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Parker"],"technology_type":["Mass Spectrometry","Bottom-up proteomics"],"software":[""],"submitter_keywords":["Protein correlation profiling","Insulin resistance","C2c12"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD075147"],"sample_protocol":["C2C12 cells were grown in Dulbecco's Modified Eagle Medium (DMEM) (GIBCO by Life Technologies; # 11995065), supplemented with 10% fetal bovine serum (FBS) (Life Technologies; #26140079), pyruvate and GlutaMAX (GIBCO by Life Technologies). Cells were kept at 37°C and 5% CO2 in a humidified incubator Direct Heat CO2 Incubator featuring Oxygen Control (In Vitro Technologies). At 90% confluence, the cells were differentiated into myotubes with the replacement of 10% FBS with 2% Horse Serum and cultured for a further 3 days. Myotubes were then incubated in 1ml of DMEM, 2% Horse Serum, Pyruvate, GlutaMAX with 0.2% BSA (fatty acid free) with or without 500µM palmitic acid for 24 hours to induce insulin resistance (IR). For IR incubation media, 5 mM palmitic acid dissolved in ethanol was conjugated with 2% BSA in DMEM, Pyruvate and GlutaMAX (GIBCO by Life Technologies) and diluted 10-fold. IR incubation media was then filter sterilised prior to incubation. On the day of harvest, cells were incubated in 1ml of DMEM, Pyruvate, GlutaMAX with 0.2% BSA with or without 500µM palmitic acid for 2 hours to achieve a basal state. Relevant cells were then stimulated with 100nM insulin for 30 minutes. Cells were washed twice with cold PBS, lysed in 100ul of native lysis buffer (0.25% Triton-X100, 10mM Tris.HCl, 50mM NaCl, 1mM TCEP, 1mM EDTA, EDTA-free PIC, phosIC, pH 7.5) and transferred to cold 1.5ml tubes and left on ice for 10 minutes. Cells were gently mixed with a pipette, spun down at 20,000 g for 20 minutes at 4°C, after which proteins were quantified with Qubit and normalized for separation via Blue Native-PAGE. Native lysates were separated on a NuPAGE 3-8% Tris-Acetate gel (Invitrogen; #EA0375BOX) at 150V for 2 hours with Tris-Glycine Native running buffer (Invitrogen; LC2672). Coomassie staining was then performed to visualize gel lanes. Briefly, gels were fixed in 40% methanol, 10% acetic acid for 10 minutes and directly incubated in 50ml of QC Colloidal Coomassie stain (BioRad; #1610803) overnight with gentle shaking at room temperature. Staining solution was removed, and gel was washed with milliQ overnight. Each gel lane was cut into 30 x 1.5mm fractions and each fraction was transferred into individual wells in a 96-well plate. Gel fractions were washed twice with 1ml of 40% acetonitrile in 50mM ammonium bicarbonate at 1500 x rpm for destaining. 500ul of acetonitrile was added and plate was shaken at 1500 x rpm to dehydrate gel fractions. Acetonitrile was removed and gel fractions were then rehydrated with 40ul of trypsin (13ng/ul; Sigma; #T6567) at 4˚C for 1.5 hours and excess removed. Gels were then covered with 50ul of 50mM ammonium bicarbonate to prevent dehydration then incubated overnight at 37˚C. The next day, 50ul of 20mM TCEP (Sigma; #75259) and 80mM CAA (Sigma; #22790) in 100mM Tris pH8.5 was added to each well and heated at 37˚C for 30 minutes for reduction and alkylation. 100ul of 1% TFA was added and mixed for 15 minutes at 100 x rpm at room temperature and the flowthrough was collected. Gel pieces were washed again with 100ul of 80% acetonitrile 0.1% TFA for 15 minutes at 100 x rpm at room temperature after which both flowthroughs containing digested peptides were purified with in-house packed SDB-RPS (Sigma; #66886-U) microcolumns. The purified peptides were resuspended in 2% acetonitrile in 0.1% TFA and stored at -80°C prior to direct injection by LC-MS/MS."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["peptides were analyzed on a Dionex 3500 nanoHPLC, coupled to an Orbitrap Ascend mass spectrometer (ThermoFischer Scientific) via electrospray ionization in positive mode with 1.9 kV at 275 °C and RF set to 40%. Separation was achieved on a 50 cm × 75 µm column packed with C18AQ (1.9 µm; Dr Maisch, Ammerbuch, Germany) (PepSep, Marslev, Denmark) over 35min at a flow rate of 300 nL/min. The peptides were eluted over a linear gradient of 3–40% Buffer B (Buffer A: 0.1% v/v formic acid; Buffer B: 80% v/v acetonitrile, 0.1% v/v FA) and the column was maintained at 50 °C. The instrument was operated in data-independent acquisition mode with an MS1 spectrum acquired over the mass range 400-1000 m/z (60,000 resolution, 250% automatic gain control (AGC) and 50 ms maximum injection time) followed by MS/MS via HCD fragmentation with 24 windows with 25 m/z isolation and a 1 m/z overlap (30,000 resolution, 2000% AGC and maximum injection time set to auto). PCP-MS DIA data were processed with Spectronaut v18.6.231127 against the mouse skeletal muscle database using DirectDIA with default parameters with precursor and protein Qvalue cutoff set to 0.01. Peptide quantification was carried out at MS2 level using 3-6 fragment ions, with automatic interference fragment ion removal as previously described (Bruderer et al., 2017). Dynamic MS1 and MS2 mass tolerance was enabled, and retention time calibration was accomplished using local (non-linear) regression. The default dynamic extracted ion chromatogram window size was performed."],"omics_type":["Proteomics"],"labhead":["Benjamin Parker"],"instrument_platform":[""],"labhead_affiliation":["The University of Melbourne"],"submission_type":["PARTIAL"],"species":["Mus Musculus (mouse)"],"submitter_mail":["ben.parker@unimelb.edu.au"],"publication":["Not available"],"submitter_affiliation":["The University of Melbourne"],"submitter_country":["Australia"],"additional_accession":[]},"is_claimable":false,"name":"PCP-MS analysis of insulin resistance in C2C12 myotubes with or without acute insulin stimulation","description":"To study dynamic remodelling of protein complexes, C2C12 myotubes were treated with 0.5 mM palmitic acid for 24 h to induce insulin resistance (IR) and then stimulated with or without acute 100 nM insulin for 30 min followed by PCP-MS(n=3 each. Samples were lysed in non-denaturing buffer, separated by BN-PAGE, and each lane cut into 30 x 1.5mm fractions. Each fraction underwent in-gel digestion and was analysed with liquid chromatography – tandem mass spectrometry (LC-MS/MS) using data-independent acquisition (DIA) resulting in the analysis of 360 individual runs.","dates":{"publication":"2026-05-29","submission":"2026-03-03"},"accession":"PXD075147","cross_references":{"TAXONOMY":["NEWT:3555","NEWT:71647","NEWT:330879","NEWT:260201","NEWT:35554","NCBITaxon:12939","NEWT:32046","NEWT:544496","NEWT:59529","NEWT:2042546","NEWT:32049","NEWT:1392696","NEWT:259447","NEWT:295546","NCBITaxon:2719036","NEWT:45351","NEWT:445974","NEWT:43179","NEWT:180454","NEWT:5722","NCBITaxon:1280","NEWT:112503","NEWT:1129","NEWT:309807","NEWT:10245","NEWT:55153","NEWT:89184","NEWT:309800","NEWT:281395","NEWT:1211601","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:351581","NEWT:43186","NEWT:428146","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:1590","NEWT:498019","NEWT:1351","NEWT:1438992","NEWT:1352","NEWT:2649997","NEWT:1147161","NEWT:661410","NEWT:638632","NEWT:263737","NEWT:224326","NEWT:1333499","NEWT:376619","NCBITaxon:79857","NEWT:1096976","NEWT:5702","NEWT:95648","NEWT:1589","NEWT:135622","NEWT:1349","NEWT:35786","NEWT:1580","NEWT:399784","NEWT:96731","NEWT:383379","NEWT:1401257","NEWT:418106","NEWT:10029","NEWT:913645","NEWT:44491","NEWT:641809","NEWT:27933","NEWT:556484","NEWT:317447","NEWT:4688","NEWT:7955","NEWT:7719","NEWT:7959","NEWT:2261","NEWT:868565","NEWT:94323","NEWT:31156","NEWT:3112","NEWT:79329","NEWT:4442","NEWT:192875","NEWT:1214915","NEWT:30069","NEWT:12637","NEWT:59729","NEWT:2164133","NEWT:284218","NEWT:295105","NEWT:108061","NEWT:60711","NEWT:1316931","NEWT:224308","NEWT:3347","NEWT:511145","NEWT:160621","NEWT:212790","NEWT:931281","NEWT:4432","NEWT:5762","NEWT:5763","NEWT:658457","NEWT:1310161","NEWT:77133","NEWT:295358","NEWT:145481","NEWT:1310165","NEWT:29058","NCBITaxon:79824","NEWT:2246","NEWT:2652724","NEWT:5757","NEWT:68415","NCBITaxon:4563","NEWT:5755","NEWT:44688","NEWT:44689","NEWT:44447","NEWT:752555","NEWT:498211","NEWT:5759","NEWT:347256","NEWT:1736231","NEWT:5518","NEWT:398007","NEWT:1527468","NEWT:1392","NEWT:498217","NEWT:498216","NEWT:2242","NEWT:11320","NEWT:286","NEWT:391619","NEWT:246196","NEWT:287","NEWT:246197","NEWT:145458","NEWT:633149","NEWT:1149786","NEWT:10239","NEWT:68895","NEWT:44685","NEWT:161934","NEWT:4897","NEWT:1148","NEWT:5744","NEWT:72901","NEWT:213863","NEWT:5508","NEWT:3329","NEWT:5507","NEWT:410661","NEWT:55571","NEWT:35500","NEWT:1235816","NEWT:1140","NCBITaxon:2157","NEWT:1143","NEWT:4896","NEWT:1287689","NEWT:1390","NEWT:11557","NEWT:1094343","NEWT:1462472","NEWT:1336795","NEWT:644042","NEWT:294","NEWT:1773","NEWT:1182590","NEWT:3712","NEWT:82380","NEWT:3711","NEWT:935293","NEWT:375146","NEWT:270643","NEWT:2065263","NEWT:177437","NEWT:1772","NEWT:10418","NEWT:118698","NEWT:1616117","NEWT:263","NEWT:118696","NEWT:34865","NEWT:52283","NEWT:11988","NEWT:284812","NEWT:8175","NEWT:400667","NEWT:43330","NEWT:1603293","NEWT:408169","NEWT:44664","NEWT:47946","NEWT:459758","NEWT:3702","NEWT:1245466","NEWT:243277","NEWT:1246791","NEWT:7067","NEWT:118694","NEWT:586722","NEWT:2850","NEWT:118691","NEWT:34871","NEWT:33548","NEWT:274","NEWT:7070","NCBITaxon:287","NEWT:408172","NEWT:408170","NEWT:96794","NEWT:3708","NEWT:332648","NEWT:44670","NEWT:536231","NEWT:376219","NEWT:219813","NEWT:1510","NEWT:1436733","NEWT:460519","NEWT:1247411","NEWT:1515","NEWT:572307","NEWT:1432138","NEWT:1424507","NEWT:1194599","NEWT:272844","NEWT:1303443","NEWT:9483","NEWT:333760","NEWT:34607","NEWT:56636","NEWT:1513458","NEWT:1233681","NCBITaxon:40559","NEWT:506599","NEWT:2853422","NEWT:81077","NEWT:84588","NEWT:1679718","NEWT:480","NEWT:65349","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:300852","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:95486","NEWT:508771","NEWT:1883446","NEWT:253","NCBITaxon:2759","NEWT:1233435","NEWT:93061","NEWT:93062","NEWT:34613","NCBITaxon:4932","NEWT:109760","NEWT:29031","NEWT:943274","NEWT:644223","NEWT:235443","NEWT:108458","NEWT:5936","NEWT:272623","NEWT:272624","NEWT:320637","NEWT:886559","NEWT:3750","NEWT:55521","NEWT:983964","NEWT:118499","NEWT:11706","NEWT:32644","NEWT:527796","NEWT:1130798","NEWT:55529","NEWT:499175","NEWT:109779","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:3988","NEWT:1116234","NEWT:561007","NEWT:1255228","NEWT:649908","NEWT:410289","NEWT:373153","NEWT:3983","NEWT:203696","NEWT:472","NEWT:352472","NEWT:97441","NEWT:1071661","NEWT:1202531","NEWT:1202532","NEWT:360094","NEWT:1408223","NEWT:470","NEWT:1408224","NEWT:41364","NEWT:5911","NEWT:1313","NEWT:1218097","NEWT:1080348","NCBITaxon:50557","NEWT:1314","NEWT:39251","NEWT:39491","NEWT:212142","NCBITaxon:5811","NEWT:29491","NEWT:101841","NEWT:153481","NEWT:2014887","NEWT:33952","NEWT:445","NEWT:153009","NEWT:261756","NEWT:571256","NEWT:40483","NEWT:63366","NEWT:63367","NEWT:688333","NEWT:215402","NCBITaxon:548681","NEWT:318586","NCBITaxon:5820","NEWT:1547","NEWT:272634","NEWT:198107","NEWT:9031","NEWT:27292","NEWT:1872122","NEWT:1308","NEWT:7091","NEWT:1034015","NEWT:760568","NEWT:141262","NEWT:108931","NEWT:1293497","NEWT:1055524","NEWT:4087","NEWT:9778","NEWT:306","NEWT:150475","NEWT:303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