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Kalocsay"],"technology_type":["Data-dependent acquisition","Mass Spectrometry"],"software":[""],"submitter_keywords":["Therapy","Cardiac fibrosis","Gpcr signaling"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD054513"],"tissue":["Heart","Embryonic Stem Cell"],"sample_protocol":["PBS-rinsed cell pellets were stored at −80°C. Pellets were lysed by addition of SDS lysis buffer and reduced cysteine residues were alkylated with iodoacetamide followed by methanol chloroform precipitation. Aspirated, wet precipitates were solubilized in freshly prepared 8M urea in 200 mM EPPS, pH 8.5 and 60 μg of solubilized total protein from each sample was then used for tryptic digest and TMTpro labelling. Digested peptides were labeled with TMT 18plex (TMTpro, ThermoFisher cat# A52045) reagents in enzyme digestion reactions (0.8M urea, 200mM EPPS pH 8.5) after addition of 30% acetonitrile (v/v). Dried de-salted TMTpro labelled multiplexed peptides were subjected to phospho-peptide enrichment using immobilized metal affinity chromatography (IMAC) with the High Select Phosphopeptide enrichment kit (Thermo Fisher Scientific, Catalog Number A32992) following manufacturer’s instructions. All flowthroughs containing TMT labelled peptides de-enriched for phospho-peptides were pooled, dried to completion, and subjected to another C18 SepPak cleanup. This phospho de-enriched sample multiplex was used for subsequent HPLC fractionation. Eluted phospho-peptides were dried and fractionated by alkaline reversed phase chromatography using the Pierce High pH Reversed Phase peptide fractionation kit (Thermo Fisher Scientific, Catalog Number 84868). HPLC revered phase C18 fractionation was performed using an Agilent 1260 Infinity II Series instrument with a flow rate of 600 μl/minute over a period of 75 minutes with a 250 mm column length packed with 3.5 μm Zorbax 300 Extend-C18 matrix (Agilent, custom order). Peptides were collected in a 96-well plate over a 65 min-gradient of 13-44 %B with Buffer A comprising 5% acetonitrile, 10 mM ammonium bicarbonate, pH 8 and Buffer B comprising 90% acetonitrile,10 mM ammonium bicarbonate, pH 8. Fractions were then pooled into 24 samples, followed by sample clean-up using the Stage Tip protocol. Peptides were injected onto a 60 cm, 75 μm (internal diameter) Aurora Frontier TS nanoflow UHPLC column (IonOpticks, HTS9027205060) and separated using an VanquishNeo uHPLC system (Thermo Fisher). The column contained 1.7 μm C18 beads and was heated to 60°C using a column heater (IonOpticks). Peptides were separated over 180-minute gradients with increasing concentrations of acetonitrile from 4% to 80% in 0.1% formic acid. Samples were injected into an Orbitrap Ascend mass spectrometer (Thermo Fisher) using a high-field asymmetric-waveform ion mobility separation device (FAIMS Pro Duo, Thermo Fisher) with an MS2 method with 3 alternating FAIMS compensation voltages (-40V, -60V and -80V; 25 scans per cycle, 30 scans per cycle for phospho). MS scans were performed in the Orbitrap over a scan range of 350–1400 m/z (375-1500m/z for phospho runs) with and injection time of 50 ms (100 ms for phospho) and a standard automatic gain control (AGC) setting (AGC target 200% for phospho) with a dynamic exclusion of 120 seconds. Intensity thresholds were 2e4 (1e4 for phospho) and charge states of 2 till 6 were selected for further analysis. TMT quantification was performed after isolation with a window of 0.7 m/z from MS2 scans recorded in the Orbitrap with a scan range of >110 m/z and an HCD collision energy of 38% (HCD 33% for phospho) with dynamic injection times of up to 91 ms (400 ms and 150% AGC target for phospho). MS2 orbitrap scan resolution was set to 45,000 (dimensionless units)."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["A compilation of commercially available software (Core software program) was used to convert mass spectrometric data (Thermo “.RAW” files) to mzXML format and to correct monoisotopic m/z measurements and erroneous peptide charge state assignments. Orbitrap ppm was corrected post-search. Assignment of MS/MS spectra was performed using COMET and the Human UniProt reference proteome database (downloaded July 2022). The database search included reversed protein sequences and known contaminants such as human keratins which were excluded for subsequent analyses. Linear discriminant analysis was used to distinguish forward and reverse hits. Peptides were identified using MS2 spectra and a false discovery rate (FDR) < 1% and was achieved by applying a target-decoy database search strategy. Variable extents of modification including the presence of oxidized methionine residues were considered during peptide assignment for whole-protein experiments and for phospho-peptide enriched samples data were searched with dynamic oxidized methionine modification and phospho-modification on S, T and Y residues dynamically. For protein identification and quantification, shared peptides were collapsed into the minimally sufficient number of proteins using rules of parsimony. Peptides with a total TMT signal to noise value of > 200 and an isolation specificity of > 0.7 were included for quantification. Data were normalized to total protein amounts and the same normalization factors were applied to quantified phospho-peptides. For cluster- and downstream analysis data were scaled for each relative protein quantification value which is the summed relative TMT signal/noise quantification values for each peptide mapping to that protein. Phospho-peptides were consolidated into individual sites for quantification. Further details of the proteomic analysis workflow can be found in PMID: 37542042."],"omics_type":["Proteomics"],"labhead":["Joe Wu"],"instrument_platform":[""],"labhead_affiliation":["Joseph C. Wu, MD, PhD Director, Stanford Cardiovascular Institute Simon H. Stertzer, MD, Professor of Cardiovascular Medicine & Radiology Stanford University School of Medicine Lorry I. Lokey Stem Cell Research Building 265 Campus Drive, Rm G1120B Stanford, CA 94305-5454 Phone: (650) 736-2246"],"submission_type":["PARTIAL"],"species":["Homo Sapiens (human)"],"publication":["42784676 Zhang H, Shivnaraine RV, Ren L, Thai PN, Janicot R, Zhu W, Huang R, Siepe DH, Tu C, Liu W, Maziarz M, Deutsch JC, Liu Y, Liu C, Shin DH, Kim H, Chandy M, Kalocsay M, Chiamvimonvat N, Garcia-Marcos M, Kobilka BK, Wu JC. Targeting an atypical G protein-coupled receptor signaling pathway for cardiac fibrosis therapy. Science. 2026 393(6818):eaej5896 10.1126/science.aej5896"],"submitter_mail":["mkalocsay@mdanderson.org"],"submitter_affiliation":["The University of Texas MD Anderson Cancer Center\nDepartment of Experimental Radiation Oncology"],"submitter_country":["United States"],"pubmed_abstract":["Cardiac fibrosis independently predicts adverse outcomes in heart failure (HF), yet no Food and Drug Administration-approved therapy directly targets fibrotic remodeling in the heart. To address this unmet clinical need, we used a multidimensional drug discovery pipeline centered on a human induced pluripotent stem cell (iPSC)-based platform. Through high-throughput screening, we identified CGS15943 (CGS) as the lead antifibrotic compound and validated its activity in human cardiac fibroblasts, three-dimensional engineered heart tissues, and animal models of HF. Mechanistic studies revealed an atypical adenosine receptor (AR)-dependent signaling pathway in which AR subtypes converge on Gβγ (the βγ subunits of heterotrimeric GTP-binding proteins) to activate phosphoinositide 3-kinase (PI3K)-AKT and yes-associated protein (YAP). CGS suppressed this signaling axis, thereby reducing fibrotic gene expression and fibroblast activation. These findings establish AR-driven Gβγ signaling as a potential therapeutic target for cardiac fibrosis."],"pubmed_title":["Targeting an atypical G protein-coupled receptor signaling pathway for cardiac fibrosis therapy."],"pubmed_authors":["Zhang Hao H, Shivnaraine Rabindra V RV, Ren Lu L, Thai Phung N PN, Janicot Remi R, Zhu Wenjuan W, Huang Rihua R, Siepe Dirk H DH, Tu Chengyi C, Liu Wenqiang W, Maziarz Marcin M, Deutsch Jonathan C JC, Liu Yu Y, Liu Chun C, Shin Danielle H DH, Kim Hyeonyu H, Chandy Mark M, Kalocsay Marian M, Chiamvimonvat Nipavan N, Garcia-Marcos Mikel M, Kobilka Brian K BK, Wu Joseph C JC"],"additional_accession":[]},"is_claimable":false,"name":"Targeting an atypical G protein–coupled receptor signalingpathway for cardiacfibrosis therapy","description":"Cardiac fibrosis independently predicts adverse outcomes in heart failure (HF), yet no Food and Drug Administration–approved therapy directly targets fibrotic remodeling in the heart. To address this unmet clinical need, we used a multidimensional drug discovery pipeline centered on a human induced pluripotent stem cell (iPSC)–based platform. Through high-throughput screening, we identified CGS15943 (CGS) as the lead antifibrotic compound and validated its activity in human cardiac fibroblasts, three-dimensional engineered heart tissues, and animal models of HF. Mechanistic studies revealed an atypical adenosine receptor (AR)–dependent signaling pathway in which AR subtypes converge on Gβγ (the βγ subunits of heterotrimeric GTP-binding proteins) to activate phosphoinositide 3-kinase (PI3K)–AKT and yes-associated protein (YAP). CGS suppressed this signaling axis, thereby reducing fibrotic gene expression and fibroblast activation. These findings establish AR-driven Gβγ signaling as a potential therapeutic target for cardiac fibrosis.","dates":{"publication":"2026-09-27","submission":"2024-08-02"},"accession":"PXD054513","cross_references":{"TAXONOMY":["NEWT:68239","NEWT:108018","NEWT:377960","NEWT:1131","NEWT:190486","NEWT:2042546","NEWT:148942","NEWT:295546","NEWT:258594","NEWT:1126","NEWT:1129","NEWT:89187","NEWT:309807","NEWT:89184","NEWT:309800","NEWT:57577","NEWT:1323429","NEWT:876138","NEWT:44271","NEWT:193516","NEWT:2315688","NEWT:592321","NEWT:1073882","NEWT:6819","NEWT:77173","NEWT:428386","NEWT:1117","NEWT:498257","NEWT:10036","NEWT:32264","NEWT:10034","NEWT:661410","NEWT:488003","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:418106","NEWT:10029","NEWT:913645","NEWT:2492962","NEWT:641809","NEWT:44250","NEWT:317447","NEWT:5537","NEWT:5530","NEWT:868565","NEWT:135674","NEWT:1562698","NEWT:12637","NEWT:2164133","NEWT:1180","NEWT:2767846","NEWT:1343074","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:33554","NEWT:1310173","NEWT:37919","NEWT:161934","NEWT:156587","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:65121","NEWT:3715","NEWT:2065263","NEWT:1549675","NEWT:177437","NEWT:9014","NEWT:10891","NEWT:118221","NEWT:34869","NEWT:226126","NEWT:1616117","NEWT:34865","NEWT:28174","NEWT:550537","NEWT:284812","NEWT:980415","NEWT:3701","NEWT:3702","NEWT:66440","NEWT:1245466","NEWT:3704","NEWT:1246791","NEWT:34875","NEWT:9005","NEWT:34871","NEWT:9006","NEWT:33548","NEWT:1883426","NEWT:657321","NEWT:34878","NEWT:10886","NEWT:3708","NEWT:536231","NEWT:219813","NEWT:2305479","NEWT:215451","NEWT:460519","NEWT:272843","NEWT:272844","NEWT:1348799","NEWT:55784","NEWT:485","NEWT:2709072","NEWT:2499525","NEWT:467144","NEWT:142786","NEWT:2853422","NEWT:487","NEWT:1679718","NEWT:480","NEWT:67767","NEWT:46835","NEWT:216778","NEWT:376686","NEWT:237610","EFO:0001352","NEWT:1385720","NEWT:219809","NEWT:219808","NEWT:119562","NEWT:1883446","NEWT:1233435","NEWT:118223","NEWT:141469","NEWT:272831","NEWT:987059","NEWT:491","NEWT:460523","NEWT:2423","NEWT:133894","NEWT:85694","NEWT:85692","NEWT:911045","NEWT:3750","NEWT:291146","NEWT:29466","NEWT:233271","NEWT:3745","NEWT:1715989","NCBITaxon:4751","NEWT:3747","NEWT:278161","NEWT:415978","NEWT:410289","NEWT:191701","NEWT:9054","NEWT:472","NEWT:29459","NEWT:1380096","NEWT:28128","NEWT:470","NEWT:41122","NEWT:1328300","NEWT:748693","NCBITaxon:50557","NEWT:3735","NEWT:29491","NEWT:911022","NEWT:446","NEWT:153481","NEWT:714485","NEWT:445","NEWT:2003758","NEWT:63366","NEWT:63367","NEWT:29488","NEWT:215402","NEWT:3726","NEWT:30455","NEWT:28141","NEWT:9030","NEWT:9031","NEWT:3728","NEWT:108931","NEWT:219799","NEWT:1325292","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:8673","NEWT:550","NEWT:10804","NEWT:554","NEWT:451516","NEWT:552","NEWT:1325291","NEWT:42410","NEWT:1225786","NEWT:771870","NEWT:8665","NEWT:51338","NEWT:9995","NEWT:28112","NEWT:49012","NEWT:28110","NEWT:10832","NEWT:311339","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:595495","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","NEWT:51329","NCBITaxon:1763","NEWT:443906","NEWT:13037","NEWT:519","NEWT:941638","NEWT:7394","NEWT:6063","NEWT:404330","NEWT:1328388","NEWT:13064","NEWT:7375","NEWT:300641","NEWT:39655","NEWT:38323","NEWT:206411","NEWT:80869","NEWT:214053","NEWT:80863","NEWT:665890","NEWT:52639","NEWT:52638","NEWT:367774","NEWT:8697","NEWT:861109","NEWT:8695","NEWT:1196866","NEWT:6039","NEWT:510516","NEWT:1045775","NEWT:1123869","NCBITaxon:1335626","NEWT:7370","NEWT:280406","NEWT:83906","NEWT:99488","NEWT:99487","NEWT:1134506","NEWT:255470","NEWT:762903","NEWT:27697","NEWT:86185","NEWT:9935","NEWT:266940","NEWT:287889","NEWT:1028307","NEWT:52603","NEWT:92867","NEWT:92866","NEWT:1344414","NEWT:202950","NEWT:741158","NEWT:1293091","NEWT:202956","NEWT:483179","NEWT:315750","NEWT:99490","NEWT:632957","NEWT:9925","NCBITaxon:9606","NEWT:1436183","NEWT:1479235","NEWT:4232","NEWT:9922","NEWT:416348","NEWT:105812","NEWT:11298","NEWT:4233","NEWT:4236","NEWT:398031","NEWT:196627","NEWT:523840","NEWT:1076878","NEWT:200308","NEWT:200302","NEWT:9917","NEWT:95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