Development And Application Of Proteomics Technology For Identification And Quantitation Of Glycopeptides

Glycosylation is one of the most common post-translational modifications(PTMs)of proteins with complex structures and diverse functions.It plays an important role in the biological function of proteins,and the alternation of glycoprotein has a close relationship with the occurrence and development of many diseases.Site-specific analysis of the structure of the glycopeptide is of great significance to reveal the biological function of glycoprotein.The traditional approach in glycoproteome is to cleave the glycans from glycopeptides,and to derive the site of glycosylation through deamination of Asn.The approach lose the glycan chain information on a glycosites,and the naturally occurred deamination of non-glycosylated peptides also cause false assignment.Therefore,it is necessary to develop a strategy to characterize glycoprotein structure at the level of intact glycopeptide with large scale and high confidence.This paper consists of three chapters.In the first chapter,the effects of glycosylation on protein function and the structural characteristics of glycosylation were introduced.The common methods used to separate and enrich glycopeptides and the mass spectrometry-based detection methods were summarized.Finally,the biological significance of protein core fucosylation(CF)was also introduced.In chapter 2,we introduce the establishment of a strategy that combining fragment ion filtering with sequential endoglycosidase processing for the high confident identification of intact glycopeptides.For this work,we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples.In the first step,tryptic intact glycopeptides were enriched using ZIC-HILIC.Secondly,a portion of the glycopeptides was treated with endoglycosidase H(Endo H)to remove high mannose(Man)and hybrid-type N-linked glycans.Thirdly,a fraction of the Endo Htreated glycopeptides was further subjected to PNGase F treatment in 18 O water to remove the remaining complex glycans.The intact glycopeptides and the two-step of deglycosylated peptides were analyzed by nano-RPLC-MS/MS,and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools.Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptides,thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification.We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line.We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells.The intensity of different glycoforms at a specific glycosite was obtained to obtain the occupancy ratios of site-specific glycoforms.These results indicate that our method can be used for characterizing sitespecific protein glycosylation in complex samples,both qualitatively and quantitatively.In the third chapter,the identification strategy of CF peptides was established.The peptide obtained from protein digestion were first enriched by HILIC and then the CF peptide was enriched from the obtained glycopeptide using LCH lectin and then digested with Endo F3,and finally detected by LC-MS.The CF peptides sequences was obtained through the pFind database search.In this work,the method was first applied to the identification of CF peptides in liver and serum samples from C57 mice.The results showed good reproducibility.Then this method is applied to the identification of the CF protein in the serum samples from healthy human,liver cirrhosis and liver cancer patients,a total of 405 proteins and 879 peptides with core fucose-modified proteins were identified from 18 samples.We confirmed the high level of CF modification on AFP for HCC patients.And also,the CF modification level of GP73,PON1 etc.were found to be changed in HCC or liver cirrhosis patients,and provides novel candidates for serum biomarker discovery.