The Strategy Development For Structure Elucidation Of Glycans On Glycoproteins Based On Mass Spectrometry

Protein glycosylation plays an important role in the physiological and pathological. But the analytical methods for the glycan structure of glycoproteins are still under developing. Site-specific glycoform structures altered significantly among different patho- and physiological processes. The tranditional PNGase F digestion approch is hard to link glycosites to differnet glycoforms, therefore, site-specific glycoform structure analysis has became an important goal of glycoproteome researches.This paper has three chapters, the first chapter, we introduced the present status on glycoproteomics research and the importance of glycosylation sites and glycoform structural analysis. And we also briefly reviewd the acquisition method and strategies for glycosylation sites, glycoforms and intact glycopeptides.The second chapter introduces the immobilized Pronase E digestion of glycoprotein, and the establishment of methods for site-specific glycoform structural analysis. In the study, we monitored the in-solution Pronase E digestion of proteins and the ratio of enzyme to proteinsused, digestion time, material selection for Pronase E immobilized enzyme, and the storage time of immobilized enzyme. We compared two enrichment methods between the the hydrophilic interaction chromatography and porous graphitized carbon, and the use of α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxybenzoic acid as matrix. In addition, the differences of MALDI-TOF instruments and Q-Exactive instrument for the detection of the glycoform structure were compaired. Finally, we chose protein digestion enzyme ratio of 1:1 or more, 48 h on Pronase E in solution; selected CNBr Beads as Pronase E immobilized material, and the enzymes and materials mass ratio 1:10. The binding efficiency of Pronase E to the the immobilizing beads was 40%, and the digestion duration for 24 h. We selected the porous graphite carbon enrichment, DHB as matrix for the glycans generated by Pronase E digestion. And DHB + 1 mM NaCl was used as the substrate for the PNGase F released glycans when subjected to MALDI-TOF detection.The third chapter, we established an analysis strategies to obtain qualitative and quantitative of intact glycopeptides from the high-abundance serum glycoproteins. Using IgG as standard glycoproteins, after trypsin digestion and HILIC enrichment, the intact glycopeptides were obtained. PNGase F digestion helped to obtain information of glycosites, combined with the information of intact glycopeptide, together with Byonic(Proteomics Discover 2.0) and data integration softwares, we obtained the glycosites and glycoforms information. The site-specific glycopeptide quantitative information was botained using the intensity information. This method is applied on the analysis of 11 high abundance glycoproteins in human serum, and the qualitative and quantitative information of the intact glycopeptids were obtained. The work demonstrated the components of site-specific glycoforms, and paved a way for the exploration of glycan disease biomarkers.