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.

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

“…Advantageously,A CTIS can be combined with any detection method which 1) can be coupled with acapillary,2)uniformly integrates the signal through the capillary cross-section, 3) has sufficiently high signal readout speed, and 4) has ac oncentration limit of quantitation below the K d values of the studied complexes.Acombination of ACTIS with MS appears to be the most suitable one for protein-small molecule complexes as it allows label-free detection of small molecules.However, because of matrix effects on ionization, [26] ACTIS-MS requires reasonably pure protein solutions.W hile TIS is not sensitive to buffer composition, MS is.T herefore,v olatile buffers are preferable over nonvolatile buffers for ACTIS-MS.I th as been previously shown that volatile buffers are suitable for studies of biomolecular interactions provided that they can support required pH and ionic strength. [27] If TIS is conducted in an onvolatile buffer, the sample should be diluted at the capillary exit with as heath liquid suitable for both dissociation of the protein-ligand complex and efficient ionization of the ligand. In general, we foresee that most of the future technical development of ACTIS will deal with its coupling to different detection systems and satisfying the four above-listed ACTIS requirements for detection systems.…”

Capillary electrophoresis‐based approaches for the study of affinity interactions combined with various sensitive and nontraditional detection techniques

“…Three volatile BGEs (ammonium acetate, ammonium bicarbonate, ammonium formate) were tested for the protein-ligand pair and compared to a physiological Tris-acetate BGE. Equilibrium-dissociation constants were obtained, and thus the possibility of coupling a volatile KCE method with MS detection was proved [78].…”

Transient Incomplete Separation Facilitates Finding Accurate Equilibrium Dissociation Constant of Protein–Small Molecule Complex

“…Therefore, volatile buffers are preferable over nonvolatile buffers for ACTIS‐MS. It has been previously shown that volatile buffers are suitable for studies of biomolecular interactions provided that they can support required pH and ionic strength . If TIS is conducted in a nonvolatile buffer, the sample should be diluted at the capillary exit with a sheath liquid suitable for both dissociation of the protein–ligand complex and efficient ionization of the ligand.…”