New Strategy Research On Large-scale Enrichment And Identification For O-GlcNAc Protein Glycosylation And Phosphorylation

Post-translational modifications are covalent modifications on proteins,which are widely distributed in cells and tissues of mammals.They have vital functional roles for the regulation of almost of all kinds of biological life process such as cell-cell recognition,signal transmission,immune response and gene transcription.Large scale protein post-translational modification identificaiotn will provide key information for the understanding of the biological system as a whole and facilitate the machasim study of the occurecen and development of many major human diseases.Biological-mass spectrometry based strategy offers a new way for the large scale protein post-translational modification research.While,large scale identification of protein post-translational modification identification still faces several technology challenges.First,most of the protein post-translational modifications are present in very low abundance in cells or tissues and therefore are easily supressd by the non-modified peptides in the "buttom up" based mass spectrometry analysis.Second,many post-translational modifications are highly dynamic in cell.Third,some of the post-translational modifications such as phosphorylation and O-glycosylation,the linkage between the modification and proteins is relatively weak and unstable.Therefore,these kinds post-translational modifications may be lost during sample preparation and leads to fauls negative results.As a result,efficient,fast and high throughput enrichment methods is in ugent need to achieve more comprehensive identifiacation of protein post-translational modification which is vital for revealing their functional roles in biological and pathological processes.Among the known 300 types of protein post-translational modification,glycosylation and phosphorylation are the most common and important ones.Therefore,in chapter one,we synthesized a new TMT-Staudinger reagent for the efficient labeling of the azide tagged O-GlcNAc protein/peptides via the highly specific azide-Staudinger ligation.Combined with the immobilized TMT-antibody enrichment,we successfully establish a new O-GlcNAc modified protein/peptide enrichment method.The experimental procedure is simple and the azide-Staudinger ligation is fast,highly efficient and stable.The immobilized TMT-antibody has high enrichment specificity towards the TMT group,thus particularly suitable for the enrichment of the extremely low abundant O-GlcNAc proteins/peptides.In chapter one,we studied protein phosphorylation.We used C57BL/6J mouse liver protein as the samples and analyzed in detail the key factors affect the phosphorylation identification results,including the amount of the digested peptides as the staring material,the ratio between the digested peptides and TiO2,choice of the pre-fractionation method and liquid chromatography separation time.The results showed that TiO2 combined with tyrosine phosphorylation specific antibody can significantly increase the enrichment specificity of tyrosine phosphorylation peptides.This method is well fit for the enrichment and identification of low abundant tyrosine phosphorylation peptides by mass spectrometry.