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.