{"database":"Pride","file_versions":[{"headers":{"Content-Type":["application/json"]},"body":{"files":{"Xlsx":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_Report.xlsx"],"Txt":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/checksum.txt"],"Raw":["ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_A2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_A3.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_A1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_G1.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_G2.raw","ftp://ftp.pride.ebi.ac.uk/pride/data/archive/2026/06/PXD073401/Astral_14min_G3.raw"]},"type":"primary"},"statusCode":"OK","statusCodeValue":200}],"scores":null,"additional":{"labhead_mail":["jingwu.kang@sioc.ac.cn"],"submitter":["ying zhu"],"technology_type":["Mass Spectrometry","Data-independent acquisition"],"software":[""],"submitter_keywords":["Affinity purification-mass spectrometry","Proteomics","Transcriptional regulation mechanisms","C-myc"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD073401"],"tissue":["Cell Culture"],"sample_protocol":["MCF-7 cells were cultured in DMEM medium, which contained 10% fetal bovine serum and 1% 100× Penicillin-Streptomycin Solution at 37 ℃ in 5% CO2. The cells were collected by centrifugation. The cells were washed with PBS 3 times, and the cell precipitates were collected and stored at -80 ℃ for later use. Suspended the harvested cells in the cold NP40 cell lysate buffer (containing 1% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail). Stand on ice for 10 minutes. Then sonicated at 400 S5 W for 120 s, with 2 s of sonication followed by 4 s of rest. After centrifugation of the cell lysate, the supernatant was collected. Protein concentration was determined by Bradford assay. All affinity purification experiments were performed with centrifugation at 4 ℃. The sample loading and elution time were controlled by centrifugal speed. The Ni2+active m-IMAC columns were equilibrized with 70 μL (about 14 column volumes) PBS buffer for 10 min prior to loading the His6-tagged bait protein. A total of 50 μg of His6-Max was mixed with 500 μg of MCF-7 cell lysate in advance, and incubated at 4 ℃ for 1 h (final protein concentration was 2.5 μg/μL). Then, 220 μL of protein mixture was loaded onto the m-IMAC column by centrifugation at 4 ℃ with 100 g. The loading process lasted about 1 h. The column was then washed with 70 μL of PBS for 15 min to remove the unbound proteins, and the washing procedure was repeated three times. The 5× SDS-PAGE protein loading buffer was diluted to 1× with H2O and used as the elution buffer. The bound proteins were eluted with 20 μL of eluting buffer. The control experiment was performed using the same procedure except use of His6-GFP as immobilized protein. The eluted bound proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, and the gels were visualized by Coomassie Brilliant Blue staining. All gel lanes were excised and subjected to in-gel tryptic digestion for mass spectrometric analysis. The proteins were reduced with 10 mM DTT and alkylated with 55 mM iodoacetamide. The samples were then digested with trypsin (0.025 mg/mL) at 37 ℃ for 16 h. Peptides were extracted from the gel pieces with 0.5% formic acid in 50% acetonitrile and vacuum-dried at 45 ℃. After desalting, the samples were dried again and reconstituted in 0.1% (v/v) formic acid."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The peptides were re-dissolved in solvent A (A: 0.1% formic acid in water) and analyzed by Vanquish Neo UHPLC system (Thermo Fisher Scientific, MA, USA) coupled to Orbitrap Astral mass spectrometer (Thermo Fisher Scientific, MA, USA). Peptide sample was loaded onto PepMap C18 column (15 cm length, 75 μm i.d, 2 μm particle size, Thermo Fisher) and separated with 14 min gradient starting at 4% buffer B (80% ACN with 0.1% FA), followed by a stepwise increase to 5% in 0.5 min, 8.5% in 0.4 min, 25% in 9 min, 35% in 2.3 min, 55% in 0.4 min, 99% in 0.5 min and stayed there for 0.9 min. The flow rate is 800 nL/min from 0.9 to 12.2 minutes, and 2000 nL/min for the rest of the time with the column temperature of 55°C. The electrospray voltage was set to 2 kV.  The mass spectrometer was run under data independent acquisition mode (DIA). For Orbitrap experiments, a survey scan was acquired at 380-980 m/z, the resolution was 240K, Normalized AGC target of 500% and a maximum injection time of 5ms. For Astral experiments, the Precursor Massrange was 380-980 m/z, Normalized AGC target of 500% and a maximum injection time of 3ms. The HCD collision energy was 25%. The isolation Window was 2 m/z and the overlap between every window was 0. Loop time was 0.6s. Raw Data of DIA were processed and analyzed by Spectronaut 19.7 (Biognosys AG, Switzerland) with default settings. The database was Human (version2024,20412 entries) which download from Uniprot database. Trypsin was the digestion enzyme and specific was the digest type. Carbamidomethyl on cysteine was specified as the fixed modification. Oxidation on methionine, Acetyl on protein N-term were specified as the variable modifications. Retention time prediction type was set to dynamic iRT. Data extraction was determined by Spectronaut based on the extensive mass calibration. Spectronaut will determine the ideal extraction window dynamically depending on iRT calibration and gradient stability. Q value (FDR) cutoff on precursor level was 1%, peptide level was 1% and protein level was 1%. Decoy generation was set to mutated which is like scrambled but will only apply a random number of AA position swamps (min=2, max=length/2). Normalization strategy was set to Local normalization. Peptides which passed the 1% Q value cutoff were used to calculate the major group quantities with MaxLFQ method. LFQ data were further analyzed using Perseus software (version 1.6.2.2). Reverse hits, contaminants, and proteins identified only by site were filtered out. Only protein groups with at least one group containing more than one unique peptide and three valid values were retained for subsequent analysis. LFQ intensities were log2-transformed, and missing values were imputed from a normal distribution (width = 0.3, downshift = 1.8). Volcano plots were generated using log₂(fold change) and −log₁₀(p-value). Statistical significance was assessed by a two-sample t-test (false discovery rate FDR < 0.5%, s0 = 2). Proteins significantly enriched relative to background were identified based on the combined criteria of t-test significance and log₂(fold change)."],"omics_type":["Proteomics"],"labhead":["Jingwu Kang"],"instrument_platform":[""],"labhead_affiliation":["State Key Laboratory of Chemical Biology, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["zhuying3@sioc.ac.cn"],"publication":["10.1021/ACS.JPROTEOME.6C00156"],"submitter_affiliation":["School of Pharmacy, East China University of Science and Technology"],"submitter_country":["China"],"additional_accession":[]},"is_claimable":false,"name":"Mapping Myc Interactome by Micro-Affinity Purification Coupling Mass Spectrometry","description":"Affinity purification-mass spectrometry (AP-MS) is a powerful approach for protein-protein interaction (PPI) analysis. Here, we report the development of a micro-scale immobilized metal affinity chromatography (m-IMAC) platform for rapid and reproducible purification of protein complexes prior to mass spectrometric analysis. The method was applied for mapping PPIs of Myc protein which regulates the expression of thousands of genes and plays a central role in tumorigenesis. Systematic characterization of Myc interactome is essential for understanding how a single transcription factor orchestrates such diverse biological processes. Affinity purification was performed using m-IMAC columns fabricated by photoinitiated polymerization within pipette tips (spin-tip columns), enabling efficient enrichment of Myc interacting proteins. Analysis with Label-Free Quantification (LFQ) with Data-Independent Acquisition (DIA) proteomics identified 574 Myc interacting proteins, the majority of which have not been previously reported as the Myc interactors. Two newly identified Myc associated factors were validated by biochemical assays, and the overall dataset showed good concordance with the reported Myc interactors. Gene Ontology (GO) enrichment analysis of the Myc associated proteome revealed significant enrichment in macromolecular metabolism (particularly RNA processing), regulation of gene expression, chromatin organization and remodeling. This work provides new insights into Myc mediated transcriptional regulation in proteomic level. Importantly, owing to its straightforward, rapid operation, the m-IMAC platform provides a general and efficient analytical strategy for high-throughput interactome analysis.","dates":{"publication":"2026-06-09","submission":"2026-01-22"},"accession":"PXD073401","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:5722","NEWT:55153","NCBITaxon:10407","NEWT:1736309","NEWT:309800","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:1096976","NEWT:95648","NEWT:3885","NEWT:3888","NEWT:1589","NEWT:135622","NCBITaxon:4896","NEWT:6915","NEWT:3649","NEWT:101510","NEWT:3880","NEWT:272559","NEWT:3641","NEWT:383379","NEWT:466585","NEWT:10029","NEWT:1000589","NEWT:85963","NEWT:85962","NEWT:317447","NEWT:7955","NEWT:7959","NEWT:2261","NEWT:31156","NEWT:398580","NEWT:4565","NEWT:1264690","NEWT:515619","NEWT:192875","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:84645","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:209285","NEWT:5888","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:286","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:3712","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:1603293","NEWT:408169","NEWT:44544","NEWT:4911","NEWT:645463","NEWT:3702","NEWT:129249","NEWT:243277","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:460519","NEWT:1187947","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:1182263","NEWT:2711","NEWT:376686","NEWT:95486","NEWT:9103","NEWT:29159","NEWT:253","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:272624","NEWT:411483","NEWT:884019","NEWT:198215","NEWT:411490","NEWT:983964","NEWT:169963","NEWT:32644","NEWT:499175","NEWT:109779","NEWT:476272","NEWT:3747","NEWT:195051","NEWT:367830","NEWT:1255228","NEWT:178616","NEWT:410289","NEWT:373153","NEWT:352472","NEWT:357","NEWT:360094","NEWT:470","NEWT:1313","NEWT:411469","NEWT:84023","NEWT:559292","NEWT:39491","NCBITaxon:5811","NEWT:411464","NEWT:411460","NEWT:2014887","NEWT:2762","NEWT:1174673","NEWT:562","NEWT:411470","NEWT:33952","NEWT:2094720","NCBITaxon:2697049","NEWT:571256","NEWT:28038","NEWT:1663","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:303","NEWT:9417","NEWT:7111","NEWT:347515","NEWT:1216979","NEWT:5180","NEWT:256737","NEWT:115104","NEWT:1121114","NEWT:663","NEWT:1081927","NEWT:1238993","NEWT:67825","NEWT:185579","NEWT:941442","NEWT:220668","NEWT:13076","NEWT:1249668","NEWT:7108","NEWT:317","NEWT:7227","NEWT:7469","NEWT:885318","NEWT:9402","NEWT:415540","NEWT:550","NEWT:675060","NEWT:4081","NEWT:334542","NEWT:554","NEWT:98334","NEWT:426428","NEWT:7574","NEWT:1715256","NEWT:7215","NEWT:575412","NEWT:29204","NEWT:2172103","NEWT:507601","NEWT:643680","NCBITaxon:6157","NEWT:746360","NEWT:6239","NEWT:470150","NEWT:216257","NEWT:102169","NEWT:9986","NEWT:4054","NEWT:73239","NEWT:226186","NEWT:1268063","NEWT:8782","NEWT:1263854","NEWT:435590","NEWT:1902","NEWT:160488","NEWT:28104","NEWT:1908","NCBITaxon:2","NEWT:985076","NEWT:1215323","NEWT:52641","NEWT:7038","NEWT:6192","NEWT:28532","NCBITaxon:38727","NEWT:353152","NEWT:2829","NEWT:366581","NEWT:216599","NEWT:216595","NEWT:51329","NEWT:243230","NEWT:8355","NEWT:9685","NEWT:7029","NEWT:1080772","NEWT:8479","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","NEWT:105231","NEWT:1216981","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:4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