A Dual-Gradient Chromatography-Tandem Mass Spectrometry Assay for Evaluating HTS Hits for Inhibitions of the Antibacterial Target MurB

摘要

1. We have developed a novel, rapid, sensitive dual LC-ESI-MS-MS method to quantify MurB enzymatic reaction product. 2. Baseline separation was achieved between substrate and reduced product, and the LC/MS-based assay has nanomolar sensitivity with a much wider dynamic range than fluorescence assay. 3. The LC/MS method has been validated for IC_(50) values and stability with competitive inhibitor NADP. 4. The developed LC/MS assay has been applied to evaluation of MurB HTS hits and can be expanded for focused library screening.