Identification of post-translational modifications resulting from LHbeta polymorphisms by matrix-assisted laser desorption time-of-flight mass spectrometric analysis of pituitary LHbeta core fragment.

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.

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

BACKGROUND: Metabolism 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. METHODS: hCGbetacf, 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. RESULTS: Mass 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. CONCLUSIONS: hCGbetacf 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.