Fig. 1

Conventional DUB ABPs label DUBs on both catalytic and non-catalytic Cys residues. a Structure of probe-labeled peptide fragment generated by trypsin digestion of Ub-VS-labeled proteins. In these ABPs, an HA tag is attached to residues 1–75 of ubiquitin by a spacer of two aminohexanoyl units. b Detection of probe-labeled DUB peptides in HEK 293T lysate after anti-HA immunoprecipitation and filter-assisted sample preparation (FASP), using HA-tagged Ub-VS, Ub-VME, or Ub-PA probes. Peptides were searched using the probe fragment as a variable modification on Cys. (Modification on other residues could not be detected.) Non-catalytic Cys residues are shown in red. # = Ub-probe fragment; * = carbamidomethyl modification; @ = methionine oxidation. c Reactive-site-centric chemoproteomic strategy to identify probe targets and labeling sites. Lysates are treated with an alkyne-containing ABP. Click chemistry is used to attach a cleavable biotin-azide tag and tagged proteins are enriched on streptavidin beads, using stringent washing to remove non-covalent binders. Proteins are digested on-bead to provide peptides for protein identification. The beads are then subject either to immediate linker cleavage to release probe-labeled peptides or to a second proteolysis before linker cleavage. The yellow triangle represents an electrophilic group, the pale blue rectangle represents a cleavable linker, and the green circle represents biotin