Comparative study of sialyl glycoprotein with multiple glycosylation sites using isotope labeling and capillary...

A comparative strategy has been demonstrated using RNase B, a single-site N-linked high-mannose glycoprotein. Glycoproteins are more common with multiple glycosylation sites and with complex glycans. A strategy capable of differentiating the changes caused by glycoprotein concentration, glycosylation site occupancy, and a glycoform profile of complex glycoproteins would be beneficial.

Tryptic-digested glycoproteins were labeled using ¹² C,H-formaldehyde and ¹³ C, D-formaldehyde, purified, and then analyzed using capillary reversed-phase liquid chromatography/mass spectrometry (RPLC/MS). The relative intensity of non-glycosylated peptides provided information on glycoprotein concentration variation. A site-specific glycoform profile variation was obtained by comparing the glycoform profile of CH₂ and ¹³ CD₂ glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated.

A strong correlation between the observed and prepared ratios for fetuin glycopeptides from 0.2 to 5 was obtained. Two fetuin samples with different glycoprotein concentrations (4-fold change), glycoform profiles (normal and modified), and glycosylation site occupancies (100% and 50%) were prepared, labeled, mixed, purified, and analyzed using RPLC/MS. The results of the comparative study had a strong correlation with the prepared values.

In this report, we demonstrated a comparative analysis of fetuin, a glycoprotein with multiple glycosylation sites and complex sialyl glycans. Compared to our previous approach, we made several modifications including the use of RPLC, a larger mass difference isotope tag, and isotope overlapping correction. The modified approach is expected to be applicable to most glycoproteins. Copyright © 2013 John Wiley & Sons, Ltd.