Screening for Noncovalent Ligand−Receptor Interactions by Electrospray Ionization Mass Spectrometry-Based Diffusion Measurements

Abstract

The application of a novel method for the identification of low-molecular-weight noncovalent ligands to a macromolecular target is reported. This technique is based on the measurement of analyte diffusion coefficients by electrospray mass spectrometry (ESI-MS) (Clark et al., Rapid Commun. Mass Spectrom. 2002, 16, 1454−1462). Potential ligands have large diffusion coefficients as long as they are free in solution. Binding to a macromolecular target, however, drastically reduces the diffusional mobility of any ligand species. Mixtures containing six different saccharides [ribose, rhamnose, glucose, maltose, maltotriose, and N,N‘,N‘ ‘-triacetylchitotriose (NAG₃)] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG₃ is known to bind to the protein. In “direct” binding tests, NAG₃ shows a significantly reduced diffusion coefficient in the presence of the protein. No changes were observed for any of the other saccharides. In a second set of experiments, the use of a “competition” screening method was explored in which mixtures of candidate saccharides were tested for their ability to displace a reference ligand from the target. The addition of NAG₃-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG₄ (N,N‘,N‘ ‘,N‘ ‘‘-tetraacetylchitotetrose), whereas mixtures that did not contain NAG₃ had no effect. These data clearly indicate the potential of ESI-MS-based diffusion measurements as a novel tool to screen compound libraries for binding to proteins and other macromolecular targets. In contrast to conventional ESI-MS-based ligand−receptor binding studies, this method does not rely on the preservation of noncovalent interactions in the gas phase.