Phosphopeptide enrichment was performed as previously described [17] . Briefly, equal amounts of proteins from untreated ( 13 C 6 -lysine) and bortezomib-treated ( 12 C 6 -lysine) U266 cells were mixed (1 mg in total) and subjected to disulfide reduction with 10 mM DTT (37uC, 3 h) and alkylation with 20 mM iodoacetamide (room temperature, 1 h in dark). The protein mixtures were mixed with four volumes of ice-cold acetone to precipitate proteins. Precipitated proteins were collected by centrifugation and washed with ethanol two times. The pellet was re-dissolved in 50 mM ammonium bicarbonate and then digested with sequencing grade modified trypsin (1:25 w/w) (Promega, Madison, WI) at 37uC for 20 h and then quenched by addition of TFA to a final concentration of 0.5%. The digests were evaporated to about 20 mL in SpeedVac centrifuge. The phosphopeptides from digested peptides were enriched by using Phosphopeptide Enrichment TiO 2 Kit (Calbiochem, San Diego, CA) according to the manufacturer's instruction with slight modifications. Briefly, the tryptic digest was dried, re-dissolved in 200 mL TiO 2 Phosphobind buffer containing 50 g/L 2,5-dihydroxybenzoic acid and then mixed with 50 mL TiO 2 Phosphobind Resin. After 30 min incubation, the supernatant was discarded, and TiO 2 was washed three times with the wash buffer. After that, 30 mL elution buffer was added two times to elute the phosphopeptides. The elutions were combined and acidified with 5 mL of 10% formic acid for SCX-LC-MS/MS analysis. All the buffers and the phosphopeptides purification resin were provided in the kit by the manufacturer.