Ultracentrifugation-based multi-target affinity selection mass spectrometry

Abstract

We have developed a new approach of affinity selection based on the strategy of rate-zonal density gradient centrifugation combined with the application of an ultra-performance liquid chromatography coupled quadrupole time-of-flight mass spectrometer. In this method, a discontinuous gradient of sucrose solution is used as the centrifugal medium, and the mixed proteins and compounds are laid on the top of it; an applied centrifugal driving force is then used to separate the mixed proteins and their respective ligands in the gradient. Ligand binding ability is defined by comparing the concentration distribution of compounds with the respective concentration distribution of targets after centrifugation once the solution is fractionated. Ideally, a specific ligand would essentially distribute identically with its targeted proteins. This method could be used to screen multiple targets simultaneously, and it would be especially helpful to screen multi-target directed ligands that can interact with multiple targets for specific pathogenesis.