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Schenk"],"technology_type":["Data-dependent acquisition","Mass Spectrometry"],"software":[""],"submitter_keywords":[""],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD062674"],"sample_protocol":["For global proteomics, cell pellets from one well of a 6-well plate per biological repeat (n=5) were lysed in 200 µL of preheated (95°C) buffer (2.5% [v/v] SDS in 100 mM Tris-HCl, pH 8.5). DNA was hydrolysed with the addition of 2 µL neat TFA and lysates were neutralised to pH 8.5 by addition of 1 M Tris-HCl as previously described (Dagley et al, 2019). Protein concentration was determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. For global proteomics, cell lysates (20 µg protein per replicate) were transferred to 0.5 mL LoBind deep well plate (Eppendorf) prepared for MS analysis using the modified SP3 protocol (Hughes et al, 2019), with some modifications. Briefly, samples were subjected to simultaneous reduction and alkylation with a final concentration of 10 mM TCEP and 40 mM 2-chloracetamide (CAA) followed by heating at 95°C for 10 min. Prewashed magnetic PureCube Carboxy agarose beads (20 µL, Cube Biotech) were added to all the samples along with ACN (70% [v/v] final concentration) and incubated at ambient temperature for 20 min. Samples were placed on a magnetic rack and supernatants were discarded, and beads were washed twice with 70% (v/v) ethanol and once with neat ACN. ACN was completely evaporated from the tubes using a CentriVap (Labconco) before the addition of digestion buffer (50 mM Tris-HCl, pH 8) containing 1 µg each of enzymes Lys-C (Wako, 129–02541) and SOLu-Trypsin (Sigma-Aldrich, EMS0004). Trypsin-LysC on-bead digestion was performed with agitation (400 rpm) for 1 h at 37°C on a ThermoMixer C (Eppendorf). For global proteomics, the samples were transferred to pre-equilibrated C18 StageTips (2× plugs of 3M Empore resin, no. 2215) following digestion for sample clean-up. The eluates were lyophilised to dryness before being reconstituted in 150 µL 0.1% (v/v) FA / 2% (v/v) ACN ready for MS analysis.Peptides were loaded on a 15 cm C18 fused silica column with an integrated emitter tip (IonOpticks, ID 75 µm, OD 360 µm, 1.6 µm C18 beads) which was maintained at 50°C using a column oven. siRNA KD samples were acquired with a Neo Vanquish (Thermo Fisher Scientific) directly coupled online with an Astral mass spectrometer (Thermo Fisher Scientific) and peptides were separated with a binary buffer system of buffer A (0.1% [v/v] FA) and buffer B (80% [v/v] ACN plus 0.1% [v/v] FA), at a flow rate of 400 nL/min. The gradient started at 2% B and increased to 34% B in 30 min before increasing to 100% (v/v) B within 0.1 min and held for 3 min prior to returning to 2% (v/v) B and re-equilibration. The mass spectrometer was operated in positive polarity mode with a capillary temperature of 275°C. The DIA methods consisted of a MS1 scan (m/z = 380-980) with an AGC target of 5 × 106 and a maximum injection time of 5 ms (R = 240,000). DIA scans were acquired with the Astral detector with an AGC target of 8 × 104 and 3 ms maximum time. Fragmentation occurred in the higher-energy collisional dissociation (HCD) cell with a normalised stepped collision energy was 25% and the spectra were recorded in profile mode. 199 non-uniform DIA windows across 380-980 were collected with a maximum injection time of 3 ms and a 0.6 s loop control which achieved an average of five data points per peak. Global proteomic samples were acquired using a custom nano-flow high-performance liquid chromatography (HPLC) system (Thermo Fisher Scientific Ultimate 300 RSLC Nano-LC, PAL systems CTC autosampler). The HPLC was coupled to a timsTOF Pro (Bruker) equipped with a CaptiveSpray source. Peptides were loaded directly onto the column at a flow rate of 600 nL/min with buffer A (99.9% Milli-Q H2O, 0.1% [v/v] FA) and eluted at 400 nL/min on a 30 min linear analytical gradient of increasing buffer B (90% [v/v] ACN, 0.1% [v/v] FA) from 2% (v/v) to 34% (v/v). The timsTOF Pro MS was operated in diaPASEF mode using Compass Hystar 5.1. The settings on the thermal ionization MS (TIMS) analyser were as follows: Lock Duty Cycle to 100% with equal accumulation and ramp times of 100 ms, and 1/K0 Start 0.6 Vs/cm2 End 1.6 Vs/cm2, Capillary Voltage 1400 V, Dry Gas 3 L/min, Dry Temp 180°C. The DIA methods were set up using the instrument firmware (timsTOF control 2.0.18.0) for data-independent isolation of multiple precursor windows within a single TIMS scan. The method included two windows in each diaPASEF scan, with window placement overlapping the diagonal scan line for doubly and triply charged peptides in the m/z – ion mobility plane across 16 × 25 m/z precursor isolation windows (resulting in 32 windows) defined from m/z 400 to 1,200, with 1 Da overlap, and collision induced dissociation (CID) collision energy ramped stepwise from 20 eV at 0.8 Vs/cm2 to 59 eV at 1.3 Vs/cm2."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["MS raw files were processed using DIA-NN 1.8.1 as described previously (Steger et al, 2021). Global raw files were searched with the following settings: Trypsin specificity, peptide length of 7-30 residues, cysteine carbamidomethylation enabled as a fixed modification, variable modifications set to N-terminal protein acetylation and oxidation of methionine, the maximum number of missed cleavages at two (global proteomics) and match between runs (MBR) enabled within individual cell lines. Downstream processing and differential expression analysis was performed in R using limma (Ritchie et al, 2015) and DEP packages. For global proteomics quantities were determined using MaxLFQ (Cox et al, 2014b) for proteins with at least two peptides, data were filtered for proteins that were observed in at least four out of five replicates in at least one condition. VSN normalised and imputed using missing values imputed using a mixed BPCA and MinD method. For global proteomics samples, significance testing of log2-transformed intensities was performed, and Q-values from limma <0.05 were considered significant."],"omics_type":["Proteomics"],"labhead":["David Komander"],"instrument_platform":[""],"labhead_affiliation":["Head, Ubiquitin Signalling Division Walter and Eliza Hall Institute of Medical Research Australia"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["schenk.p@wehi.edu.au"],"publication":["41946992 Schenk P, Devine SM, Cobbold SA, Geoghegan ND, Kyran EL, Ang CS, Alexandrovics JA, Calleja DJ, Multari DH, Vaibhav V, Lu BGC, Klemm TA, Dagley LF, Lowes KN, Williamson NA, Eichhorn PJA, Ng AP, Feltham R, Komander D. Global analysis of cancer cell responses to USP9X inhibition. EMBO J. 2026 10.1038/s44318-026-00742-y"],"submitter_affiliation":["WEHI"],"submitter_country":["Australia"],"additional_accession":[]},"is_claimable":false,"name":"Global analysis of cancer cell responses to USP9X inhibition","description":"Global proteomics experiment using USP9X siRNA (and control) MDA-MB-231 cells with (10 µM, 24 h) and without USP9X inhibitor treatment using WEHI-092 and untreated (DMSO) control (P5448, see sample key). Global proteomics experiment profiling cancer cell lines treated with USP9X inhibitor WEHI-092 (10 µM, 24 h) and untreated (DMSO) control.P4971 1-5 UO-31 cells, untreatedP4971 6-10 UO-31 cells, WEHI-092P4971 11-15 A549 cells, untreatedP4971 16-20 A549 cells, WEHI-092P4971 21-25 DLD-1 cells, untreatedP4971 26-30 DLD-1 cells, WEHI-092P4971 41-45 OVCAR8 cells, untreatedP4971 46-50 OVCAR8 cells, WEHI-092P4971 51-55 SK-MEL-2 cells, untreatedP4971 56-60 SK-MEL-2 cells, WEHI-092P5080 11-15 HMEC-1 cells, untreatedP5080 16-20 HMEC-1 cells, WEHI-092","dates":{"publication":"2026-04-09","submission":"2025-04-07"},"accession":"PXD062674","cross_references":{"TAXONOMY":["NEWT:108018","NEWT:377960","NEWT:1131","NEWT:2042546","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:224326","NEWT:376619","NEWT:3765","NEWT:3767","NEWT:433727","NEWT:135622","NEWT:672127","NEWT:656061","NEWT:37965","NEWT:656064","NEWT:3760","NEWT:36630","NEWT:381198","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:44250","NEWT:317447","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:12637","NEWT:2164133","NEWT:2767846","NEWT:314344","NEWT:108061","NEWT:224308","NEWT:49928","NEWT:160621","NEWT:110368","NEWT:1916073","NEWT:1310161","NEWT:1230383","NEWT:77133","NEWT:1310165","NEWT:114727","NEWT:5516","NEWT:498211","NEWT:347256","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:498217","NEWT:498216","NEWT:11320","NEWT:1310173","NEWT:161934","NEWT:1148","NEWT:5508","NEWT:419481","NEWT:5507","NEWT:1140","NCBITaxon:2157","NEWT:2066563","NEWT:1143","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:709964","NEWT:3712","NEWT:3711","NEWT:3714","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:550537","NEWT:284812","NEWT:3701","NEWT:3702","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:215451","NEWT:460519","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2499525","NEWT:142786","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:987059","NEWT:491","NEWT:460523","NEWT:85694","NEWT:85692","NEWT:3750","NEWT:291146","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:445","NEWT:2003758","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:219798","NEWT:548","NEWT:150475","NEWT:256737","NEWT:51350","NEWT:8694","NEWT:8692","NEWT:542","NEWT:394503","NEWT:13076","NEWT:219785","NEWT:451515","NEWT:550","NEWT:10804","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:1225786","NEWT:771870","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:507601","NEWT:520","NEWT:64657","NEWT:186441","NEWT:2585209","NEWT:643680","NEWT:214092","NEWT:40697","NEWT:13095","NEWT:162425","NEWT:242124","NEWT:9986","NEWT:8654","NEWT:8658","NEWT:1268063","NEWT:8657","NEWT:9983","NEWT:8655","NEWT:80833","NEWT:479096","NEWT:28108","NEWT:28104","NEWT:1114957","NEWT:568703","NEWT:568708","NEWT:52641","NEWT:353152","NEWT:353153","NEWT:227377","NEWT:1774284","NEWT:27689","NEWT:207769","NEWT:40674","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:83906","NEWT:99488","NEWT:1134506","NEWT:255470","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:287889","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:315750","NEWT:632957","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:11298","NEWT:4236","NEWT:398031","NEWT:196627","NEWT:523840","NEWT:1076878","NEWT:200308","NEWT:200302","NEWT:9913","NEWT:9915","NEWT:105841","NEWT:1170227","NEWT:2666255","NEWT:999810","NEWT:28995","NEWT:1392998","NEWT:1150621","NEWT:1085791","NEWT:745531","NEWT:226900","NEWT:231490","NEWT:590651","NEWT:449239","NEWT:244704","NEWT:9909","NEWT:9902","NEWT:430615","NEWT:563041","NEWT:36599","NEWT:41949","NEWT:9900","NEWT:326423","NEWT:326424","NEWT:13013","NEWT:749906","NEWT:418985","NEWT:749907","NEWT:431947","NEWT:142809","NEWT:1095183","NEWT:302409","NEWT:9979","NEWT:130821","NEWT:2320211","NEWT:27606","NEWT:290610","NEWT:1519788","NEWT:59202","NEWT:9975","NEWT:8643","NEWT:1801631","NEWT:1159899","NEWT:502780","NEWT:860688","NEWT:35231","NEWT:1774208","NEWT:8644","NEWT:1125630","NEWT:202914","NEWT:222523","NEWT:27602","NEWT:502779","NEWT:297246","NEWT:483139","NEWT:101491","NEWT:8639","NEWT:8637","NEWT:1114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