Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.634098
Title: Functional group imprinted polymers : towards a novel method of phosphopeptide enrichment using molecular imprinting technology
Author: Ewen-Al-Kashi, Sally
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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Abstract:
A range of techniques exists for the characterisation of the phosphoproteome, the most effective of which is mass spectrometry (MS); however, enrichment of phosphorylated peptides is a necessary prerequisite. Current enrichment strategies are inadequate, either due to low selectivity or because they involve chemical modification of the sample. Adapting molecular imprinting technology, we have designed and synthesised a series of prototype synthetic phospho-receptors, termed "Functional Group Imprinted Polymers" (FIPs). We define 'FIPs' as imprinted polymers that selectively recognise molecules due to the presence of a particular functional group - in this case, the phosphate monoester - irrespective of the type, class, substructure or size of the molecule. Thus, two types of polymeric binding site have been investigated, one based on a novel isothiouronium monomer and the other based on a novel gallium-chelated species. A water-soluble crosslinker, completely new to the field of molecular imprinting, has been shown to be an excellent crosslinker for water compatible [functional group or molecular] imprinting applications, superior to currently used crosslinkers. Initial binding experiments, using a range of simple test systems, indicate that the FIPs - in particular, those based on Ga(III) - bind phosphorylated species with selectivity over other oxyanionic functional groups (carboxylate, sulfate, sulfonate); the phosphorylated analytes are subsequently released using mild conditions. Furthermore, because the selectivity of the imprinted polymers is not confined to the molecule originally used as template (cf. conventional MIPs), but is instead for the phosphate monoester functional group substructure, this is excellent initial evidence in favour of the functional group imprinting concept. Comparison with the phosphoselective recognition of non-imprinted, control polymers, clearly demonstrates a functional group imprinting effect. However, initial binding experiments using peptide mixtures, indicate that further optimisation must be carried out to lessen the co-extraction of nonphosphorylated peptides containing multiple carboxylic residues. Phosphoproteomic analysis affords a means of elucidating new therapeutic targets as well as furthering physiological understanding. Protein phosphorylation underlies the regulation and dysregulation of virtually all cellular processes, and kinase substrates are implicated in many high impact diseases including cancer and Alzheimer's disease. An optimised realisation of the FIP concept would greatly facilitate progress in phosphoproteomic research.
Supervisor: Woscholski, Rudiger Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.634098  DOI: Not available
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