Chen Cheng, Zhang Xiaofei, Dong Xuefang, Zhou Han, Li Xiuling, Liang Xinmiao
Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
University of Chinese Academy of Sciences, Beijing, China.
Front Chem. 2021 Aug 11;9:703176. doi: 10.3389/fchem.2021.703176. eCollection 2021.
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 TiO, 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 TiO and may shed light on simultaneously monitoring protein multiple post-translational modifications.
可逆蛋白糖基化和磷酸化紧密调节重要的细胞过程，并以相互依赖的方式密切参与病理过程。由于其重要性以及糖肽和磷酸肽的低丰度，已开发出多种策略来同时富集和共洗脱糖肽和磷酸肽。然而，去糖基化肽和磷酸肽的共存加剧了质谱分析的难度。在此，我们开发了一种基于用TiO同时富集、在线去糖基化和收集去糖基化肽以及随后洗脱磷酸肽来分析糖肽和磷酸肽的新策略。为了优化在线去糖基化条件，研究了溶液pH值、缓冲液类型和浓度以及去糖基化时间。将这种新策略应用于100μg小鼠脑，得到了355个糖肽和1975个磷酸肽，分别是用报道方法富集得到的糖肽和磷酸肽数量的2.5倍和1.4倍。本研究将扩展TiO的应用，并可能为同时监测蛋白质多种翻译后修饰提供线索。
