The Synthesis And Application Of Derivatization Reagents For N-Glycopeptides

Protein glycosylation effect generally exists in eukaryotic cells and has very important influence on the structure and the biological function of the protein, such as the discrimination ability of the cells. In recent years, the study found that the occurrence of many diseases are associated with the change of sugar on the protein, such as inflammation, the disease caused by immune medium, tumor, congenital glycosylation syndrome and so on. So it is very necessary on glucide of qualitative and quantitative analysis for the discovery of disease-related characteristics information. For the less of the standard substances, so the relative quantitative technology is widely focused. On N-sugar chain, the chemical tags firstly and then mass spectrum detection, that is the most effective analysis method and the best isotope internal standard generally is 13C, D,15N. For more in-depth research of glycoproteins, we not only study the sugar chain but also essentially study the peptides.Combination of peptides and sugar chain research, that is meaningful to the analysis of glycoprotein.Recent reports, Endo-M enzyme with its variation Endo-M-N175Q, could cut N-sugar chain on the glycoproteins between two GlcNAc of the beta 1-4 glycosidic bond and released the N-sugar chain and the peptides or protein with GlcNAc. At the same time, it could transfer the oligosaccharide chains to the receptors with the groups of GlcNAc to form a new glycosidic bond between two new GlcNAc, eventually create a new labeling glycoprotein or glycopeptide derivatives. Based on this method, in the second chapter of this paper, we designed and synthesized 4 pairs of light and heavy stable isotope labeled receptors. They could improve the mass spectrum detection sensitivity and resolution. Characterization of all receptors and simple application in enzyme reaction, we got the ideal sugar-based products.For more in-depth study of glycoproteins. peptides study of N-glycopeptide is also very important. In the third chapter of this paper, we designed the 5 kinds of isotope labeling derivatization reagents, that could solve the bottleneck of more than four tags to the analysis of peptides in the level of mass spectrometry. In the experiments, we successfully synthesized two heavy isotope labeling derivatization reagents d0/d3-MPPZ, by the reaction with the stable isotope labeling derivative reagent and various amino acid under the same conditions, and explored the best reaction conditions. Due to the amino terminal on amino acids, we synthesized a new type of the isotope labeling derivative reagent do/d3-MPPZ for its amino terminal reaction in response. In the study of peptides, this paper takes the sialoglycopeptide (SGP) as the sample of N-sugar peptide, with the same amount of the isotope labeling derivative products on LC-MS analysis. We could get the stable derivative products, mass spectrometry abundance and extraction chromatographic peak area of 1:1 and also could prove that the derivative reagent can be applied to the relative quantitative analysis in peptides. In the derivation process, the chemical tags simply, derivative reaction condition mildly, reaction degree relative completely, less by-products, labeling products stably, reagents cheaply etc. For the future, molecular greater, structure more complicated in glycoprotein research provides the experimental basis.