Determination Of N-linked Glycans Based On Endoglycosidase

The sugar chain has the characteristics of diversification, The sugar chain carries complex biological information, which is directly related to heredity, pathology and human life activities. The glycosylation process is closely related to the physiological and biochemical process, and it is related to the occurrence and pathological changes of multiple diseases, such as cancer, which has practical significance for the research of the sugar chain. In this paper, two kinds of endoglycosidase Endo M and Endo M-N-175Q were used to analyze the enzyme reaction of sugar chain.Endo M-N-175Q as a mutant of Endo M, while retaining the activity of the conversion of sugar at the same time almost lost the hydrolysis activity, so in the extension of the reaction time of the enzyme with a higher conversion efficiency. This paper introduces the development of desalination technology, focuses on the instructions of the graphite carbon column purification and enrichment of simple.efficient and this method was applied to the experiment. Based on graphitized carbon column enrichment oligosaccharide elution mode selection, the selection of the enzyme which cut glycoprotein to establish a method for analysis of glycoprotein N-glycan,succeed to use the application of this method to the experiment for the analysis of complex glycoprotein ovalbumin and successfully detected eight kinds of oligosaccharide.According to reports, oxazoline both in carbohydrate chemistry based method and chemical enzyme glycosylation method above is known very well the glycosyl donor. In order to oxazoline as substrate, using Endo M-N-175Q transglycosylation reaction rate can reach above 90%. For premise, in order to further improve the transfer rate of enzyme reaction, through the synthesis of sugar oxazoline explore improve enzyme reaction transfer rate method.In this paper, four groups of experiments on the N-sugar chain analysis. First of all, SGP hydrolysis by Pronase E, enzymatic hydrolysis products enriched by PGC and react with Endo M-N-175Q monitored by LC-ESI-MS of product formation and not the final from three different elution mode selectively neutral elution mode as the enrichment process of oligosaccharides, application of this method in the subsequent experiments.Secondly, the two groups of standard glycoproteins bovine pancreatic Ribo B respectively by Pronase E and PNGase F solution, enzyme solution is obtained for the oligosaccharide PGC after enrichment and react with Endo M-N-175Q and detected by LC-ESI-MS. The former obtains a kind of oligosaccharide M5N2, the latter obtains three kinds of oligosaccharide M5N2,M6N2,M8N2 and the content is higher than the former, therefore selects the PNGase F. Then.the former methods used in the analysis of complex glycoprotein ovalbumin N-glycan, with PNGase F enzyme solution ovalbumin, enzymolysis products by graphite carbon column enrichment, Endo M-N-175Q enzyme reaction get eight kinds of oligosaccharide through LC-ESI-MS detection. Finally, use GlcNAc successful synthesis of sugar GlcNAc-oxa, this method is applied to SGP and synthetic SG-oxa were conducted Endo M-N-175Q enzyme reaction. Reaction results and SGP directly comparing the results of the Endo M enzyme reaction, the former peak area integral is 14.2 times than that of the latter, to sugar oxazolines as substrate for Endo M-N-175Q enzyme reaction will significantly improve the enzyme reaction rate.