title: Slow-Equilibration Approximation in Kinetic Size Exclusion Chromatography.
abstract: Kinetic size exclusion chromatography with mass spectrometry detection (KSEC-MS) is a solution-based label-free approach for studying kinetics of reversible binding of a small molecule to a protein. Extraction of kinetic data from KSEC-MS chromatograms is greatly complicated by the lack of separation between the protein and protein-small molecule complex. As a result, a sophisticated time-consuming numerical approach was used for the determination of rate constants in the proof-of-principle works on KSEC-MS. Here, we suggest the first non-numerical (analytical) approach for finding rate constants of protein-small molecule interaction from KSEC-MS data. The approach is based on the slow-equilibration approximation, which is applicable to KSEC-MS chromatograms that reveal two peaks. The analysis of errors shows that the slow-equilibration approximation guarantees that the errors in the rate constants are below 20% if the ratio between the characteristic separation and equilibration times does not exceed 0.1. The latter condition can typically be satisfied for specific interactions such as receptor-ligand or protein-drug. The suggested analytical solution equips analytical scientists with a simple and fast tool for processing KSEC-MS data. Moreover, a similar approach can be potentially developed for kinetic analysis of protein-small molecule binding by other kinetic-separation methods such as nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM).
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , Toronto, Ontario M3J 1P3, Canada.
authors.forename: Leonid T
authors.initials: LT
authors.lastname: Cherney
authors.affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University , Toronto, Ontario M3J 1P3, Canada.
authors.forename: Sergey N
authors.initials: SN
authors.lastname: Krylov
mesh_terms: D002850:Chromatography, Gel; D019075:Electrophoresis, Capillary; D007700:Kinetics; D013058:Mass Spectrometry; D004364:Pharmaceutical Preparations / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D004364:Pharmaceutical Preparations; D011506:Proteins