Large-scale Release And Separation Of N-glycans Based On Ammonia Catalysis

Glycosylation is a type of protein post-translational modification,which plays an important role in the regulation of a series of cellular biological activities.Different types of oligosaccharides are linked to the protein backbone during glycosylation,including O-glycans attached to Ser/Thr residues and N-glycans attached to the Asn residues usually in the Asn-X-Ser/Thr sequence.The glycan realizes its biological function through the interaction with the glycoconjugate protein.To study the relationship between the structure and function of the glycan,it is necessary to obtain a large number of clear and diverse structure glycan monomer.At present,the release method of N-glycans are mainly enzymatic and chemical methods.Enzymatic hydrolysis is mainly carried out by using a highly specific glycosidase(such as PNGase F and PNGase A)or proteolytic enzyme Pronase E combined with glycosylasparaginase(GA)to obtain a free glycans.But enzymes are expensive,generally used for the analysis of glycans and not for the large scale preparation of glycans.Based on the advantages of low cost and high availability of chemical methods,the preparation of N-glycans is often carried out by chemical methods.However,the existing chemical methods such as hydrazinosis have harsh reaction conditions,high toxicity,and formation of by-products;concentrated aqueous ammonia containing saturated ammonium carbonate(60 ? water bath reaction for 40 hours)can only release part of the intact N-glycans non-reducing.Therefore,they have not been applied to large-scale preparation of glycans.Although sodium hypochlorite oxidation is applied to large-scale preparation of N-glycans,however,the GlcNAc at the reducing end of part of the N-glycan is lost.Therefore,it is of great significance to develop versatile chemical method to release N-glycan in the large-scale preparation glycans.In this study,a simple,rapid,versatile and cost-effective method was developed for the dissociation of educing N-glycan.We applied the method to the large-scale preparation of N-glycan.The prepared glycan was derivatized and subjected to 2D-HPLC separation to obtain an N-glycan monomer.The research results are as follows: 1.A new chemical method for releasing N-glycans was established.Glycoproteins were dissolved in concentrated aqueous ammonia,reacted in a water bath at 60 ? for 16 hours in an airtight container,and then purified by protein removal and impurity removal steps to obtain reduced N-glycans.The method is cheap,simple,and can be used in neutral,sialylated and core ?-1,3-fucosylation N-glycans.Reducing N-glycan can be obtained by the method,and the dissociated N-glycans can be labeled with fluorescent reagent for separation and analysis by HPLC.We have successfully applied this method to biological samples such as RNase B,egg white protein,fetal bovine serum albumin,peanut protein,ginkgo seed protein,soy protein and egg white.2.Based on the advantages of this method,such as simple operation,low cost,strong versatility,and can be used for the large-scale preparation of the reducing N-glycan.In this study,the preparation of the reducing N-glycan is carried out by this method.On the basis of the established method,the reaction system was expanded.After the reaction was completed,a large amount of protein was removed by acid precipitation,and the N-glycan was purified by a self-made C18 and PGC solid phase extraction column(20 g/300 mL).The step of acid precipitation of proteins is particularly important in the subsequent industrial preparation,which reduces the cost of subsequent sample purification.The currently prepared egg white protein,fetal bovine serum albumin,soy protein and N-glycan in egg white have reached the gram level.3.After benzenesulfonyl hydrazide(BSH)conjugation,the N-glycans from chicken albumin and soybean protein can be primary separated and purified by hydrophilic chromatography column one-dimensional HPLC,and then de-labeled to obtain different reducing N-glycan components.Reducing N-glycans of different components can be coupled with different labeling reagents as needed,and combined with two-dimensional HPLC chromatography separation to obtain high-purity N-glycan derivative monomers for various studies.In this study,we selected the bifunctional reagent 2-amino-N-(2-aminoethyl)-benzamide(AEAB)for labeling and combined with C18 column(RP-HPLC)for two-dimensional chromatographic separation.21 N-glycan-AEAB conjugates were obtained from chicken albumin,and 8 N-glycan-AEAB conjugates were obtained from soybean protein.The obtained N-glycan-AEAB conjugates having a function primary alkylamine can be effectively immobilized on the surface of microarray,and is further used for functional research of glycans.