Clinical Chemistry
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Clinical Chemistry 43: 1172-1181, 1997;
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(Clinical Chemistry. 1997;43:1172-1181.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Mass spectrometric characterization of nicked fragments of the ß-subunit of human chorionic gonadotropin

Chuanliang Liu1 and Larry D. Bowersa

Athletic Drug Testing and Toxicology Laboratory, Department of Pathology and Laboratory Medicine, Indiana University Medical Center, 635 Barnhill Dr., Indianapolis, IN 46202-5120.
1 Present address: Pacific Northwest National Laboratory, Richland, WA.
a Author for correspondence. Fax (317)278-2018; e-mail lbowers{at}iupui.edu

We describe the use of an HPLC/MS technique for the characterization of nicked fragments of hCG ß-subunit. After reductive alkylation of the nicked hCG ß-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 and45Leu. 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 ß-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.


Key Words: indexing terms: chromatography, liquid • mass spectrometry • peptide sequencing • proline • oligosaccharide microheterogeneity




The following articles in journals at HighWire Press have cited this article:


Home page
 Clin. Chem.Home page
E. S. Jacoby, A. T. Kicman, P. Laidler, and R. K. Iles
Determination of the Glycoforms of Human Chorionic Gonadotropin {beta}-Core Fragment by Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
Clin. Chem., November 1, 2000; 46(11): 1796 - 1803.
[Abstract] [Full Text] [PDF]


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Copyright © 1997 by the American Association for Clinical Chemistry.