Two-step Bioorthogonal Activity-Based Protein Profiling of Individual Human Proteasome Catalytic Sites

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. This paper describes a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes designed for β1i, β2i, β5c and β5i proved to be useful two-step ABPs that effectively label their designed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (IEDDA) ligation. The compound designed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro . The compound designed for β2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of β2c via azide-alkyne click ligation chemistry both in vitro and in situ . In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which reporting on proteasome activities in living cells