Patent ID: 8927297
Filing Date: 2015-01-06
Classification: G01N

Abstract:
1. A method to measure the dissociation rate of a ligand that forms a reversible covalent bond with a protein, wherein said protein is an Estrogen Related Receptor alpha (ERR-α), comprising the steps of: (a) measuring by LC/MS a mass for the protein, a mass for a first ligand that forms a reversible covalent thioether bond with the protein at Cys325, and a mass for a competing second ligand that forms a reversible covalent bond with the protein, wherein the competing second ligand has a different mass than the first ligand; (b) mixing the protein and the first ligand in a solution with the first ligand in molar excess of the protein; (c) incubating the protein and the first ligand in the solution to allow for a protein:ligand complex to form; (d) removing an aliquot of the solution and measuring by LC/MS the mass for the protein:ligand complex; (e) adding the competing second ligand to the solution containing the protein:ligand complex, wherein the competing second ligand is in molar excess to the protein:ligand complex and in molar excess to the first ligand; (f) removing aliquots of the solution at time 0 and at regular time intervals; (g) measuring the time-dependent change in the mass of the protein:ligand complex equal to the mass difference of the first ligand and the competing second ligand; and, (h) determining the dissociation rate for the first ligand.