Table6_TiO2 Simultaneous Enrichment, On-Line Deglycosylation, and Sequential Analysis of Glyco- and Phosphopeptides.xlsx

Reversible protein glycosylation and phosphorylation tightly modulate important cellular processes and are closely involved in pathological processes in a crosstalk dependent manner. Because of their significance and low abundances of glyco- and phosphopeptides, several strategies have been developed to simultaneously enrich and co-elute glyco- and phosphopeptides. However, the co-existence of deglycosylated peptides and phosphopeptides aggravates the mass spectrometry analysis. Herein we developed a novel strategy to analyze glyco- and phosphopeptides based on simultaneous enrichment with TiO2, on-line deglycosylation and collection of deglycosylated peptides, and subsequent elution of phosphopeptides. To optimize on-line deglycosylation conditions, the solution pH, buffer types and concentrations, and deglycosylation time were investigated. The application of this novel strategy to 100 μg mouse brain resulted in 355 glycopeptides and 1,975 phosphopeptides, which were 2.5 and 1.4 folds of those enriched with the reported method. This study will expand the application of TiO2 and may shed light on simultaneously monitoring protein multiple post-translational modifications.

可逆蛋白质糖基化与磷酸化可精准调控重要细胞过程，并以串扰依赖的方式深度参与病理进程。鉴于二者的生物学重要性以及糖肽与磷酸肽的低丰度特性，学界已开发出多种可同时富集并共洗脱糖肽与磷酸肽的策略。然而，去糖基化肽与磷酸肽的共存会加重质谱分析的复杂度。在此研究中，我们开发了一种全新的糖肽与磷酸肽分析策略：基于二氧化钛（TiO₂）同步富集、在线去糖基化并收集去糖基化肽，随后洗脱磷酸肽。为优化在线去糖基化条件，我们对溶液pH值、缓冲液类型与浓度以及去糖基化时长开展了系统性研究。将该策略应用于100 μg小鼠脑组织样本，共鉴定得到355条糖肽与1975条磷酸肽，分别为已有报道方法富集所得结果的2.5倍与1.4倍。本研究将拓展二氧化钛的应用场景，同时为同时监测蛋白质的多种翻译后修饰提供新的研究思路。