β Scribed by Joanne Charlwood; J. Mark Skehel; Patrick Camilleri
No coin nor oath required. For personal study only.
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 timeof-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 outlined 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.
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This paper reports an extension of the in-gel technique for releasing N-linked glycans from glycoproteins for analysis by matrix-assisted laser desorption/ ionization (MALDI) mass spectrometry reported by B. Ku Β¨ster, S. F. Wheeler, A. P. Hunter, R. A. Dwek, and D. J. Harvey (1997, Anal. Biochem. 25
## Abstract This paper describes an improved scheme for the identification of antigens in crude extracts recognized by specific antibodies when analyzed by a combination of twoβdimensional gel electrophoresis and immunoblotting. First, protein components in gels are electrophoretically transferred