蛋白质磷酸化位点富集与鉴定方法的建立与应用

The development and application for the enrichment and identification of protein phosphorylation

建立了一种用Ti O2填料,自制色谱小柱,从而实现磷酸化肽段富集的方法,填料负载量可根据需求调整等优势。质谱分析显示,Ti O2小柱可以实现标准磷酸化蛋白质修饰位点的识别,针对alpha-casein的分析显示,结合液相色谱串联质谱鉴定,可以获得15个磷酸化修饰位点。方法用于突触核蛋白(α-synuclein)磷酸化修饰位点的鉴定,确认了数据库中的S87,T125 2个修饰位点,同时发现了11个新的修饰位点。有望促进磷酸化蛋白质质谱分析技术在基础与临床研究实验室的推广应用。

The phosphorylation of reversible protein plays an important role in regulating cell signal transduction,gene expression,metablism and so on. It also involved key disease such as tumor,neurodegeneration disease and cadiovascular disease. The study of protein phosphorylation site could aid to a better understanding of the protein function in pathphyosigical processes. An easy option to identify phosphosites is using mass spectrometry.Whereas,the low level of phosphorylational modification made it easy to be suppressed by the non-modificated peptides during mass spectrometric analysis. In this research,we established a facile approach to enrich phosphopeptides,by home-made tip-column using titandium dioxide（ Ti O2） media. Merits of the approach included low cost,easy to operate,and the easily controlled scale. We used standard phosphoprotein to evaluate the method. The result showed that 15 phosphosites from alpha-casein could be covered by liquid chromatography tandem mass spectrometry. Finally,we used this method to the phosphosites analysis of α-synuclein. We found11 new phosphorsites as well as confirmed 2 well-known sites at S87 and T125. We believed the research method here would facilitate the clinical and basic application of mass spectrometry-based technique for phosphoprotein analysis.