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Title: Investigating redox posttranslational modifications in proteins using mass spectrometry
Author: Thurlow, Sophie Erica
ISNI:       0000 0004 5991 7200
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2015
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Redox potential, a measure of how oxidising or reducing an environment is, is tightly regulated by cells to minimise detrimental chemical oxidation and reduction reactions. In proteins, it is the sulfur containing cysteine residues that can be post-translationally modified through specific redox reactions, for example, the formation of disulfide bonds between cysteine residues can be crucial to protein structure. It has recently been hypothesised that signalling pathways utilising redox regulated proteins may be arranged into electrochemical series. The characterisation of the redox properties of specific cysteine residues in proteins has proven difficult using traditional redox characterisation methods such as cyclic voltammetry. A number of biochemical methods have been developed for studying the effect of the redox environment on proteins, many making use of mass spectrometry and allowing for localisation of the site of the modification to specific cysteine residues. However, fewer methods have been reported that facilitate accurate quantification for the determination of the mid-point potential of these redox regulated cysteine residues. Here, a differential labelling protocol using high resolution mass spectrometry techniques for the study of redox chemistry of cysteine residues in proteins will be reported. The protocol exploits the novel chemistry of thiol groups for specific alkylation and allows for both qualitative and quantitative experiments. Thioredoxin-1 from E. coli and human systems was used as a model protein and a novel disulfide bond was characterised. The reducing potential of the active site cysteine residues of human thioredoxin were found to be very similar to those of the E. coli proteoform, -276 ± 1 and -281.4 ± 0.3 mV respectively. The remaining three cysteine residues of human thioredoxin were found to be regulated at more oxidising potentials. The protocol developed was applied to a protein from the cell death pathway of apoptosis; human caspase-3 is an executioner protease from the caspase cascade. Caspase-3 was found to contain three redox sensitive cysteine residues. The catalytically active cysteine residue was redox regulated via two mechanisms, glutathionylation and disulfide bond formation. One of these mechanisms gives the active site cysteine residue a calculated reducing potential of -165 ± 6 mV supporting the correlation between caspase-3 activity and its observed role in the apoptotic pathway but not in necrotic cell death.
Supervisor: Langridge-Smith, Patrick ; Campbell, Colin Sponsor: Engineering and Physical Sciences Research Council (EPSRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: mass spectrometry ; proteins ; redox