Craig, Sarah Jane; (1995) Analysis of complex glycoproteins by capillary electrophoresis and mass spectrometry. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Natural and recombinant proteins are now being produced for biomedical research and therapeutic applications. Extensive purification and characterisation of the protein or glycoprotein can be required. Chromatographic and electrophoretic techniques can be used to isolate components and evaluate purity. In this study the modem analytical technique of capillary zone electrophoresis (CZE) has been used to investigate the complexity of the human α-interferon, Wellferon. Seventeen UV absorbing components were resolved by optimisation of conditions. This was compared to an established reverse phase high performance liquid chromatography (RP-HPLC) separation which identified fifteen UV absorbing components. Further analysis of RP-HPLC fractions by CZE showed some fractions to contain more than one component. Structural characterisation of proteins using mass spectrometry (MS), X-ray crystallography (XRC), nuclear magnetic resonance (NMR) and other spectroscopic techniques together with traditional Edman degradation with amino acid analysis is essential to identify primary, secondary and tertiary structure as well as post-translational modifications. Electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption mass spectrometry (MALDI-MS) have been used to analyse Wellferon and its RP- HPLC fractions in order to confirm the proposed sequence. The antibodies (IgG1) Campath-1H and an Anti CD4 were analysed by CZE, ES-MS and MALDI-MS. Mass spectrometry showed Campath and anti CD4 to have molecular weights of ~149,000 Da. Peak shape indicated carbohydrate microheterogeneity for both molecules. Further analysis on fragments of the antibodies produced from papain, N-glycanase and DTT treatments confirmed the protein sequence and indicated the carbohydrate structures. These studies show that CZE and mass spectrometry are essential techniques in the structural analysis of proteins and glycoproteins and can readily identify blocked and modified amino acid sequences within a molecule.

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