Comparative study of sialyl glycoprotein with multiple glycosylation sites using isotope labeling and capillary liquid chromatography/mass spectrometry.

RATIONALE 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. METHODS Tryptic-digested glycoproteins were labeled using 12C,H-formaldehyde and 13C, 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 13CD2 glycopeptides. Determining the protein concentration and glycoform profile variations allows the glycosylation site occupancy variation to be calculated. RESULTS 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. CONCLUSIONS 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. [ABSTRACT FROM AUTHOR]