{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T12:52:53Z","timestamp":1791895973257,"version":"4.3.4"},"reference-count":49,"publisher":"American Chemical Society (ACS)","issue":"40","license":[{"start":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T00:00:00Z","timestamp":1790380800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001869","name":"Academia Sinica","doi-asserted-by":"publisher","award":["AS-GCP-113-L01"],"award-info":[{"award-number":["AS-GCP-113-L01"]}],"id":[{"id":"10.13039\/501100001869","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100020950","name":"National Science and Technology Council","doi-asserted-by":"publisher","award":["NSTC 114-2628-M-001-014"],"award-info":[{"award-number":["NSTC 114-2628-M-001-014"]}],"id":[{"id":"10.13039\/501100020950","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,10,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>G protein-coupled receptors (GPCRs) are central physiological signal transducers and represent a primary class of therapeutic targets. While emerging evidence highlights a critical link between ligand-induced receptor conformations and pharmacological outcomes, the structural diversity dictating GPCR-G protein interface interactions remains incompletely understood. High-resolution structural studies reveal discrete, ligand-stabilized states, but these static snapshots do not fully capture the dynamic processes underlying GPCR activation and ligand efficacy. To address this, we established a native mass spectrometry (nMS) platform to probe ligand-induced conformational heterogeneity at the GPCR intracellular interface. Using the \u03b21-adrenergic receptor (\u03b21AR) as a model, we demonstrated a strong correlation between ligand efficacy and receptor complex formation with diverse active-state-specific nanobodies. Notably, leveraging the quantitative capacity of nMS, we uncovered distinct propensities of two highly similar nanobodies, Nb80 and Nb6B9, and showed that Nb6B9 engages weak-agonist-stabilized receptor states more effectively than Nb80. These interactions indicate that Nb6B9 recognizes ligand-specific conformations distinct from active states induced by full agonists. To gain structural insights, we utilized Nb6B9 chimeras with exchanged complementarity-determining regions (CDRs) and identified CDR1 and CDR3 as the primary drivers for ligand-specific recognition, implicating the involvement of receptor transmembrane motifs TM5 and TM6 in this conformational diversity. Finally, we demonstrated that nanobody-based nMS can distinguish receptor responses induced by the two salbutamol enantiomers, implying stereochemistry-dependent intracellular interface heterogeneity. Together, our study highlights the utility of nanobody-based nMS for evaluating the structural heterogeneity and dynamic landscapes of GPCRs.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c04571","type":"journal-article","created":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T15:01:27Z","timestamp":1790434887000},"page":"29378-29388","source":"Crossref","is-referenced-by-count":0,"title":["Native Mass Spectrometry Reveals Ligand-Specific Conformational\nHeterogeneity of the \u03b21-Adrenergic Receptor\nUsing Nanobody Probes"],"prefix":"10.1021","volume":"98","author":[{"given":"Yi-Quan","family":"Wang","sequence":"first","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guan-Ting","family":"Lian","sequence":"additional","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]},{"name":"National Taiwan University , , ,","place":["Taipei, Taiwan, 106319"]},{"name":"Taiwan International Graduate Program, Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi-An","family":"Chen","sequence":"additional","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rong","family":"Chen","sequence":"additional","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]},{"name":"National Taiwan University , , ,","place":["Taipei, Taiwan, 106319"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng-Yi","family":"Wu","sequence":"additional","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]},{"name":"National Taiwan University , , ,","place":["Taipei, Taiwan, 106319"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hsiang-Hui","family":"Peng","sequence":"additional","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6612-3324","authenticated-orcid":true,"given":"Hsin-Yung","family":"Yen","sequence":"additional","affiliation":[{"name":"Academia Sinica , , ,","place":["Taipei, Taiwan, 115201"]},{"name":"National Taiwan University , , ,","place":["Taipei, Taiwan, 106319"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,9,26]]},"reference":[{"issue":"4","key":"2026101308071016700_cit1","doi-asserted-by":"publisher","first-page":"251","DOI":"10.1038\/s41573-024-01083-3","article-title":"Functional\ndynamics of G protein-coupled receptors\nreveal new routes for drug discovery","volume":"24","author":"Conflitti","year":"2025","journal-title":"Nat. Rev.\nDrug Discovery"},{"issue":"1","key":"2026101308071016700_cit2","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-024-01803-6","article-title":"G protein-coupled receptors (GPCRs):\nadvances in structures, mechanisms,\nand drug discovery","volume":"9","author":"Zhang","year":"2024","journal-title":"Signal Transduction Targeted\nTher."},{"issue":"2","key":"2026101308071016700_cit3","doi-asserted-by":"publisher","first-page":"C583","DOI":"10.1152\/ajpcell.00464.2021","article-title":"Recent progress in\nassays for GPCR drug discovery","volume":"323","author":"Guo","year":"2022","journal-title":"Am. J. Physiol."},{"issue":"3","key":"2026101308071016700_cit4","doi-asserted-by":"publisher","first-page":"372","DOI":"10.1038\/aps.2011.173","article-title":"Tools for GPCR drug discovery","volume":"33","author":"Zhang","year":"2012","journal-title":"Acta Pharmacol.\nSin."},{"issue":"7366","key":"2026101308071016700_cit5","doi-asserted-by":"publisher","first-page":"549","DOI":"10.1038\/nature10361","article-title":"Crystal structure\nof the \u03b22 adrenergic receptor-Gs protein complex","volume":"477","author":"Rasmussen","year":"2011","journal-title":"Nature"},{"issue":"6442","key":"2026101308071016700_cit6","doi-asserted-by":"publisher","first-page":"775","DOI":"10.1126\/science.aau5595","article-title":"Molecular\nbasis for high-affinity agonist binding in GPCRs","volume":"364","author":"Warne","year":"2019","journal-title":"Science"},{"issue":"7329","key":"2026101308071016700_cit7","doi-asserted-by":"publisher","first-page":"175","DOI":"10.1038\/nature09648","article-title":"Structure of a nanobody-stabilized\nactive state of the \u03b2(2) adrenoceptor","volume":"469","author":"Rasmussen","year":"2011","journal-title":"Nature"},{"issue":"7472","key":"2026101308071016700_cit8","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1038\/nature12572","article-title":"Adrenaline-activated structure of\n\u03b22-adrenoceptor stabilized by an engineered nanobody","volume":"502","author":"Ring","year":"2013","journal-title":"Nature"},{"issue":"7614","key":"2026101308071016700_cit9","doi-asserted-by":"publisher","first-page":"104","DOI":"10.1038\/nature18966","article-title":"Structure\nof the adenosine A(2A) receptor bound to an engineered G protein","volume":"536","author":"Carpenter","year":"2016","journal-title":"Nature"},{"issue":"1","key":"2026101308071016700_cit10","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-022-31823-1","article-title":"Structures\nof \u03b2(1)-adrenergic receptor in complex with Gs and ligands of\ndifferent efficacies","volume":"13","author":"Su","year":"2022","journal-title":"Nat. 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Pharmacol."},{"issue":"1","key":"2026101308071016700_cit31","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-14424-8","article-title":"Modular detergents\ntailor the purification and structural analysis of membrane proteins\nincluding G-protein coupled receptors","volume":"11","author":"Urner","year":"2020","journal-title":"Nat.\nCommun."},{"issue":"12","key":"2026101308071016700_cit32","doi-asserted-by":"publisher","first-page":"583","DOI":"10.1093\/protein\/gzw049","article-title":"Engineering a minimal G protein to\nfacilitate crystallisation\nof G protein-coupled receptors in their active conformation","volume":"29","author":"Carpenter","year":"2016","journal-title":"Protein Eng., Des. 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U.S.A."},{"issue":"W1","key":"2026101308071016700_cit48","doi-asserted-by":"publisher","first-page":"W521","DOI":"10.1093\/nar\/gkae241","article-title":"The EMBL-EBI Job Dispatcher\nsequence analysis tools framework in 2024","volume":"52","author":"Madeira","year":"2024","journal-title":"Nucleic\nAcids Res."},{"issue":"W1","key":"2026101308071016700_cit49","doi-asserted-by":"publisher","first-page":"W320","DOI":"10.1093\/nar\/gku316","article-title":"Deciphering key features in protein structures with the new ENDscript\nserver","volume":"42","author":"Robert","year":"2014","journal-title":"Nucleic Acids Res."}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c04571","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/40\/29378\/68666031\/acs.analchem.6c04571.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/40\/29378\/68666031\/acs.analchem.6c04571.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T12:29:19Z","timestamp":1791894559000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/40\/29378\/5439315\/Native-Mass-Spectrometry-Reveals-Ligand-Specific"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,26]]},"references-count":49,"journal-issue":{"issue":"40","published-online":{"date-parts":[[2026,9,26]]},"published-print":{"date-parts":[[2026,10,13]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c04571","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,26]]}}}