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Mass Spectrometric Characterization of the β‐Subunit of Human Chorionic Gonadotropin
Author(s) -
Liu ChuanLiang,
Bowers Larry D.
Publication year - 1997
Publication title -
journal of mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.475
H-Index - 121
eISSN - 1096-9888
pISSN - 1076-5174
DOI - 10.1002/(sici)1096-9888(199701)32:1<33::aid-jms446>3.0.co;2-x
Subject(s) - chemistry , high performance liquid chromatography , chromatography , oligosaccharide , mass spectrometry , glycosylation , protein subunit , trifluoroacetic acid , reversed phase chromatography , human chorionic gonadotropin , alkylation , biochemistry , hormone , catalysis , gene
A high‐performance liquid chromatographic/electrospray mass spectrometric (HPLC/MS) technique is described for the characterization of the β‐subunit of the glycopeptide human chorionic gonadotropin (hCG). The β‐subunit of hCG was dissociated from the α‐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 β‐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 α‐ or β‐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 127 Ser and 132 Ser could not be documented. Microheterogeneity of the carbohydrate moiety on both N‐glycosylation sites on the β‐subunit could be readily observed. A larger degree of heterogeneity was observed on 13 Asn. Differences were also observed in the oligosaccharide distribution in three commerial preparations of hCG. Detection of the C‐terminal portion of the β‐subunit required enzymatic deglycosylation prior to HPLC/MS analysis. © 1997 by John Wiley & Sons, Ltd.