Abstract
Electrospray ionization (ESI) mass spectrometry (MS) is a crucial method for rapidly determining the interactions between small molecules and proteins with ultrahigh sensitivity. However, nonvolatile molecules and salts that are often necessary to stabilize the native structures of protein-ligand complexes can readily adduct to protein ions, broaden spectral peaks, and lower signal-to-noise ratios in native MS. ESI emitters with narrow tip diameters (∼250 nm) were used to significantly reduce the extent of adduction of salt and nonvolatile molecules to protein complexes to more accurately measure ligand-protein binding constants than by use of conventional larger-bore emitters under these conditions. As a result of decreased salt adduction, peaks corresponding to protein-ligand complexes that differ in relative molecular weight by as low as 0.06% can be readily resolved. For low-molecular-weight anion ligands formed from sodium salts, anion-bound and unbound protein ions that differ in relative mass by 0.2% were completely baseline resolved using nanoscale emitters, which was not possible under these conditions using conventional emitters. Owing to the improved spectral resolution obtained using narrow-bore emitters and an analytically derived equation, K (d) values were simultaneously obtained for at least six ligands to a single druggable protein target from one spectrum for the first time. This research suggests that ligand-protein binding constants can be directly and accurately measured from solutions with high concentrations of nonvolatile buffers and salts by native MS.