Determination of Dissociation Constants for Protein−Ligand Complexes by Electrospray Ionization Mass Spectrometry

Abstract

A fully automated biophysical assay based on electrospray ionization mass spectrometry (ESI-MS) for the determination of the dissociation constants (KD) between soluble proteins and low molecular mass ligands is presented. The method can be applied to systems where the relative MS response of the protein and the protein-ligand complexes do not reflect relative concentrations. Thus, the employed approach enables the use of both electrostatically and nonpolar bound complexes. The dynamic range is wider than for most biological assays, which facilitates the process of establishing a structure-activity relationship. This fully automated ESI-MS assay is now routinely used for ligand screening. The entire procedure is described in detail using hGHbp, a 25-kDa extracellular soluble domain of the human growth hormone receptor, as a model protein.

Which electrospray-based ionization method best reflects protein-ligand interactions found in solution? A comparison of ESI, nanoESI, and ESSI for the determination of dissociation constants with mass spectrometry

“…A lot of effort is currently put into developing approaches to quantitatively measure noncovalent interaction strengths of protein/peptide-ligand systems by ESI-MS [33][34][35][36][37]. Improvements for accurate determination of dissociation constants (K D )s have been presented, e.g., taking into account different ionization efficiencies of free protein and noncovalent complexes [38,39], or correcting for nonspecific ligand binding [40,41].…”

Electrospray mass spectrometry of neuac oligomers associated with the c fragment of the tetanus toxin

“…The specific interactions taking place in this study occur between the negatively charged carboxylic groups of the sialic acids and positive (or partially positive) regions on the toxin's surface. Based on the conclusions that have been drawn from previous work, it appears reasonable to assume such a complex could maintain its conformation throughout the ionization process and this MS study might be expected to provide [12,13].…”

“…These results show that the dimer is not an artifact produced by the electrospray process and that its stability is disrupted by moderate methanol concentrations. The observation that the dimer is always observed in lower abundance than the monomer in the ESI-MS spectra suggests that the stability of the hydrophobic interactions is more easily disrupted in the gas phase [13][14][15].…”

“…Electrospray ionization (ES) has been established as a soft-ionization technique for introducing noncovalent complexes into the gas phase [6 -11], and previous studies have demonstrated the utility of ESI MS for determining affinity constants [12,13] of protein-ligand complexes. These experiments have evoked considerable discussion in the literature about whether or not the gas phase is a valid representation of solution conditions that govern the rates of association (or dissociation) between proteins and ligands.…”

“…The conclusions that have been drawn from these discussions are that the bound state is thermodynamically favored in the gas phase for noncovalent complexes dominated by ionic or polar interactions. Conversely, nonpolar or hydrophobic interactions between molecules are favored as dissociated entities in the gas phase [13][14][15]. For this reason, ESI MS is not recommended as the method of choice studying protein/ligand interactions [12,15].…”