Screening neutral and acidic IgG N-glycans by high density electrophoresis.
IgG carries bi-antennary N-linked glycans which differ in degrees of galactosylation, core fucosylation and bisecting N-acetyl glucosamine. The majority of these are non-sialyated closely related neutral structures which can be resolved by HPLC analysis, but which are difficult to separate in techniques such as fluorophore-coupled carbohydrate electrophoresis. Derivatisation with the singly charged fluorophore, 2-amino benzoic acid and separation in gels with a 30% monomer content in tris/glycine buffer enabled separation of neutral glycans. In particular, agalactosyl glycans with either a core fucose substitution or bisecting N-acetyl galactosamine could be resolved. Good separation of mono- and di-galactosylated glycans was also achieved with this system. It was shown that IgG can be separated from serum by size-exclusion and anion exchange chromatography with minimal contamination, with complete glycan release accomplished by the enzyme peptide-N-glycosidase F (F. meningosepticum). This method of resolving IgG glycans could be used to monitor patients in which glycosylation changes may have a diagnostic value, as in rheumatoid arthritis. It could also be used to monitor recombinant IgG glycosylation where routine screening is required in the biotechnology industry.
Frears
E R
ER
Academic Unit of Musculoskeletal Disease, St. George's Hospital Medical School, London. e.frears@sghms.ac.uk
Merry
A H
AH
Axford
J S
JS
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D011134:Polysaccharides / Q000097:blood / Q000737:chemistry*; D062367:ortho-Aminobenzoates
D005456:Fluorescent Dyes; D007074:Immunoglobulin G; D011134:Polysaccharides; D062367:ortho-Aminobenzoates; C031385:anthranilic acid