title: 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.
authors: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario M3J 1P3, Canada.
authors: Jiayin
authors: J
authors: Bao
authors: Sergey N
authors: SN
authors: Krylov
mesh_terms: D052157:Aptamers, Nucleotide / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001896:Boron Compounds / Q000378:metabolism; D002021:Buffers; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D029968:Escherichia coli Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009961:Orosomucoid / Q000378:metabolism; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D014835:Volatilization
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D052157:Aptamers, Nucleotide; D001896:Boron Compounds; D002021:Buffers; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D008024:Ligands; D009961:Orosomucoid; D011506:Proteins