Identification of Peroxisome-Derived Hydrogen Peroxide-Sensitive Target Proteins Using a YAP1C-Based Genetic Probe

As the reversible oxidation of protein cysteine thiols is an important mechanism in signal transduction, it is essential to have access to experimental approaches that allow for spatiotemporal indexing of the cellular sulfenome in response to local changes in H₂O₂ levels. Here, we provide a step-by-step guide for enriching and identifying the sulfenome of mammalian cells at the subcellular level in response to peroxisome-derived H₂O₂ by the combined use of (i) a previously developed cell line in which peroxisomal H₂O₂ production can be induced in a time- and dose-dependent manner; (ii) YAP1C, a genetically encoded yeast AP-1-like transcription factor-based probe that specifically reacts with S-sulfenylated cysteines and traps them through mixed disulfide bonds; and (iii) mass spectrometry. Given that this approach includes differential labeling of reduced and reversibly oxidized cysteine residues, it can also provide additional information on the positions of the modified cysteines. Gaining more in-depth insight into the complex nature of how alterations in peroxisomal H₂O₂ metabolism modulate the cellular sulfenome is key to our understanding of how these organelles act as redox signaling hubs in health and disease.