Extensively used in previous peptidomic studies: HEK293T and human neuroblastoma-derived SH-SY5Y cells. Cells were treated with a sub-toxic level of bortezomib for 1, 6, or 16 hours, or with higher concentrations for 30, 60, or 90 minutes, and then the peptidome examined using a quantitative peptidomics approach. Levels of some peptides were reduced by treatment with bortezomib, Paclitaxel clinical trial consistent with the hypothesis that the proteasome produces these peptides. However, many other peptides were elevated by bortezomib treatment, including a large Cycloheximide number that contained hydrophobic residues in the cleavage sites. This raises the possibility that bortezomib affects the cellular peptidome by changing the processing pathways. The global change in peptide levels caused by bortezomib may contribute to the physiological effects of this important anticancer drug. In contrast, incubating longer with the same concentration of bortezomibor for the same time with higher concentrationscauses a change in the level of many peptides. As expected, the bortezomib treatment led to a decrease in the levels of some peptides, consistent with the hypothesis that the proteasome was responsible for the generation of many of the observed cellular peptides. However, an unexpected finding was that a very large number of peptides were elevated by the bortezomib treatment, especially when HEK293T cells were incubated with 500 nM of the drug for 1 hour. A second cell line, the human neuroblastoma SHSY5Y cell line was also treated with 500 nM bortezomib for 1 hour and examined by peptidomics, and an increase in many peptides was also detected. The HEK293T cells incubated with 500 nM bortezomib for a total time of 30 or 90 minutes showed changes similar to those observed with the 60 minute treatment. The analysis shown in Figure 2 represents every group of cells within each experiment, and does not provide information on the variability of a peptide within the replicates of each experiment or between experiments. For this, we analyzed the data using a heat map-type plot. The color scheme used in the heat map is identical to the color scheme used in Figure 2, with white squares representing missing data. Only peptides found in three or more experiments were included in the heat map, for a total of 173 peptides. Each of the 25 columns represents a separate group of cells within the various experiments. For the majority of peptides, the relative level of peptide in the replicates of bortezomib-treated cells was fairly close and the resulting color of the squares was either the same for all replicates, or reflected minor differences that caused one of the replicates to be in a different bin than the other replicates. In addition to showing that the results from the replicates in each experiment are generally close, the heat map shows that many of the peptides altered by bortezomib in one experiment are similarly affected in other experiments. For example, most of the peptides altered by treatment of HEK293T cells with 500 nM bortezomib for 1 hour are similarly affected in HEK293T cells treated with 500 nM bortezomib for 30 or 90 minutes, or with 50 nM bortezomib for 1 hour. The SHSY5Y cell line treated with 500 nM bortezomib for 1 hour 
also showed many of the same changes, although the peptidomes of the different cell lines were not identical, as previously noted. The long-term treatment of HEK293T cells with 5 nM bortezomib caused some of the same changes as those observed with higher concentrations for shorter time periods. The heat map analysis was sorted by different parameters. When sorted by peptide mass, there is no clear correlation between the changes in peptide levels and peptide size.