A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrix-assisted laser desorption/ionization mass spectrometry.
N-linked oligosaccharides were released from human and bovine polyclonal immunoglobulin G (IgG) obtained from commercial sources and also from a monoclonal IgG(1) secreted by murine B-lymphocyte hybridoma cells (CC9C10) grown under different serum-free conditions. These conditions differed according to their steady-state dissolved oxygen concentrations. This work is based on a previous quantitative study where released glycans were characterized by fluorophore-assisted carbohydrate electrophoresis (FACE) and high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) (J. P. Kunkel, D. C. H. Jan, J. C. Jamieson, and M. Butler, 1998, J. Biotechnol. 62, 55-71). In the present article, peptide-N-glycosidase F-released glycans from different species of polyclonal IgG and murine monoclonal IgG were characterized qualitatively by high-performance liquid chromatography (HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). The glycans were also analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). The MALDI mass spectrometer used allowed acquisition of MS and tandem MS data, which were useful in structural investigations at a more detailed level than allowed by FACE and HPAEC-PAD. Predominant N-linked structures, as determined by all techniques, were core-fucosyl asialyl biantennary chains with varying galactosylation. Minor amounts of afucosyl, bisected, and monosialyl oligosaccharides were also detected. In contrast to FACE and HPAEC-PAD, MALDI-double quadrupole/time-of-flight MS and HPLC/ESI-MS also detected low-abundance high-mannose and hybrid structures in some of the species under investigation.
Saba
Julian A
JA
Department of Chemistry, University of Manitoba, 144 Dysart Road, Winnipeg, Manitoba R3T 2N2, Canada.
Kunkel
Jeremy P
JP
Jan
David C H
DC
Ens
Werner E
WE
Standing
Kenneth G
KG
Butler
Michael
M
Jamieson
James C
JC
Perreault
Hélène
H
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D000983:Antipyrine / Q000031:analogs & derivatives / Q000737:chemistry; D002240:Carbohydrate Sequence; D002417:Cattle; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange; D000077553:Edaravone; D004586:Electrophoresis; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010100:Oxygen / Q000032:analysis; D011134:Polysaccharides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D011134:Polysaccharides; D000077553:Edaravone; D010100:Oxygen; D000983:Antipyrine