Probing the proteasome cavity in three steps: bio-orthogonal photo-reactive suicide substrates.

Tri-functional activity-based protein probes that encompass an electrophilic trap, a photo-reactive group and a bio-orthogonal ligation handle are described. With these, and in a three-step chemical proteomics approach, proteasomal catalytic sites are covalently and irreversibly modified, followed by photocrosslinking of these to flanking subunits and Staudinger-Bertozzi ligation for visualization and identification of the resulting conjugates.