Comparative studies of Protein Glycosylation Using Isotope Labeling and Electrospray Ion Trap Mass Spectrometry

In the analysis of glycoprotein, glycoprotein was often digested with trypsin before MS analysis. Several causes including glycoprotein concentration, glycosylation site occupancy and glycan profile may account for the difference if the signal of a specific glycopeptide was found to be different. A strategy was proposed for comparative analyses of glycoprotein in which the change of glycoprotein concentration, the change of glycosylation site occupancy and the change of glycan profile could be differentiated. Comparative analysis was performed by stable isotope labeling using formaldehyde-d0 and formaldehyde-d2 along with cellulose microcrystalline enrichment and mass spectrometry analysis. The utility of the proposed strategy was demonstrated with ribonuclease B. The change of glycoprotein concentration was studied using samples prepared with different quantity of ribonuclease B. Level of glycosylation site occupancy was studied by mixing different ratios of ribonuclease B and ribonuclease A. The change of the site-specific glycan profile was studied by mixing ribonuclease B treated with α-mannosidase and intact ribonuclease B. ESI-MS analysis of glycopeptides was performed on a linear ion trap mass spectrometer. With the capability of MS2 and MS3 of the instrument, the site of glycosylation and the glycan sequence may also be obtained.