STRUCTURAL CHARACTERIZATION OF SITE-SPECIFIC N-GLYCOSYLATION OF RECOMBINANT HUMAN FACTOR-VIII BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

The present study addresses the site occupancy and the site-specific c arbohydrate microheterogeneity of N-linked oligosaccharides in recombi nant human factor VIII, expressed in Chinese hamster ovary cells. The four factor VIIIa polypeptides, formed upon incubation with human thro mbin, were isolated and separately subject to proteolysis with trypsin . These tryptic digests were analyzed by reversed-phase high-performan ce liquid chromatography/electrospray ionization mass spectrometry. Se lected ion monitoring of diagnostic carbohydrate ions was utilized to identify glycopeptide-containing chromatographic peaks. Oligomannose a nd complex carbohydrates were detected at the glycosylation sites of t he 50 and 73 kDa polypeptides, while all the oligosaccharides identifi ed on the B-domain were complex-type structures. Only the 43 kDa polyp eptide was found nonglycosylated. These studies established a biantenn ary core-fucosylated carbohydrate as the major substituent, consistent with the conclusions of the analyses on the entire N-linked carbohydr ate pool (Kumar, H. P. M.; Hague, C.; Haley, T.; Starr, C. M.; Besman, M. J.; Lundblad, R.; Baker, D. Biotechnol. Appl. Biochem. 1996, 24, 2 07-216.). In addition, this mass spectrometric investigation revealed the presence of a complex nonfucosylated oligosaccharide not reported previously for this glycoprotein.