Label-free kinetic measurements of non-covalent interactions between macromolecule and small molecule achieved by kinetic capillary electrophoresis-mass spectrometry and kinetic capillary electrophoresis-UV

Non-covalent macromolecule-small molecule interactions control various biological processes. Understanding biomolecular interactions is essential in modern drug discovery. Most drug targets are macromolecules such as proteins and nucleic acids, most drug compounds are small molecules. Consequently to study kinetic interactions between small molecule and macromolecule is fundamental in controlling biological processes and determining efficacies of drug compounds. Up to now majority of kinetic analysis methods involve in chemical labelling such as fluorescent labelling and surface immobilization. However modifying either macromolecule or small molecule changes natural structure of molecule alters binding parameters and also chemical modification by itself is difficult carry out. Hence new generation analytical approach which holds great sensitivity without affecting binding activity is in demand. Here we demonstrated the label-free solution-based approaches for studying the kinetics of macromolecule-small molecule interactions. In this proof-of-principle work we applied kinetic capillary electrophoresis -- mass spectrometry (KCE-MS) and kinetic capillary electrophoresis -- UV (KCE-UV) to study kinetics of interactions for protein-small molecule and aptamer-small molecule pairs. The calculated kinetic and equilibrium parameters were successfully validated by using isothermal titration calorimetry (ITC). Our study has also demonstrated that the dissociation rather than binding controls the equilibrium of the interaction complex. Due to their label-free and solution based features, KCE-MS and KCE-UV promise to become practical tool for kinetic analysis of macromolecule-small molecule interactions.