Cellular synthesis of ubiquitin-based probes for spatially resolved deubiquitinase profiling

摘要

Deubiquitinases (DUBs) orchestrate ubiquitin homeostasis, yet their compartment-specific activities remain underexplored. We developed a genetically encoded strategy to assembly activity-based ubiquitin probes in cellulo via sortase A (SrtA)-mediated transpeptidation. Co-targeting SrtA and its ubiquitin substrate to the outer mitochondrial membrane reduced off-target transpeptidation and enabled spatially resolved profiling in live cells. Quantitative proteomics identified distinct sets of cytosolic, nuclear, and cell line specific DUBs in HEK293T and MCF7 cells. This localization-tunable, delivery-free strategy expands the chemical biology toolkit for mapping DUB activity within native cellular environments.