The heat map analysis shown in XL-184 Figure 5 excludes AM114, which did not substantially inhibit the proteasome at the concentration used in the peptidomics study. Peptides selected for inclusion in the heat map were chosen based on the number of times found in each of the distinct experiments; peptides found in at least five separate runs are included. Only a handful of peptides were detected in every replicate in every experiment. The failure to detect a peptide doesn’t necessarily mean it isn’t present; there are multiple reasons for the absence of a signal. In general, the studies with MG132, MG262, and clasto-Lactacystin b-lactone resulted in fewer detectable peptides than the other studies. Despite this limitation, several trends were detected in the heat map analysis. First, many of the same peptides elevated upon treatment of cells with bortezomib are also elevated by MG262. In contrast, most of the other proteasome inhibitors do not cause these peptides to be elevated. One exception is carfilzomib, which produces an increase in some but not all of the peptides elevated by bortezomib and MG262. Another trend revealed by the heat map analysis is that some peptides show similar responses to all of the proteasome inhibitors. One set of peptides was decreased in at least five of the runs and had an average ratio in all runs of #0.65. In some replicates, these peptides were in the “no change” group, but never showed an increase in any of the replicates. Altogether there were 18 peptides in this set. The majority of these peptides represent the N-terminus or C-terminus of the protein, and therefore only a single cleavage is required to produce the peptide. All of the peptides in this set are produced by cleavages attributed to the beta 5 proteasome subunit, based on the presence of a hydrophobic amino acid residue in the P1 positions of the cleavage sites required to generate the peptide. Another set of peptides was not greatly affected by the proteasome inhibitors in any of the replicates. The average ratio for these peptides ranged from 0.85 to 0.99 in all of the studies, and in no case was a large change found in any of the replicates. One member of this group is the small protein thymosin beta-10, which only undergoes removal of the initiation methionine and would not be expected to be altered by treatment with proteasome inhibitors. A third set of peptides was found to increase in at least five of the experiments, with an average ratio.3.0 for all experiments, and no replicate WY 14643 showing a decrease in any of the experiments. Of the 11 peptides in this group, the majority represented internal fragments of the protein and therefore required two cleavages to be produced. Of the 18 cleavage sites required to produce these peptides, only 11 match the consensus site for beta 5 cleavages, the rest match the consensus site for beta 1 or beta 2 cleavages. There was no substantial difference in the average mass or peptide length for the peptides in set 1 versus set 3. The finding that bortezomib and other compounds increase the levels of some peptides can be explained by one of two possible mechanisms; either the compounds increase the formation of the peptides or the compounds block the degradation of the peptides. A recent study predicted that bortezomib could inhibit TPP2. TPP2 is thought to play a major role in peptide degradation within the cell. To test whether bortezomib inhibited TPP2, we first assayed HEK293T cell extracts with the TPP2 substrate AlaAla-Phe-AMC. Because this substrate is not specific for TPP2 and can be degraded by other cellular peptidases, we examined the activity in the presence of various concentrations of the TPP2-selective 
inhibitor butabindide.