{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:58:40Z","timestamp":1790945920029,"version":"4.1.0"},"reference-count":40,"publisher":"American Chemical Society (ACS)","issue":"10","license":[{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T00:00:00Z","timestamp":1788912000000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["CA282268"],"award-info":[{"award-number":["CA282268"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,2]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Thermal proteome profiling (TPP) and its higher-throughput derivative, the proteome integral solubility alteration (PISA) assay, measure changes in protein thermal stability upon ligand binding or other perturbations and have been widely adopted in drug discovery and biomedical research. Though the PISA workflow is straightforward, key parameters, including detergent concentration, methods for removing denatured aggregates, and temperature range selection, vary across studies and can markedly influence assay outcomes. Yet these factors have not been systematically evaluated, limiting rational experimental design and data interpretation. Here, through a combined use of TPP, PISA, tandem mass tag (TMT)-based multiplexing, and computational simulation, we systematically characterize these parameters based on the melting behavior of \u223c9000 proteins. We find that reducing detergent concentration elevates apparent Tm by 1.5\u20132 \u00b0C proteome-wide, and aggregate removal by filtration versus centrifugation further alters measurements. We leverage these observations to characterize how these parameters shape PISA and then apply selected conditions to identify the aminopeptidase NPEPPS as a previously uncharacterized binding partner of angiotensin II, a key vasoactive peptide hormone in blood pressure regulation. Together, this work provides a general framework for assay design and data interpretation and extends the utility of PISA beyond small molecules to dissecting peptide\u2013protein interactions, an increasingly important modality in drug discovery.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00125","type":"journal-article","created":{"date-parts":[[2026,9,9]],"date-time":"2026-09-09T20:02:48Z","timestamp":1788984168000},"page":"5114-5126","source":"Crossref","is-referenced-by-count":0,"title":["Systematic\nCharacterization of Thermal Stability Assay\nParameters and Application in Discovery of Peptide\u2013Protein\nInteractions"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1876-1700","authenticated-orcid":true,"given":"Daniel M.","family":"Richards","sequence":"first","affiliation":[{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]},{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangyi Coco","family":"Zhai","sequence":"additional","affiliation":[{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]},{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]},{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaoxian","family":"Li","sequence":"additional","affiliation":[{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]},{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0468-5353","authenticated-orcid":true,"given":"Qing","family":"Yu","sequence":"additional","affiliation":[{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]},{"name":"University of Massachusetts Chan Medical School , , , ,","place":["Worcester, Massachusetts, United States, 01605"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,9]]},"reference":[{"issue":"9","key":"2026100208414240000_cit1","doi-asserted-by":"publisher","first-page":"2212","DOI":"10.1038\/nprot.2007.321","article-title":"The Use of Differential Scanning\nFluorimetry to Detect Ligand Interactions That Promote Protein Stability","volume":"2","author":"Niesen","year":"2007","journal-title":"Nat. Protoc."},{"issue":"6141","key":"2026100208414240000_cit2","doi-asserted-by":"publisher","first-page":"84","DOI":"10.1126\/science.1233606","article-title":"Monitoring Drug Target\nEngagement in Cells and Tissues Using the Cellular Thermal Shift Assay","volume":"341","author":"Molina","year":"2013","journal-title":"Science"},{"issue":"6205","key":"2026100208414240000_cit3","doi-asserted-by":"publisher","DOI":"10.1126\/science.1255784","article-title":"Tracking Cancer Drugs in Living Cells by Thermal Profiling\nof the Proteome","volume":"346","author":"Savitski","year":"2014","journal-title":"Science"},{"issue":"6","key":"2026100208414240000_cit4","doi-asserted-by":"publisher","first-page":"1495","DOI":"10.1016\/j.cell.2018.03.053","article-title":"Pervasive Protein\nThermal Stability Variation During the Cell Cycle","volume":"173","author":"Becher","year":"2018","journal-title":"Cell"},{"issue":"9","key":"2026100208414240000_cit5","doi-asserted-by":"publisher","first-page":"894","DOI":"10.1038\/s41592-019-0499-3","article-title":"High Throughput\nDiscovery of Functional Protein Modifications by Hotspot Thermal Profiling","volume":"16","author":"Huang","year":"2019","journal-title":"Nat. Methods"},{"issue":"11","key":"2026100208414240000_cit6","doi-asserted-by":"publisher","first-page":"4027","DOI":"10.1021\/acs.jproteome.9b00500","article-title":"Proteome Integral\nSolubility Alteration:\nA High-Throughput Proteomics Assay for Target Deconvolution","volume":"18","author":"Gaetani","year":"2019","journal-title":"J. Proteome Res."},{"issue":"1","key":"2026100208414240000_cit7","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-53240-2","article-title":"Streamlined\nAnalysis of Drug Targets\nby Proteome Integral Solubility Alteration Indicates Organ-Specific\nEngagement","volume":"15","author":"Batth","year":"2024","journal-title":"Nat. Commun."},{"key":"2026100208414240000_cit8","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.95595","article-title":"Large-Scale Characterization of Drug\nMechanism of Action Using Proteome-Wide Thermal Shift Assays","volume":"13","author":"Van Vranken","year":"2024","journal-title":"eLife"},{"issue":"22","key":"2026100208414240000_cit9","doi-asserted-by":"publisher","DOI":"10.21769\/BioProtoc.4556","article-title":"Proteome Integral\nSolubility Alteration (PISA) Assay in Mammalian Cells for Deep, High-Confidence,\nand High-Throughput Target Deconvolution","volume":"12","author":"Zhang","year":"2022","journal-title":"Bio-Protoc."},{"issue":"12","key":"2026100208414240000_cit10","doi-asserted-by":"publisher","first-page":"1129","DOI":"10.1038\/nmeth.3652","article-title":"Thermal Proteome Profiling Monitors\nLigand Interactions with Cellular Membrane Proteins","volume":"12","author":"Reinhard","year":"2015","journal-title":"Nat. Methods"},{"issue":"10","key":"2026100208414240000_cit11","doi-asserted-by":"publisher","first-page":"1567","DOI":"10.1038\/nprot.2015.101","article-title":"Thermal\nProteome Profiling for Unbiased Identification of Direct and Indirect\nDrug Targets Using Multiplexed Quantitative Mass Spectrometry","volume":"10","author":"Franken","year":"2015","journal-title":"Nat. Protoc."},{"issue":"5","key":"2026100208414240000_cit12","doi-asserted-by":"publisher","first-page":"2159","DOI":"10.1021\/acs.jproteome.0c00063","article-title":"Selection\nof Heating Temperatures Improves the Sensitivity of the Proteome Integral\nSolubility Alteration Assay","volume":"19","author":"Li","year":"2020","journal-title":"J. Proteome Res."},{"issue":"4","key":"2026100208414240000_cit13","doi-asserted-by":"publisher","first-page":"309","DOI":"10.1038\/s41573-020-00135-8","article-title":"Trends in Peptide\nDrug Discovery","volume":"20","author":"Muttenthaler","year":"2021","journal-title":"Nat. Rev. Drug Discovery"},{"key":"2026100208414240000_cit14","doi-asserted-by":"publisher","first-page":"62","DOI":"10.1016\/j.peptides.2018.04.011","article-title":"Apelin and\nElabela\/Toddler; Double Ligands for APJ\/Apelin Receptor in Heart Development,\nPhysiology, and Pathology","volume":"111","author":"Kuba","year":"2019","journal-title":"Peptides"},{"issue":"10","key":"2026100208414240000_cit15","doi-asserted-by":"publisher","first-page":"2700","DOI":"10.1016\/j.bmc.2017.06.052","article-title":"Therapeutic Peptides:\nHistorical Perspectives, Current\nDevelopment Trends, and Future Directions","volume":"26","author":"Lau","year":"2018","journal-title":"Bioorg.\nMed. Chem."},{"issue":"5","key":"2026100208414240000_cit16","doi-asserted-by":"publisher","first-page":"495","DOI":"10.1038\/s41592-020-0801-4","article-title":"Meltome Atlas-Thermal Proteome Stability Across the\nTree of Life","volume":"17","author":"Jarzab","year":"2020","journal-title":"Nat. Methods"},{"issue":"1","key":"2026100208414240000_cit17","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1038\/s41596-018-0082-x","article-title":"Single-Pot,\nSolid-Phase-Enhanced Sample Preparation\nfor Proteomics Experiments","volume":"14","author":"Hughes","year":"2019","journal-title":"Nat. Protoc."},{"issue":"6","key":"2026100208414240000_cit18","doi-asserted-by":"publisher","first-page":"2226","DOI":"10.1021\/acs.jproteome.8b00217","article-title":"Streamlined Tandem Mass Tag (SL-TMT)\nProtocol: An Efficient Strategy for Quantitative (Phospho)proteome\nProfiling Using Tandem Mass Tag-Synchronous Precursor Selection-MS3","volume":"17","author":"Navarrete-Perea","year":"2018","journal-title":"J. Proteome Res."},{"issue":"11","key":"2026100208414240000_cit19","doi-asserted-by":"publisher","first-page":"5153","DOI":"10.1021\/acs.jproteome.4c00668","article-title":"Achieving a 35-Plex\nTandem Mass Tag Reagent Set Through Deuterium Incorporation","volume":"23","author":"Zuniga","year":"2024","journal-title":"J. Proteome Res."},{"issue":"9","key":"2026100208414240000_cit20","doi-asserted-by":"publisher","first-page":"6478","DOI":"10.1021\/acs.analchem.9b05685","article-title":"Benchmarking\nthe Orbitrap Tribrid Eclipse for Next Generation Multiplexed Proteomics","volume":"92","author":"Yu","year":"2020","journal-title":"Anal. Chem."},{"issue":"1","key":"2026100208414240000_cit21","doi-asserted-by":"publisher","first-page":"22","DOI":"10.1002\/pmic.201200439","article-title":"Comet:\nAn Open-Source MS\/MS Sequence\nDatabase Search Tool","volume":"13","author":"Eng","year":"2013","journal-title":"Proteomics"},{"issue":"3","key":"2026100208414240000_cit22","doi-asserted-by":"publisher","DOI":"10.1016\/j.xinn.2021.100141","article-title":"clusterProfiler 4.0:\nA Universal Enrichment Tool for Interpreting Omics Data","volume":"2","author":"Wu","year":"2021","journal-title":"Innovation (Cambridge (Mass.))"},{"issue":"1","key":"2026100208414240000_cit23","doi-asserted-by":"publisher","first-page":"155","DOI":"10.1037\/\/0033-2909.112.1.155","article-title":"A Power Primer","volume":"112","author":"Cohen","year":"1992","journal-title":"Psychological Bulletin"},{"issue":"7873","key":"2026100208414240000_cit24","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1038\/s41586-021-03819-2","article-title":"Highly Accurate Protein\nStructure Prediction with AlphaFold","volume":"596","author":"Jumper","year":"2021","journal-title":"Nature"},{"issue":"21","key":"2026100208414240000_cit25","doi-asserted-by":"publisher","first-page":"2695","DOI":"10.1093\/bioinformatics\/btl461","article-title":"Bio3d:\nAn R Package for the Comparative Analysis of Protein Structures","volume":"22","author":"Grant","year":"2006","journal-title":"Bioinformatics"},{"issue":"7873","key":"2026100208414240000_cit26","doi-asserted-by":"publisher","first-page":"590","DOI":"10.1038\/s41586-021-03828-1","article-title":"Highly Accurate Protein Structure Prediction for the\nHuman Proteome","volume":"596","author":"Tunyasuvunakool","year":"2021","journal-title":"Nature"},{"issue":"4","key":"2026100208414240000_cit27","doi-asserted-by":"publisher","first-page":"985","DOI":"10.1006\/jmbi.1998.1645","article-title":"Contact Order,\nTransition State Placement\nand the Refolding Rates of Single Domain Proteins","volume":"277","author":"Plaxco","year":"1998","journal-title":"J. Mol. Biol."},{"issue":"8016","key":"2026100208414240000_cit28","doi-asserted-by":"publisher","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate Structure\nPrediction of\nBiomolecular Interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"issue":"5","key":"2026100208414240000_cit29","doi-asserted-by":"publisher","first-page":"1385","DOI":"10.1038\/sj.emboj.7601592","article-title":"Puromycin-Sensitive\nAminopeptidase\nIs the Major Peptidase Responsible for Digesting Polyglutamine Sequences\nReleased by Proteasomes During Protein Degradation","volume":"26","author":"Bhutani","year":"2007","journal-title":"EMBO J."},{"issue":"7","key":"2026100208414240000_cit30","doi-asserted-by":"publisher","DOI":"10.15252\/msb.20188242","article-title":"Thermal Proteome\nProfiling in Bacteria: Probing Protein State in Vivo","volume":"14","author":"Mateus","year":"2018","journal-title":"Mol. Syst. Biol."},{"issue":"6327","key":"2026100208414240000_cit31","doi-asserted-by":"publisher","DOI":"10.1126\/science.aai7825","article-title":"Cell-Wide\nAnalysis of Protein Thermal Unfolding Reveals Determinants of Thermostability","volume":"355","author":"Leuenberger","year":"2017","journal-title":"Science"},{"issue":"40","key":"2026100208414240000_cit32","doi-asserted-by":"publisher","first-page":"13711","DOI":"10.1074\/jbc.RA120.014183","article-title":"Dipeptidyl Peptidase 3 Modulates the Renin-Angiotensin\nSystem in Mice","volume":"295","author":"Jha","year":"2020","journal-title":"J. Biol. Chem."},{"issue":"1","key":"2026100208414240000_cit33","doi-asserted-by":"publisher","first-page":"114","DOI":"10.1074\/jbc.M111.273052","article-title":"Identification of\nMembrane-Bound Variant of Metalloendopeptidase Neurolysin (EC 3.4.24.16)\nas the Non-Angiotensin Type 1 (Non-AT1), Non-AT2 Angiotensin Binding\nSite","volume":"287","author":"Wangler","year":"2012","journal-title":"J. Biol. Chem."},{"issue":"6","key":"2026100208414240000_cit34","doi-asserted-by":"publisher","first-page":"1048","DOI":"10.1093\/hmg\/ddac290","article-title":"Whole-Exome\nSequencing Study Identifies Four Novel Gene Loci Associated with Diabetic\nKidney Disease","volume":"32","author":"Pan","year":"2023","journal-title":"Hum. Mol. Genet."},{"issue":"1","key":"2026100208414240000_cit35","doi-asserted-by":"publisher","first-page":"238","DOI":"10.1210\/en.2017-00759","article-title":"Obligatory Metabolism\nof Angiotensin II to Angiotensin\nIII for Zona Glomerulosa Cell-Mediated Relaxations of Bovine Adrenal\nCortical Arteries","volume":"159","author":"Kopf","year":"2018","journal-title":"Endocrinology"},{"issue":"7","key":"2026100208414240000_cit36","doi-asserted-by":"publisher","first-page":"1171","DOI":"10.1093\/aob\/mcq265","article-title":"The Role\nof Multifunctional M1Metallopeptidases in Cell Cycle Progression","volume":"107","author":"Peer","year":"2011","journal-title":"Ann. Bot."},{"key":"2026100208414240000_cit37","doi-asserted-by":"publisher","DOI":"10.1016\/j.cophys.2026.100898","article-title":"The Role\nof Aminopeptidases in Angiotensin Peptide Processing","volume":"47","author":"Fitzgibbon","year":"2026","journal-title":"Curr. Opin. Physiol."},{"issue":"6380","key":"2026100208414240000_cit38","doi-asserted-by":"publisher","first-page":"1170","DOI":"10.1126\/science.aan0346","article-title":"Thermal Proximity Coaggregation\nfor System-Wide\nProfiling of Protein Complex Dynamics in Cells","volume":"359","author":"Tan","year":"2018","journal-title":"Science"},{"issue":"5","key":"2026100208414240000_cit39","doi-asserted-by":"publisher","first-page":"2587","DOI":"10.1172\/JCI133270","article-title":"GPR160 de-Orphanization Reveals Critical\nRoles in Neuropathic Pain in Rodents","volume":"130","author":"Yosten","year":"2020","journal-title":"J. Clin.\nInvest."},{"issue":"2","key":"2026100208414240000_cit40","doi-asserted-by":"publisher","first-page":"236","DOI":"10.1016\/S0003-9861(03)00123-1","article-title":"Mutation of Active Site Residues\nof the Puromycin-Sensitive Aminopeptidase: Conversion of the Enzyme\ninto a Catalytically Inactive Binding Protein","volume":"413","author":"Thompson","year":"2003","journal-title":"Arch. Biochem. Biophys."}],"container-title":["Journal of Proteome Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.jproteome.6c00125","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5114\/68464999\/acs.jproteome.6c00125.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/jprobs\/article-pdf\/25\/10\/5114\/68464999\/acs.jproteome.6c00125.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T12:51:54Z","timestamp":1790945514000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/jprobs\/article\/25\/10\/5114\/5427298\/Systematic-Characterization-of-Thermal-Stability"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,9]]},"references-count":40,"journal-issue":{"issue":"10","published-online":{"date-parts":[[2026,9,9]]},"published-print":{"date-parts":[[2026,10,2]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.jproteome.6c00125","relation":{},"ISSN":["1535-3893","1535-3907"],"issn-type":[{"value":"1535-3893","type":"print"},{"value":"1535-3907","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,9]]}}}