Quantifying labile protein—Ligand interactions using electrospray ionization mass spectrometry
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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.
KeywordsLigand Complex Electrospray Ionization Mass Spectrometry Isothermal Titration Calorimetry Reference Ligand Single Chain Fragment
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