Quantitative Proteomics of Cysteine Redox Post-Translational Modifications

Abstract

Cardiovascular disease is the leading cause of death worldwide with a mortality rate of 17 million people per annum. Ischemic heart disease is the largest contributor to this mortality rate. Ischemia is the loss of an adequate supply of oxygenated blood to the heart. This severely impairs heart function. During ischemia / reperfusion (I/R) the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is sufficient to cause cellular damage. ROS/RNS can alter proteins via posttranslational modification (PTM) of Cys residues. Redox PTM of Cys can be broken down into two groups. Those that are considered biologically ‘reversible’ and those that are considered biologically ‘irreversible’. Irreversible modifications are associated with protein dysfunction and proteolytic degradation. Current techniques to measure Cys PTMs are limited to few methods. To overcome this, we applied an enrichment technique for irreversibly modified Cys peptides to profile sites of irreversible Cys redox PTM in rat hearts subjected to ex vivo Langendorff perfusion and I/R injury. Quantitation was achieved using a novel parallel reaction monitoring mass spectrometry (PRM-MS) strategy on Cys peptides. We correlated changes to specific Cys peptide redox status with a targeted metabolomics approach. We next applied this approach to investigate ROS/RNS imbalance in the diabetic heart. Increased oxidative stress via ROS/RNS has been implicated in the aetiology of the diabetic heart, however, the targets of ROS/RNS are poorly defined. We utilised a rodent model of T2DM to examine hearts by Langendorff perfusion. Irreversibly modified Cys were largely increased in abundance in diabetic hearts under baseline conditions. This thesis has developed methods suitable for the routine identification and quantification of sites of irreversible Cys redox PTMs. Identification of such sites provides targets for specific antioxidant therapy to preserve enzyme functions during pathogenesis.