Mass spectrometric characterization of nicked fragments of the beta-subunit of human chorionic gonadotropin.
We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG beta-subunit. After reductive alkylation of the nicked hCG beta-subunit with vinylpyridine, endoproteinase Glu-C or trypsin was used to digest the protein to produce peptides that could be analyzed by HPLC/electrospray ionization MS. Human leukocyte elastase digestion was used to produce an experimentally nicked hCG. Two nicking sites were observed, between amino acids 42Thr and 43Arg and between 44Val and 45Leu. The former site has not been previously reported for elastase digestion. The structures of the fragments were confirmed by HPLC/MS after removal of the oligosaccharide by direct mass measurement and by mass determination of their proteolytic digests. Without the glycopeptidase treatment, the microheterogeneity of the two N-linked oligosaccharides could be deduced from the spectra of the proteolytic fragments. Nicking with elastase was found to alter the oligosaccharide structures. Nicked beta-subunit samples isolated from the urine of choriocarcinoma patients were also analyzed and the location of the nicking site(s) agreed with that determined by classical techniques. Important differences in the oligosaccharide structures were also observed in these samples, including the presence of triantennary oligosaccharides not found in hCG from healthy subjects. These findings demonstrate the potential of HPLC/MS for characterization of glycoprotein standard preparations.
Liu
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Department of Pathology and Laboratory Medicine, Indiana University Medical Center, Indianapolis 46202-5120, USA.
Bowers
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LD
D000478:Alkylation; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002822:Choriocarcinoma / Q000652:urine; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000032:analysis / Q000737:chemistry* / Q000652:urine; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry; D011247:Pregnancy; D011725:Pyridines / Q000737:chemistry; D012697:Serine Endopeptidases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014594:Uterine Neoplasms / Q000652:urine
D018997:Chorionic Gonadotropin, beta Subunit, Human; D009844:Oligosaccharides; D011725:Pyridines; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D019272:Leukocyte Elastase; D014357:Trypsin