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<p>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 (<i>Kd</i>) using frontal analysis capillary electrophoresis coupled with time-of-flight mass spectrometry (FACE-TOF-MS). This method enables the rapid determination of the <i>Kd</i> 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 <i>Kd</i> values determined by FACE-TOF-MS and BLI were 3.32&#xa0;μM and 0.60&#xa0;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 <i>Kd</i> values of multiple ligands in complex samples, particularly in the areas of biomarker discovery, high-throughput lead compound screening, and therapeutic target validation.</p> Graphical Abstract <p></p>

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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

  • 期刊论文

  • 发表日期：

    2025年5月19日

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  • Jia Tang,
  • Fan Shui,
  • Li Deng,
  • Yujie Zhang,
  • Qibing Mei,
  • Min Wang,
  • Jing Zeng

摘要

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.

Graphical Abstract