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Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry Open Access
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Kitova, Elena N.
Klassen, John S.
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A new electrospray ionization mass spectrometry (ES-MS) approach for quantifying protein—ligand complexes that are prone to in-source (gas-phase) dissociation is described. The method, referred to here as the reference ligand ES-MS method, is based on the direct ES-MS assay and competitive ligand binding. A reference ligand (Lref), which binds specifically to the protein (P), at the same binding site as the ligand (L) of interest, with known affinity and forms a stable protein—ligand complex in the gas phase, is added to the solution. The fraction of P bound to Lref, which is determined directly from the ES mass spectrum, is sensitive to the fraction of P bound to L in solution and enables the affinity of P for L to be determined. A mathematical framework for the implementation of the method in cases where P has one or two specific ligand binding sites is given. Affinities of two carbohydrate-binding proteins, a single chain fragment of a monoclonal antibody and the lectin concanavalin A, for monosaccharide ligands are reported and the results are shown to agree with values obtained using isothermal titration calorimetry.
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- © 2010 El-Hawiet, A., Kitova, E. N., Liu, L., & Klassen, J. S. This version of this article is open access and can be downloaded and shared. The original author(s) and source must be cited.
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El-Hawiet, A., Kitova, E. N., Liu, L., & Klassen, J. S. (2010). Quantifying labile protein-ligand interactions using electrospray ionization mass spectrometry. Journal of the American Society for Mass Spectrometry, 21(11), 1893-1899. http://doi.org/10.1016/j.jasms.2010.07.008
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