Project summary/abstract Spatial organization and temporal dynamics are inherent properties of eukaryotic cell signaling pathways. Dynamic changes in the subcellular localization of proteins are programmed by post-translational modifications (PTMs), such as phosphorylation and proteolysis, which occur in response to biological stimuli. Eukaryotic signaling pathways rely on the introduction of PTMs to specific proteins to rapidly change protein function and localization, enabling cells to respond to changing internal or environmental conditions. However, despite the importance of spatial organization and temporal dynamics in biological signaling, current technologies are unable to provide a systems-level experimental mapping of the dynamic subcellular localization of post-translationally modified proteins. To meet this challenge, we propose to develop new methods for PTM proteomics with subcellular spatial and temporal resolution by engineering genetically targetable, PTM-selective proximity labeling enzymes. These enzymes will tag post-translationally modified proteins in specific subcellular locations, enabling their enrichment and analysis with mass spectrometry-based proteomics experiments for mapping PTMs with spatial and temporal resolution. We will initially focus on two pervasive PTMs, proteolysis and phosphorylation, both of which play critical roles in numerous biological signaling pathways relevant to human health and disease. We will apply protein engineering approaches to develop three distinct classes of enzymes for spatiotemporally resolved capture of proteolytic neo-C termini, phosphoserine/phosphothreonine, and phosphotyrosine, respectively. We will deploy these tools to dissect the spatiotemporal dynamics of proteolysis during apoptosis; phosphorylation during growth factor signaling; and crosstalk between proteolysis and phosphorylation during the cellular decision between life and death. The biological pathways and states that can be probed with the tools that we will develop are nearly limitless, ensuring that they will have a broad and transformative impact across the biomedical sciences. Completion of the proposed work will transform our understanding of how cellular signaling unfolds across space and time and has the potential to reveal new paradigms for therapeutic intervention in PTM-based signaling pathways.