Immobilisation of antibodies in gels allows the improved release and identification of glycans.
Human IgG and IgM, bovine IgM and three therapeutic IgG monoclonal antibodies have been separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Carbohydrates were then released from these immobilised proteins by direct enzymatic digestion, derivatised with a highly fluorescent probe and analysed by high performance liquid chromatography and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. This procedure not only allowed measurement of the purity of the intact antibodies but also provided detailed analysis of the complex mixtures of oligosaccharides covalently attached to these glycoproteins. The methodology out-lined allows the simultaneous processing of a number of glycoproteins separated on one single gel. In contrast to the release of carbohydrate from glycoproteins in solution, this procedure can also be conveniently applied when only impure glycoprotein is available.
Charlwood
J
J
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK.
Skehel
J M
JM
Camilleri
P
P
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D005782:Gels; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007075:Immunoglobulin M / Q000737:chemistry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D020543:Proteome; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000906:Antibodies; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D005782:Gels; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011134:Polysaccharides; D020543:Proteome; D012967:Sodium Dodecyl Sulfate