Application Of A Novel Alkynyl Modified Magnetic Beads To Enrich Azido-glycoproteins

Glycosylation is a widespread posttranslational modification of proteins. Glycosylation defect is a sign of many diseases, such as tumor development, autoimmune diseases, and certain genetic diseases. It has become an active area of genomics research. Because the glycan structure is very complex and micro-heterogeneity, enrich glycoproteins form complex biological samples remains have much difficulties. This paper focus on studying glycan metabolic labeling, enrichment and analysis, provide a new method for glycomics research. Our laboratory has developed a kind of Disulfide- and terminal alkyne-modified magnetic silica particles(DA-MSPs), which can specifically enrich azide modified glycoproteins through click chemistry. This thesis using the alkyne modifided magnetic nanoparticles as a tool, explored DA-MSPs enrichment processes, optimized the reaction conditions of enrichment, deceloped a novel enrichment method of glycoproteins. Secondly, we established the metabolic labeling method and detection method of glycan. The azide modified monosaccharides such as tetra acetylated azido galactosamine(Ac4GalNAz) can enter the intracellular glucose metabolism pathway of cells, so that the intracellular glycoprotein marked the azide group, and then by the click reaction between azide and phosphine or alkynyl group can realize the detection of the labeled proteins. Last, use DA-MSPs to enrich azido glycoproteins / glycopeptides from metabolic labeled cells. The interference of the hybrid peptide proved the enrich specifity of DA-MSPs, and when combined with silver staining and lectin blot, it is suggest that the DA-MSPs can also enrich azido glycoproteins directly. Finally, we use mass spectrometry to analysis and identify glycoproteins after DA-MSPs enrichment. This technique laid the foundation for further studies of the O-glycosylation enrichment studies.