Research On The Large Scale Enrichment And Identification Of Protein O-GlcNAc Modification

As an important type of post translational modification,O-GlcNAc glycosylation widely exists in the nucleus and cytoplasm of eukaryote cells. It consists of a single N-acetylglucosamine linked to the serine/threonine residues of a protein. The attachment and removle of O-GlcNAc glycosylation by O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA) is an important way of intracellular signal transduction regulation. It is involved in numerous biological processes, such as cell cycle regulation, signal transduction, transcription and translation regulation and stress responds. The abnormal O-GlcNAc glycosylation is found closely related to a variety of diseases sush as cancer, alzheimer’s disease, parkinson’s disease and diabetes. O-GlcNAc modification is highly dynamic, present in very low abundance in the cells and has poor responds in mass spectromers. Therefore, efficient enrichment is a prerequest for successful identification of O-GlcNAc in real biological samples, so it is difficult to identify the O-GlcNAcylated glycoproteins.In this thesis, we established a novel O-GlcNAc glycoproteins/glycopeptides enrichment strategy using GO-WGA/HILIC materials. The feasibility of the latter enrichment strategy was proved by the successful applications in the enrichment and identification O-GlcNAc peptides of HeLa cells. Furthermore, the HILIC enrichment method was successfully applied in the enrichment and identification of O-GlcNAc modification in RNA/DNA binding proteins and histones. The parameters in the database searching were also investigated and optimized for we studied crosstalk between O-GlcNAcylation and phosphorylation modifications.The first chapter of this thesis is the reviews on the current separation and enrichment methods for glycoproteins O-GlcNAc identificaitonby mass spectrometry. The second chapter described the experimental details of the enrichment of standard O-GlcNAc protein of a-Crystallin by GO-WGA and the enrichment of O-GlcNAc peptides of HeLa cells by HILIC materials. Totally, we identified 1145 O-GlcNAc glycoproteins corresponding to 1770 O-GlcNAc glycopeptides and 2612 O-GlcNAc sites in HeLa cells.In the third chapter we applied the HILIC enrichment method for the identification of O-GlcNAc modification in RNA binding proteins (RBPs), DNA binding proteins (DBPs) and histones. By tandem enrichment of RBPs/DBPs/histones and O-GlcNAc peptides, we successfully identified 55 O-GlcNAc modified RNA binding proteins,26 O-GlcNAc modified DNA binding proteins and 24 new O-GlcNAc sites on the histons. In the fourth chapter of this study, we investigated and optimized the parameters of database searching for study of the crosstalk between O-GlcNAcylation and phosphorylation. We found that the identified sites of O-GlcNAc would increase by 6.6%-31.5% if phosphorylation was included increased to variable modification. Furhermore, we found that phosphorylated and O-GlcNAcylation usually occurs in the same peptides in the adjacent amino acid residues.We successfully established a new HILIC based method for efficient enrichment of O-GlcNAc peptides and successful applications were demostraed in O-GlcNAc analysis of functional proteins, therefore provides a powerful way for proteomics and glycomics studies.