Fig. 4. Concepts underlying REX techniques: competition between reactivity vs. native diffusion propensity of electrophiles or covalent ligands, and associated controls. (A) In T-REX, after the stoichiometric anchoring of Ht-Pre-LDEs, washing away of excess Ht-Pre-LDEs, and subsequent low-energy UV light-induced photo-uncaging (365 nm, 5 mW cm⁻² over 1–5 min), LDEs are liberated. This liberation forms a transient “encounter complex” (dotted circle). When POI is a kinetically privileged sensor for the liberated LDE, the POI can intercept the LDE before its diffusion, leading to POI-specific fractional labeling and (likely) a functional response. When POI is not a kinetically privileged sensor of the specific LDE, the liberated LDE (<5 μM)⁷⁰ is not intercepted and diffuses into the cellular environment. The POI is not labelled, and no response is triggered. (B) The electrophile-sensor cysteine within the POI can be mutated into serine (shown) or alanine to generate a sensing-defective mutant-POI (that is otherwise validated to be functional). This functional mutant-POI fails to capture the LDE when T-REX is replicated under otherwise identical conditions. In this scenario, the LDE released diffuses from the POI (see below) is averaged across the cell, leading to mimimal perturbation of the cell. For instance, the low (<5 μM)⁷⁰ amount of LDE released does not affect the overall cellular GSH/GSSG pools (present at mM levels).^69,72 Generally, such a mutation silences downstream signaling changes that are otherwise measured by T-REX using wild-type POI that senses electrophiles during T-REX.^65,66,70,71.