Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.693939
Title: Functional polymers and gels for the purification of phosphorylated and thiophosphorylated proteins
Author: Patel, Chirag Bharatkumar
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
Reversible protein phosphorylation is a universal means of regulating many processes in cell such as division, development and differentiation. Aberrant phosphorylation often causes or exacerbates disease progression (e.g. cancer). While significant progress has been made in the identification and biochemical characterisation of the protein kinases and phosphatases themselves, it has proven significantly more problematic to identify their substrates. Thus, the development o f techniques for the separation and enrichment of phosphoproteins or phosphopeptides is vital to assist substrate identification. Advances have been made however; the techniques remain limited for a number of reasons: 1-3 - Phosphorylation levels are low. - Any given protein may have a number of different phosphorylated forms. - The abundance of signalling molecules in the cell in very little. - Current analytical techniques do not have sufficient sensitivity and thus sometimes miss the identification of minor phosphorylation sites. The results presented in this thesis explore the three techniques of: (1) affinity gel electrophoresis, (2) affinity bead chromatography and (3) molecularly imprinted polymers for the improvement of phosphorylation analysis by utilising polymeric materials that incorporate synthetic receptors known to selectively bind phosphorylated species. The approaches taken towards each technique's synthesis and recognition of the selected biologically active compounds have been discussed. Furthermore several studies were carried out to elucidate all three techniques' performances under a variety of condition and also reveal their limitations. Affinity gel electrophoresis and affinity bead chromatography results have shown separation and enrichment in model mixtures and more importantly, in highly complex cell lysate samples. Moreover both techniques - selectivity for phosphorylated proteins can be tuned to thiophosphorylated proteins by simply changing the metal in receptor from manganese or zinc to cadmium. Furthermore PAGE imprinted gels and molecularly imprinted polymer monoliths in aq. buffered solutions were synthesised and tested. PAGE imprinted gels using phosphoprotein and phosphoepitopes as templates proved unsuccessful with no improvement in the separation of phosphoproteins. However, molecularly imprinted polymer monoliths prepared using a phosphoepitope as a template have displayed strong imprinting effects for the first time. However, selectivity at the level of phosphoepitopes remains an on-going challenge.
Supervisor: Mann, David ; Vilar, Ramon ; Steinke, Joachim Sponsor: Institute of Chemical Biology ; Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.693939  DOI: Not available
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