{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T12:19:17Z","timestamp":1788351557319,"version":"build-2803163510"},"reference-count":26,"publisher":"American Chemical Society (ACS)","issue":"20","license":[{"start":{"date-parts":[[2019,10,31]],"date-time":"2019-10-31T00:00:00Z","timestamp":1572480000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/pubs.acs.org\/page\/policy\/authorchoice_ccby_termsofuse.html"}],"funder":[{"DOI":"10.13039\/100010661","name":"Horizon 2020 Framework Programme","doi-asserted-by":"publisher","award":["731077"],"award-info":[{"award-number":["731077"]}],"id":[{"id":"10.13039\/100010661","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100008530","name":"European Regional Development Fund","doi-asserted-by":"publisher","award":["A70135"],"award-info":[{"award-number":["A70135"]}],"id":[{"id":"10.13039\/501100008530","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Biocenter Kuopio","doi-asserted-by":"publisher","award":["NA"],"award-info":[{"award-number":["NA"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2019,11,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Thyroid hormones are important regulatory hormones, acting on nearly every cell in the body. The two main thyroid hormones are l-thyroxine (tetraiodo-l-thyronine, T4) and 3,3\u2032,5-triiodo-l-thyronine (T3), which are produced in the thyroid gland and secreted into the blood stream. Other important thyroid hormone metabolites are 3,3\u2032-diiodo-l-thyronine (T2) and l-thyronine (T0), which may show increased levels in circulation due to dietary iodine deficiency or other medical disorders. Owing to their central role in cellular functions, sensitive and specific detection methods for thyroid hormones are needed. In this work, native mass spectrometry (MS) was used to quantitate thyroid hormone binding to the anti-T4 antibody Fab fragment. First, the binding affinity for T2 was determined via direct ligand titration experiments. Then, the affinities for the other ligands were determined by competition experiments using T2 as the \u201clow-affinity\u201d reference ligand. The highest affinity was measured for T3, followed by T4, T2, and T0 (Kd = 29, 3.4, and 260 nM and 130 \u03bcM, respectively). Thus, it is evident that the number and positions of the iodine substituents within the thyronine rings are important for the ligand binding affinity of anti-T4 Fab. Surprisingly, structurally related tetrahalogen bisphenols were also able to bind to anti-T4 Fab with nanomolar affinities.<\/jats:p>","DOI":"10.1021\/acsomega.9b02659","type":"journal-article","created":{"date-parts":[[2019,10,31]],"date-time":"2019-10-31T10:45:17Z","timestamp":1572518717000},"page":"18718-18724","source":"Crossref","is-referenced-by-count":9,"title":["Quantitation of Thyroid Hormone Binding to Anti-Thyroxine\nAntibody Fab Fragment by Native Mass Spectrometry"],"prefix":"10.1021","volume":"4","author":[{"given":"Senthil K.","family":"Thangaraj","sequence":"first","affiliation":[{"name":"University of Eastern Finland , , P.O. Box 111, ,","place":["Joensuu, Finland, FI-80101"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henri","family":"Arola","sequence":"additional","affiliation":[{"name":"VTT Technical Research Centre of Finland Ltd. , P.O. Box 1000, ,","place":["Espoo, Finland, FI-02044 VTT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Antti","family":"Tullila","sequence":"additional","affiliation":[{"name":"VTT Technical Research Centre of Finland Ltd. , P.O. Box 1000, ,","place":["Espoo, Finland, FI-02044 VTT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tarja K.","family":"Nevanen","sequence":"additional","affiliation":[{"name":"VTT Technical Research Centre of Finland Ltd. , P.O. Box 1000, ,","place":["Espoo, Finland, FI-02044 VTT"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1843-5718","authenticated-orcid":true,"given":"Juha","family":"Rouvinen","sequence":"additional","affiliation":[{"name":"University of Eastern Finland , , P.O. Box 111, ,","place":["Joensuu, Finland, FI-80101"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8446-4704","authenticated-orcid":true,"given":"Janne","family":"Ja\u0308nis","sequence":"additional","affiliation":[{"name":"University of Eastern Finland , , P.O. Box 111, ,","place":["Joensuu, Finland, FI-80101"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2019,10,31]]},"reference":[{"key":"2026090207445918900_cit1","doi-asserted-by":"publisher","first-page":"1097","DOI":"10.1152\/physrev.2001.81.3.1097","article-title":"Physiological\nand Molecular Basis of Thyroid Hormone Action","volume":"81","author":"Yen","year":"2001","journal-title":"Physiol. Rev."},{"key":"2026090207445918900_cit2","doi-asserted-by":"publisher","first-page":"582","DOI":"10.1038\/nrendo.2014.143","article-title":"Thyroid\nHormone Receptors and Resistance\nto Thyroid Hormone Disorders","volume":"10","author":"Ortiga-Carvalho","year":"2014","journal-title":"Nat. Rev. Endocrinol."},{"key":"2026090207445918900_cit3","doi-asserted-by":"publisher","first-page":"55801","DOI":"10.1074\/jbc.M410124200","article-title":"Thyroxine-Thyroid Hormone Receptor\nInteractions","volume":"279","author":"Sandler","year":"2004","journal-title":"J. Biol. Chem."},{"key":"2026090207445918900_cit4","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1093\/clinchem\/42.1.146","article-title":"Analytical Performance of Free and Total Thyroxine\nAssays","volume":"42","author":"Nelson","year":"1996","journal-title":"Clin. Chem."},{"key":"2026090207445918900_cit5","doi-asserted-by":"publisher","first-page":"14930","DOI":"10.1073\/pnas.1203574109","article-title":"Structural and Mechanistic Insights into Bisphenols\nAction Provide Guidelines for Risk Assessment and Discovery of Bisphenol\nA Substitutes","volume":"109","author":"Delfosse","year":"2012","journal-title":"Proc. Natl. Acad. Sci."},{"key":"2026090207445918900_cit6","doi-asserted-by":"publisher","first-page":"226","DOI":"10.1093\/toxsci\/kfy028","article-title":"The Affinity\nof Brominated Phenolic Compounds for Human and Zebrafish Thyroid Receptor\n\u03b2: Influence of Chemical Structure","volume":"163","author":"Kollitz","year":"2018","journal-title":"Toxicol.\nSci."},{"key":"2026090207445918900_cit7","doi-asserted-by":"publisher","first-page":"10481","DOI":"10.3390\/s150510481","article-title":"Surface Plasmon Resonance:\nA Versatile Technique for Biosensor Applications","volume":"15","author":"Nguyen","year":"2015","journal-title":"Sensors"},{"key":"2026090207445918900_cit8","doi-asserted-by":"publisher","first-page":"79","DOI":"10.1016\/S0091-679X(07)84004-0","article-title":"Isothermal Titration\nCalorimetry: Experimental Design,\nData Analysis, and Probing Macromolecule\/Ligand Binding and Kinetic\nInteractions","volume":"84","author":"Freyer","year":"2008","journal-title":"Methods Cell Biol."},{"key":"2026090207445918900_cit9","doi-asserted-by":"publisher","first-page":"319","DOI":"10.1002\/jmr.951","article-title":"Label-Free\nDetermination\nof Protein-Ligand Binding Constants Using Mass Spectrometry and Validation\nUsing Surface Plasmon Resonance and Isothermal Titration Calorimetry","volume":"22","author":"Jecklin","year":"2009","journal-title":"J. Mol. Recognit."},{"key":"2026090207445918900_cit10","doi-asserted-by":"publisher","first-page":"927","DOI":"10.1038\/nmeth.1265","article-title":"Native\nMass Spectrometry : A Bridge between Interactomics and Structural\nBiology","volume":"5","author":"Heck","year":"2008","journal-title":"Nat. Methods"},{"key":"2026090207445918900_cit11","doi-asserted-by":"publisher","first-page":"4325","DOI":"10.1021\/ac0497914","article-title":"Determination\nof Dissociation Constants for Protein-Ligand Complexes by Electrospray\nIonization Mass Spectrometry","volume":"76","author":"Tjernberg","year":"2004","journal-title":"Anal. Chem."},{"key":"2026090207445918900_cit12","doi-asserted-by":"publisher","first-page":"5370","DOI":"10.1021\/ac050511+","article-title":"Estimates of Protein Surface Areas in Solution by Electrospray\nIonization Mass Spectrometry","volume":"77","author":"Kaltashov","year":"2005","journal-title":"Anal.Chem."},{"key":"2026090207445918900_cit13","doi-asserted-by":"publisher","first-page":"1424","DOI":"10.1016\/j.jasms.2004.05.005","article-title":"Features of the ESI Mechanism That\nAffect the Observation of Multiply Charged Noncovalent Protein Complexes\nand the Determination of the Association Constant by the Titration\nMethod","volume":"15","author":"Peschke","year":"2004","journal-title":"J. Am. Soc. Mass Spectrom."},{"key":"2026090207445918900_cit14","doi-asserted-by":"publisher","first-page":"133","DOI":"10.1021\/bc960088z","article-title":"Immunoassay Reagents for Thyroid\nTesting. 2. Binding Properties and\nEnergetic Parameters of a T4 Monoclonal Antibody and Its Fab Fragment\nwith a Library of Thyroxine Analog Biosensors Using Surface Plasmon\nResonance","volume":"8","author":"Adamczyk","year":"1997","journal-title":"Bioconjugate Chem."},{"key":"2026090207445918900_cit15","doi-asserted-by":"publisher","first-page":"23","DOI":"10.1021\/bc9701353","article-title":"Immunoassay\nReagents for Thyroid Testing. 3. Determination of the Solution Binding\nAffinities of a T4 Monoclonal Antibody Fab Fragment for a Library\nof Thyroxine Analogs Using Surface Plasmon Resonance","volume":"9","author":"Adamczyk","year":"1998","journal-title":"Bioconjugate Chem."},{"key":"2026090207445918900_cit16","doi-asserted-by":"publisher","first-page":"139","DOI":"10.1210\/er.2009-0007","article-title":"Molecular\nAspects of Thyroid Hormone\nActions","volume":"31","author":"Cheng","year":"2010","journal-title":"Endocr. Rev."},{"key":"2026090207445918900_cit17","doi-asserted-by":"publisher","first-page":"111","DOI":"10.1038\/nrendo.2015.205","article-title":"Nongenomic Actions of Thyroid Hormone","volume":"12","author":"Davis","year":"2016","journal-title":"Nat. Rev. Endocrinol."},{"key":"2026090207445918900_cit18","doi-asserted-by":"publisher","first-page":"1169","DOI":"10.3390\/ijms18061169","article-title":"Utilization of Multi-Immunization\nand Multiple Selection\nStrategies for Isolation of Hapten-Specific Antibodies from Recombinant\nAntibody Phage Display Libraries","volume":"18","author":"Tullila","year":"2017","journal-title":"Int. J. Mol.\nSci."},{"key":"2026090207445918900_cit19","doi-asserted-by":"publisher","first-page":"2446","DOI":"10.1021\/acs.analchem.5b04591","article-title":"Specific Noncompetitive Immunoassay\nfor HT-2 Mycotoxin\nDetection","volume":"88","author":"Arola","year":"2016","journal-title":"Anal. Chem."},{"key":"2026090207445918900_cit20","doi-asserted-by":"publisher","first-page":"248","DOI":"10.1016\/0003-2697(76)90527-3","article-title":"A Rapid\nand Sensitive Method for the Quantitation of Microgram Quantities\nof Protein Utilizing the Principle of Protein-Dye Binding","volume":"72","author":"Bradford","year":"1976","journal-title":"Anal. Biochem."},{"key":"2026090207445918900_cit21","doi-asserted-by":"publisher","first-page":"525","DOI":"10.1016\/0005-2795(78)90398-7","article-title":"Quantitation\nof Submicrogram Quantities of Protein\nby an Improved Protein-Dye Binding Assay","volume":"533","author":"Bearden","year":"1978","journal-title":"Biochim.\nBiophys. Acta, Protein Struct."},{"key":"2026090207445918900_cit22","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/S1387-3806(02)00585-7","article-title":"Quantitative\nDetermination of Noncovalent Binding Interactions Using Soft Ionization\nMass Spectrometry","volume":"216","author":"Daniel","year":"2002","journal-title":"Int. J. Mass Spectrom."},{"key":"2026090207445918900_cit23","doi-asserted-by":"publisher","first-page":"3867","DOI":"10.1021\/ac3005082","article-title":"Quantifying Ligand\nBinding to Large Protein Complexes\nUsing Electrospray Ionization Mass Spectrometry","volume":"84","author":"El-Hawiet","year":"2012","journal-title":"Anal. Chem."},{"key":"2026090207445918900_cit24","doi-asserted-by":"publisher","first-page":"1219","DOI":"10.1111\/j.1476-5381.2009.00604.x","article-title":"Ligand\nBinding Assays at Equilibrium: Validation and\nInterpretation","volume":"161","author":"Hulme","year":"2010","journal-title":"Br. J. Pharmacol."},{"key":"2026090207445918900_cit25","doi-asserted-by":"publisher","first-page":"8859","DOI":"10.1021\/ja00139a023","article-title":"Using Electrospray\nIonization FTICR Mass Spectrometry To Study Competitive Binding of\nInhibitors to Carbonic Anhydrase","volume":"117","author":"Cheng","year":"1995","journal-title":"J. Am. Chem.\nSoc."},{"key":"2026090207445918900_cit26","doi-asserted-by":"publisher","first-page":"600","DOI":"10.1002\/jms.1355","article-title":"Binding Constant Determination of\nHigh-Affinity Protein\n\u2013 Ligand Complexes by Electrospray Ionization Mass Spectrometry\nand Ligand Competition","volume":"43","author":"Wortmann","year":"2008","journal-title":"J. Mass Spectrom."}],"container-title":["ACS Omega"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsomega.9b02659","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/4\/20\/18718\/67718475\/acsomega.9b02659.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/4\/20\/18718\/67718475\/acsomega.9b02659.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T11:45:06Z","timestamp":1788349506000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acsodf\/article\/4\/20\/18718\/1445928\/Quantitation-of-Thyroid-Hormone-Binding-to-Anti"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,10,31]]},"references-count":26,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2019,10,31]]},"published-print":{"date-parts":[[2019,11,12]]}},"URL":"https:\/\/doi.org\/10.1021\/acsomega.9b02659","relation":{},"ISSN":["2470-1343"],"issn-type":[{"value":"2470-1343","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,10,31]]}}}