Screening for noncovalent ligand-receptor interactions by electrospray ionization mass spectrometry-based diffusion measurements.
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 (NAG(3))] were screened for noncovalent binding to lysozyme. Of these six compounds, only NAG(3) is known to bind to the protein. In "direct" binding tests, NAG(3) 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 NAG(3)-containing mixtures significantly increased the diffusion coefficient of the reference ligand NAG(4) (N,N',N'',N'''-tetraacetylchitotetrose), whereas mixtures that did not contain NAG(3) 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.
Clark
Sonya M
SM
Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Konermann
Lars
L
D000818:Animals; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000032:analysis* / Q000737:chemistry; D002645:Chickens; D004058:Diffusion; D008024:Ligands; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013997:Time Factors
D002241:Carbohydrates; D008024:Ligands; D009113:Muramidase