To reduce the contribution from secondary changes due to altered protein levels, cell stress, or cell death; these do not occur upon short exposure of cells to proteasome inhibitors. Peptide levels were measured using a quantitative peptidomics technique that uses stable isotopic labels to compare up to five samples in a single experiment. For all of these analyses, 2�C3 replicates of inhibitor-treated cells were compared to 2 replicates of control cells. Relative levels of peptides were quantified by measurement of peak height for each of the isotopic peaks detected in the MS spectra, and peptides were subsequently identified by MS/MS analysis using rigorous criteria previously established for peptidomics. Because the peptide levels are expressed as a relative ratio, any peptide not detected in one of the groups of replicates was capped at a level 1/5th that of the observed peptide; this means that peptides only detected in the control groups and not in the treated samples are listed with ratios #0.20 while those found only in the treated groups are listed with ratios of $5. In addition to including all data in a supplementary file, the results are graphically represented in rank order plots. To generate these plots, the ratio of the level of peptide in each of the biological replicates was compared to the average level in the control replicates and then sorted by rank order and plotted. The y-axis represents the relative level of peptide in the indicated replicate and the x-axis is the rank order of the peptides. In most of the control replicates, each individual replicate did not differ by more than 2-fold from the average of the two controls, with an average ratio of 1.0. In contrast, 
very few of the peptides in the inhibitor-treated groups had ratios Masitinib around 1.0, and most peptides were either much higher or lower than this ratio. Treatment of HEK293T cells with MG132, clasto-Lactacystin b-lactone, or MLN2238 produced changes in the peptidome that were generally similar to those caused by the treatment with 0.2 mM epoxomicin; the majority of peptides was greatly decreased by the proteasome inhibitor and few peptides were elevated. Similar changes were observed with MG132, clasto-Lactacystin b-lactone, and MLN2238 when tested with SHSY5Y cells. In contrast, treatment of the cells with MG262 produced changes that were generally similar to those caused by 500 nM bortezomib, which were also similar to those produced by 50 nM bortezomib. Carfilzomib decreased the levels of many peptides but also Dabrafenib elevated levels of a number of other peptides in HEK293T cells and SHSY5Y cells. Because AM114 did not produce a substantial change in levels of peptides in HEK293T cells and did not substantially inhibit the proteasome, this compound was not further tested in SH-SY5Y cells. While the summary plots shown in Figures 3 and 4 provide a visual representation of the overall pattern of peptide levels, these plots do not provide information about specific peptides. Table S1 contains data on every peptide detected in each experiment, both identified and unknowns, but due to the size of this table it is difficult to compare trends among different peptides. To compare levels of specific peptides between datasets, heat maps were created. For these analyses, peptides that were found in multiple experiments were placed into a single table and the relative levels of peptide in each of the experimental replicates were color-coded, with green indicating peptides that were decreased in the treated cells, red indicating peptides that were elevated in the treated cells, gray indicating peptides that were not greatly affected by the treatment, and missing data in white. Table S2 shows the data with values and peptides sequences, while Figure 5 shows only the color-coded results.