New Method And Application For Identification Of N-glycan By Mass Spectrometry

Glycosylation is one of the most important post-translational modification in the animal system.Glycan presenting on the cell-cell interface are linked to many functionally important proteins,such as enzymes,immune receptors and growth factors.There is increasingly evidence that the glycans coupling with their attached proteins play an important role in cell growth and development,tumor metastasis,immune recognition,cell-cell communication and microbial pathogenesis.The glycan structures with numerous anomeric linkages and branching could be greater diverse than linear nucleic acid and polypeptide structure.The diversity of the glycan structure arises from the nontemplatebased biosynthesis pathway which involves many glycosyltransferases and isoforms of these enzymes.Competition between glycosyltransferases for the same glycan substrates can strongly influence the relative abundance of specific glycan structure in the total glycome.There have many technical challenges in identification of glycosylation structure,sites and relative ratio of the specific structure.In thesis,the sialylated Nglycans and their isomers was derivatized on the solid phase agarose and analyzed by matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS)for funding the disease related biomarkers.In addition,we explored to use the chemical-enzyme and solid phase method to enrich the N-glycan peptide.MALDI-MS has been a major approach for glycan analysis.However,the preferential cleavage of the sialic acid moiety by in-and post-source decay influences the determination of sialylated glycans by MALDI-MS.Among reported derivatization methods,methylamidation could completely derivatize both ?(2,3)and ?(2,6)sialic acids.Here,a novel derivatization method was developed,in which proteins were conjugated on the solid phase support in order to stabilize the sialic acid by methylamidation.The results showed that solid phase methylamidation could reduce sample lost and contamination.The amount of fetuin glycan could be identified as low as 0.5 ?g on each MALDI spot.When compared with the conventional methylamidation method,the solid phase method would enhance the sensitivity and signal to noise ratio.The developed method was also applied to the N-glycan profiling of human serum from patient and healthy volunteer and some noticeable alterations of the core-fucosylated structures were also observed in the high mass region of the spectrum.HPLC-fluorescence detection verified the potential of the method in identification of glycan biomarkers.On the foundation of solid phase methylamidation,we further developed a method to achieve identification of sialylated isomers.After the glycoprotein conjugated to the solid phase agarose,two step derivatization method was introduced to convert the ?(2,6)-linked sialic acid into ethyl esters and the ?(2,3)-inked counterparts into N-methyl amides,respectively.Ultimately,the ethyl esterified ?(2,6)-linked sialic acids was with a mass increment of 28 Da,and the methylamidated ?(2,3)-linked sialic acids was with a mass shift of 13 Da.The sialic linkage could be distinguished through the 15 Da difference in molecular mass.The new method overcome the instability of ?(2,3)-lactones and improve the reproducibility.When applied our method to analysis monoclonal antibody drug,we found that the sialic acids of the antibody were all with ?(2,3)-linkage.Finally,we used the solid phase two derivatization method to investigate the biomarker of hepatocellular carcinoma.Through the receiver operating characteristic(ROC)curve analysis,we identified 6 overall glycan abundance related biomarkers and 2 sialylated glycan isomer related glycans.Compared with PNGase F,endoglycosidase H(ENDO-H)could identify specific type glycans,such as high-mannose and hybrid glycan.we identified RNAse B and avidin glycan with using the endoglycosidase H.In addition,there would retain one GlcNAc residue on the glycopeptide.The glycopeptide was enriched by combining of the chemical-enzyme and solid phase method.The enriched peptide was with characteristic disaccharide label which was beneficial to confirm the glycopeptide.Finally,our method identified the glycan structure and peptide at the same time.In our thesis,we achieved the derivatization of sialic acids on the solid phase agarose and identification of the sialylated isomers.As the derivatives were more stable than the previous method,our method was able to achieve accurate relative quantification of Nglycan as well as their corresponding sialylated isomers.The glycan structure and glycopeptide were initially analyzed with using the endoglycosidase H.The identification of monoclonal drug and liver cancer samples demonstrated that our method had great potential in the area of pharmaceutical industry and funding glycan related biomarkers.