Endoglycosidase-mediated incorporation of 18O into glycans for relative glycan quantitation.
ABSTRACT Stable isotopic labeling coupled with mass spectrometry analysis is a promising method of detecting quantitative variations in glycans, which may result in aberrant glycosylation in many disorders and diseases. Although various isotopic labeling methods have been used for relative glycan quantitation, enzymatic (18)O labeling, which offers advantages for glycomics similar to those by protease-catalyzed (18)O labeling for proteomics, has not been developed yet. In this study, endoglycosidase incorporated (18)O into the N-glycan reducing end in (18)O-water as N-glycans were released from glycoproteins, rendering glycan reducing-end (18)O labeling (GREOL) a potential strategy for relative glycan quantitation. This proposed method provided good linearity with high reproducibility within 2 orders of magnitude in dynamic range. The ability of GREOL to quantitatively discriminate between isomeric hybrid N-glycans and complex N-glycans in glycoproteins was validated due to the distinct substrate specificities of endoglycosidases. GREOL was also used to analyze changes in human serum N-glycans associated with hepatocellular carcinoma.