Redox regulation is a phenomenon in which signals are relayed through oxidative and electrophilic modifications of specific redox-responsive proteins. These chemical events in turn orchestrate unique cytoprotective responses downstream. The deregulation of redox signaling pathways underlies many human diseases, including cancers, Alzheimer's, and cardiovascular disorders. However, the links between disease and individual redox signaling events remain shrouded in mystery. Currently, decoding the consequences of redox-linked modifications of specific proteins in cells induced by a given reactive small molecule is impossible. Conventional strategies limited to whole-cell bathing with reactive entities target simultaneously many, if not most, re- dox-sensitive proteins in cells. Although these multihit approaches model oxidative stress, they do not allow study of physiological redox signaling. My laboratory has developed a chemistry-driven innovation-targetable reactive electrophiles and oxidants (T-REX)-ultimately aimed at directly linking individual downstream biological responses to the chemical redox alteration of specific target proteins. T-REX mimics endogenous signaling, enabling selective and spatiotemporally controlled perturbation of any redox-responsive protein in cells with any reactive entity. Toward this ultimate goal, we recently showed proof of concept in which ligand-directed intramolecular delivery enabled selective targeting of a bioactive lipid electrophile, 4-hydroxynonenal (HNE), to two distinct redox-responsive proteins of biomedical interest in living cells. We herein exploit T-REX to probe mechanistic relationships between specific protein-electrophile perturbation and downstream signal propagation within a major disease-implicated redox signaling cascade. The proposed experiments will (1) profile the target-specific mechanisms by which HNE and analogous electrophiles operate in cells, (2) define the extent to which physiologic HNEylation prompts response in an otherwise native cell, and (3) pinpoint specific residues of the upstream target genuinely responsible for electrophile sensing. A multidisciplinary combination of synthetic chemistry, chemical biology, mechanistic biochemistry, mammalian cell biology and structural biology approaches will be used. Success will establish T-REX as the first chemical biology platform capable of mimicking non-enzyme-mediated posttranslational modifications in redox signaling. The utility of T-REX is far reaching: the strategy described herein can perturb any disease-implicated redox-responsive protein with any bioactive small-molecule inducer such that sophisticated redox-information-processing mechanisms within individual redox-modulated pathways can be clearly understood. T-REX is an ambitious yet transformative approach to understanding redox regulation. As with phosphoregulatory pathways wherein mechanistic understanding of temporal dynamics within individual phosphoryl transfer events has yielded multiple medical breakthroughs, T-REX strategy has the same long-term potential in impacting modern biomedical research.