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.
Department of Chemistry, The University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Sonya M
SM
Clark
Lars
L
Konermann
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