| Glycosylation is a typical post-translational modification of protein in human.It has been known as an significant process which closely related to the life activities of organism and physiological and biochemical processes.The procedure of forming glycoprotein need under the control of enzyme making sugar compounds as an adjunction append to protein or lipid.Glycoprotein associated with diverse dissimilar biological events,for instance,identification and adhesion between the cells,protein folding and structure stability,immune response,etc.Research demonstrate that the occurrence and development of many diseases are mainly associated with the change of the expression quantity and structural loci of glycoprotein glycans,such as malignant tumor,cancer,stomach and alimentary trace problems.Therefore,do qualitative and quantitative analysis of glycoprotein glycans have positive practical significance in the diagnosis and treatment of disease.By using enzymatic transglycosylation of Endo-M-N175Q which have specific function that could complete incision and transfer two process in one step,to combined with stable isotope labeling acceptors based on LC/ESI-MS,we establish an analysis method for N-linked glycans of glycoprotein.First of all,according to the transglycosylation feature of Endo-M-N175Q,it could cut off the bond between two GlcNAcs and transfer the entire oligosaccharides of various types of N-glycans onto various acceptors who having an N-acetylglucosamine(GlcNAc)residue easily.Based on this feature and predecessor research in our laboratory,we have synthesized glycosyl acceptors PDPZ-Boc-Asn-GlcNAc with Boc-Asn-GlcNAcs which is a part of glycoprotein as basic structure for the application of analyzing oligosaccharides in glycoprotein.Furthermore,according to the previous work,we have found that there exist chemical reagent(NP-40)factor which can affact the reaction and the LC-MS test results during the process of glycoprotein denatured and oligosaccharide digested.Therefore,using method of exclusion,discussed and study the whole procedure of dealing glycoprotein and test the source of impurities and removal method to find out which step could influence reaction and detection.At the same time,for optimizing and simplifying the procedure of dealing glycoprotein,our laboratory create a new simple and highly effective method which avoid adding additional chemical reagents,reduce reaction time and process and avoid the reaction liquid loss during the transfer process.Finally,using high mannose type glycoprotein ribonuclease-B as study sample,based on the Endo-M-N175Q and glycosyl acceptors,to test the practicality of the optimized analysis method of glycoprotein glycans,and we detected 5 different kinds of oligosaccharides M5-9N2 successfully.Then analysis the hybrid type glycoprotein ovalbumin and complex type glycoprotein fetuin,successfully detected 9 different kinds of oligosaccharides:M5-7N2,M4-5N3,M3N4-6,M4M4 and 7 different kinds of oligosaccharides:N2M5,S1-2G2N4M3,S1-4G3N5M3,respectively.Three types of glycoprotein gain great testing results which illustrate that the new method has extensive applicability and practicability and will provide analytic technical support for glycoprotein glycans research. |
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