Determination Of N-linked Glycans In Glycoproteins Based On Endo-M N175Q Transglycosylation Reaction

In recent years,the analysis of sugar chains using mass spectrometry as an analytical platform has made great progress.The sample preparation methods such as the release and derivatization of sugar chains have been continuously enriched and improved,and various analysis strategies for sugar chains have been successively established.This paper mainly analyzes and summarizes the qualitative analysis methods of sugar chains.Chemical derivatization first synthesizes various glycosyl donors and glycosyl acceptors as a substrate for enzyme-catalyzed reactions by chemical methods,and then catalyzes the binding of glycosyl donors to glycosyl acceptors by enzymatic methods to obtain the corresponding products..However,this method requires the introduction of additional chemical reagents,cumbersome procedures,the destruction of sugar chain structure and the occurrence of side reactions and other shortcomings.So this paper uses the chemical enzyme labeling method,enzyme labeling is one of the most promising methods of glycosylation analysis Because of its simplicity:The labeling occurs during the enzymatic digestion and additional reagents,extra steps and side reactions can be avoided.Because of its mild reaction conditions and high spatial regioselectivity,the chemical enzymatic method has become a research hotspot since it avoids the tedious protection and deprotection steps of chemical synthesis.It does not have the disadvantages of chemical derivatization methods.It is used to study N in glycoproteins.-Sugar chains provide new ideasThe first chapter in the first chapter is about the status of glycoprotein carbohydrates and their relationship with diseases,and details the composition and application of glycoproteins.The PNGaseF enzyme is commonly used in the study of peptides.Currently,the method of investigating sugar chains is relatively scarce.Therefore,the introduction part briefly introduces and explains these two principles,and explains the point that acetone enriches glycopeptides.At the same time,a detailed explanation of the role and advantages of the Tang-based enzyme Endo-M N175Q in the final step was given.The second chapter of the experimental part focuses on the preparation of the various solutions required before the experiment,as well as the desalting,tryptic digestion,acetone enrichment of glycopeptides,enzyme reaction conditions and methods involved in the experimental process,and through the final determination of the method The qualitative analysis and analysis of the three standard products showed good results,all of which were reflected in the third chapter.In the third chapter of the results and discussion,we focused on the analysis of three glycoproteins.One of them is to transfer oligosaccharides directly to the receptors under the action of Endo-M N175Q.The second method is to transfer the oligosaccharide to the receptor under the action of the PNGaseF enzyme in comparison with the commonly used oligosaccharide chain analysis method.The third method is the method discussed in this paper.The trypsin is used to cleave the glycoprotein,and the peptide chain is enriched in acetone.Now the general method of most papers is to analyze the peptides,and the method in this paper is novel.Using the method of acetone enrichment of glycopeptides,the oligosaccharides were transferred to the receptors and reacted with the 175Q enzyme.After spectrogram analysis,it was found that the method of enriching glycopeptides with acetone was very effective.Afterwards,the optimization of reaction conditions in each step was carefully discussed.,to determine the optimal method.This method was then applied to the three standard glycoproteins,bovine pancreatic ribonuclease B,ovalbumin,and fetuin,which were qualitatively shown after the spectral results.Among them,it was highlighted that acetone is the best in the-25 degree freezer,and that 5 times the volume of the enriched glycopeptides is the best,with the most detected glycopeptides.Through the different solubility of the sugar chain in acetone and water,overnight and precipitation are formed,and then the precipitate is enriched with acetone to obtain abundant sugar chain information.Finally,the 175Q enzyme of the mutant Endo-M was used for the transfer.The efficiency was high and the result was good.Five sugar chains were detected with bovine pancreatic ribonuclease B,which were M5N2,M6N2,M7N2,M8N2,and M9N2.Five types of ovalbumin were detected,namely M5N2,M6N2,M8N2,M3N4,and M3N6.Six kinds of fetuin were detected,namely M3N4G2,M3N4G2Ac2,M3N4G2Ac3,M3N5G3Ac,M3N5G3Ac2,and M3N6G4Ac4.Now the general study of the N-oligosaccharides method uses chemical derivatization after oligosaccharides are cut off,but this requires the purification of the denatured glycoproteins.This process will be lost and the sugar chain structure will be easily changed.Therefore,another innovation in this paper is the use of chemistry.Enzymatic analysis of sugar chains has become a research hotspot due to its mild reaction conditions and high spatial selectivity,avoiding the tedious protection and deprotection steps of chemical synthesis,and it is worth further studying the analysis of sugar chains in glycoproteins.