A New Method For Analysis Of N-Glycosylation Sites Based On Cotton Hydrophilic Chromatography Enrichment Of Glycopeptides

Glycosylation modification is one of the most common and complex post-translational modifications of proteins.About 1/2 of the proteins in animals are glycosylated.Studies have shown that protein glycosylation plays an important role in many life activities,such as signal transduction,immune response,and sperm-egg binding.Abnormal protein glycosylation is directly related to the occurrence of disease,and changes in the degree of protein glycosylation modification are detected in the development of many diseases such as diabetes,infectious diseases,and cancer.A comprehensive analysis of glycosylation sites will help to better understand the biological functions of protein glycosylation.At present,research methods for glycosylation sites have been reported.However,the method of enriching glycopeptides by hydrophilic interaction,combined with trifluoromethanesulfonic acid method and enzymatic deglycosylation for comprehensive analysis of N-glycosylation sites has not been reported.In this study,The innovation of research is that the use of medical absorbent cotton to prepare cotton hydrophilic column for glycopeptides enrichment,which effectively reduces the experimental cost.At the same time,the deglycosylation treatment was carried out by using the trifluoromethanesulfonic acid method to avoid the interference of the O-glycosylation site,and a GlcNAc was left as a mass label at the glycosylation site,reducing the sensitivity of the mass spectrometer.Meanwhie,the N-glycosylation site was comprehensively analyzed by the obtained glycan dates using enzymatic deglycosylation.The method was validateds using sialylated glycopeptide and bovine pancreatic ribonuclease B as standard proteins.At the same time,the method was successfully applied to the identification of glycosylation site of soybean?-conglycinin protein,which is of great significance for glycoproteomics research.The results obtained are as follows:1.The cotton serotonin column was used to separate and purify the sialylated glycopeptide from the egg yolk.Compared with the previous method,the method greatly reduced the experimental cost and shortened the time.We purified from 600 egg yolks to 600 mg sialogopeptide in just one week,and the method can be used for preparation,which has a good application prospect.2.The glycosylation site of the sialylated glycopeptide was successfully identified as:KVAN~#KT.The glycopeptides of bovine pancreatic ribonuclease B after trypsin digestion were successfully enriched,and the glycosylation site was identified as:N~#LTK,and the five N-glycans at the glycosylation site were resolved.The N-glycans are:H5N2,H6N2,H7N2,H8N2,H9N23.The 22 glycopeptides of soybean?-conglycinin protein after trypsin enzyme and pepsin enzyme complexase were successfully enriched by cotton hydrophilic column,and three subunits of?-conglycinin protein were successfully identified:?subunit,?,sub Five glycosylation sites of the base and?subunit:ILN~#GTA,PVMVN~#A,VVN~#ATSNL,LIVILN~#GTA,PVVVN~#ATSNL.In addition,we also analyzed the sugar chain at the glycosylation site,and found that four high-mannose-type N-glycans were determined:H5N2,H6N2,H7N2,H8N2.