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

• DOI: 10.1002/cpch.43
• Publication Date: 2018-08-07T14:03:36Z
• Authors: Alexandra Van Hal...; Yi Zhao; Daniel A. Urul; Marcus J. C. Long; Yimon Aye
• Keywords: Streptavidin; Bioorthogonal Chemistry

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