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Title: Capillary zone electrophoresis-based protein size determination and development of an offline CZE/EVV-2DIR interface platform for proteome-on-chip applications
Author: Loeffeld, Christian B.
ISNI:       0000 0004 2723 9839
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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The development of an offline interface platform for continuous fraction collection from capillary zone electrophoresis (CZE) is presented. The platform is specified for Proteome-On-Chip applications that employ the protein quantification and differentiation technology Electron-Vibration-Vibration Two-Dimensional Infrared (EVV-2DIR) spectroscopy as the primary protein analysis tool. It is demonstrated that the interface platform facilitates preservation of an initial CZE separation effort and enables generation of single and multi-component tracks that are accessible to examination using EVV-2DIR spectroscopy and intrinsic fluorescence imaging (IFI) microscopy. With the introduction of a novel signal detection substrate, it is shown that EVV-2DIR spectroscopy facilitates detection and differentiation of post-translational modifications, and in particular phosphorylations of tyrosine moieties in polypeptides. The results of analytical and bioinformatical procedures performed on data generated with the interface platform-connected tool set suggest that a continued development of this modular proteomics-driven strategy may offer potential to successfully engage in proteomics projects. Furthermore, it was found that CZE, in addition to electrophoretic separation, also facilitates molecular sizing of the components constituting a complex mixture in a single process step. Despite the absence of an externally applied hydrodynamic flow field, a direct proportionality between protein molecular weight and dispersion coefficient is observed. This qualitative resemblance to the phenomenology of Taylor-Aris dispersion, together with a derived radial temperature gradient (dT)R > 103 K/m and resulting Peclet numbers Pe > 102, is interpreted to be due to Joule heating-induced Taylor-Aris dispersion. For single, as well as for multi-protein solutions, the employed experimental conditions facilitate a mean accuracy of the method of 70 %. The method is independent of the type of solvent or buffer systems employed, and thus experimental parameter optimisation may enhance the accuracy to a degree in order to be useful in the application of proteomics strategies as stated above.
Supervisor: Klug, David ; Willison, Keith Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral