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

Rookyard AW1,2, Li DK2, White MY1,2,3 and Cordwell SJ1,2,3

  1. School of Life and Environmental Sciences.
  2. Charles Perkins Centre.
  3. Discipline of Pathology.

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, induces cellular damage. Notably mitochondrial dysfunction occurs, increasing 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 protein post translational modifications (PTMs). The most redox active amino acid is Cysteine (Cys) and Cys redox PTMs can be broken down into two classes, those that are biologically reversible (e.g. S-glutathionylation) or irreversible (sulfinic and sulfonic acid; Cys-SO2H/SO3H). Irreversible Cys redox PTMs occur with sufficient exposure to high levels of ROS/RNS and are associated with protein dysfunction and/or degradation. A mass spectrometry technique based on parallel reaction monitoring was employed to detect changes in irreversible Cys modification in a Langendorff model of myocardial ischemia reperfusion injury (I/R). Due to the low abundance of Cys, and Cys PTMs, an enrichment strategy was used to better profile the changes in irreversible Cys PTM. I/R significantly increased Cys-SO2H/SO3H-modified peptides from proteins involved in mitochondrial fatty acid biosynthesis and the tricarboxylic acid cycle. The addition of an aminothiol antioxidant MPG (N-2-mercaptopropionylglycine) in reperfusion attenuated irreversible modification of Cys and improved functional recovery of hearts.