备工作流程，这些工作流程与串联质谱方法相结合，可鉴定单糖肽种类。在本章中，我们提供了详细的方法描述，包括以下步骤：（i）使用特定蛋白酶进行蛋白水解消化，（ii）使用亲水相互作用液相色谱进行可选的糖肽富集，（iii）纳米 LC-MS/MS 分析糖肽，以及（iv）数据分析以鉴定糖肽。我们的工作流程为系统地分析来源于复杂生物或临床样本的 N-和 O-糖肽的位点特异性分析提供了一个框架。
