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Human α‐fetoprotein produced from hep G2 cell line: Structure and heterogeneity of the oligosaccharide moiety
Author(s) -
Ferranti Pasquale,
Pucci Pietro,
Marino Gennaro,
Fiume Immacolata,
Terrana Benedetto,
Ceccarini Costante,
Malorni Antonio
Publication year - 1995
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/jms.1190300415
Subject(s) - chemistry , oligosaccharide , moiety , mass spectrometry , chromatography , fast atom bombardment , glycosylation , glycoprotein , fucose , residue (chemistry) , hydrolysis , peptide , high performance liquid chromatography , molecular mass , biochemistry , stereochemistry , enzyme
Abstract The carbohydrate moiety of human α‐fetoprotein, an RMM 67000 glycoprotein produced in a hepatoma cell line (Hep G2), was investigated by the combined use of high‐resolution chromatographic techniques and mass spectrometry. Fast atom bombardment mass spectrometric (FABMS) and reversed‐phase high‐performance liquid chromatographic analysis of α‐fetoprotein obtained from a large‐scale cell culture following tryptic and peptide N ‐glycanase F hydrolysis demonstrated that the protein contains a single glycosylation site at level of asparagine 232. Further, electrospray mass spectrometric measurement of the intact protein molecular mass showed that two main glycoforms are present. The complete definition of the structural heterogeneity of the oligosaccharide moiety was achieved by high‐performance anion‐exchange chromatography with pulsed amperometric detection together with carbohydrate mapping by FABMS of the released oligosaccharides demonstrating (i) that the glycosylation produced by cell culture is of the biantennary complex type typical of human hepatoma α‐fetoprotein and (ii) the presence of two main structures in a ratio of about 2:1 differing in the presence of a fucose residue in the N ‐acetylglucosamine in the non reducing portion of the molecule.