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Cells were incubated for 4 hours at 37ºC and then additionally treated with media containing 1 μM erlotinib and 300 ng/mL AXL activating antibody for 10 minutes. The cells were then immediately placed on ice, washed with ice-cold phosphate-buffered saline, and lysed with cold 8 M urea containing Phosphatase Inhibitor Cocktail I and Protease Inhibitor Cocktail I (Boston BioProducts). The lysates were centrifuged at 20,000 x g and 4°C to pellet cell debris, and the supernatants were removed and stored at - 80°C. A bicinchoninic acid (BCA) protein concentration assay (Pierce) was performed according to the manufacturer’s protocol to estimate the protein concentration in each lysate. Cell lysates were reduced with 10 mM DTT for 1 hour at 56℃, alkylated with 55 mM iodoacetamide for 1 hour at RT shielded from light, and diluted 5-fold with 100 mM ammonium acetate, pH 8.9, before trypsin (Promega) was added (20:1 protein:enzyme ratio) for overnight digestion at RT. The resulting solutions were acidified with 1 mL of acetic acid (HOAc) and loaded onto C18 Sep-Pak Plus Cartridges (Waters), rinsed with 10 mL of 0.1% HOAc, and eluted with 10 mL of 40% Acetonitrile (MeCN)/0.1% HOAc. Peptides were divided into 200 aliquots, and sample volume was reduced using a vacuum centrifuge (Thermo) before lyophilization to dryness for storage at -80℃. TMT labeling for multiplexed analysis was performed according to the manufacturer’s protocol. Samples, each containing ~200 μg of peptides, were resuspended in 35 μL HEPES (pH 8.5), vortexed, and spun down at 13,400 rpm for 1 minute. 400 μg of a given channel of TMT10plex (Thermo) in anhydrous MeCN was added per sample. Samples were shaken at 400 rpm for 1 hour, after which the labeling reaction was quenched using 5% hydroxylamine (50%, Thermo). After another 15 minutes of shaking, all samples were combined using the same pipette tip to minimize sample loss, and sample aliquots were washed twice with 40 μL of 25% MeCN/0.1% HOAc, which was added to the collection tube to improve yield. Sample volume was reduced using a vacuum centrifuge and then lyophilized to dryness for storage at -80℃.Immunoprecipitation (IP) and IMAC were used sequentially to enrich samples for phosphotyrosine-containing peptides. TMT-labeled samples were incubated in IP buffer consisting of 1% Nonidet P-40 with protein G agarose beads conjugated to 24 μg of 4G10 V312 IgG and 6 μg of PT-66 (P3300, Sigma) overnight at 4℃. Peptides were eluted with 25 μL of 0.2% trifluoroacetic acid for 10 minutes at room temperature; this elution was performed twice to improve yield. Eluted peptides were subjected to phosphopeptide enrichment using immobilized metal affinity chromatography (IMAC)-based Fe-NTA spin column to reduce non- specific, non-phosphorylated peptide background. A High-Select Fe-NTA enrichment kit (Pierce) was used according to the manufacturer’s instructions with the following modifications. Eluted peptides from IP were incubated with Fe-NTA beads containing 25 μL binding washing  33  buffer for 30 minutes. Peptides were eluted twice with 20 mL of elution buffer into a 1.7 mL microcentrifuge tube. The eluates were concentrated in a SpeedVac until approximately 1 μL of sample remained, and then resuspended in 10 μL of 5% acetonitrile in 0.1% formic acid. Samples were loaded directly onto an in-house constructed fused silica capillary column (50 μm inner diameter (ID) x 10 cm) packed with 5 μm C18 beads (YMC gel, ODS-AQ, AQ12S05) and with an integrated electrospray ionization tip (~2 μm tip ID)."],"repository":["Pride"],"labhead":["Forest White"],"instrument_platform":[""],"submission_type":["COMPLETE"],"labhead_affiliation":["Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA, 02139, USA; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge MA, 02139, USA; Center for Precision Cancer Medicine, Massachusetts Institute of Technology, Cambridge MA, 02139, USA"],"publication":["Not available"],"submitter_affiliation":["Koch Institute, Massachusetts Institute of Technology"],"labhead_mail":["fwhite@mit.edu"],"technology_type":["Data-dependent acquisition","Mass Spectrometry","Bottom-up proteomics"],"disease":["Lung Non-squamous Non-small Cell Carcinoma"],"submitter_keywords":["Computational modeling","Axl","Systems biology","Kinase biology","Luad","Drug resistance","Egfr"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD079076"],"quantification_method":[""],"modification":[""],"data_protocol":["LC-MS/MS of pTyr peptides was performed on an Agilent 1260 LC system coupled to a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific). Peptides were separated using a 140-minute gradient with 70% acetonitrile in 0.2 mol/L acetic acid at a flow rate of 0.2 mL/minute and an approximate split flow of 20 nL/minute. The mass spectrometer was operated in data-dependent acquisition with the following settings for MS1 scans: m/z range: 350 to 2,000; resolution: 60,000; AGC target: 3 x 10 6 ; maximum injection time (maxIT): 50 ms. The top 15 most abundant ions were isolated and fragmented by higher energy collision-induced dissociation with the following settings: resolution: 60,000; AGC target: 1x10 5 ; maxIT: 350 ms; isolation width: 0.4 m/z; collisional energy (CE): 33%; dynamic exclusion: 20 seconds. Crude peptide analysis was performed on a Q Exactive Plus mass spectrometer to correct for small variations in peptide loadings across the TMT channels. Approximately 30 ng of the supernatant from pTyr IP was loaded onto an in-house packed precolumn (100 μm ID x 10 cm) packed with 10 mm C18 beads (YMC gel, ODS-A, AA12S11) and analyzed with a 70-minute LC gradient. MS1 scans were performed at the following settings: m/z range: 350 to 2,000; resolution: 70,000; AGC target: 3x10 6 ; maxIT: 50 ms. The top 10 most abundant ions were isolated and fragmented with a collision energy (CE) of 33% at a resolution of 35,000. Mass spectra were processed using Proteome Discoverer, version 2.5 (Thermo Fisher Scientific), and searched against the human SwissProt database with Mascot, version 2.4 (MatrixScience, RRID:SCR_014322). MS/MS spectra were searched with a mass tolerance of 10 ppm for precursor ions and 20 mmu for fragment ions. Cysteine carbamidomethylation, TMT-labeled lysine, and TMT-labeled peptide N-termini were set as fixed modifications. Oxidation of methionine and phosphorylation of serine, threonine, and tyrosine were searched as dynamic modifications. TMT reporter quantification was extracted and isotope corrected in Proteome Discoverer. Peptide spectrum matches (PSM) were filtered according to the following parameters: rank=1, Mascot ion score &gt; 15, isolation interference &lt; 40%, and average TMT signal &gt; 1,000. Peptides with missing values across any channel were filtered out. We performed three biological replicates for phosphoproteomic analysis of PC9 AXL Y-to-F mutants treated with EA. The three datasets were concatenated, mean-centered across cell lines, and log2-transformed. To exclude phosphosites with unreliable measurements across replicates, all recurrent phosphosites in two or three biological replicates were identified. For those appearing in two biological replicates, peptides with a Pearson correlation coefficient less than 0.55 were filtered out; for those appearing in all three biological replicates, peptides with standard deviations of 0.5 or more were discarded. To exclude peptides with minimal variation across cell lines, we filtered out phosphosites showing less than a 0.5-fold change between the maximum and minimum values in each cell line. Overlapping peptides were then averaged across replicates. The resulting preprocessed phosphoproteomic data set was then fit using DDMC."],"omics_type":["Proteomics"],"species":["Homo Sapiens (human)"],"submitter_mail":["jacqueline.s.gerritsen@gmail.com"],"curator_keywords":["Human proteome project"],"submitter_country":["United States"],"doi":["10.6019/PXD079076"],"additional_accession":[]},"is_claimable":false,"name":"Dissecting early AXL signaling regulators and associated phenotypes in erlotinib-treated EGFR mutant lung cancer by phophosite perturbations","description":"Targeted therapies for receptor tyrosine kinases are effective but invariably limited by drug resistance. In EGFR-mutant lung cancer, AXL activation drives resistance to erlotinib by restoring cell survival and migration. To map these signaling mechanisms, we generated a panel of lung adenocarcinoma PC9 cell lines with phenylalanine substitutions at each intracellular AXL tyrosine residue. By integrating phosphorylation data with phenotypic changes via multivariate modeling, we found that AXL signaling organizes into two clusters enriched for Abl1 and SFK substrate motifs. A peptide specificity screen identified FAK1 as a top proximal substrate of AXL. Downstream, AXL-mediated YAP1 activation was found to sustain drug tolerance, while combined inhibition eliminated persister cells in vitro. These AXL and YAP pathways correlate with disease progression and poor clinical outcomes in erlotinib-treated patients. Collectively, this study dissects the specific signaling regulators by which AXL drives erlotinib resistance.","dates":{"publication":"2026-06-14","submission":"2026-05-30"},"accession":"PXD079076","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:2042546","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:59753","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:1073882","NEWT:475174","NEWT:111205","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:10034","NEWT:661410","NEWT:638632","NEWT:224326","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:1589","NEWT:135622","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:656064","NEWT:383379","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:38815","NEWT:317447","NEWT:4688","NEWT:111225","NEWT:7719","NEWT:868565","NEWT:135674","NEWT:79329","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:224308","NEWT:3347","NEWT:160621","NEWT:212790","NEWT:110368","NEWT:1310161","NEWT:77133","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:44688","NEWT:44689","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:246196","NEWT:246197","NEWT:145458","NEWT:44685","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:3329","NEWT:419481","NEWT:5507","NEWT:410661","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:1287689","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:1182590","NEWT:3712","NEWT:3711","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:284812","NEWT:8175","NEWT:43330","NEWT:1603293","NEWT:44664","NEWT:43346","NEWT:3702","NEWT:1245466","NEWT:244366","NEWT:1246791","NEWT:118694","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:52299","NEWT:8165","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:460519","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1303443","NEWT:9483","NEWT:485","NEWT:56636","NEWT:2853422","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1185650","NEWT:216778","NEWT:294607","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:109760","NEWT:29031","NEWT:317047","NEWT:460523","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:108458","NEWT:5936","NEWT:320637","NEWT:1078283","NEWT:3750","NEWT:983964","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:1116234","NEWT:1255228","NEWT:410289","NEWT:373153","NEWT:472","NEWT:940825","NEWT:529507","NEWT:1071661","NEWT:470","NEWT:5911","NCBITaxon:50557","NEWT:39251","NEWT:29491","NEWT:101841","NEWT:446","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:166313","NEWT:63366","NEWT:63367","NEWT:215402","NEWT:1547","NEWT:906914","NEWT:9031","NEWT:27292","NEWT:1050722","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:150475","NEWT:267872","NEWT:172269","NEWT:9534","NEWT:5180","NEWT:256737","NEWT:9541","NEWT:8694","NEWT:33936","NEWT:8692","NEWT:2903","NEWT:185579","NEWT:13076","NEWT:1006581","NEWT:8673","NEWT:33940","NEWT:550","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:36185","NEWT:1054211","NEWT:1225786","NEWT:51338","NEWT:575412","NEWT:28112","NEWT:6493","NEWT:6494","NEWT:6491","NEWT:507601","NEWT:520","NEWT:186441","NEWT:643680","NEWT:214092","NCBITaxon:6157","NEWT:13095","NEWT:162425","NEWT:104105","NEWT:216257","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:5147","NEWT:28108","NEWT:28104","NEWT:407821","NEWT:568703","NCBITaxon:2","NEWT:568708","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:298176","NEWT:40674","NEWT:1194669","NEWT:51329","NEWT:443906","NEWT:519","NEWT:2510939","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","NEWT:475932","NEWT:256318","NEWT:206411","NCBITaxon:6191","NEWT:229533","NEWT:214053","NEWT:80863","NEWT:90675","NEWT:52638","NEWT:57075","NEWT:1666905","NEWT:8697","NEWT:8695","NEWT:884204","NEWT:1123869","NEWT:9544","NEWT:9545","NEWT:979","NEWT:7370","NEWT:83906","NEWT:99488","NEWT:1134506","NEWT:255470","NEWT:38783","NEWT:6426","NEWT:33090","NEWT:9935","NEWT:287889","NEWT:305959","NEWT:13489","NEWT:29829","NEWT:92867","NEWT:92866","NCBITaxon:3055","NEWT:51750","NEWT:202950","NEWT:295027","NCBITaxon:11320","NEWT:632957","NEWT:9925","NCBITaxon:9606","NEWT:90690","NEWT:1436183","NEWT:300273","NEWT:4232","NEWT:416348","NEWT:11298","NEWT:4236","NEWT:7742","NEWT:196627","NEWT:200308","NEWT:200302","NEWT:870435","NEWT:9913","NEWT:9915","NEWT:105841","NEWT:2666255","NEWT:999810","NCBITaxon:5693","NEWT:28995","NEWT:1392998","NEWT:380394","NEWT:226900","NEWT:1266738","NEWT: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