Structure Analysis Of N-glycosylation Of Pregnant Mare Serum Gonadotropin Based On High Resolution Mass Spectrometry

Pregnant mare serum gonadotropin(PMSG)is a very important glycoprotein hormone that can promote estrus and ovulation in animals and has been widely used in animal husbandry.At present,the research on PMSG hormone is mostly focused on the purification of PMSG hormone from pregnant horse serum,while the research on N-glycosylation and sugar chain structure is less.A large number of studies have shown that N-glycosylation is a ubiquitous protein post-translational modification in organisms,and it has an important impact on the biological activity of proteins.Therefore,this project takes PMSG glycoprotein as the research object,analyzes its glycosylation site and sugar chain structure,in order to provide a reference for understanding the biological activity of PMSG glycoprotein.The main research results of this subject are as follows:1.Preparation and enrichment performance evaluation of λ-Carr-Ag-SA nanocomposites.Since the glycopeptide content in protease hydrolysate is very low and the ionization efficiency of glycopeptides is low,it is necessary to enrich the glycopeptides before mass spectrometry analysis.This topic uses λ-carrageenan and sodium alginate to reduce silver ions by one-step method to synthesize a new type of nanocomposite λ-Carr-Ag-SA.The standard protein and human serum were used as experimental objects to evaluate its enrichment performance.The results showed that after enriching with λ-Carr-Ag-SA,the number of glycopeptides detected by mass spectrometry and the signal intensity were significantly improved.Therefore,this study provides a new research strategy for N-glycopeptide enrichment.2.Enrichment of N-glycopeptides from PMSG glycoprotein and sugar chain structure analysis.In this study,three different purification stages of PMSG glycoprotein samples were pre-processed.By optimizing chromatographic and mass spectrometry conditions,a highresolution mass spectrometry analysis method for PMSG peptide sequences was established.Based on this method,this study analyzed the unenriched and enriched PMSG glycoproteolysis solutions,and analyzed the glycopeptide oxonium ion and Y0(peptide),Y1(peptide+1Hex NAc)or Y2(peptide+2Hex NAc).4 and 11 N-glycopeptide sequences and sugar chain structure information of PMSG glycoprotein were finally obtained,separately.Compared with the nonenriched case,the N-glycosylation structure information after enrichment is more abundant,and the signal intensity is also greatly improved.3.Further verification of N-glycosylation sites of PMSG glycoprotein under different collision energies.Due to the different collision energy of high-energy induced collision dissociation(HCD),the fragmentation of the intact glycopeptide in the glycosidic bond and polypeptide backbone is different.However,the information of peptide backbone in the currently obtained glycopeptide secondary spectrum is less.In order to further verify the glycosylation sites,the established high-resolution mass spectrometry analysis method was used and the intact N-glycosylated peptides of the enriched PMSG glycoprotein were analyzed by changing the collision energy.By comparing the experimental results,the two N-glycosylation modification sites of PMSG(Asn 80 on α subunit and Asn 33 on β subunit respectively)were finally determined.The results will provide an experimental basis for further exploring the biological activity of PMSG glycoprotein.