Post-translational modifications (PTMs) control the structure, activity, localization, and lifetime of nearly all proteins and are often dysregulated in human disease. Identification of PTMs has far outpaced assignment of their biological functions, an endeavor that requires proteome-scale information about where and when these modifications occur within the cell that is unobtainable with current technologies. To meet this challenge, my lab integrates principles from chemical biology, organic chemistry, protein engineering, and mass spectrometry-based proteomics to develop innovative enzymatic tools for systems-level mapping of PTMs with subcellular spatial resolution. These technologies will advance our fundamental understanding of how post-translational modifications exert spatiotemporal control over protein activity to program biological function, opening up new opportunities for fundamental biological discovery and the identification of new therapeutic targets.