{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_1uM_LF-268_1hr_phosphokinomics_w-Annotation.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L1-1uM-LF-268_1hr_H1-DMSO_Phospho_tech2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L-DMSO_H-1uM-LF-268_1hr_Phospho_tech2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L1-1uM-LF-268_1hr_H1-DMSO_Phospho_tech1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L2-1uM-LF-268_1hr_H2-DMSO_Phospho_tech2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L2-1uM-LF-268_1hr_H2-DMSO_Phospho_tech1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/04/PXD075307/111718_HCT116_L-DMSO_H-1uM-LF-268_1hr_Phospho_tech1.raw"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["djmaly@uw.edu"],"submitter":["Daniel Brush"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Human","Dda","Lc-msms","Phosphoproteomics","Kinase"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD075307"],"tissue":["Colon"],"sample_protocol":["HCT-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."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Raw 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."],"omics_type":["Proteomics"],"labhead":["Dustin James Maly"],"instrument_platform":[""],"labhead_affiliation":["Department of Chemistry, University of Washington"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["10.1038/S41589-026-02212-2"],"submitter_mail":["dbrush@uw.edu"],"submitter_affiliation":["University of Washington"],"submitter_country":["United States"],"additional_accession":[]},"is_claimable":false,"name":"Phosphoproteomic profiling of kinobead-enriched fractions from LF-268-treated HCT-116 cells via SILAC-based quantification","description":"SILAC-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.","dates":{"publication":"2026-04-20","submission":"2026-03-06"},"accession":"PXD075307","cross_references":{"TAXONOMY":["NEWT:6945","NEWT:3555","NEWT:2","NEWT:157546","NEWT:35554","NEWT:38942","NEWT:307972","NEWT:32046","NEWT:544496","NEWT:2042546","NEWT:45351","NEWT:43179","NEWT:4513","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:1211601","NEWT:876138","NEWT:237561","NEWT:5833","NEWT:6928","NEWT:10036","NEWT:36745","NEWT:1351","NEWT:1438992","NEWT:2649997","NEWT:272563","NEWT:224326","NCBITaxon:79857","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:10029","NEWT:1000589","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:1283","NEWT:931281","NEWT:4550","NEWT:1000561","NEWT:197","NCBITaxon:79824","NEWT:4787","NCBITaxon:4563","NEWT:5755","NEWT:44689","NEWT:3218","NEWT:5759","NEWT:1736231","NEWT:1270","NEWT:2242","NEWT:4784","NEWT:11320","NEWT:360106","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1735272","NEWT:83334","NEWT:83332","NEWT:44685","NEWT:317513","NEWT:1148","NEWT:580240","NEWT:294128","NEWT:11676","NEWT:55571","NEWT:100226","NEWT:4530","NEWT:4896","NEWT:75058","NEWT:13616","NEWT:1094343","NEWT:296543","NEWT:1773","NEWT:1895","NEWT:1182590","NEWT:935293","NEWT:64152","NEWT:4924","NEWT:749200","NEWT:990346","NEWT:145953","NEWT:257309","NEWT:100816","NEWT:263","NEWT:230741","NEWT:52283","NEWT:284812","NCBITaxon:1313","NEWT:43330","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:990119","NEWT:408172","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:257313","NEWT:400772","NEWT:3708","NEWT:128161","NEWT:332648","NEWT:106592","NEWT:536231","NEWT:1432138","NEWT:10312","NEWT:1424507","NCBITaxon:1773","NEWT:9598","NEWT:8030","NEWT:1639","NEWT:188229","NEWT:3818","NEWT:480","NEWT:4909","NEWT:67767","NEWT:432359","NEWT:46835","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:10306","NCBITaxon:2759","NEWT:1233435","NEWT:8022","NEWT:145943","NCBITaxon:4932","NEWT:595536","NEWT:240906","NEWT:593117","NEWT:3635","NEWT:5811","NEWT:235443","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:499175","NEWT:476272","NEWT:3747","NEWT:367830","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:28038","NEWT:1423","NEWT:4932","NEWT:3603","NEWT:2759","NEWT:3847","NEWT:327159","NEWT:178876","NEWT:327160","NEWT:573","NEWT:9031","NEWT:7091","NEWT:241368","NEWT:42528","NEWT:190802","NEWT:9778","NEWT:150475","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:13076","NEWT:1249668","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:4081","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:507601","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:2829","NEWT:216599","NEWT:216595","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:1283300","NEWT:6183","NEWT:6063","NEWT:334747","NEWT:61235","NEWT:6289","NEWT:436486","NEWT:6287","NEWT:300641","NEWT:727","NEWT:9796","NEWT:725","NEWT:170187","NEWT:260707","NCBITaxon:6191","NEWT:1836","NEWT:185431","NEWT:29760","NEWT:260704","NEWT:703612","NEWT:260705","NEWT:80863","NEWT:2697049","NCBITaxon:6073","NEWT:884204","NEWT:6279","NEWT:1123869","NEWT:9544","NEWT:7370","NEWT:83906","NEWT:607699","NEWT:6282","NEWT:208964","NEWT:1134506","NEWT:575584","NEWT:38783","NEWT:8727","NEWT:4006","NEWT:8726","NEWT:6426","NEWT:6669","NEWT:10090","NEWT:4120","NEWT:51515","NEWT:5693","NEWT:8724","NEWT:51511","NEWT:92867","NEWT:8723","NEWT:5334","NEWT:37334","NEWT:382352","NCBITaxon:10359","NEWT:242619","NEWT:632957","NEWT:373995","NEWT:544404","NEWT:9925","NEWT:8839","NEWT:4232","NEWT:2758385","NEWT:4113","NEWT:837","NEWT:11298","NEWT:171101","NEWT:714","NEWT:421932","NEWT:196627","NEWT:5691","NEWT:48479","NEWT:627025","NEWT:1097677","NEWT:61674","NEWT:1117957","NEWT:9913","NEWT:4100","NEWT:1076","NEWT:6763","NEWT:803","NEWT:29722","NEWT:380394","NEWT:1692259","NEWT:180066","NEWT:135588","NEWT:1843183","NEWT:58002","NEWT:326423","NEWT:4577","NEWT:1416333","NEWT:5664","NEWT:21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