Epithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta 5t subunit that has an... Show moreEpithelial cells of the thymus cortex express a unique proteasome particle involved in positive T cell selection. This thymoproteasome contains the recently discovered beta 5t subunit that has an uncharted activity, if any. We synthesized fluorescent epoxomicin probes that were used in a chemical proteomics approach, entailing activity-based profiling, affinity purification, and LC-MS identification, to demonstrate that the beta 5t subunit is catalytically active in the murine thymus. A panel of established proteasome inhibitors showed that the broad-spectrum inhibitor epoxomicin blocks the beta 5t activity and that the subunit-specific antagonists bortezomib and NC005 do not inhibit beta 5t. We show that beta 5t has a substrate preference distinct from beta 5/beta 5i that might explain how the thymoproteasome generates the MHC class I peptide repertoire needed for positive T cell selection. Show less