Spatiotemporally Resolved Protein Tagging for Dynamic and Tissue‐Specific Secretome Profiling In Vivo

The dynamic and tissue-specific nature of protein secretion underlies a wide range of physiological and pathological processes, yet tools for profiling the in vivo secretome with high spatial and temporal resolution remain limited. Here, we present STePTag (Spatio-Temporal Protein Tagging), a conditional proximity labeling (PL) system for profiling secreted proteins in live animals. STePTag integrates the rapid labeling kinetics of the PL enzyme TurboID with a destabilized dihydrofolate reductase (DHFR) domain, enabling tight post-translational control of labeling activity through the small-molecule stabilizer trimethoprim (TMP). Targeting STePTag to the endoplasmic reticulum (ER) confines labeling to the secretory pathway and enables robust labeling within 10 min of TMP administration. Furthermore, we design tissue-specific lipid nanoparticles (tsLNPs) to enable programmable, in vivo delivery of STePTag to the mouse liver. Using this approach, we identified 93 liver-derived secretory proteins under physiological conditions and uncovered 40 dynamically regulated proteins in a model of acetaminophen-induced acute liver injury (ALI), including Aldh1a1, which we functionally validated as a protective factor in ALI. Together, STePTag provides a versatile platform for spatiotemporally resolved secretome profiling, enabling the discovery of context-dependent biomarkers and tissue-derived signaling molecules in native physiological environments.