Mass spectrometric characterization of the beta-subunit of human chorionic gonadotropin.
A high-performance liquid chromatographic/electrospray mass spectrometric (HPLC/MS) technique is described for the characterization of the beta-subunit of the glycopeptide human chorionic gonadotropin (hCG). The beta-subunit of hCG was dissociated from the alpha-subunit using 0.1% trifluoroacetic acid (TFA) and separated by reversed-phase HPLC using a 0.1% TFA-acetonitrile gradient. Although reductive alkylation with 4-vinylpyridine allowed direct observation of the intact beta-subunit of hCG by HPLC/MS due to the increase in charge, the heterogeneity of the carbohydrate fractions resulted in poor detection limits and extremely complex spectra. After reductive alkylation with either iodoacetate or 4-vinylpyridine, tryptic fragments of either the alpha- or beta-subunit can be observed using reversed-phase HPLC/MS. HPLC/MS data were consistent with the reported primary sequence, although oligosaccharide attachment sites at both 127Ser and 132Ser could not be documented. Microheterogeneity of the carbohydrate moiety on both N-glycosylation sites on the beta-subunit could be readily observed. A larger degree of heterogeneity was observed on 13Asn. Differences were also observed in the oligosaccharide distribution in three commercial preparations of hCG. Detection of the C-terminal portion of the beta-subunit required enzymatic deglycosylation prior to HPLC/MS analysis.
Liu
C
C
Department of Pathology and Laboratory Medicine, Indiana University Medical Center, Indianapolis 46202-5120, USA.
Bowers
L D
LD
D006063:Chorionic Gonadotropin / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D009439:Neuraminidase / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D006063:Chorionic Gonadotropin; D006020:Glycopeptides; D009844:Oligosaccharides; D010446:Peptide Fragments; D006596:Hexosaminidases; D009439:Neuraminidase; C060638:glycopeptide alpha-N-acetylgalactosaminidase; D014357:Trypsin