A) Classical DUB probes versus chemically synthesized probes. B) Ub-based probes react with the active-site cysteine residue present in most DUBs. C) Table of probes synthesized via the two reaction pathways. See Figure S2 for structures of incorporated tags and linkers. D) Synthesis scheme: two reaction pathways to chemically synthesize active-site-directed Ub-based probes i) 1. PyBOP, DIPEA, R-COOH, NMP, 16 h, RT; 2. Piperidine, NMP, 3×10 min., RT; ii) HFIP/DCM, 30 min, RT; iii) PyBOP, Et3N, GlyVME, DCM, 16 h, RT; iv) TFA, TIPS, H2O, 3 h, RT; v) PyBOP, DIPEA, TMR, DCM, 16 h, RT. PG=protecting group.