© 2013 Nature America, Inc. All rights reserved. PROTOCOL NATURE PROTOCOLS | VOL.8 NO.6 | 2013 | 1155 INTRODUCTION Focusing on proteasome activities The proteasome is an evolutionarily conserved proteolytic com- plex that is responsible for the degradation of most proteins in eukaryotic cells ranging from yeast to human. It is essential for protein homeostasis and production of major histocompatibility complex (MHC) class I restricted epitopes. Protein degradation is necessary for the turnover of damaged or misfolded proteins and regulation of biochemical pathways by lowering enzyme activity or messenger concentration. Obviously, the proteasome is a central protease in various cellular processes, including transcrip- tion, translation, DNA repair, cell division and antigen presenta- tion 1,2 . In the past decade, the proteasome became an attractive clinical target after the approval of the proteasome inhibitor bortezomib (Velcade) by the US Food and Drug Administration for the treatment of multiple myeloma 3 . Encouraged by the clinical success of bortezomib, a series of new-generation protea- some inhibitors are being investigated as therapeutics of various diseases; thus, both (pre)clinical and fundamental knowledge of the activity of proteasomes is required. The challenge of determining proteasome activity by a robust and high-throughput method is substantial. The proteasome is not a single protease, but a multisubunit protease cluster that, in eukaryotes, contains active subunits with different cleavage preferences. Mammalian 30S proteasomes contain the catalytic 20S barrel-shaped core particle (CP) capped on both sides by 19S regulatory particles (Fig. 1). The 20S CP consists of four hepta- meric rings assembled from α- or β-subunits (α 1–7 ,β 1–7 ,β 1–7 ,α 1–7 ) and harbors three different peptidase activities on each β-ring 4,5 . Crystallographic and substrate specificity studies 6 show that the active site pockets of the β5 subunits can accommodate and cut at the C terminus of bulky, hydrophobic amino acid residues in a manner that resembles chymotrypsin activity. β2 subunits prefer cleaving after basic residues and are referred to as bearing ‘trypsin- like’ activities, whereas β1 cuts after acidic residues and is known to have ‘caspase-like’ activity. This constitutive 20S proteasome CP is present in all eukaryo- tic cells. In immunocompetent tissues, three additional catalyti- cally active β-subunits are expressed: β1i (low-molecular-weight protein-2, LMP2), β2i (multicatalytic endopeptidase complex– like-1, MECL1) and β5i (LMP7) 2 . The immuno-β-subunits show comparable substrate cleavage preference, share around 50% protein sequence identity and have different functional roles compared with the constitutive β1, β2 and β5 subunits when replacing them in newly assembled 20S CP, yielding the so-called immunoproteasomes 1 . Recently, a β5t subunit was identified that is exclusively expressed in cortical thymus epithelial cells, in which it is incorporated in immunoproteasomes instead of β5i, yielding the thymo-proteasome 7 . Despite this diversity, the activity of the β-subunits is conveyed by the same mechanism of nucleophilic attack of the N-terminal threonine (Thr1) γ-hydroxyl on the peptide backbone 8 . A commonly used technique to determine the proteasome activity is by means of fluorogenic substrates. Short peptides optically quench the aminocoumarin at their C termini, which upon cleavage by the proteasome is released and starts to fluoresce in solution 9 . Excellent subunit-specific fluorogenic substrates for each of the three constitutive β-subunit activities are commercially available; however, these substrates cannot discriminate between constitutive and immunoproteasome activities when present in the same cellular system. This problem is encountered during immunological studies of antigen presentation or (pre)clinical research in immune cells, such as leukemia and myeloma cells. Relative quantification of proteasome activity by activity-based protein profiling and LC-MS/MS Nan Li, Chi-Lin Kuo, Guillem Paniagua, Hans van den Elst, Martijn Verdoes, Lianne I Willems, Wouter A van der Linden, Mark Ruben, Eric van Genderen, Jacob Gubbens, Gilles P van Wezel, Herman S Overkleeft & Bogdan I Florea Gorlaeus Laboratories, Leiden Institute of Chemistry and Netherlands Proteomics Centre, Leiden, The Netherlands. Correspondence should be addressed to B.I.F. (b.florea@chem.leidenuniv.nl) Published online 23 May 2013; doi:10.1038/nprot.2013.065 Activity-based protein profiling (ABPP) is a functional proteomics technique for directly monitoring the expression of active enzymes in cell extracts and living cells. The technique relies on irreversible inhibitors equipped with reactive groups (warheads) that covalently attach to the active site of enzymes and fluorescent or affinity tags for imaging and purification purposes, respectively. Here, a high-throughput and robust protocol for high-resolution quantitative activity-based proteasome profiling is described. We use both panreactive and subunit-specific fluorescent activity-based probes (ABPs) to quantify the proteasome activity in living cells, in the presence or absence of the potent proteasome inhibitor bortezomib. Active proteasome subunits from cell lysates are affinity-purified via a biotinylated ABP. Purification from live cells involves a two-step ABP approach using a reagent with a cell-permeable azide-warhead and postlysis installation of biotin. By means of liquid chromatography–mass spectrometry (LC-MS)-based proteomics, we can accurately identify the enriched proteins and the active site peptides of the enzymes, and relatively quantify all the proteasome activities in one experiment. The fluorescence ABPP protocols takes 2–3 d, and approximately 8–10 d are needed to complete the entire protocol.