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Biswas"],"technology_type":["Data-dependent acquisition","Mass Spectrometry"],"software":["Not available"],"submitter_keywords":["Dcas9","P300","Clasp","Hat","Smad","Mass spectrometry","Enchip"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD065551"],"tissue":["Epithelial Cell"],"sample_protocol":["The eluted samples were precipitated using 1ml isopropanol, 50 µl 3M sodium acetate and 5 µl of 20 mg/ml glycogen at -20°C overnight. The immunoprecipitated samples were mixed with 40 µl of 2x sample loading dye and incubated at 100°C for 30min before subjecting to polyacrylamide gel electrophoresis (PAGE). Thirty samples were taken from the gel for the protein digestion. The bands were then rinsed and destained in 50% ethanol and 5% acetic acid followed by drying with acetonitrile. Before the in-gel digestion, samples were reduced with DTT and alkylated with iodoacetamide. Every band was then completely digested using trypsin in-gel by adding 10 μl of the 5 ng/μl of the enzyme to 50 mM ammonium bicarbonate and incubated overnight at room temperature. Following formation of the peptides, the polyacrylamide was extracted using two aliquots of 30 μl of 50% acetonitrile mixed with 5% formic acid. Together, these extracts were evaporated in a speed-vac to about 10 μl. The peptides were reconstituted in 0.1% formic acid to yield a final volume of around 30 μl for LC-MS analysis. The LC-MS was performed in the Proteomics core of the Lerner Research Institute of the Cleveland Clinic, Cleveland, OH, USA. The LC-MS system consisted of a Captive-Spray ion source and a Bruker Times-Tof Pro2Q-Tof mass spectrometry system, both from Bruker Daltonik, Bremen, Germany in positive ion mode. The reverse phase capillary chromatography column used in the HPLC analysis was a Bruker 15cm×75µm (i.d.) C18 Reprosil AQ, 1.9 µm, 120 Å. A single microliter of the extract from the column was added to the mass spectrometer’s online source using a gradient of acetonitrile and 0.1% formic acid at a flow rate of 0.3 microliters per minute. A Parallel Accumulation-Serial Fragmentation approach was employed to select precursor ions for fragmentation with a TIMS-MS scan and ten PASEF MS/MS scans, respectively, in the digest analysis. With a ramp time of 166 ms, the TIMS-MS survey scan was obtained between 0.60 and 1.6 Vs/cm2 and 100-1,700 m/z. The PASEF scans took 1.2 seconds to complete in total, and the collision energy used in the MS/MS studies ranged from 20eV (0.6 Vs.cm2). With the target value set to 20,000 a.u and the intensity threshold set to 2,500 a.u, precursors with two to five charges were chosen. For 0.4 seconds, precursors were dynamically excluded."],"repository":["Pride"],"quantification_method":["Not available"],"modification":[""],"data_protocol":["The LC-MS/MS data were searched against the human SwissProtKB database downloaded on 3-23-2022 (26,576 entries) using the program PEAKSOnline v11.  These searches were performed considering full tryptic peptides with no more than 2 missed cleavage sites.  The MS1 and MS2 mass accuracies was set to 20ppm and 0.06 Da respectively, carbamidomethylating was considered as a fixed modification, and oxidation of methionine and protein acetylation were considered as variable modifications.  The results were filtered using a reverse decoy database strategy, Peptide, and protein FDR rates were set to 1%.  Positively identified proteins were required to be identified by a minimum of 2 peptides with at least one of these being a unique peptide identification."],"omics_type":["Proteomics"],"labhead":["Kaushik Biswas"],"instrument_platform":[""],"labhead_affiliation":["Department of Biological Sciences, Bose Institute, Kolkata, West Bengal, 700091, India"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"submitter_mail":["kblabomicsdata@gmail.com"],"publication":["10.1096/FJ.202502746R"],"submitter_affiliation":["Professor"],"submitter_country":["India"],"additional_accession":[]},"is_claimable":false,"name":"enChIP Mediated proteome profiling reveals the role of SMAD-p300 regulatory axis in GM2 synthase transcriptional regulation.","description":"Aberrant expression of sialic acid containing glycolipids, or gangliosides and their corresponding synthases have been associated with a variety of cancers. GM2-synthase,the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be overexpressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (EMT) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic mechanism underlying the transcriptional regulation of GM2-synthase gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a modified CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn’t clear. This study demonstrated that the histone acetyltransferase (HAT), p300 acts a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand modulates SMAD2/4 to have a direct positive impact on GM2 synthase gene transcription. We identified p300 to have an activator role in GM2 synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns shows the important role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2--synthase transcriptional regulation.","dates":{"publication":"2026-03-27","submission":"2025-06-27"},"accession":"PXD065551","cross_references":{"TAXONOMY":["NEWT:330879","NEWT:377960","NEWT:2042546","NEWT:259447","NEWT:295546","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: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:383379","NEWT:10029","NEWT:913645","NEWT:641809","NEWT:317447","NEWT:4688","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: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: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:485","NEWT:56636","NEWT:2853422","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:109757","NEWT:582580","NEWT:1502","NEWT:128017","NEWT:376686","NEWT:95486","NEWT:1883446","NEWT:1233435","NEWT:109760","NEWT:29031","NEWT:235443","NEWT:1757312","NEWT:85694","NEWT:108458","NEWT:5936","NEWT:320637","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:63366","NEWT:63367","NEWT:215402","NEWT:1547","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:33940","NEWT:550","NEWT:554","NEWT:451516","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:8655","NEWT:5147","NEWT:28104","NEWT:407821","NCBITaxon:2","NEWT:568708","NEWT:986","NEWT:52641","NEWT:28532","NEWT:353152","NEWT:40674","NEWT:1194669","NEWT:443906","NEWT:519","NEWT:7394","NEWT:6063","NEWT:1328388","NEWT:1548728","NEWT:667127","NEWT:9557","NEWT:377586","NEWT:300641","NEWT:39655","NEWT:9554","NEWT:38323","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:1134506","NEWT:255470","NEWT:38783","NEWT:6426","NEWT:33090","NEWT:9935","NEWT:287889","NEWT:305959","NEWT:13489","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:4232","NEWT:416348","NEWT:11298","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:231490","NEWT:244704","NEWT:7725","NEWT:9909","NEWT:9902","NEWT:430615","NEWT:72664","NEWT:563041","NEWT:36599","NEWT:41949","NEWT:326423","NEWT:452467","NEWT:198822","NEWT:36111","NEWT:326424","NEWT:1678078","NEWT:749906","NEWT:418985","NEWT:749907","NEWT:150847","NEWT:431947","NEWT:69014","NEWT:142809","NEWT:302409","NEWT:130821","NEWT:27606","NEWT:1519788","NEWT:59202","NEWT:9975","NEWT:8643","NEWT:1159899","NEWT:50278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