Development of a frontal analysis capillary electrophoresis coupled with time-of-flight mass spectrometry for determining the equilibrium dissociation constant between cyclophilin A and cyclosporin A

Exploring the interactions between ligands and proteins in vitro and in vivo is of irreplaceable significance for disease prevention, diagnosis, therapeutic development, and drug discovery. Herein, we have developed a novel method for determining the equilibrium dissociation constant (Kd) using frontal analysis capillary electrophoresis coupled with time-of-flight mass spectrometry (FACE-TOF-MS). This method enables the rapid determination of the Kd value for the interaction between Cyclophilin A and cyclosporin A. Furthermore, the reliability of this method was validated by comparing it with bio-layer interferometry (BLI). The Kd values determined by FACE-TOF-MS and BLI were 3.32 μM and 0.60 μM, respectively, which fall within an acceptable range when considering previous reports using various methods. This study introduces a novel approach for analyzing molecular interactions, offering potential specificity for simultaneously determining the Kd values of multiple ligands in complex samples, particularly in the areas of biomarker discovery, high-throughput lead compound screening, and therapeutic target validation.

Bio-layer interferometry; Cyclophilin A; Cyclosporin A; Equilibrium dissociation constant; FACE-TOF-MS; Intermolecular interactions.