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, developed for the treatment of Rh(D) haemolytic disease of the newborn, was performed. Capillary electrophoresis (CE) has been employed for peptide mapping of the IgG heavy chain and glycopeptide identification. The combination of the high resolution and low solvent consumption of CE and the ultrasensitive detection and precise identification properties of mass spectrometry led to a complete glycosylation analysis of the protein. Glycopeptides were easily isolated from a single injection in a 100 microns i.d. capillary of the preparative CE system and collected for molecular mass determination using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The off-line CE-MS characterization revealed the presence of different oligosaccharides linked to the unique N297-S-T glycosylation site of the IgG heavy chain. The differences between calculated and experimental masses of the glycopeptides suggested the presence of a fucosylated biantennary structure containing one or two galactose units as major oligosaccharide, together with similar species bearing a bisecting N-acetylglucosamine. CE conditions were optimized to allow the MS identification of sialylated forms.
Bihoreau
N
N
Laboratoire Français du Fractionnement et des Biotechnologies, Les Ulis, France.
Ramon
C
C
Lazard
M
M
Schmitter
J M
JM
D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000276:immunology; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary; D006020:Glycopeptides / Q000032:analysis; D006026:Glycoside Hydrolases; D006031:Glycosylation; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000032:analysis / Q000302:isolation & purification; D007147:Immunoglobulin Light Chains / Q000032:analysis; D010449:Peptide Mapping; D012204:Rh-Hr Blood-Group System / Q000276:immunology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000911:Antibodies, Monoclonal; D006020:Glycopeptides; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D012204:Rh-Hr Blood-Group System; D006026:Glycoside Hydrolases