Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_1uM_LF-268_1hr_phosphokinomics_w-Annotation.xlsxftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/checksum.txtftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L1-1uM-LF-268_1hr_H1-DMSO_Phospho_tech2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L-DMSO_H-1uM-LF-268_1hr_Phospho_tech2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L1-1uM-LF-268_1hr_H1-DMSO_Phospho_tech1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L2-1uM-LF-268_1hr_H2-DMSO_Phospho_tech2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L2-1uM-LF-268_1hr_H2-DMSO_Phospho_tech1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L-DMSO_H-1uM-LF-268_1hr_Phospho_tech1.rawprimaryOK200djmaly@uw.eduDaniel BrushData-dependent acquisitionMass SpectrometryBottom-up proteomicsHumanDdaLc-msmsPhosphoproteomicsKinasehttps://www.ebi.ac.uk/pride/archive/projects/PXD075307ColonHCT-116 cells grown in SILAC DMEM supplemented with 10% FBS, labeled with heavy (Arg10/Lys8) or light (Arg0/Lys0) isotopes. Treated with LF-268 (1 µM) or DMSO for 1 hour. Lysed with ice-cold modified RIPA buffer (50 mM Tris pH 7.8, 150 mM NaCl, 10 mM NaF, 1% IGEPAL CA-630, 0.25% sodium deoxycholate, 5% glycerol, protease inhibitors, 1 mM PMSF, phosphatase inhibitor cocktails 2 & 3) on 15-cm plates (250 µL), cleared at 17,000×g for 10 min at 4°C, and diluted to 2.0 mg/mL by Pierce 660 nm assay. Lysate (150 µL) incubated with kinobeads (10 µL 50% slurry, pre-washed 2× with modified RIPA) for 3 h at 4°C with end-over-end rotation. Beads washed 2× modified RIPA and 3× ice-cold TBS (50 mM Tris pH 7.8, 150 mM NaCl). On-bead denaturation in 25 µL denaturing buffer (6 M guanidinium chloride, 100 mM Tris pH 8.5, 5 mM TCEP, 10 mM CAM) at 95°C for 5 min. Diluted with 25 µL 100 mM TEAB, pH adjusted to 8–9 with 1 N NaOH. Sequential digestion: LysC (0.4 µg, 37°C, 2 h, 1400 rpm), diluted with 50 µL 100 mM TEAB, trypsin (0.4 µg, 37°C, overnight, 800 rpm). Quenched with Buffer A (0.1% TFA/5% acetonitrile, 1% formic acid), supernatant loaded onto in-house C18 column, washed with Buffer A (50 µL), eluted with Buffer B (80% acetonitrile/0.1% TFA, 50 µL), resuspended in Buffer A (20 µL). Phosphopeptide enrichment: peptides resuspended in 80% aq. acetonitrile/0.1% TFA and incubated with Fe³⁺-NTA loaded beads (pre-washed 10× with 80% aq. acetonitrile/0.1% TFA). Eluted twice with 50 mM KH₂PO₄/NH₄OH (pH 10), combined, pH neutralized, dried, C18 cleanup, reconstituted in 80:20 ACN:water. n=3 biological replicates.PrideRaw data analyzed with MaxQuant (Andromeda search engine) against UniProt human database (July 2015). Settings: multiplicity=2; SILAC labels Arg0/Lys0 (light) and Arg10/Lys8 (heavy); enzyme=trypsin/P; variable modifications: oxidation (M), acetyl (protein N-term), phospho (STY); LFQ minimum ratio count=2; LFQ minimum neighbors=3; LFQ average neighbors=6; MS/MS tolerance=10 ppm; FDR=0.01. Perseus downstream analysis: proteins classified as "only identified by site," "reverse," or "contaminant" excluded; proteins with ≤1 unique peptide excluded. Intensities log2-transformed; missing values imputed from a downshifted normal distribution (shift=1.3, width=0.2). H/L ratios reported as log2[LF-268/DMSO]. Significantly regulated phosphosites defined by competition score ≥ 1.5 and confidence value (−log10 p-value) ≥ 1.5 from two-tailed two-sample t-test, FDR=0.05.ProteomicsDustin James MalyDepartment of Chemistry, University of WashingtonPARTIALHomo Sapiens (human)10.1038/S41589-026-02212-2dbrush@uw.eduUniversity of WashingtonUnited StatesfalsePhosphoproteomic profiling of kinobead-enriched fractions from LF-268-treated HCT-116 cells via SILAC-based quantificationSILAC-based phosphoproteomic profiling of kinase-enriched fractions from HCT-116 cells treated with the DFG-out pan-RAF inhibitor LF-268 to characterize kinase signaling responses. Cells were metabolically labeled and treated with 1 µM LF-268 or DMSO for 1 hour, followed by serial kinobead enrichment and Fe³⁺-NTA IMAC phosphopeptide enrichment prior to LC-MS/MS.2026-04-202026-03-06PXD075307NEWT:6945NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:1211601NEWT:876138NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NEWT:6915NEWT:3649NEWT:101510NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:10029NEWT:1000589NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:3635NEWT:5811NEWT:235443NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:499175NEWT:476272NEWT:3747NEWT:367830NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:28038NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:115104NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:13076NEWT:1249668NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:415540NEWT:4081NEWT:554NEWT:98334NEWT:426428NEWT:7574NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:507601NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NCBITaxon:2NEWT:985076NEWT:1215323NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:2829NEWT:216599NEWT:216595NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:1283300NEWT:6183NEWT:6063NEWT:334747NEWT:61235NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:260707NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NCBITaxon:6073NEWT:884204NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NEWT:607699NEWT:6282NEWT:208964NEWT:1134506NEWT:575584NEWT:38783NEWT:8727NEWT:4006NEWT:8726NEWT:6426NEWT:6669NEWT:10090NEWT:4120NEWT:51515NEWT:5693NEWT:8724NEWT:51511NEWT:92867NEWT:8723NEWT:5334NEWT:37334NEWT:382352NCBITaxon:10359NEWT:242619NEWT:632957NEWT:373995NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:418985NEWT:146479NEWT:1911079NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT:72407NEWT:349741NEWT:9605NEWT:9606NEWT:157295NEWT:641501NEWT:7668NEWT:915099NEWT:74940NEWT:9721NEWT:137221NEWT:1450511NEWT:1143193NEWT:411901NEWT:9838NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:3055NEWT:6326NEWT:6689NEWT:5476NEWT:1274432NEWT:1274426NEWT:70448NEWT:9825NEWT:1274423NEWT:393765NEWT:698936NEWT:39946NEWT:9823NEWT:9940NEWT:521001NEWT:39947NEWT:1274420NEWT:5784580009-0009-2046-4973