Single‐Protein‐Specific Redox Targeting in Live Mammalian Cells and C. elegans

T‐REX (targetable reactive electrophiles and oxidants) enables electrophile targeting in living systems with high spatiotemporal precision and at single‐protein‐target resolution. T‐REX allows functional consequences of individual electrophile signaling events to be directly linked to on‐target modifications. T‐REX is accomplished by expressing a HaloTagged protein of interest (POI) and introducing a Halo‐targetable bioinert photocaged precursor to a reactive electrophilic signal (RES). Light exposure releases the unfettered RES on demand, enabling precision modification of the POI due to proximity. Using alkyne‐functionalized 4‐hydroxynonenal (HNE) as a representative RES, this protocol delineates optimized strategies to (1) execute T‐REX in live human cells and C. elegans, (2) quantitate the POI's RES‐sensitivity by either azido‐fluorescent‐dye conjugation or (3) enrich using biotin‐azide/streptavidin pulldown procedure in both model systems, and (4) identify the site of RES‐labeling on the POI using proteomics. Built‐in T‐REX controls that allow users to directly confirm on‐target/on‐site specificity of RES‐sensing are also described. © 2018 by John Wiley & Sons, Inc.

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