The proteasome, the primary protease for ubiquitin-dependent proteolysis in eukaryotes, is usually found as a mixture of 30S, 26S, and 20S complexes. These complexes have common catalytic sites, which makes it challenging to determine their distinctive roles in intracellular proteolysis. Here, we chemically synthesize a panel of homogenous ubiquitinated proteins, and use them to compare 20S and 26S proteasomes with respect to substrate selection and peptide-product generation. We show that 20S proteasomes can degrade the ubiquitin tag along with the conjugated substrate. Ubiquitin remnants on branched peptide products identified by LC-MS/MS, and flexibility in the 20S gate observed by cryo-EM, reflect the ability of the 20S proteasome to proteolyze an isopeptide-linked ubiquitin-conjugate. Peptidomics identifies proteasome-trapped ubiquitin-derived peptides and peptides of potential 20S substrates in Hi20S cells, hypoxic cells, and human failing-heart. Moreover, elevated levels of 20S proteasomes appear to contribute to cell survival under stress associated with damaged proteins.

Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site. Here, we demonstrate the tuning of palladium chemoselectivity in aqueous medium for on-demand deprotection of several Cys-protecting groups that are useful in protein synthesis and modification. These tools allow the preparation of highly complex analogues as we demonstrate in the synthesis of the copper storage protein and selectively modified peptides with multiple Cys residues. We also report the synthesis of an activity-based probe comprising ubiquitinated histone H2A and its incorporation into nucleosomes and demonstrate its reactivity with deubiquitinating enzyme to generate a covalent nucleosome–enzyme complex.