Poster Presentation 23rd Annual Lorne Proteomics Symposium 2018

Quantitative proteomics of cysteine redox post-translational modifications in myocardial ischemia / reperfusion (I/R) using parallel reaction monitoring mass spectrometry (#122)

Alexander W Rookyard 1 2 , Desmond K Li 2 3 , Melanie Y White 1 2 3 , Stuart J Cordwell 1 2 3 4
  1. School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, Australia
  2. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  3. Discipline of Pathology, School of Medical Sciences, The University of Sydney, Sydney, NSW, Australia
  4. Sydney Mass Spectrometry, The University of Sydney, Sydney, NSW, Australia

Ischemic heart disease involves the occlusion of blood vessels resulting in a cessation of oxygenated blood flow to the heart. This hypoxia, and the necessary reperfusion to salvage surviving myocytes, induce various biochemical changes that cause cellular damage. Notably this includes mitochondrial dysfunction that increases the production of reactive oxygen and reactive nitrogen species (ROS/RNS). This increase in ROS/RNS overwhelms cellular antioxidant defence mechanisms and can alter protein structure / function via various post translational modifications (PTMs). The most common protein PTM induced by ROS/RNS occurs to cysteine (Cys) residues. The broad range of PTM that can occur on Cys can be broken down into two groups, those that are considered enzymatically or chemically reversible and those that are considered ‘irreversible’. Reversible modifications include S-nitrosylation, S-glutathionylation, S-acylation, sulfenic acid (Cys-SOH) and intra- and inter-molecular disulfide bonds that influence protein structure, induce redox signalling, act as molecular ‘switches’ and/or protect Cys residues from subsequent irreversible modification. Irreversible Cys redox PTMs (sulfinic and sulfonic acid; Cys-SO2H/SO3H) however are associated with protein dysfunction and/or degradation. A mass spectrometry (MS) technique based on parallel reaction monitoring (PRM) was employed to detect changes in Cys redox PTM in a Langendorff model of myocardial ischemia reperfusion injury (I/R). Due to the low abundance of Cys, and Cys PTM, methods to enrich reversible and irreversible Cys PTM were employed to better profile the changes that occur in I/R. Several Cys sites from a range of proteins underwent a dynamic response in reversible redox PTM during I/R, and irreversible oxidation of some of these Cys sites was also affected. Additionally, the presence of an aminothiol antioxidant (N-mercaptoproionylglycine [MPG]) during reperfusion attenuated I/R injury and irreversible redox modifications. PRM-MS is an effective approach to measuring specific redox modification site abundances during I/R.