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Medientyp: E-Artikel Titel: Binding constant determination of high‐affinity protein–ligand complexes by electrospray ionization mass spectrometry and ligand competition Beteiligte: Wortmann, Arno; Jecklin, Matthias C.; Touboul, David; Badertscher, Martin; Zenobi, Renato Erschienen: Wiley, 2008 Erschienen in: Journal of Mass Spectrometry, 43 (2008) 5, Seite 600-608 Sprache: Englisch DOI: 10.1002/jms.1355 ISSN: 1076-5174; 1096-9888 Schlagwörter: Spectroscopy Entstehung: Anmerkungen: Beschreibung: <jats:title>Abstract</jats:title><jats:p>We describe an approach for the determination of binding constants for protein–ligand complexes with electrospray ionization mass spectrometry, based on the observation of unbound ligands competing for binding to a protein target. For the first time, dissociation constants lower than picomolar could be determined with good accuracy by electrospray ionization mass spectrometry. The presented methodology relies only on the determination of signal intensity ratios for free ligands in the low mass region. Therefore, all the advantages of measuring low masses with mass spectrometry, such as high resolution are preserved. By using a reference ligand with known binding affinity, the affinity of a second ligand can be determined. Since no noncovalently bound species are observed, assumptions about response factors are not necessary. The method is validated with ligands binding to avidin and applied to ligands binding to p38 mitogen‐activated protein kinase. Copyright © 2007 John Wiley & Sons, Ltd.</jats:p>