Platform for Large-Scale Determination of Protein-Ligand Binding
EMSL Project ID
49167
Abstract
Noncovalent protein-ligand interactions play a major role in many biological processes including signal transduction, enzymatic catalysis and immune response and determining those interactions is essential for applications ranging from biofuel optimization to drug development. The overall objective of this proposal is to develop and evaluate a new ultrasensitive, high-throughput and versatile protein/ligand interaction platform based on droplet-based microfluidics coupled with ion mobility spectrometry-mass spectrometry (IMS-MS) that will broadly advance fundamental and applied biological research. Currently, methods used to monitor binding kinetics either require fluorescent labeling or immobilization of protein or ligand. With our platform, measurements will be completely solution-based, label-free, consume minimal amounts of sample and have orders of magnitude higher throughput than existing methodologies. We will demonstrate the “soft” ionization and transport in the electrospray IMS-MS instrument such that weak binding in solution will be accurately reflected in the gas phase measurements. In the microfluidic platform, we will develop quantitative dilution, rapid mixing of multiple components, and variable incubation times prior to delivery of analyte to the ionization source. The capabilities developed will enable rapid, low cost activity profiling of proteins against panels of ligands to determine both equilibrium binding as well as rate constants. We will characterize the platform using a panel of kinases and small molecule inhibitors but the platform will be broadly applicable for monitoring biological interactions for a wide range of applications.
Project Details
Start Date
2015-10-30
End Date
2016-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members