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

Abstract

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.

Direct Analysis of hCGβcf Glycosylation in Normal and Aberrant Pregnancy by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

“…In order to determine the masses of the carbohydrate moieties, a previously described method was used [28,39]: reduced hCGβcf spectra were calibrated by using the β 55–92 non-glycosylated peptide as described above. The inferred masses were determined by subtracting the mass of the glycosylated peptide of hCGβcf (β 6–40), which was calculated from its given primary sequence at a mass of 3752.4 Da.…”

Direct and rapid mass spectral fingerprinting of maternal urine for the detection of Down syndrome pregnancy

“…Recently, we have used MALDI-ToF MS to identify variation in the glycosylation that occurs in human chorionic gonadotrophin beta core fragment (hCGβcf), following reports of an increase in hCG hyper glycosylation in Down syndrome pregnancies [ 21 - 23 ]. We adopted a non-direct approach to analyse molecules in the high-resolution range of 3,000 -5,000 m/z [ 22 , 24 ]. Differences in the mass spectral profile in this mass region were indicative of disorders in pregnancy [ 25 ].…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2003–2004

“…Sometimes, with large glycopeptides, peaks are poorly resolved but can often be improved by chemical treatments. Thus, for example, the core fragment of human luteinizing hormone gave a broad, poorly resolved peak (m/z 8,700-10,700) when examined from sinapinic acid with a linear TOF instrument (Jacoby, Kicman, & Iles, 2003). The compound consisted of two peptide chains, only one of which was glycosylated, linked with four disulfide bonds.…”

“…MALDI-TOF effective for determination of glycoprotein (Mokrzycki-Issartel, et al, 2003) Luteinizing hormone Human DTT L-TOF (Sinapinic acid), Glycoprotein Complex, truncated, Asn-30 Ident. of glycans resulting from LHβ polymorphisms (Jacoby, et al, 2003) Luteinizing (Bousfield, et al, 2004) Lyc e 2 (Westphal, et al, 2003) Lysozyme mutant (G49N)…”