Immobilisation of antibodies in gels allows the improved release and identification of glycans

Human IgG and IgM, bovine IgM and three therapeutic IgG monoclonal antibodi es have been separated by sodium dodecyl sulfate polyacrylamide gel electro phoresis. Carbohydrates were then released from these immobilised proteins by direct enzymatic digestion, derivatised with a highly fluorescent probe and analysed by high per formance liquid chromatography and matrix-assisted laser desorption/ionisation time of-flight mass spectrometry. This procedu re 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 outlined allow s 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 impu re glycoprotein is available.