The glycosylation of
human chorionic gonadotropin (hCG) plays an important role in reproductive
tumors. Detecting hCG N-glycosylation alteration may significantly improve the diagnostic accuracy and sensitivity of related
cancers. However, developing an immunoassay directly against the N-linked
oligosaccharides is unlikely because of the heterogeneity and low immunogenicity of
carbohydrates. Here, we report a
hydrogen/
deuterium exchange and MS approach to investigate the effect of N-glycosylation on the binding of
antibodies against different hCG glycoforms. Hyperglycosylated hCG was purified from the urine of
invasive mole patients, and the structure of its N-linked
oligosaccharides was confirmed to be more branched by MS. The binding kinetics of the anti-hCG
antibodies MCA329 and MCA1024 against hCG and hyperglycosylated hCG were compared using biolayer interferometry. The binding affinity of MCA1024 changed significantly in response to the alteration of hCG N-linked
oligosaccharides.
Hydrogen/
deuterium exchange-MS reveals that the
peptide β65-83 of the hCG β subunit is the
epitope for MCA1024. Site-specific N-glycosylation analysis suggests that N-linked
oligosaccharides at Asn-13 and Asn-30 on the β subunit affect the binding affinity of MCA1024. These results prove that some
antibodies are sensitive to the structural change of N-linked
oligosaccharides, whereas others are not affected by N-glycosylation. It is promising to improve
glycoprotein biomarker-based
cancer diagnostics by developing combined immunoassays that can determine the level of
protein and measure the degree of N-glycosylation simultaneously.