To obtain a global view of the changes of protein phosphorylation in bortezomib-treated myeloma cells, we compared the phosphoproteome of U266 cells treated with or without bortezomib. The workflow is outlined in Figure 1 . Cells in normal medium (light culture) were treated with bortezomib, and cells grown in medium containing stable isotopes (heavy culture) were treated with vehicle. These two populations of cells were lysed, mixed at a 1:1 ratio, and subjected to TiO 2 purifications followed by LC-MS/MS analysis. After LC-MS/MS analysis on the enriched phosphopeptides, all MS/MS spectra were searched, respectively, against the forward and reversed human protein sequence databases to estimate rates of false-positive matches. Search results were filtered based on peptide score of MASCOT and PTM score. In total 1024 phosphopeptides (redundant) from the target database passed our criteria, allowing 14 decoy matches. The phosphopeptide false-positive rate was therefore estimated to be 1.4%. Multiple filtering criteria were established to validate search results. For each of the phosphorylated peptides identified in this work, peptide sequences were manually confirmed. After validation we identified 418 unique phosphorylation sites from 244 unique phosphopeptides corresponding to 233 protein groups. This entire dataset is provided as Table S1 in which hyperlinks are built up to view all the MS/MS spectra. Using the PTM score, we could localize the phosphor groups with high confidence (Class I phosphorylation sites) in 197 cases (Table S1), indicating that the phosphorylation sites detected by current strategy are of high confidence. Figure 2A shows a representative MS/MS spectrum for a phosphosite-containing peptide in the detection, and all other MS/MS spectra are available via the hyperlinks in the Table S1 .