Technique And Strategy In Phosphopeptides Enrichment And Their Application

Protein phosphorylation is one of the most important post-translational modifications in cells, and the reversible phosphorylation of proteins regulates most aspect of cell life such as cell cycle, differentiation and development, metabolism, nerve activity, muscle constriction, and cellular regulation. So the detection of phosphoproteins and the conforming of phosphorylation sites are very important to understand the mechanism of phosphorylated protein in a biological pathway. The study of phosphoproteomics involves the identification of phosphoproteins and phosphopeptides, the localization of the exact residues which are phosphorylated and the quantitation of phosphorylation. The detection of phosphopeptides remains challenging because of the low abundance of phosphoproteins. So the enrichment of phosphoproteins and phosphopeptides before MS analysis is one of the important steps to improve the MS response of phosphopeptide signals. We report here an optimized strategy of enrichment of phosphopeptides by using TiO2 particle. And we also report the phosphoproteomics analysis of mitochondrial proteins from mouse liver by using combined phosphopeptides enrichment strategy.In the first part of the article, we developed the enrichment method of phosphopeptides. TiO2 particle was used to selectively enrich phosphopeptides from alpha-casein digest. As the optimization of the enrichment method, we investigated the concentration of acetonitrile, the variety of acid and its concentration in the loading buffer. We also changed the ionic strength of solution by adding salt, so as to discuss the reason of co-elution of non-phosphopeptides. After that, in order to enhance the selectivity of TiO2 particle, the aspartic acid was added in the solution as the non-phosphopeptides excluder so that the number of unique phosphopeptides which were detected was increased. This method was validated in the enrichment of 9 standard protein digest mixtures, and we found that the effect of enrichment of phosphopeptides was correlated with the complexity of the sample. In our research, the method we developed could be used in micro or nano TiO2 enrichment experience, and couldn't work in IMAC enrichment experience. In the second part, a combined phosphopeptides enrichment strategy (SCX- TiO2 - IMAN) was used to characterize the phosphoproteome of the mitochondrial proteins from mouse liver and probe the role of protein phosphorylation. 37 phosphoproteins, 37 phosphopeptide sequences are determined, 68 phosphorylation sites are defined in SCX- TiO2 analysis strategy, and in SCX-Fe3+-IMAN analysis strategy we identified 40 phosphoproteins, 44 phosphopeptide with 63 phosphorylation sites. A total of 71 phosphoproteins and 77 phosphopeptides were identified.