Experiments using MIB-MS were performed as previously described (13). Briefly, cells or tumors were lysed on ice in buffer containing 50 mM HEPES (pH 7.5), 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, 2.5 mM sodium orthovanadate, 1× protease inhibitor cocktail (Roche), and 1% each of phosphatase inhibitor cocktails 2 and 3 (Sigma-Aldrich). Particulate was removed by centrifugation of lysates at 21,000g for 15 min at 4°C and filtration through 0.45-μm syringe filters. Protein concentrations were determined by bicinchoninic acid analysis (Thermo Fisher Scientific). To compare kinome signatures within and HGSOC tumors, we designed a super-SILAC method consisting of a cocktail of cancer cell lines that encompasses the activity of the “cancer kinome” to be used as a control for individual samples. Five cancer cell lines were selected from the NCI-60 panel (UACC257, MOLT4, COLO205, ACHN, and PC3) that differed in their origin, gene expression patterns, and mutation status. The cancer cell lines were labeled using SILAC and mixed equally providing a diverse SILAC {[13C6, 15N4]arginine (Arg10) and [13C6]lysine (Lys8)} heavy reference standard (s-SILAC) to be used repeatedly in kinome profiling assays. An equal amount of the s-SILAC reference (5 mg) lysate was added to our nonlabeled (5 mg) sample (cell or tumor tissue) and analyzed on MIB beads. Endogenous kinases were isolated by flowing lysates over kinase inhibitor–conjugated Sepharose beads (purvalanol B, VI16832, PP58, and CTx-0294885 beads) in 10-ml gravity-flow columns. After 2 × 10 ml column washes in high-salt buffer and 1 × 10 ml wash in low-salt buffer [containing 50 mM Hepes (pH 7.5), 0.5% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM sodium fluoride, and 1 M NaCl or 150 mM NaCl, respectively], retained kinases were eluted from the column by boiling in 2 × 500 μl of 0.5% SDS, 0.1 M tris-HCl (pH 6.8), and 1% 2-mercaptoethanol. Eluted peptides were reduced by incubation with 5 mM dithiothreitol (DTT) at 65°C for 25 min and alkylated with 20 mM iodoacetamide at room temperature for 30 min in the dark, and alkylation was quenched with DTT for 10 min. For MIB competition experiments related to Fig. 6C and fig. S5B, heavy-labeled (drug-treated or control) or light-labeled (drug-treated or control) lysates were run on MIB columns separately, eluted, and then mixed before reduction and alkylation. Samples were concentrated to about 100 μl with Millipore 10-kDa cutoff spin concentrators. Detergent was removed by chloroform/methanol extraction, and the protein pellet was resuspended in 50 mM ammonium bicarbonate and digested with sequencing-grade modified trypsin (Promega) overnight at 37°C. Peptides were cleaned with PepClean C18 spin columns (ThermoFisher Scientific), dried in a SpeedVac, resuspended in 50 μl of 0.1% formic acid, and extracted with ethyl acetate (10:1, ethyl acetate:H2O). Briefly, 1-ml ethyl acetate was added to each sample, vortexed, centrifuged at a maximum speed for 5 min, and then removed. This process is repeated four more times. After removal of ethyl acetate after the fifth centrifugation, samples were placed at 60°C for 10 min to evaporate residual ethyl acetate. The peptides were dried in a SpeedVac, and subsequent liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was performed.

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