COMBINATION OF CAPILLARY ELECTROPHORESIS AND MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY FOR GLYCOSYLATION ANALYSIS OF A HUMAN MONOCLONAL ANTI-RHESUS(D) ANTIBODY

Characterization of a human anti-Rhesus(D) monoclonal antibody, develo ped for the treatment of Rh(D) haemolytic disease of the newborn, was performed, Capillary electrophoresis (CE) has been employed for peptid e mapping of the Ige heavy chain and glycopeptide identification. The combination of the high resolution and low solvent consumption of CE a nd the ultrasensitive detection and precise identification properties of mass spectrometry led to a complete glycosylation analysis of the p rotein. Glycopeptides were easily isolated from a single injection in a 100 mu m I.D. capillary of the preparative CE system and collected f or molecular mass determination using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The off-line CE-MS character ization revealed the presence of different oligosaccharides linked to the unique N-297-S-T glycosylation site of the IgG heavy chain. The di fferences between calculated and experimental masses of the glycopepti des suggested the presence of a fucosylated biantennary structure cont aining one or two galactose units as major oligosaccharide, together w ith similar species bearing a bisecting N-acetylglucosamine, CE condit ions were optimized to allow the MS identification of sialylated forms . (C) 1997 Elsevier Science B.V.