Determination of the glycoforms of human chorionic gonadotropin beta-core fragment by matrix-assisted laser desorption/ionization time-of-flight massspectrometry

Background: Metabolism of human chorionic gonadotropin (hCG) in the serum a nd kidney yields the terminal urinary product hCG beta -core fragment (hCG beta cf), comprising two disulfide-linked peptides (beta6-beta 40 and beta 55-beta 92) of which one (beta6-beta 40) retains truncated N-linked sugars. Hyperglycosylated hCG beta cf may indicate choriocarcinoma or Down syndrom e, but the glycosylation profile of hCG beta cf has not been thoroughly eva luated. Methods: hCG beta cf, purified from pregnancy urine, was reduced by "on-tar get" dithiothreitol (DTT) reduction and analyzed by matrix-assisted laser d esorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The m ass ([M+H](+)) of the primary sequence of the glycosylated peptide beta6-be ta 40 was subtracted from the m/z values of the discrete peaks observed to give the masses of the carbohydrate moieties. Carbohydrate structure was pr edicted by sequentially subtracting the masses of the monosaccharide residu es corresponding to N-linked carbohydrates of the hCG beta -subunit reporte d in the literature. Results: Mass spectra of hCG beta cf revealed a broad triple peak at m/z 87 00-11300. After reduction, the triple peak was replaced by a discrete set o f peaks between m/z 4156 and 6354. A peak at m/z 4156.8 corresponded to the nonglycosylated peptide (beta 55-beta 92). The remaining nine peaks indica ted that urinary hCG beta cf comprises a set of glycoforms smaller and larg er than the trimannosyl core. Conclusions: hCG beta cf comprises a wider set of glycoforms than reported previously. Peaks of highest mass indicate evidence of hyperglycosylated ca rbohydrate moieties. The data support previous reports that hCG beta cf oli gosaccharides lack sialic acid and galactose residues. No indication was fo und of a beta 36-beta 40 peptide that was entirely devoid of carbohydrate. (C) 2000 American Association for Clinical Chemistry.