Localis-REX: a general protocol for time-resolved spatiofunctional profiling in live cells and C. elegans

We describe Localis-REX, a method for identifying organ or subcellular locale-specific innate reactive electrophilic metabolite (REM) responsive proteins in live Caenorhabdtis elegans and cultured cells. Localis-REX mimics the natural spatiotemporal production of REMs by expressing HaloTag transiently or stably in a specific organ or tissue of live worms or in a designated organelle or subcellular compartment in cultured cells. Following treatment with a submicromolar photocaged REM and washout, light-triggered uncaging generates brief, localized REM pulses near Halo. REM responsive proteins within the vicinity compete to capture the liberated REM before endogenous clearance processes, including glutathione adduction and redox metabolism, enabling selective identification of the most REM-responsive targets under minimally perturned conditions. Localis-REX can be coupled with 1) proteomics to map chemically-actionable local proteomes or 2) live cell and in vivo functional readouts to define biological responses to locale-specific REM-accumulation, such as translation efficiency monitoring through genetic code expansion. The workflow requires 3-6 h, with downstream data acquisition and analysis requiring an additional 24-48 h, depending on the application 1) or 2. We further describe integration of in vivo Localis-REX with ultraID spatial profiling in live C. elegans, enabling expression normalization across profiled locales. Mechanistic validation of newly-identified responder and REM pairs is facilitated using established T-REX and Z-REX target engagement and on-target REM signaling interrogation protocols. Notably, the workflow was initially established as part of a masters thesis project by a student co-author with no prior experience in C. elegans or cell culture research by the master student, highlighting its accessibility to well-trained junior researchers pursuing interdisciplinary studies.