Proximity labeling (PL) has emerged as a powerful approach for elucidating subcellular proteomes. In PL, a catalyst is localized to a subcellular region of interest, where it tags nearby endogenous proteins; the tagged proteins are then isolated and identified by mass spectrometry. Although PL catalysts have enabled numerous biological discoveries, new PL catalysts are needed to enhance the sensitivity and specificity of spatially resolved proteomic mapping. For example, genetically encoded enzyme catalysts (such as APEX and BioID) can be conveniently targeted to cellular locations of interest, but they are limited in their chemical mechanisms of tagging, which hampers control over the labeling radius (limiting specificity) and restricts which amino acids can be tagged (limiting sensitivity). Recently, synthetic PL catalysts have enabled 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. My group has developed three classes of hybrid biological-abiotic PL catalysts, offering improved sensitivity and specificity for spatially-resolved proteomic mapping. Each class of catalyst offers complementary advantages, and the three aims will be pursued independently. 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 compared to the widely used APEX/biotin phenol methodology, allowing for enhanced spatial resolution. Additionally, we are developing peroxidase-based PL methodologies to 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. For all three research areas, we are applying the novel 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 l...