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.

Background: Metabolism of human chorionic gonadotropin (hCG) in the serum and kidney yields the terminal urinary product hCG β-core fragment (hCGβcf), comprising two disulfide-linked peptides (β6-β40 and β55-β92) of which one (β6-β40) retains truncated N-linked sugars. Hyperglycosylated hCGβcf may indicate choriocarcinoma or Down syndrome, but the glycosylation profile of hCGβcf has not been thoroughly evaluated. Methods: hCGβcf, 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 β6-β40 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 β-subunit reported in the literature. Results: Mass spectra of hCGβcf 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 (β55-β92). The remaining nine peaks indicated that urinary hCGβcf comprises a set of glycoforms smaller and larger than the trimannosyl core. Conclusions: hCGβcf 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 hCGβcf oligosaccharides lack sialic acid and galactose residues. No indication was found of a β6-β40 peptide that was entirely devoid of carbohydrate.

Metabolism of the human chorionic gonadotrophin (hCG)-and LHβ-subunits (hCGβ, LHβ) terminates with the urinary excretion of core fragment (hCGβcf, LHβcf) molecules that retain antigenic shape and constituent N-linked carbohydrate moieties. We have previously demonstrated the resolved mass spectra of hCGβcf, from which the carbohydrate moieties present at two N-linked glycosylation sites were identified. LHβcf was subjected to the same mass spectrometric analysis. As LHβ shares 82% homology with hCGβ but possesses only one glycosylation consensus site a simpler spectral fingerprint of LHβcf glycoforms was expected. LHβcf 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 hCGβ N-linked carbohydrates reported in the literature. The mass spectra of LHβcf 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 LHβ6-40. A peak at m/z 4252·2 corresponded to the non-glycosylated peptide LHβ55-93. Remaining peaks indicated that the pooled sample comprised a wide set of glycoforms, contained LHβcf 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.

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