Broad-spectrum kinase profiling in live cells with lysine-targeted. sulfonyl fluoride probes
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1 Broad-spectrum kinase profiling in live cells with lysine-targeted sulfonyl fluoride probes Qian Zhao, Xiaohu Ouyang, Xiaobo Wan, Ketan S. Gajiwala, John C. Kath, Lyn H. Jones, Alma L. Burlingame, and Jack Taunton Supplementary Figures Figure S1. LC-MS analysis of purified SRC kinase domain treated with probes 1-3. Figure S2. LC-MS/MS analysis of SRC/probe 2 adduct. Figure S3. Proportion of kinases and nonkinases identified from cells treated with probe 2. Figure S4. In-gel fluorescence analysis of proteins labeled by probe 2, added to cells in the presence of increasing concentrations compound 4. Figure S5. Kinome coverage of probe 2, as determined by enzymatic assays of 375 protein kinases. X-ray data collection and refinement statistics Synthesis and Characterization of Kinase Probes Scheme S1. Chemical synthesis and characterization of probes 1-3 and compound 4 General Methods and Materials S1
2 Figure S1. LC-MS spectra showing adducts of probes 1-3 with SRC kinase domain after incubation for 1 h at room temperature. (A) LC-MS analysis of unmodified SRC (32,705 Da). (B-D) LC-MS analysis of SRC + probes 1-3, showing the expected increase in MW (±2 Da): 1, +533 Da; 2, +519 Da; 3, +519 Da. S2
3 Figure S2. LC-MS/MS analysis of SRC/probe 2 adduct after trypsinization. The following fragment ions have a mass consistent with the addition of probe 2 (minus fluoride): b4, b6, b7, b11, b12, b14, b15, b16. S3
4 Figure S3 (related to Figure 2B, Supplementary Table 2). Proportion of (A) protein number, (B) total MS signal intensity, (C) median MS signal intensity of kinases and nonkinases identified from cells treated with probe 2. Figure S4 (related to Figure 3). Fluorescence scan of SDS-PAGE gel resolving proteins labeled by probe 2 in the presence of increasing concentrations of compound 4. S4
5 Figure S5 (related to Supplementary Table 4). Probe 2 (1 µm) was tested against a panel of 375 kinases in enzymatic assays (Invitrogen). Kinases inhibited by 50% were plotted on a kinome phylogenetic tree. Kinome trees (Fig. 5 and S5) were produced using Kinome Render ( courtesy of Cell Signaling Technology ( S5
6 X-ray Data Collection and Refinement Statistics SRC EGFR (T790M/L858R) Space group P1 P Cell dimensions a, b, c (Å) 42.0,63.3, ,70.0,110.4 α, β, γ (º) 78.85,88.75, ,90,90 Wavelength (Å) Resolution range(å) a ( ) ( ) Unique reflections Redundancy 1.5 (1.2) 6.5 (6.5) I/σ (1.58) 13.9 (4.7) Completeness (%) (55.14) 99.9 (99.1) b R merge (0.848) (0.525) Structure refinement Resolution range (Å) ( ) ( ) No. reflections (2743) (6611) c R work (0.3432) (0.250) d R free (0.3598) (0.252) Average B factor (Å 2 ) Rmsd bond length (Å) Rmsd bond angles (º) PROCHECK statistics e Ramachandran favored (%) Ramachandran outliers (%) a Values in parentheses are for the data in the highest resolution shell. b R merge = I i I m / I i, where I i is the intensity of the measured reflection and I m is the mean intensity of all symmetry related reflections. c R work = F o F c / F o, where F o and F c are the observed and calculated structure factor amplitudes. d R free is the same as R work, but calculated on 5% reflections not used in refinement. e Analyzed by PROCHECK S6
7 Synthesis and Characterization of Kinase Probes Scheme S1. Chemical synthesis and characterization of probes 1-3 and compound 4. Scheme S1: Reagents and conditions: (a) 5-cyclopropyl-1H-pyrazol-3-amine, DIPEA, THF (57%); (b) NaOH, dioxane, H 2 O (44%); (c) Propargylamine, HATU, DIPEA, DMF (86%); (d) N-Boc-piperazine, DMF, 80 o C (89%); (e) TFA, DCM (98%); (f) 4-(fluorosulfonyl)benzoyl chloride, DIPEA, DCM, DMF (39%); (g) 4-(bromomethyl)benzenesulfonyl fluoride, DIPEA, DMF (72%); (h) 3- (bromomethyl)benzenesulfonyl fluoride, DIPEA, DMF (63%); (i) N-Boc-piperazine, DMF, 130 o C (94%); (j) TFA, DCM (91%); (k) 4-(bromomethyl)benzenesulfonyl fluoride, DIPEA, DMF (65%). Synthetic Methods NMR spectra were recorded on a Varian spectrometer at 400 MHz or on a Bruker spectrometer at 500 MHz. Chemical shifts were reported as parts per million (ppm) from an internal tetramethylsilane standard or solvent references. LC/MS was performed on a Waters Alliance HT LC/MS (0.2 ml/min) S7
8 using an Xterra MS C18 column (Waters) and a water/acetonitrile gradient (0.1% formic acid). All other solvents were of ACS grade (Fisher Scientific) and used without further purification. Commercially available reagents were used without further purification. Analytical thin-layer chromatography was performed with silica gel 60 F254 glass plates (EM Science). Silica gel chromatography was performed with mesh silica gel (Selecto Scientific). Compound 5. To a solution of methyl 2,6-dichloropyrimidine-4-carboxylate (15 g, 72 mmol) in anhydrous THF (100 ml) at 0 o C, was added 5-cyclopropyl-1H-pyrazol-3- amine (9 g, 72 mmol) and N,N-diisopropylethylamine (11 g, 85 mmol) in anhydrous THF (100 ml), and the reaction was then stirred at room temperature for 2 h. The solvents were evaporated in vacuo, and the residue was heated in methanol (100 ml) at 80 o C for 1 h. The mixture was cooled to room temperature and the precipitate was filtered and dried under vacuum overnight. Compound 5 (12 g, 41 mmol, 57%) was obtained as a light yellow solid. 1 H-NMR (400 MHz, DMSO-d6): δ (br, 1H), (d, J = 44 Hz, 1H), 8.28 (s, 0.5H), 7.34 (s, 0.5H), 6.38 (s, 0.5H), 5.67 (s, 0.5H), 3.87 (s, 3H), 1.91 (br, 1H), 0.93 (m, 2H), 0.69 (m, 2H). MS (ES+) m/z (M + H) +1. Compound 6. To a solution of compound 5 (8 g, 27 mmol) in dioxane (50 ml), was added 1 M NaOH (50 ml) over 10 min. After the reaction was stirred at room temperature for 20 min, the mixture was acidified to ph 4 with 1 M HCl, and a white precipitate formed. After filtration, the solid precipitate was washed with water (2 x 50 ml) and dried under vacuum overnight. Compound 6 (3.4 g, 12 mmol, 44%) was obtained as a white solid. 1 H-NMR (400 MHz, DMSO-d6): δ (br, 1H), (d, J = 40 Hz, 1H), 8.24 (s, 0.5H), 7.32 (s, 0.5H), 6.37 (s, 0.5H), 5.68 (s, 0.5H), 1.91 (br, 1H), 0.93 (m, 2H), 0.69 (m, 2H). MS (ES+) m/z (M + H) +1. S8
9 Compound 7. To a mixture of compound 6 (3.3 g, 11.8 mmol), propargylamine (0.65 g, 11.8 mmol), and N,N-diisopropylethylamine (6.1 g, 47.2 mmol) in anhydrous DMF (50 ml), was added HATU (5.8 g, 15.3 mmol) over 30 min. The reaction was stirred at room temperature under argon overnight. The mixture was diluted with 150 ml of water and extracted with 200 ml of ethyl acetate. The organic phase was dried over magnesium sulfate and concentrated in vacuo. Pure compound 7 (3.2 g, 10.1 mmol, 86%) was obtained as a white solid after silica gel chromatography (1:1 to 1:4 hexanes : ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (br, 1H), (d, J = 72 Hz, 1H), 9.03 (t, J = 4 Hz, 1H), 8.22 (s, 0.5H), 7.34 (s, 0.5H), 6.40 (s, 0.5H), 5.69 (s, 0.5H), 4.03 (dd, J = 8 Hz, 4 Hz, 2H), 3.09 (t, J = 4 Hz, 1H), 1.92 (m, 1H), 0.94 (m, 2H), 0.70 (m, 2H). MS (ES+) m/z (M + H) +1. Compound 8. To a solution of compound 7 (200 mg, 0.63 mmol) in DMF (10 ml), was added N-Boc-piperazine (235 mg, 1.26 mmol), and the reaction was stirred at 100 o C for 2 h. The mixture was concentrated in vacuo and dissolved in 30 ml ethyl acetate. The resulting mixture was washed with saturated ammonium chloride (30 ml), saturated sodium bicarbonate (30 ml), and brine (30 ml). The organic phase was dried over magnesium sulfate and concentrated in vacuo. Pure compound 8 (260 mg, 0.56 mmol, 89%) was obtained as a white solid after silica gel chromatography (100% ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (S, 1H), 9.79 (br s, 1H), 8.94 (t, J = 4 Hz, 1H), 6.83 (br s, 1H), 6.17 (br s, 1H), 4.02 (dd, J = 8 Hz, 4 Hz, 2H), 3.79 (s, 4H), 3.41 (s, 4H), 3.08 (t, J = 4 Hz, 1H), 1.89 (m, 1H), 1.43 (m, 9H), 0.93 (m, 2H), 0.68 (m, 2H). 13 C- NMR (500 MHz, DMSO-d6): δ , , , , , , , 94.35, 92.84, 81.29, 79.06, 72.57, 43.38, 42.69, 28.19, 28.08, 7.87, MS (ES+) m/z (M + H) +1. S9
10 Compound 9. To a solution of compound 8 (0.2 g, 0.43 mmol) in DCM (7 ml), was added trifluoroacetic acid (3 ml), and the reaction was stirred at room temperature. After 1 h, the reaction mixture was concentrated in vacuo and residual trifluoroacetic acid was azeotropically removed. Compound 9 (0.2 g, 0.42 mmol, 98%) was obtained as a light yellow solid and used in the next step without further purification. MS (ES+) m/z (M + H) +1. Compound 1. To a mixture of compound 9 (40 mg, mmol), N,Ndiisopropylethylamine (32 mg, 0.25 mmol) in DCM (4 ml) and DMF (1 ml), was added 4-(fluorosulfonyl)benzoyl chloride (22 mg, 0.1 mmol). The reaction was stirred at room temperature for 20 min, and DCM was evaporated in vacuo. The residue was partitioned between EtOAc (20 ml) and saturated aqueous sodium bicarbonate (20 ml). The organic phase was dried over magnesium sulfate and concentrated in vacuo. Pure compound 1 (18 mg, mmol, 40%) was obtained as a white solid after silica gel chromatography (1:1 to 1:6 hexanes : ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (S, 1H), 9.83 (br s, 1H), 8.93 (s, 1H), 8.24 (d, J = 8 Hz, 2H), 7.86 (d, J = 8 Hz, 2H), 6.83 (br s, 1H), 6.15 (br s, 1H), 4.02 (s, 2H), 3.95 (s, 2H), 3.82 (s, 2H), 3.75 (s, 2H), 3.36 (s, 2H), 3.09 (s, 1H), 1.89 (m, 1H), 0.90 (m, 2H), 0.67 (m, 2H). MS (ES+) m/z (M + H) +1. Compound 2 (XO44). To a mixture of compound 9 (40 mg, mmol) and 4-(bromomethyl)benzenesulfonyl fluoride (23 mg, 0.09 mmol) in DMF (1 ml) was added N,N-diisopropylethylamine (43 mg, 0.33 mmol). The reaction was stirred at room temperature for 20 min and concentrated in vacuo. The residue was partitioned between EtOAc (20 ml) and saturated aqueous sodium bicarbonate (20 ml). The organic phase was washed with brine (20 ml), dried over magnesium sulfate and concentrated in vacuo. Pure S10
11 compound 2 (32 mg, 0.06 mmol, 72%) was obtained as a white solid after silica gel chromatography (1:2 to 1:4 hexanes : ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (s, 1H), 9.79 (s, 1H), 8.91 (t, J = 4 Hz, 1H), 8.12 (d, J = 8 Hz, 2H), 7.78 (d, J = 8 Hz, 2H), 6.78 (s, 1H), 6.16 (s, 1H), 4.00 (dd, J = 8 Hz, 4 Hz, 2H), 3.82 (s, 4H), 3.72 (s, 2H), 3.09 (t, J = 4 Hz, 1H), 2.48 (s, 4H), 1.86 (m, 1H), 0.90 (m, 2H), 0.64 (m, 2H). 13 C-NMR (500 MHz, DMSO-d6): δ , , , , , , , , , , , 94.08, 92.81, 81.31, 72.59, 61.07, 52.59, 43.53, 28.17, 7.83, MS (ES+) m/z (M + H) +1. Compound 3. To a mixture of compound 9 (40 mg, mmol) and 3- (bromomethyl)benzenesulfonyl fluoride (23 mg, 0.09 mmol) in DMF (1 ml) was added N,N-diisopropylethylamine (43 mg, 0.33 mmol). The reaction was stirred at room temperature for 20 min, and the solvents were evaporated in vacuo. The residue was partitioned between EtOAc (20 ml) and saturated aqueous sodium bicarbonate (20 ml). The organic phase was then washed with brine (20 ml), dried over magnesium sulfate, and concentrated in vacuo. Pure compound 3 (28 mg, mmol, 63%) was obtained as a white foam after silica gel chromatography (1:2 to 1:4 hexanes : ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (s, 1H), 9.73 (br s, 1H), 8.87 (t, J = 4 Hz, 1H), 8.06 (s, 1H), 8.02 (d, J = 8 Hz, 1H), 7.92 (d, J = 8 Hz, 1H), 7.75 (t, J = 4 Hz, 1H), 6.81 (br s, 1H), 6.11 (br s, 1H), 3.99 (dd, J = 8 Hz, 4 Hz, 2H), 3.79 (s, 4H), 3.68 (s, 2H), 3.05 (t, J = 4 Hz, 1H), 2.46 (s, 4H), 1.85 (m, 1H), 0.90 (m, 2H), 0.64 (m, 2H). 13 C-NMR (500 MHz, DMSO-d6): δ , , , , , , , , , , , , , 94.06, 92.76, 81.26, 72.52, 60.64, 52.44, 43.55, 28.15, 7.79, MS (ES+) m/z (M + H) +1. S11
12 Compound 10. To a solution of compound 5 (0.64 g, 2.18 mmol) in DMF (10 ml), was added N-Boc-piperazine (0.81 g, 4.36 mmol), and the reaction was stirred at 130 o C for 4 h. The mixture was concentrated in vacuo and dissolved in 50 ml ethyl acetate. The resulting mixture was washed with saturated ammonium chloride (50 ml), saturated sodium bicarbonate (50 ml), and brine (50 ml). The organic phase was dried over magnesium sulfate and concentrated in vacuo. Pure compound 10 (0.91 g, 2.05 mmol, 94%) was obtained as a white solid after silica gel chromatography (100% ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (s, 1H), 9.83 (s, 1H), 6.80 (s, 1H), 6.16 (s, 1H), 3.81 (s, 3H), 3.72 (t, J = 4 Hz, 4H), 3.40 (t, J = 4 Hz, 4H), 1.91 (m, 1H), 1.42 (s, 9H), 0.93 (m, 2H), 0.68 (m, 2H). MS (ES+) m/z (M + H) +1. Compound 11. To a solution of compound 10 (100 mg, 0.23 mmol) in DCM (3.5 ml), was added trifluoroacetic acid (1.5 ml), and the reaction was stirred at room temperature. After 1 h, the reaction mixture was concentrated in vacuo and the residual trifluoroacetic acid was azeotropically removed. Compound 11 (95 mg, 0.21 mmol, 91%) was obtained as a light yellow solid and used in the next step without further purification. MS (ES+) m/z (M + H) +1. Compound 4. To a mixture of compound 11 (70 mg, mmol) and 4- (bromomethyl)benzenesulfonyl fluoride (40 mg, mmol) in DMF (1 ml) was added N,N-diisopropylethylamine (80 mg, mmol). The reaction was stirred at room temperature for 20 min, and the solvents were evaporated in vacuo. The residue was partitioned between EtOAc (20 ml) and saturated aqueous sodium bicarbonate (20 ml). The organic phase was washed with brine (20 ml), dried over magnesium sulfate, and concentrated in vacuo. Pure compound 4 (51 mg, mmol, 65%) was obtained as a white solid after silica gel chromatography S12
13 (1:2 hexanes : ethyl acetate). 1 H-NMR (400 MHz, DMSO-d6): δ (s, 1H), 9.85 (s, 1H), 8.10 (d, J = 8 Hz, 2H), 7.76 (d, J = 8 Hz, 2H), 6.76 (s, 1H), 6.19 (s, 1H), 3.80 (s, 3H), 3.76 (s, 4H), 3.68 (s, 2H), 2.46 (s, 4H), 1.87 (m, 1H), 0.90 (m, 2H), 0.65 (m, 2H). 13 C-NMR (500 MHz, DMSO-d6): δ , , , , , , ,130.29, , , , 96.96, 92.73, 61.10, 52.53, 52.33, 43.50, 7.82, MS (ES+) m/z (M + H) +1. S13
14 General Methods and Materials Protein expression and purification Hexahistidine-tagged recombinant chicken SRC kinase domain (residues ) was purified as previously described 1. The final concentrations were determined spectrophotometrically at 280 nm using an extinction coefficient of 52,370 M -1 cm -1. LC-MS analysis of SRC modification by probes 1-3 Labeling experiments were performed by treating 20 µl SRC (final concentration 5 μm) in kinase buffer (50 mm Tris ph 8, 100 mm NaCl, 5 % glycerol) with probes 1-3 (15 μm). After 1 hour, the reaction was quenched by adding 20 µl acetonitrile/0.1 % formic acid. Samples were analyzed by LC-MS (Waters Acquity UPLC/ESI-TQD, mm Acquity UPLC BEH300 C4 column). Deconvolution of multiply charged ions was performed using Waters MassLynx software (version 4.1). Protein crystallography To a solution of SRC (5 mg/ml) in kinase buffer was added 3 equiv of probe 2 in DMSO (10 mm). Complete labeling was confirmed by LC/MS after 1 hour incubation at room temperature. The mixture was clarified by centrifugation at 14,000 g for 10 min. The hanging drop crystallization method was used by mixing 1:1 protein and precipitation solution (100 mm MES, ph 6.1, 50 mm NaOAc, 16% glycerol and 4% PEG 4000). Crystals grew to their maximum size in ~5 days and were cryoprotected in the crystallization solution supplemented with 20% glycerol and stored in liquid nitrogen. Diffraction data were collected in the UCSF Macromolecular Structure Group Crystallography Facility using a Rigaku RU-200 rotating anode system fitted with Varimax-HR optics and a Raxis-IV image plate detector. The reflections were indexed, integrated and scaled using HKL2000 (HKL Research, Inc.) in the space group P1. Molecular replacement was performed using Phenix 2 with SRC kinase domain as a starting model (PDB code 3EL7). The initial model building was performed manually in the program Coot 3 and further refined with REFMAC 4 from the CCP4 suite 5. Human EGFR kinase domain containing T790M/L858R/V948R mutations was expressed, purified, and crystallized as previously described. 7 Briefly, EGFR kinase domain (T790M,L858R,V948R) at 7 mg/ml was S14
15 introduced to 1 mm probe 2 in DMSO (~ 1:5 molar ratio), and incubated at 4 for 2 hrs. The complex was then filtered with a low-protein binding 0.45 µm membrane to remove particulates, and set up for crystallization using a Phoenix (Art Robbins) robot in a 96-well SBS vapordiffusion format. Sitting drops (1:1 v/v protein : well solution, containing 0.2 M ammonium sulfate, 0.1 M HEPES ph 7.5, 20% PEG 8000, 5.7% v/v isopropanol) were incubated at 4 for 5-7 days before crystals resembling wedges grew. Crystals were harvested and placed into a cryo containing well solution + 25% glycerol before they were frozen in liquid N2 and shipped to IMCA for data collection. The protein complex crystallized in space group P Trypsinization of SRC/probe 2 adduct To a solution of SRC (5 mg/ml) in kinase buffer was added 3 equiv of probe 2 in DMSO (10 mm). Complete labeling was confirmed by LC/MS after 1 hour incubation at room temperature. The SRC/probe 2 adduct was reduced with 5 mm DTT at 56 for 30 min and alkylated with 20 mm iodoacetamide in the dark for 15 min. Then the mixture was diluted with 50 mm ammonium biocarbonate. Sequencing grade trypsin (Promega) was incubated with the mixture at 1:10 ratio at 37 C overnight. Digestion was stopped by adding 1% formic acid. The resulting peptides were enriched with C18 OMIX tips (Agilent) and eluted in 50% acetonitrile and 0.1% formic acid. The samples were dried down by SpeedVac and resuspended in 0.1% formic acid for analysis by LC-MS/MS. Cell culture Jurkat cells were cultured in RPMI media 1640 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco), 100 U/mL penicillin and 100 μg/ml streptomycin. Cells were maintained in a humidified 37 C incubator with 5% CO 2. Treatment of cells with probes and preparation of cell lysates Jurkat cells were cultured to a density of 1x10 6 /ml. Cells were first treated with compound 4, dasatinib (Selleck Chemicals), or DMSO for 60 min at 37. Probe 2 was then added to a final concentration of 2 µm and the cells were incubated for 30 min at 37. During incubation, the cell suspensions were gently mixed every min. Cells were collected by centrifugation at 300 g at 4 and then resuspended in lysis buffer containing 100 mm HEPES ph 7.5, 150 mm S15
16 NaCl, 0.1% NP-40, 1 mm PMSF, and 1X complete EDTA-free protease inhibitor cocktail (Roche). Cellular debris was removed by centrifugation at 16,000 g for 30 min at 4 C. Protein concentration was determined by the BCA assay (Novagen). Cell lysates were diluted with lysis buffer to give a final protein concentration of 5 mg/ml for the subsequent Cu(I)-catalyzed click reactions. Click conjugation with TAMRA-azide or biotin-azide Click chemistry was initiated by sequential addition of the following to each lysate (50 µl for fluorescence visualization and 1 ml for pulldowns): 1% SDS, 100 µm TAMRA-azide or biotinazide, 1 mm TCEP, 100 µm TBTA ligand in 1:4 DMSO:t-butyl alcohol, and 1 mm CuSO4. After 1.5 h at room temperature, reactions were quenched as described below. In-gel fluorescence visualization After click conjugation, 15 µl samples were treated with 6X Laemmli sample buffer and resolved by SDS-PAGE. Gels were scanned for fluorescently labeled proteins (Typhoon Imaging System, Molecular Dynamics), then stained with Coomassie. Streptavidin affinity enrichment of biotinylated proteins After click conjugation, samples (1 ml) were mixed with 10 ml cold acetone and stored overnight ( 20 C). Precipitated proteins were pelleted by centrifugation at 3,700 rpm at 4 C for 30 min and washed twice with 5 ml cold methanol. Protein pellets were resuspended in 1 ml buffer containing 100 mm TRIS ph 7.5 and 1% SDS and desalted by passing through a NAP-10 columns (GE) with 1% NP-40, 0.1% SDS in ice-cold PBS. Labeled proteins were then immobilized with magnetic streptavidin beads (Thermo Fisher Scientific). After washing with ice-cold lysis buffer and 1 ml freshly prepared 6 M urea (3 times each), beads were rinsed with buffer containing 20 mm Tris-HCl, ph 8.0 and 2 mm CaCl 2. On-bead trypsin digestion of pulldown products Proteins immobilized on magnetic streptavidin beads were suspended in trypsinization buffer (20 mm Tris-HCl, ph 8.0 and 2 mm CaCl 2 ). Sample were treated with 5 mm DTT at 56 C for 30 min, followed by alkylation with 20 mm iodoacetamide at room temperature for 30 min in the S16
17 dark. Samples were treated once more with 5 mm DTT (5 min), followed by incubation with 400 ng sequencing grade trypsin (Promega) overnight at 37 C. Digestion was stopped by adding 1% formic acid. The resulting peptides were enriched with C18 Omix Tips (Agilent Technologies) and eluted with 50% acetonitrile and 0.1% formic acid. The samples were dried down by SpeedVac and then resuspended in 0.1% formic acid for analysis by LC-MS/MS. LC-MS/MS analysis Peptides resulting from trypsinization were analyzed on an Orbitrap Velos (Thermo Scientific) connected to a NanoAcquity Ultra Performance UPLC system (Waters). An EasySpray C18 column (Thermo Scientific) was used to resolve peptides (90-min gradient with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B). The LTQOrbitrap Velos was operated in the data-dependent mode to automatically switch between MS and MS/MS. The top six precursor ions with a charge state of 2+ or higher were fragmented by CID. All generated peak lists were searched against SwissProt human database (SwissProt ) using Protein Prospector.6 The database search was performed with the following parameters: a mass tolerance of 20 ppm for precursor masses; ±0.8 Da for MS/MS, cysteine carbamidomethylation as a fixed modification and methionine oxidation as a variable modification. The enzyme was specified as trypsin with 1 missed cleavage allowed. For analysis of SRC/probe 2 adduct, probe 2 mass addition ( Da) was included as a variable modification. Spectra were manually inspected to ensure correct identification and ion assignment of the probe 2-modified peptide. Label-free quantification using mass spectrometry Only peptides uniquely identified in a single protein in the proteome database and not containing post-translational modifications were included in the analysis. Extracted peptide ion chromatographs (XICs) were created in Protein Prospector6 using a retention time window of +/20 seconds from when they were selected for MS/MS. Protein intensities were calculated from the sum of the individual unique peptide XICs using an in-house script. Intensities of undetected proteins were set to 1 (low non-zero value to avoid errors in calculating log ratios). For crosssample normalization and subsequent statistical analysis, a set of background proteins was selected based on the following criteria: (1) proteins detected in control streptavidin bead S17
18 pulldowns from untreated cells (lacking probe 2); (2) proteins whose intensity values showed a coefficient of variation of <90% across all conditions. To normalize intensity values between samples, we used a modified median centering method based on the log2 intensities of the background proteins in each sample. All subsequent data analyses were performed using the normalized protein intensities. For protein quantification, we required proteins to have a minimum of three unique peptides in three biological replicates. The percent reduction of probe 2 labeling by dasatinib was calculated for each kinase as follows: % Inhibition = 100(1 Idas/IDMSO), where Idas = intensity in dasatinib-treated cells, and IDMSO = intensity in DMSOtreated cells. To assess the statistical significance of this value for each kinase, we used Student s t-test as follows: for each kinase, the percent reduction of probe 2 labeling by 100 nm or 300 nm dasatinib (3 replicate measurements, "array 1") were compared to the percent reduction of probe 2 labeling of all background proteins (3 replicate measurements, "array 2") using the T.TEST function in Microsoft Excel (one-tailed, unequal variance). To make the volcano plots in Figure 4, the mean "% inhibition" value (percent reduction of probe 2 labeling by 100 nm or 300 nm dasatinib) was calculated for each kinase, along with the corresponding P-value from the t-test. Supplementary References (1) Seeliger, M. A.; Young, M.; Henderson, M. N.; Pellicena, P.; King, D. S.; Falick, A. M.; Kuriyan, J. Protein Sci. 2005, 14, (2) Adams, P. D.; Afonine, P. V.; Bunkoczi, G.; Chen, V. B.; Davis, I. W.; Echols, N.; Headd, J. J.; Hung, L.-W.; Kapral, G. J.; Grosse-Kunstleve, R. W.; McCoy, A. J.; Moriarty, N. W.; Oeffner, R.; Read, R. J.; Richardson, D. C.; Richardson, J. S.; Terwilliger, T. C.; Zwart, P. H. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010, 66, 213. (3) Emsley, P.; Cowtan, K. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60, (4) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240. (5) Project, C. C. Acta Crystallogr., Sect. D: Biol. Crystallogr. 1994, 50, 760. (6) Chalkley, R. J.; Baker, P. R.; Huang, L.; Hansen, K. C.; Allen, N. P.; Rexach, M.; Burlingame, A. L. Mol. Cell. Proteomics 2005, 4, (7) Gajiwala, K. S.; Feng, J.; Ferre, R.; Ryan, K.; Brodsky, O.; Weinrich, S.; Kath, J. C.; Stewart, A. Structure 2013, 21, 209. S18
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