Volatile Kinetic Capillary Electrophoresis for Studies of Protein–Small Molecule Interactions

Abstract

Kinetic capillary electrophoresis (KCE) is a toolset of homogeneous affinity methods for studying kinetics of noncovalent binding. Sensitive KCE measurements are typically done with fluorescence detection and require a fluorescent label on a smaller-sized binding partner. KCE with fluorescence detection is difficult to use for study of protein-small molecule interactions since labeling small molecules is cumbersome and can affect binding. A combination of KCE with mass-spectrometry (KCE-MS) has been recently suggested for label-free studies of protein-small molecule interactions. The major obstacle for studies by KCE-MS is a buffer mismatch between KCE and MS; MS requires volatile buffers while KCE of protein-ligand interactions is always run in near-physiological buffers. Here we asked a simple question: can protein-ligand interactions be studied with KCE in a volatile buffer? We compared three volatile buffers (ammonium acetate, ammonium bicarbonate, and ammonium formate) with a near-physiological buffer (Tris-acetate) for three protein-ligand pairs. The volatile buffers were found not to significantly affect protein-ligand complex stability; moreover, when used as CE run buffers, they facilitated good-quality separation of free ligands from the protein-ligand complexes. The use of volatile buffers instead of Tris-acetate in detection of small molecules by MS improved the detection limit by approximately 2 orders of magnitude. These findings prove the principle of "volatile" KCE, which can be easily coupled with MS to facilitate label-free kinetic studies of protein-small molecule interactions.