Data Processing to Probe the Cellular Hydrogen Peroxide Landscape

Hydrogen peroxide (H₂O₂) regulates signaling pathways by modulating the activity of redox-sensitive proteins denominated redox switches. The magnitude of the transient variations in localized H₂O₂ pools during signaling events and how these variations impact redox switches present in the cell remain elusive. A canonical model with two chemical reactions comprising the oxidation/reduction cycle of a redox switch is described. The model is dimensionless with respect to the redox switch concentration. Thus, the time-series data required to apply the equations deduced is the percentage of oxidation of a redox switch, avoiding the application of absolute concentrations that are often difficult to measure experimentally. Here, we describe detailed protocols for the processing of experimental data with the canonical model to probe the absolute concentrations of H₂O₂ found in the vicinity of redox switches and probes, as well as the kinetic parameters that describe the reduction and oxidation of redox switches. The protocols are an analytical tool that helps to depict the cellular hydrogen peroxide signaling landscape, giving new insights on H₂O₂ signaling mechanisms, and hold the potential to be a framework for a future redox kinetomics analytical platform.