This is consistent with the concept that once proteins are transported into the proteasome, they cannot exit until degraded and if the preferred enzyme is not active, then cleavage by the other subunits becomes the primary route of degradation. Another previous study examined the effect of bortezomib on the cellular peptidome. Bortezomib is a reversible proteasome inhibitor containing an active site boronate group and is FDAapproved to treat multiple myeloma and mantle cell lymphoma. Bortezomib is a potent inhibitor of the beta 5 subunit, and at higher concentrations blocks the beta 1 subunit. Because the beta 5 subunit plays a major role in the conversion of proteins into peptides, and bortezomib potently inhibits this subunit, it was expected that this drug would cause a decrease in the levels of these peptides, as found for epoxomicin. However, the opposite result was found; the majority of intracellular peptides was elevated by treatment with bortezomib, including many peptides that were predicted to be products of beta 5 cleavages. One possible explanation of this paradoxical result is that bortezomib has off-target effects on the enzymes that degrade the intracellular peptides; a previous study predicted that bortezomib may inhibit TPP2, based on the finding that bortezomib inhibited other cellular serine proteases such as cathepsins A and G. Alternatively, bortezomib is known to allosterically influence proteasome stability, gate opening, and cleavage specificity, and it is possible that these allosteric effects cause the increase in cellular peptides upon exposure to bortezomib. To study this, we used a peptidomics method to examine the effect of a variety of proteasome inhibitors on the peptidome of HEK293T and SH-SY5Y cells; these cell lines were used because their peptidomes have been well-studied. The inhibitors picked for this analysis include three boronate compounds that inhibit the proteasome reversibly, and three non-boronate compounds, one of which is an irreversible inhibitor and two of which are reversible inhibitors. Carfilzomib is an analog of epoxomicin that was recently approved for the treatment of multiple myeloma and mantle cell lymphoma. Some of these proteasome inhibitors are known to have off-target effects, such as MG132 which inhibits calpain and clasto-Lactacystin b-lactone which inhibits cathepsin A. We also tested bortezomib as an inhibitor of peptidases present in HEK293T cells using assays that detect TPP2 and puromycinsensitive aminopeptidase. Finally, we examined whether potent inhibitors of these two enzymes Niltubacin HDAC inhibitor influenced the peptidome of HEK293T cells. Although bortezomib, MG262, and one of the other boronate-containing proteasome inhibitors are weak inhibitors of HEK293T cell aminopeptidase activity, this effect does not appear to contribute to the large increase in most cellular peptides observed with bortezomib and MG262, and to a lesser extent, with carfilzomib. To identify peptides, MS/MS data were analyzed using the Mascot search 
engine and the IPI_human data base. LEE011 Searches include variable modifications of N-terminal acetylation, methionine oxidation, and the isotopic D0-, D3-, D6-, and D9TMAB tags used in our study. Results were manually interpreted to eliminate false positives, using previously described criteria. To investigate the discrepancy between the effect of epoxomicin and bortezomib on the levels of intracellular peptides, we tested six additional proteasome inhibitors. Because AM114 contains two boronate groups, it was included in subsequent studies to test if the previously observed effects of bortezomib were caused by off-target effects due to the boronate groups rather than inhibition of the proteasome.