Prideapplication/xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/phospho_mqpar.xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/total_mqpar.xmlftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction1.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230505_Audrey_PhosEnrichment_TMT_2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction5.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction8.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction6.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction3.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction4.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/20230421_AudreyLumeau_TMT_fraction2.rawftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/Reporter_channel_descriptions.jpegftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/total_txt.zipftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/05/PXD061444/phospho_txt.zipprimaryOK200s.mcclelland@qmul.ac.ukFaraz MardakhehData-dependent acquisitionMass SpectrometryBottom-up proteomicsBreast Cancerphospho-proteomicsAtr; phospho-proteomics; tmt;tmtAtrhttps://www.ebi.ac.uk/pride/archive/projects/PXD061444Permanent Cell Line CellCell CultureCells were washed with PBS and scraped in SDS 4%, 50mM Tris HCl pH 7.4 for cell lysis and protein extraction. After sonication and lysate clearing by centrifugation, protein concentration was measured using BCA protein assay kit and balanced to 55 ug for every sample. To prepare the samples for proteomics analysis, isobaric filter-aided sample preparation (iFASP), as performed previously (Azman et al., EMBO J, 2023). Briefly, samples were diluted in UA buffer (8M Urea, 100mM Tris-HCl pH 8.5 in dH2O) and reduced with the addition of 10mM DTT (VWR, M109) and incubated for 30 min at room temperature. Samples were then alkylated with 55mM IAA (Iodoacetamide, VWR, 786–228) and incubated in the dark for 30 min at room temperature. Samples were loaded on Vivacon® 500, 30,000 MWCO Hydrosart filters (Sartorius, VN01H22) and centrifuged at 14,000 g for 20 min. Sampled were washed twice with UA buffer and centrifuged at 14,000 g for 20min. Then, samples were washed three times with 100mM TEAB (Triethylammonium bicarbonate, Fisher Scientific, 15215753) before a final addition of 100μL 100nM TEAB and 1μg of Trypsin (Merck, T6567) to every sample before an overnight incubation at 37C. Each TMTpro™ 18-plex Label Reagent (ThermoFisher Scientific, A52047) was resuspended in 100% Acetonitrile and 8μg of TMT-Pro label was added to each respective sample filter before incubation for 1h at 25C at 600rpm. Excess TMT-Pro labels were quenched with the addition of 5% hydroxylamine and incubated for another 30min. Peptides were eluted by centrifugation at 14,000 g for 20 mins, the filters were washed using 100mM TEAB and a final wash of 30% acetonitrile. All labelled peptides were pooled and split 10% for the total proteomics analysis and 90% for the phospho-enrichment. The pooled peptide mixture was dried with a vacuum concentrator before downstream sample preparation. For total proteomics analysis, the TMT-labelled pool was fractionated into seven fractions using Pierce™ High pH reverse‐phase fractionation kit (Life Technologies, 84868), using the manufacturers protocol. Fractions were dried using vacuum concentration before being resuspended in Buffer A* (2% ACN, 0.1% TFA, 0.5% Acetic Acid) for LC-MS/MS analysis. For phosphoproteomics analysis, phosphopeptides were enriched for using Titansphere TiO phospho‐peptide enrichment kit (GL Sciences, 5010-21308), following the manufacturer’s instructions. Eluted enriched TMT-labelled phosphopeptides were dried using vacuum centrifugation before resuspension in Buffer A* for LC-MS/MS analysis.PrideLC–MS/MS analysis was performed on a Q Exactive‐plus Orbitrap mass spectrometer coupled with a nanoflow ultimate 3000 RSL nano HPLC platform (Thermo Fisher). Dried peptide mixtures were resuspended in A* buffer. For total proteomics analysis, equivalent of ~1 μg of protein was injected into the nanoflow HPLC from each fraction. For phospho‐proteomics analysis, ~90% of the total peptide mixture was injected. Samples were resolved at flow rate of 250 nl/min on an Easy‐Spray 50 cm × 75 μm RSLC C18 column (Thermo Fisher). Each run consisted of a 123 min gradient of 3% to 35% of Buffer B (0.1% FA in Acetonitrile) against Buffer A (0.1% FA in LC–MS gradient water), and separated samples were infused into the MS by electrospray ionization (ESI). Spray voltage was set at 1.95 kV, and capillary temperature was set to 255°C. MS was operated in data‐dependent positive mode, where MS1 scan is followed by 15 MS2 scans (top 15 methods). Full‐scan survey spectra (m/z 375–1,500) were acquired with a 70,000 resolution for MS scans and 35,000 for the MS2 scans. A 30‐s dynamic exclusion was applied. Mass spectrometry raw data files were searched and quantified against a FASTA file of the Homo sapiens proteome using MaxQuant (version 2.2.2.0). Parameter groups were set to define the phospho- or total-proteome analysis and fractions were set for the total proteomics analysis. “Reporter ion MS2” type option was selected with a reporter mass tolerance of 0.003 Da. Enzyme specificity was set to “Trypsin,” allowing up to two missed cleavages. Phosphorylation (STY) was added to variable modifications for the phospho-parameter group. False discovery rates (FDR) were calculated using a reverse database search approach and were set at 1%. “Re‐quantify” options were enabled. Default MaxQuant settings were used for all other parameters. Downstream statistical analysis was performed in Perseus (version 1.6.2.3) where data was filtered for contaminants, reverse peptides and peptides only identified by site, log2 transformed, and filtered for valid values only. The Log2 data generated were used for subsequent analysis of total protein expression and Kinase Enrichment Analysis (KSEA).ProteomicsSarah McClellandBarts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ.PARTIALHomo Sapiens (human)Not availablef.mardakheh@qmul.ac.ukDepartment of Biochemistry, University of OxfordUnited KingdomfalsePhospho-proteomics analysis of ATR inhibitor responses in sensitive and resistant human breast cancer cellsTo search in an unbiased manner for the specific mechanisms causing differential ATRi sensitivity, we performed total and phosphoproteomic analyses using tandem mass tagging (TMT) mass spectrometry. Extracted proteins were compared between three sensitive and three resistant human breast cancer cell lines, treated with DMSO, ATR inhibitor 1h or 24h, using quantitative proteomics.2026-05-192025-03-04PXD061444NEWT:6945NEWT:184922NEWT:6703NEWT:3555NEWT:2NEWT:157546NEWT:35554NEWT:38942NEWT:307972NEWT:32046NEWT:544496NEWT:2102NEWT:2042546NEWT:45351NEWT:43179NEWT:4513NEWT:5722NEWT:55153NCBITaxon:10407NEWT:1736309NEWT:309800NEWT:1211601NEWT:876138NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:10036NEWT:36745NEWT:1351NEWT:1438992NEWT:2649997NEWT:272563NEWT:224326NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:3885NEWT:3888NEWT:1589NEWT:135622NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:3641NEWT:383379NEWT:466585NEWT:10029NEWT:913645NEWT:1000589NEWT:85963NEWT:85962NEWT:317447NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:192875NEWT:34305NEWT:59729NCBITaxon:183674NEWT:224308NEWT:84645NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:1283NEWT:931281NEWT:4550NEWT:1000561NEWT:197NEWT:1390363NEWT:288705NCBITaxon:79824NEWT:4787NCBITaxon:4563NEWT:5755NEWT:44689NEWT:3218NEWT:5759NEWT:1736231NEWT:1270NEWT:374990NEWT:2242NEWT:4784NEWT:11320NEWT:360106NEWT:286NEWT:391619NEWT:287NEWT:10117NEWT:10239NEWT:10116NEWT:1280NEWT:1735272NEWT:83334NEWT:83332NEWT:44685NEWT:317513NEWT:1148NEWT:580240NEWT:294128NEWT:11676NEWT:55571NEWT:35500NEWT:100226NEWT:4530NEWT:4896NEWT:75058NEWT:13616NEWT:1390NEWT:1094343NEWT:296543NEWT:1773NEWT:1895NEWT:1182590NEWT:3712NEWT:105023NEWT:935293NEWT:64152NEWT:4924NEWT:749200NEWT:375146NEWT:990346NEWT:145953NEWT:257309NEWT:100816NEWT:263NEWT:230741NEWT:52283NEWT:284812NCBITaxon:1313NEWT:43330NEWT:1603293NEWT:408169NEWT:44544NEWT:4911NEWT:645463NEWT:3702NEWT:129249NEWT:243277NEWT:990119NEWT:408172NEWT:408170NEWT:493760NEWT:260710NEWT:257313NEWT:400772NEWT:3708NEWT:128161NEWT:332648NEWT:106592NEWT:536231NEWT:460519NEWT:1187947NEWT:1432138NEWT:10312NEWT:1424507NCBITaxon:1773NEWT:9598NEWT:8030NEWT:9483NEWT:1639NEWT:188229NEWT:3818NEWT:480NEWT:4909NEWT:67767NEWT:432359NEWT:46835NEWT:1182263NEWT:2711NEWT:376686NEWT:95486NEWT:9103NEWT:29159NEWT:253NEWT:10306NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:8022NEWT:145943NCBITaxon:4932NEWT:595536NEWT:240906NEWT:593117NEWT:89920NEWT:3635NEWT:5811NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:411483NEWT:884019NEWT:198215NEWT:411490NEWT:983964NEWT:169963NEWT:32644NEWT:225117NEWT:499175NEWT:109779NEWT:476272NEWT:3747NEWT:195051NEWT:367830NEWT:1255228NEWT:178616NEWT:410289NEWT:373153NEWT:352472NEWT:357NEWT:360094NEWT:470NEWT:1313NEWT:411469NEWT:84023NEWT:559292NEWT:39491NCBITaxon:5811NEWT:411464NEWT:411460NEWT:2014887NEWT:2762NEWT:1174673NEWT:562NEWT:411470NEWT:33952NEWT:2094720NCBITaxon:2697049NEWT:571256NEWT:28038NEWT:1663NEWT:1423NEWT:4932NEWT:3603NEWT:2759NEWT:3847NEWT:327159NEWT:178876NEWT:327160NEWT:573NEWT:9031NEWT:7091NEWT:108931NEWT:241368NEWT:42528NEWT:190802NEWT:9778NEWT:150475NEWT:303NEWT:9417NEWT:7111NEWT:347515NEWT:1216979NEWT:5180NEWT:256737NEWT:9541NEWT:115104NEWT:1121114NEWT:663NEWT:1081927NEWT:1238993NEWT:67825NEWT:185579NEWT:941442NEWT:220668NEWT:13076NEWT:1249668NEWT:7108NEWT:317NEWT:7227NEWT:7469NEWT:885318NEWT:9402NEWT:9644NEWT:415540NEWT:550NEWT:675060NEWT:4081NEWT:334542NEWT:554NEWT:27592NEWT:98334NEWT:426428NEWT:1276815NEWT:9639NEWT:7574NEWT:1715256NEWT:7215NEWT:575412NEWT:29204NEWT:2172103NEWT:7460NEWT:507601NEWT:643680NCBITaxon:6157NEWT:746360NEWT:6239NEWT:470150NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:73239NEWT:226186NEWT:1268063NEWT:8782NEWT:1263854NEWT:435590NEWT:1902NEWT:160488NEWT:28104NEWT:1908NEWT:13164NEWT:216129NCBITaxon:2NEWT:985076NEWT:1215323NEWT:52641NEWT:7038NEWT:6192NEWT:28532NCBITaxon:38727NEWT:353152NEWT:2829NEWT:366581NEWT:216599NEWT:216595NEWT:1194669NEWT:51329NEWT:243230NEWT:8355NEWT:9685NEWT:7029NEWT:1080772NEWT:8479NEWT:1283300NEWT:6183NEWT:6063NEWT:630NEWT:334747NEWT:61235NEWT:15368NEWT:6289NEWT:436486NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:170187NEWT:469008NEWT:260707NEWT:256318NCBITaxon:6191NEWT:1836NEWT:185431NEWT:29760NEWT:260704NEWT:703612NEWT:260705NEWT:80863NEWT:2697049NEWT:105231NEWT:1216981NEWT:4097NCBITaxon: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:34073NEWT:373995NEWT:5689NEWT:544404NEWT:9925NEWT:8839NEWT:4232NEWT:2758385NEWT:37349NEWT:4113NEWT:837NEWT:11298NEWT:171101NEWT:714NEWT:36015NEWT:421932NEWT:196627NEWT:5691NEWT:48479NEWT:627025NEWT:1097677NEWT:61674NEWT:1117957NEWT:9913NEWT:8709NEWT:4100NEWT:1076NEWT:6763NEWT:803NEWT:29722NEWT:380394NEWT:1692259NEWT:180066NEWT:817NEWT:135588NEWT:1843183NEWT:58002NEWT:326423NEWT:36111NEWT:4577NEWT:1416333NEWT:5664NEWT:2157NEWT:1678078NEWT:749906NEWT:418985NEWT:146479NEWT:749907NEWT:1911079NEWT:69373NEWT:264203NEWT:1480154NEWT:1274414NEWT:27606NEWT:59202NEWT:9975NEWT:9612NEWT:38865NEWT:51953NEWT:3197NEWT:9615NEWT:10299NEWT:860688NEWT:112273NEWT:36329NEWT:1147787NCBITaxon:3044782NEWT: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