A comparative study of glycoprotein concentration,glycoform profile and glycosylation site occupancy using isotope labeling and electrospray linear ion trap mass spectrometry

A strategy is presented for comparative analysis of glycoproteins in which the variation of protein concentration, variation of glycosylation site occupancy and variation of glycoform profile can be determined. A comparative study was performed using stable isotope labeling of glycopeptides and peptides by formaldehyde-H₂ and formaldehyde-D₂ and analysis by ESI-MS analysis. The relative intensity of the nonglycosylated peptide provided information about protein concentration variation. Variation of the glycoform profile was obtained by comparing the glycoform profile of d₀- and d₄-dimethyl labeled glycopeptides. By knowing the variation of protein concentration and the variation of glycoform profile, the variation of glycosylation site occupancy could be calculated. The utility of the proposed strategy was demonstrated with ribonuclease B with different protein concentrations, different levels of glycosylation site occupancy and different glycoform profiles.