Establish An Analysis Approach N-linked Glycan In Glycoprotein Using HPLC/ESI-MS

Glycosylation is one of the most common post-translational modifications of proteins in human. It has been estimated that over 50% of human proteins are glycosylated. Glycans play a vital role in protein conformation, activity, and localization, which occur in many biological processes. The development and progression of many diseases are associated with the changing structure and expression of N-glycans in glycoprotein. Therefore, the development and establishment of determination methods of N-glycans are expected to result in great progress in biological and pathological studies.Nowadays, the most convenient approachs to determine the structures of the glycoprotein N-glycans is derivatization with a suitable compound, the main disadvantage is extra reagents, additional steps, and side reactions. Enzymatic labeling is one of the most promising approaches for the glycosylation analysis due to its characteristic. First of all, with Boc-Asn-GlcNAc as a basic structure, two kinds of new isotopically labeled acceptors were synthesized for the resolution of oligosaccharides in glycopeptides. On the basis of previous work in our laboratory, we have optimized the method of synthesis reaction. DMT-MM as a condensing agent in reaction system, temperature, time, ratio of reactants to experiment was explored, respectively, yield to 95-98%. Validation of the acceptors by standard glycopeptide SGP, we have shown that the Endo-M-N175Q demonstrates noticeable transglycosidase-like activity for natural N-glycans and the productivity of Endo-M N175Q is estimated to be 2.0-fold higher than that of Endo-M. We have used glycoproteins with known glycan structures, ribonuclease B. Glycopeptide enrichment by acetone and PNGase F to release the oligosaccharides chain, which are two ways to obtain oligosaccharides, experimental results show that the latter have higher efficiency. In order to test the practicality of this method, we used glycoprotein ovalbumin as actual samples, which have studied by other researchers. A series of oligosaccharides were detected successfully (M5N2、M6N2、M7N2、M4N3、M5N3、 M3N4、M3N5、M4N4、M3N6) the same to literatures. When the ESI- mode was adopted for the detection of the PDPZ-labeled oligosaccharides in glycoprotein. many oligosaccharides were detected as bivalent ions or trivalent ion. Compared to traditional chemical derivation methods, enzymatic labeling occurs during enzymatic digestion, avoiding extra reagents, additional steps, and side reactions. Therefore, we used Boc-Asn-GlcNAc as a basic structure and developed acceptors for the transglycosylation reaction using Endo-M-N175Q, combined with HPLC/ESI-MS, which is the most effective means for detecting N-glycans. This new method for oligosaccharides analysis is efficient and feasible.