Optimization Of Titanium Dioxide Method For Enrichment Of Phosphopeptides And Its Applications On Mouse Liver Phosphoproteome Research

Protein phosphorylation is one of the most important post-translational modificationsin cells. Phosphorylation and de-phosphorylation is a reversible process, and it is themost normal and important regulations in cellular life. There are many phosphorylatedproteins in mammals, and the abnormal phosphorylations can make life into disorder.Titanium dioxide (TiO2) is a very good strategy for the phosphopeptides enrichment,and it has been widely used in phosphoproteome research recently. The advantages ofthis method are easy operation and low cost. The enrichment specificity could beimproved largely by adding materials such as glutamic acid in the loading buffer toreduce nonspecific binding of non-phosphorylated peptides. Actually there are alsomany other factors can affect the enrichment efficiency, and the parameters need to beoptimized to obtain better enrichment results.In this study, the peptide mixtures from six standard proteins are used as modelsample to evaluate and optimize the parameters such as the proportion of acetonitrileand trifluoroacetic acid in loading buffer and the peptide-to-TiO2beads ratio. Theresults showed that80%acetonitrile,1%trifluoroacetic acid and1:40(mass/mass)peptide-to-TiO2beads ratio are the optimum condition to obtain the best enrichmentselectivity and maximum phosphopeptides identification. For the first time, theoptimum enrichment conditions were applied for phosphoproteomic analysis of theThermoanaerobacter tengcongensis, an anaerobic, saccharolytic, thermophilicbacterium isolated from a hot spring in Tengcong, China, and25phosphorylatedproteins were identified in the preliminary experiment. The result provided areference for further study on this organism survived under extreme environment.Then we carried out liver phosphoproteomic analysis on the C57BL/6J mouse. Wefocused on the nuclear and membrane proteins. Firstly,7cm SDS-PAGE gel was usedto separate the proteins. And10gel slices were cut and digested by trypsin. Finally,198phosphorylated proteins,275non-redundant phosphorylated peptides and486phosphorylation sites were identified from the nuclear fraction.175phosphorylatedproteins,298non-redundant phosphopeptides and338phosphorylation sites wereobtained from the membrane fraction.