Slow-Equilibration Approximation in Kinetic Size Exclusion Chromatography.

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 consulting 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).