Metabolism of the human chorionic gonadotrophin (hCG)- and LHbeta-subunits (hCGbeta, LHbeta) terminates with the urinary excretion of core fragment (hCGbetacf, LHbetacf) molecules that retain antigenic shape and constituent N-linked carbohydrate moieties. We have previously demonstrated the resolved mass spectra of hCGbetacf, from which the carbohydrate moieties present at two N-linked glycosylation sites were identified. LHbetacf was subjected to the same mass spectrometric analysis. As LHbeta shares 82% homology with hCGbeta but possesses only one glycosylation consensus site a simpler spectral fingerprint of LHbetacf glycoforms was expected. LHbetacf was reduced with dithiothreitol and analysed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Glycoforms were predicted by subtracting the peptide mass from the m/z values of the observed peaks and then sequentially subtracting the masses of the monosaccharide residues of hCGbeta N-linked carbohydrates reported in the literature. The mass spectra of LHbetacf revealed a broad single peak ranging from m/z 8700 to 10 700. Following reduction, this peak was replaced by a set of partially resolved peaks between m/z 4130 and 5205 corresponding to glycosylated forms of the peptide LHbeta6-40. A peak at m/z 4252.2 corresponded to the non-glycosylated peptide LHbeta55-93. Remaining peaks indicated that the pooled sample comprised a wide set of glycoforms, contained LHbetacf with two N-linked carbohydrate moieties and indicated evidence of further glycosylation due to amino acid substitution in polymorphic variants. This is evidence that a single nucleotide polymorphism alters the post-translational modification of a protein and hence its structural phenotype.

Although immobilization of antigen-specific immunoglobulins onto matrix-assisted laser desorption/ionization (MALDI) targets allows the specific detection and enrichment of an antigen from complex biological fluids, the process of antibody immobilization is not optimal. The principal reason is that the antibody can bind to the template in various orientations, many of which block antigen recognition. An affinity capture MALDI mass spectrometry methodology was developed by covalently immobilizing an Fc receptor (recombinant protein G) onto MALDI gold targets for the purpose of orientating an immunoglobulin G, with the Fab domains pointing away from the target surface. The pregnancy and cancer marker, human chorionic gonadotropin beta core fragment (hCGbetacf), was our chosen test substance. To optimize the methodology, different surface densities of protein G and immunoglobulin were achieved by employing varying concentrations for immobilization. Captured amounts of hCGbetacf were compared using an external standard (cytochrome c). Orientation of immunoglobulin resulted in an approximately 3-fold increase in MALDI signal compared to using randomly immobilized antibody. Higher antibody concentrations resulted in diminished MALDI signals, which were explained by steric hindrance. Purification and enrichment of hCGbetacf was achieved from a test solution containing contaminant peptides and proteins using oriented immunoglobulins on-target.

BACKGROUNDMetabolism of human chorionic gonadotropin (hCG) in the serum and kidney yields the terminal urinary product hCG beta-core fragment (hCGbetacf), comprising two disulfide-linked peptides (beta6-beta40 and beta55-beta92) of which one (beta6-beta40) retains truncated N-linked sugars. Hyperglycosylated hCGbetacf may indicate choriocarcinoma or Down syndrome, but the glycosylation profile of hCGbetacf has not been thoroughly evaluated.METHODShCGbetacf, purified from pregnancy urine, was reduced by "on-target" dithiothreitol (DTT) reduction and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass ([M+H](+)) of the primary sequence of the glycosylated peptide beta6-beta40 was subtracted from the m/z values of the discrete peaks observed to give the masses of the carbohydrate moieties. Carbohydrate structure was predicted by sequentially subtracting the masses of the monosaccharide residues corresponding to N-linked carbohydrates of the hCG beta-subunit reported in the literature.RESULTSMass spectra of hCGbetacf revealed a broad triple peak at m/z 8700-11300. After reduction, the triple peak was replaced by a discrete set of peaks between m/z 4156 and 6354. A peak at m/z 4156.8 corresponded to the nonglycosylated peptide (beta55-beta92). The remaining nine peaks indicated that urinary hCGbetacf comprises a set of glycoforms smaller and larger than the trimannosyl core.CONCLUSIONShCGbetacf comprises a wider set of glycoforms than reported previously. Peaks of highest mass indicate evidence of hyperglycosylated carbohydrate moieties. The data support previous reports that hCGbetacf oligosaccharides lack sialic acid and galactose residues. No indication was found of a beta6-beta40 peptide that was entirely devoid of carbohydrate.

Prenatal DiagnosisVolume 19, Issue 8 p. 790-792 Letter to the Editor Dimerization of urinary β-core/hCFβcf: a cause of poor β-core assay performance in Down syndrome screening studies Ray K. Iles, Ray K. Iles Williamson Laboratory, St Bartholomew's and the Royal London School of Medicine and Dentistry, St Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, UKSearch for more papers by this authorStephen A. Butler, Stephen A. Butler Williamson Laboratory, St Bartholomew's and the Royal London School of Medicine and Dentistry, St Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, UKSearch for more papers by this authorEli Jacoby, Eli Jacoby Williamson Laboratory, St Bartholomew's and the Royal London School of Medicine and Dentistry, St Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, UKSearch for more papers by this author Ray K. Iles, Ray K. Iles Williamson Laboratory, St Bartholomew's and the Royal London School of Medicine and Dentistry, St Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, UKSearch for more papers by this authorStephen A. Butler, Stephen A. Butler Williamson Laboratory, St Bartholomew's and the Royal London School of Medicine and Dentistry, St Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, UKSearch for more papers by this authorEli Jacoby, Eli Jacoby Williamson Laboratory, St Bartholomew's and the Royal London School of Medicine and Dentistry, St Bartholomew's Hospital, West Smithfield, London, EC1A 7BE, UKSearch for more papers by this author First published: 18 August 1999 https://doi.org/10.1002/(SICI)1097-0223(199908)19:8<790::AID-PD627>3.0.CO;2-ZCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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