A straightforward method for measuring binding affinities of ligands to proteins of unknown concentration in biological tissues

The equilibrium dissociation constant (<i>K</i> _d) is a quantitative measure of the strength with which a drug binds to its receptor. Methods for determining <i>K</i> _d typically require <i>a priori</i> knowledge of protein concentration or mass. We report a simple dilution method for estimation of <i>K</i> _d using native mass spectrometry which can be applied to protein-ligand complexes involving proteins of unknown concentration, from complex mixtures, including direct tissue sampling.