|Title:||Development of Redox Proteomic Techniques to Profile Cysteine Post-translational Modifications in the Myocardium|
|Publisher:||University of Sydney.|
Faculty of Science.
School of Molecular Bioscience.
|Abstract:||Once thought to be an unfortunate consequence of aerobic life, the generation of reactive oxygen and nitrogen species (ROS / RNS) is now known to be integral to the proper functioning of the cell. Cysteine (Cys) residues in proteins show all the necessary properties to act as a sensor / responder to redox signals. The potential role for Cys residues in both the physiological function and pathological dysfunction of the cell has lead to much interest in the identification of target proteins / cellular processes and sites regulated by Cys post-translational modification (PTM). Progress in this area has been hampered however, by a relative lack of experimental methods for the study of these sites on a global scale. Thus, the aims of this thesis were to develop enrichment techniques which were robust, sensitive, reproducible and specific in the enrichment of Cys PTM sites, and to apply these technique(s) to the study of ischemia / reperfusion (IR) injury, which is known to be ameliorated by antioxidant interventions. Enrichment of reversible Cys PTM was carried out following their reduction to the thiol by the chemistry of thiol-dislufide exchange (TDE). An additional technique was developed to identify sites forming over-oxidised or irreversible Cys PTM (sulfinic and sulfonic acid) utilising orthogonal separation by strong cation exchange and hydrophilic interaction liquid chromatography (SCX-HILIC). The techniques developed in this thesis were used to profile 6559 unique reversible Cys PTM sites on 2694 proteins and 188 Cys sites on 142 proteins. The techniques were then adapted and utilised to measure changed Cys PTM occurring during IR injury in the presence and absence of antioxidant intervention. Overall, these techniques have lead to a substantial increase in the knowledge of Cys PTM sites in the literature as well as potential processes affected by redox.|
|Type of Work:||PhD Doctorate|
|Type of Publication:||Doctor of Philosophy Ph.D.|
|Appears in Collections:||Sydney Digital Theses (Open Access)|
|Paulech_J_thesis.pdf||PhD Thesis||4.69 MB||Adobe PDF|
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