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Title: Capillary electrophoresis with multiple readout techniques for protein analysis
Author: Goyder, Miriam Sarah
ISNI:       0000 0004 2706 1655
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2011
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In the era of proteomics, new technologies in separation and identification are required. Separation methods, such as capillary electrophoresis or liquid chromatography, are a crucial part of high throughput proteomic workflows. In this thesis, novel approaches to proteomics using capillary electrophoresis are presented. A platform of technologies based on capillary electrophoresis with continuous deposition of separated proteins onto metallic substrates enables subsequent analyses and identification. Since sample deposition and identification are decoupled, multiple readout techniques can be explored. Readout techniques used include matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS), electron-vibration-vibration two dimensional infrared spectroscopy (EVV 2DIR) and fluorescence microscopy. This technology was used without the deposition interface, to achieve advances in ribosomal separations or with the deposition interface, to develop new proteomic strategies of separation and readout. The eukaryotic ribosomal proteins were separated using capillary electrophoresis for the first time. Over 26 peaks were resolved in less than 10 minutes. An outstanding RSD migration time of < 0.5% was achieved, demonstrating that the readout could provide a ribosomal ' fingerprint'. Separations of proteins were successfully analysed using a standard MALDIMS instrument. This work was advanced by the offline coupling of CE to MALDI-imaging and applied to the ribosomal proteins to demonstrate a novel workflow from cell culture to protein identification. Quantitative analysis of protein levels is an important part of proteomics, but is difficult to achieve using mainstream technologies with high throughput and accuracy. EVV 2DIR is a non-linear spectroscopy which is able to achieve absolute quantification of proteins.[1] Coupling of EVV 2DIR to CE (CE- 2DIR) was demonstrated through the deposition and analysis of peptide and proteins. CE-2DIR offers great promise as a new proteomic tool.
Supervisor: de Mello, Andrew ; Ces, Oscar ; Klug, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral