Cysteine (Cys) is a crucial site for redox post-translational modification (PTM). Some Cys remain modified for the life of the protein (e.g. disulfides), while others vary in PTM in response to the redox environment. There are many different Cys redox PTM, including nitrosylation, glutathionylation and acylation, and these are considered ‘reversible’ as cells contain an array of enzymes that reduce these modifications (e.g. thioredoxins, glutaredoxin) back to their free thiol form. In conditions where the oxidant:antioxidant balance in the cell is in favour of oxidants, thiols can be over-oxidised via Cys-sulfenic acid (Cys-SOH) to either Cys sulfinic or sulfonic acids (Cys-SO2H/Cys-SO3H), which are considered enzymatically ‘irreversible’ and mark the protein for unfolding and ultimately, degradation. We describe a method for peptide-based, enrichment, identification and quantification of Cys-SO2H/Cys-SO3H based on negative selection by strong cation exchange (SCX) chromatography and positive selection by hydrophilic interaction liquid chromatography (HILIC), coupled to parallel reaction monitoring-mass spectrometry (PRM-MS). We identified >300 irreversibly modified Cys sites in an animal model of myocardial ischemia / reperfusion (I/R) injury, and quantified them in comparison to non-ischemic time controls and I/R in the presence of an antioxidant intervention mediated by N-mercaptopropionylglycine (MPG). Functional analysis showed that Cys sites oxidised during I/R and ‘protected’ by MPG were largely associated with metabolic processes, particularly the tricarboxylic acid cycle, and these data correlated with perturbations in TCA cycle flux as assayed by LC-MS/MS-based metabolomics. Cys sites identified here are targets of reactive oxygen species (ROS) that contribute to protein dysfunction during I/R.