Chemoproteomic Methods to Evaluate Cysteine Oxidation in the Mitochondria

Abstract

Reactive oxygen species (ROS) modulate protein function through cysteine oxidation. Identifying protein targets of ROS can provide insight into uncharacterized ROS-regulated pathways especially within ROS generating organelles such as the mitochondria. There are several known examples of mitochondrial cysteine targets that alter protein and pathway activity resulting in pathological effects. Several chemoproteomic workflows, including ABPP and OxICAT, can be used to identify sites of cysteine oxidation. However, determining ROS targets localized within subcellular regions and ROS hotspots remains challenging with existing workflows. Here, we present combined cysteine- monitoring chemoproteomic platforms (isoTOP-ABPP and OxICAT) with mitochondrial enrichment (organelle isolation and proximity labeling) to monitor cysteine oxidation events within the mitochondria. First, we profile redox- sensitive cysteines under exogenous and endogenous peroxide in isolated mitochondria using isoTOP-ABPP and OxICAT. Next, we introduce PL-OxICAT which combines enzymatic proximity labeling (PL) (TurboID/APEX) and OxICAT to monitor localized cysteine oxidation events within subcellular compartments such as the mitochondrial matrix and intermembrane space as well as ROS hotspots. Together, these platforms further hone our ability to monitor cysteine oxidation events within specific subcellular locations and ROS hotspots and provide a deeper understanding of the protein targets of endogenous and exogenous ROS.