Purification Of N-Glycans Or Glycopeptides And Quantitative Analysis Of Immunoglobulin G Fc Glycosylation

N-linked glycosylation of protein is a fundamental and important post-translational modification that resulting from the covalent attachment of a glycan onto specific asparagine residues of polypeptide chains.Glycans play an important role in indirect function by regulating the properties(such as conformation)of the proteins as well as molecular recognition between glycan and its receptor,including cellular adhesion,signal transduction,and immune response.At present,abnormal glycosylation analysis,structural characterization and biofunctional studies of N-glycans attract a wide attention.The availability of structurally defined N-glycan or N-glycopeptide libraries is essential for structural and functional glycomics and accurate quantitative analysis of glycosylation.In this dissertation,the two-dimensional liquid chromatography separation and purification method of N-glycan/glycopeptide was developed based on the HILIC stationary phase independently-developed by our group.Combined with tandem mass spectrometry,purification and structural characterization of natural N-glycan/glycopeptide compounds were achieved.Based on the well-defined N-glycan and glycopeptide standards,glycan microarray analysis and the accurate quantitative analysis of glycopeptides in human sera and monoclonal antibody based on mass spectrometry were carried out.Firstly,a purification strategy was established based on two-dimensional liquid chromatography for the acquisition of underived neutral N-glycan standards from natural source.A total of thirty-one N-glycan compounds including seven pairs of isomers and five unreported glycan structures with the amounts from 1.3-254.3 μg were isolated from ovalbumin as the model glycoprotein.The tag-free glycan compounds with well-defined structures,purity and amounts were finally assembled on the glass slide through neoglycolipid technology.Higher binding affinity of hybrid-type N-glycans with a bisecting GlcNAc residue with biotinylated wheat germ agglutinin lectin indicated the potential of the established strategy in application for structural and functional glycomics.Secondly,a two-dimensional hydrophilic purification system was developed.Combined with tandem mass spectrum,a total of eleven IgG-1 and nine IgG-2 Fc-glycopeptides from model human IgG protein were purified and characterized.Absolute amounts of the purified glycopeptide standards were achieved according to the principle of molar equivalence between the glycopeptides and its deglycosylated peptides.The amount of the purified glycopeptides was from 1.07 to 335.69 μg,which was acquired by the corresponding molar concentrations of deglycosylated peptides through internal standard curve method.Further,based on the acquired IgG-1 Fc-glycopeptide standards,an absolute quantitation analysis of high abundant IgG-1 glycopeptides from pooled human sera was developed and applied to the determination and comparison of 11 high abundant IgG-1 Fc-glycopeptides between acute myocardial infarction patients and healthy persons.Compared to conventional relative quantitation by using peak area normalization,this calibrated method overcome the difference MS response of glycopeptide with different glycoforms and provided more accurate distribution profiles of 11 high abundant Fc-glycopeptides.In order to expand the standard library of Fc-glycopeptide,a modified two-dimensional HILIC purification method was developed to purified low abundant glycopeptides from IgG1 Fc fusion protein.Additional fifteen glycopeptides with mAbs specific glycoforms were purified and characterized by negative ESI-MS/MS.Taking advantage of the common twenty-six IgG-1 Fc-glycopeptides,the differences of different MS-based quantitative methods of Fc-glycopeptides from trastuzumab were systematically compared.The introduction of Fc-glycopeptide calibration standard has remarkably improved the accuracy and transferability of calibrated HILIC-MRM method for Fc-glycopeptide.Individual amount of glycopeptides,degree of glycosylation and glycosylation site coverage of six IgG1 monoclonal antibodies were achieved.This strategy may contribute to the consistency evaluation of IgG1-type therapeutic mAbs.