R3 microcolumns for desalting The Poros Oligo R3 reversed phase resin was suspended in 70% acetonitrile. The R3 beads were loaded onto constricted GELoader tips EPZ-5676 chemical structure containing a C8 microdisc and gentle air pressure was applied to pack the beads in order to obtain R3 microcolumns of 3 mm. Each acidified sample was loaded onto an R3 microcolumn. The R3 microcolumns were subsequently washed with 30 ul of 0. 1% TFA, and the peptides were eluted from the Poros R3 col umn using 30 ul of 70% acetonitrile, 0. 1% TFA. The phosphopeptides were subsequently ressuspended in 0. 5 ul of 100% formic acid and 10 ul of prior to nanoLC MS analysis. Dimethyl labeling After digestion, the total protein extract was quantified by the BCA method and the volume was adjusted to 100 ul of 100 mM TEAB.

CH2O or 4% CD2O or 4% 13CD2O was added, followed by the addition of 4 ul of 600 mM NaBH3CN or 4 ul of 600 mM NaBD3CN. The mixture was incubated for 1 h at room temperature. The reaction was quenched with 16 ul of 1% ammonia and 8 ul formic acid was added. The differen tially labeled samples from three different time points were pooled and desalted using microcolumns filled with Poros R3 beads. This sample was subjected to vacuum centrifu gation and stored at ?20 C for further use. Titanium dioxide chromatography The pooled samples were subjected to the phosphoenrichement procedure by mixing with TiO2 beads, which were ressuspended in loading buffer. 15 mg of TiO2 beads were washed in loading buffer and loaded into the sample tube. The mixture was incubated for 15 min at ambient temperature under agitation.

The mixture was centrifuged for 60 s at 12,000 g and the supernatant was collected, dessalted, and lyophilized. The TiO2 beads, complexed with phosphopeptides, were washed twice with 500 ul of loading buffer and, subsequently, with 30 ul of washing buffer. The phosphopeptides were eluted using 50 ul of ammonium water followed by 10 ul of 30% acetonitrile. The eluent was acid ified by adding 5 ul of 100% formic acid prior to the dessalting step. Offline TSK amide 80 HILIC peptide fractionation Peptide fractionation was performed using a neutral TSK Amide 80 HILIC and a mobile phase containing TFA. The purified peptides were ressuspended in 90% acetonitrile, 0. 1% TFA and loaded onto a 320 um inner 450 um outer diameter �� 17 cm microcapillary column packed with TSK Amide 80 using an Agilent 1200 Series HPLC.

The HPLC gradient was 100 60% of solvent 90% acetonitrile 0. 1% TFA in water for 42 min at a flow rate of 6 uL min. Fractions were collected every minute and com bined into 8 12 fractions depending on the intensity of UV detection measured at 210. Carfilzomib 8 nm. The fractions were dried by vacuum centrifugation. Nano LC MS Nano LC MS experiments were performed using a 7 tesla LTQ FT mass spectrometer. The sample was applied onto an EASY nano LC system.