Supporting Information Galan et al. 10.1073/pnas.1405601111 SI Experimental Procedures Sample Preparation and Trypsin Digestion. Cells were lysed in 1% GST-14-3-3 Fusion Proteins, Subtractive Fractionation, and Pull-Down sodium deoxycholate in 50 mM ammonium bicarbonate (pH 7.8) Assays. Fifty milliliters of overnight cultures of BL21 Escherichia and heated to 99 °C for 5 min. The mixture was then allowed to coli transformed with pGEX-4T-14-3-3e WT or K49E mutant cool down on ice and centrifuged at 13,000 × g for 10 min 5 mM were diluted into 500 mL and culture was induced with 1 mM DTT was added to the supernatant and incubated for 30 min at isopropyl β-D-1-thiogalactopyranoside overnight at 25 °C. Cells 56 °C, followed by alkylation with 15 mM iodoacetamide for 1 h were pelleted and resuspended in 40 mL of bacterial lysis buffer at 25 °C in the dark. Excess iodoacetamide was neutralized using (1× PBS/10 mM EDTA/0.1% Triton X, 1 mM PMSF, and 1× 5 mM DTT incubated for 15 min at 25 °C. Proteins were digested protease inhibitor mixture). Extracts were placed in a 50-mL overnight with sequencing-grade modified trypsin (enzyme: conical tube on ice and sonicated using a probe sonicator six protein ratio of 1:50) at 37 °C. The digested mixture was acid- times for 30 s with 30-s delays between blasts. After sonication, ified with 1% formic acid (FA) final concentration. The samples the extracts were centrifuged at 13,000 × g for 30 min and ali- were desalted using C18 (Waters) cartridge following the manufacturer’s instructions and then evaporated to dryness in a quoted into 1-mL tubes to be stored at −80 °C until further use. SpeedVac. For subtractive fractionation, we used a method previously de- scribed (1). Briefly, serum-starved A375 cells were lysed as de- Phosphopeptide Isolation, Offline Strong Cation Exchange Fractionation, scribed above. Cellular debris were removed by centrifugation for and Nanoflow Liquid Chromatography-MS/MS. The peptide samples 10 min at 13,000 × g. The supernatant, corresponding to 20 mg of were subjected to the TiO2 enrichment protocol as described in protein, was precleared by pouring it sequentially over glutathi- ref. 2. Briefly, sample loading, washing, and elution steps were one-Sepharose (GE/Amersham/Pharmacia), glutathione-Sephar- performed in homemade spin columns assembled following the ose bound to 500 μg of GST, and glutathione-Sepharose bound StageTip principle (3, 4) and composed of a 200-μL pipette tip with μ e to 250 g of GST-14-3-3 K49E. The flow-through was divided frit made of SDB-XC membrane (3M) and filled with TiO2 beads. equally and poured over GST-14-3-3e K49E (250 μg) or GST- SDB-XC material has similar hydrophobic properties to C18 and 14-3-3e WT (250 μg) (as shown in the schematic of Fig. 4A). The allows for combining phosphopeptide enrichment and desalting beads were then washed two times with 2 mL lysis buffer and steps. Centrifugation speed was set to 2,000 × g. Before peptide a final wash of 2 mL of lysis buffer lacking Nonidet P-40 and Brij loading, columns were equilibrated with 100 μL of loading buffer 35. Cellular proteins bound to each column were eluted stepwise [250 mM lactic acid in 70% acetonitrile (ACN) and 3% TFA]. μ with 400 μL of 400 mM MgCl2 in 50 mM Hepes, 400 μLof Peptides were solubilized in 100 L of loading buffer and applied μ 800 mM MgCl2 in 50 mM Hepes, and finally chased with 200 μL on a TiO2 column. Each column was washed with 100 Lof of 50 mM Hepes. Eluates were combined and precipitated with loading buffer followed by 2 × 100 μL of 125 mM asparagine and 15% trichloroacetic acid. Precipitated proteins were pelleted, glutamine in 70% ACN and 3% TFA and 100 μL of 70% ACN μ washed with acetone, resuspended in reducing sample buffer, and 3% TFA. Subsequent washing with 50 Lof1%FAwasused pH-adjusted with one-sixth volume of 1 M Tris base, boiled, and to equilibrate SDB-XC frit material. Phosphopeptides were × μ subjected to SDS/PAGE for Coomassie staining or immuno- eluted from TiO2 with 2 50- L portions of 500 mM Na2HPO4, blotting. For smaller-scale GST pull-downs, cell lysates were in- pH 7, and retained on SDB-XC frit. Peptides were desalted in 50 μ μ cubated with 10 μg of GST-14-3-3e WT or K49E for 2 h and L of 1% FA and subsequently eluted from SDB-XC in 50 Lof 50% ACN and 0.5% FA. Eluates were dried on a speedvac and washed four times with lysis buffer before elution with reducing − sample buffer, SDS/PAGE, and immunoblotting. stored at 80 °C. To increase phosphoproteome coverage before MS analysis, Stable Isotope Labeling by Amino Acids in Cell Culture. HEK293 and phosphopeptides were fractionated offline by strong cation ex- 12 14 12 14 change (SCX) chromatography. Peptides were solubilized in 100 A375 cells were grown in light ([ C6 N2]Lys, [ C6 N4]Arg) 13 15 13 15 μL of loading buffer (0.2% FA and 15% ACN) and loaded onto and heavy ([ C6 N2]Lys, [ C6, N4]Arg) DMEM (Cambridge Isotope Labs) where required. Both light and heavy cell types StageTips containing 6 mg of PolySULFOETHYL A SCX phase (5 μm, 300 angstrom). Then columns were washed with were supplemented with 10% dialyzed fetal bovine serum (In- μ vitrogen). For the use of pharmacological inhibitors, HEK293 an additional 50 L of the loading buffer and peptides were eluted in 100-μL salt steps with 40, 70, 100, 150, and 500 mM cells were serum-starved for 24 h and cells cultured in heavy NaCl dissolved in loading buffer. Flow-through and salt fractions media were treated with PD184352 (10 μM) or BI-D1870 were collected, dried on a speedvac, resuspended in 15 μLof4% (10 μM) for 30 min followed by phorbol-12-myristate-13-acetate FA, and analyzed by nano liquid chromatography-MS/MS. (PMA) (50 ng/mL) treatment for 30 min in both light and heavy SCX fractions obtained after offline fractionation were analyzed conditions. In A375 cells, cells were serum-starved for 24 h and by online reverse-phase chromatography coupled with an elec- μ μ treated with PD184352 (10 M) or BI-D1870 (10 M) for 2 h in trospray ionization interface to acquire MS (measuring intensity the heavy media. For the use of shRNA to target RSK1/2, and m/z ratio for peptides) and MS/MS (fragmentation spectra of HEK293 cells in heavy media were virally infected as described peptides) scans. A nanoflow HPLC system (Eksigent; Thermo above, serum-starved for 24 h, and stimulated with PMA (50 ng/ Fisher Scientific) was used for online reversed-phase chromato- mL) treatment for 30 min in both light and heavy conditions. graphic separation; peptides were loaded on a 5-mm-long trap A375 cells in heavy media were virally infected as described column (inner diameter 300 μm) in buffer A (0.2% FA) and above and serum-starved for 24 h. We performed two biological separated on 18-cm-long fused silica capillary analytical column replicates for each experimental design detailed above with (inner diameter 150 μm), both packed with 3 μm 200 Å Magic cross-labeling (changing the treatment condition on the light AQ C18 reverse-phase material (Michrom). Peptides were eluted cultured cells instead of heavy cultured cells) without significant by an increasing concentration of buffer B (0.2% FA in ACN) differences. from 5 to 40% in 100 min. Following the gradient elution, the Galan et al. www.pnas.org/cgi/content/short/1405601111 1of10 column was washed with 80% buffer B and reequilibrated with also added to the database as well as reversed versions of all 5% buffer B. Peptides were eluted into the mass spectrometer at sequences. For searching, the enzyme specificity was set to a flow rate of 600 nL/min. The total run time was ∼125 min, trypsin with the maximum number of missed cleavages set to 2. including sample loading and column conditioning. Peptides were The precursor mass tolerance was set to 20 ppm for the first search analyzed using an automated data-dependent acquisition on a [used for nonlinear mass recalibration (6)] and then to 6 ppm LTQ-Orbitrap Elite mass spectrometer. Each MS scan was for the main search. Search criteria included a static modifica- acquired at a resolution of 240,000 FWHM (at 400 m/z)for + mass range 300–2,000 Th with the lock mass option enabled tion of cysteine residues of 57.0214 Da; a variable modification + (m/z: 445.120025) and was followed by up to 12 MS/MS data- of 15.9949 Da to include potential oxidation of methionines; and dependent scans on the most intense ions using collision- a modification of +79.966 on serine, threonine, or tyrosine for the induced activation (CID). AGC target values for MS and MS/ identification of phosphorylation. The false discovery rate for pep- MS scans were set to 1E6 (maximum fill time 500 ms) and 1E5 tide, protein, and site identification was set to 1%, the minimum (maximum fill time 50 ms), respectively. The precursor isolation peptide length was set to 6, and the “peptide requantification” window was set to 2 Th with CID normalized collision energy of function was enabled. To transfer identifications across different 35; the dynamic exclusion window was set to 60 s. runs, the “match between runs” option in MaxQuant was enabled with a retention time window of 1 min.