Genovis AB, Lund, Sweden.
Methods Mol Biol. 2021;2271:155-167. doi: 10.1007/978-1-0716-1241-5_11.
O-glycosylation is a difficult posttranslational modification to analyze. O-glycans are labile and often cluster making their analysis by LC-MS very challenging. OpeRATOR is an O-glycan specific protease that cleaves the protein backbone N-terminally of glycosylated serine and threonine residues. This enables the generation of glycopeptides of suitable size for mapping O-glycosylation sites in detail by bottom-up LC-MS analysis. In this chapter we demonstrate a simple workflow for in-depth analysis of O-glycosylation sites on heavily glycosylated proteins using OpeRATOR digestion and HILIC-MS/MS analysis.
O-糖基化是一种难以分析的翻译后修饰。O-聚糖不稳定，并且经常聚集，使得通过 LC-MS 进行分析极具挑战性。OpeRATOR 是一种 O-聚糖特异性蛋白酶，可在糖基化丝氨酸和苏氨酸残基的 N 端切割蛋白质骨架。这使得能够生成适合通过自上而下的 LC-MS 分析详细映射 O-糖基化位点的糖肽。在本章中，我们展示了一种使用 OpeRATOR 消化和 HILIC-MS/MS 分析对高度糖基化蛋白质上的 O-糖基化位点进行深入分析的简单工作流程。
