A powerful technology for characterizing subcellular proteomes is “proximity labeling” (PL), in which a catalyst is localized to a specific cellular location, followed by promiscuous tagging of endogenous proteins in the vicinity. The tagged proteins are then isolated and identified by mass spectrometry. Although PL catalysts are powerful, new PL catalysts are needed to enhance the sensitivity and specificity of spatially resolved proteomic mapping. Genetically encoded enzymes can be conveniently targeted to cellular locations of interest, but they are limited in their mechanisms of tagging, which hampers control over the labeling radius (limiting specificity) and restricts which amino acids can be tagged (limiting sensitivity). Synthetic PL catalysts have recently introduced a greater diversity of chemical labeling mechanisms, but new approaches are needed for selective activation of these catalysts in highly specific subcellular regions of interest. We propose that hybrid biological-abiotic PL catalysts can achieve improved sensitivity as well as specificity in spatially-resolved proteomic mapping. We are pursuing this hypotheses through the development of three classes of hybrid PL catalysts, which offer complementary advantages. In Aim 1, we have used directed evolution to discover heme peroxidase enzymes capable of generating highly reactive radicals, which exhibit a shorter diffusion radius and label chemically diverse amino acids, in contrast to the APEX approach that almost exclusively labels tyrosines. This ability to react with more amino acids will enhance sensitivity for detecting proximal proteins. In Aim 2, we have developed hybrid DNA- synthetic PL catalysts that become activated only in highly specific subcellular locations. We are applying these switchable catalysts for activation of PL selectively at protein–protein interactions (PPIs) on the surface of cancer cells, and we will extend this approach for activation at intercellular PPIs in neuronal synapses. In Aim 3, we have developed hybrid DNA-synthetic catalysts that tag proteins through contact-dependent mechanisms, instead of generating diffusible reactive species. We will attach these contact-dependent catalysts to DNA nano- rod structures with tunable lengths and rigidities, enabling precise control over the labeling radius in the range of ~1–50 nm. We are applying all three classes of PL catalysts for proteomic mapping in living mammalian cells, in collaboration with Prof. Lloyd Smith, an expert in high-resolution biomolecular mass spectrometry. Additionally, we are collaborating with Prof. Edwin Chapman to employ these PL tools in cultured neurons to benchmark their performance against existing tools. Throughout the next five years, my laboratory will continue to develop new mechanisms for PL using hybrid abiotic-biological catalysts. I envision that these technologies will be employed not only in my laboratory, but also in the broader community, to elucidate novel prot...