Single‐Protein‐Specific Redox Targeting in Live Mammalian Cells and <i>C. elegans</i>

Abstract

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.