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Title: Chemical labelling strategies for mass spectrometric peptide analysis
Author: Robinson, Helen
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2015
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The work in this thesis describes the use of self-assembled monolayers (SAMs) to capture peptides on a gold-coated MALDI chip for mass spectrometric analysis. A SAM was formed on the gold-coated MALDI chip, onto which 4-bromophenylalanine was coupled (giving peaks due to 4-bromophenylalanine-SAM species a characteristic isotopic distribution). Simple single peptides and tryptic peptides from single proteins have been analysed using the developed technology. The capture of peptides on SAMs for mass spectrometric analysis has many advantages; all steps (chemistry and analysis) take place in situ on the gold-coated MALDI chip. This means that there are no sample transfer steps, reducing sample loss. Additionally, unreacted reagents and buffers can be removed from the surface of the gold-coated MALDI chip through washing. Buffers which are not compatible with mass spectrometric analysis (e.g. phosphate-based buffers) can be used for peptide capture as the buffer is washed away prior to analysis. This thesis describes an alternative cleaning strategy for the removal of organic material from the surface of the gold-coated MALDI chips. Two plasma instruments were investigated for their abilities to remove organic material from the gold-coated MALDI chips, and both were shown to be a suitable safe alternative cleaning strategy to the caustic, potentially explosive piranha solution currently used. Finally, in-solution dimethyl labelling was investigated, as a labelling strategy which could be adapted for used with the SAM technology. Quantification was performed using standard solutions of light and heavy labelled simple protein digests mixed in a range of ratios. Two different commercially-available software packages were investigated to assess their ability to analyse the generated data in order to determine whether one is more suitable for dimethyl labelling quantification. A set of complex standard samples was dimethyl labelled and analysed, to define parameters for most effectively determining quantification ratios, before application to a set of ‘real’ samples. Muscle protein samples were analysed and a set of potentially differentially abundant proteins identified for further validation.
Supervisor: Routledge, Anne ; Thomas-Oates, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available