%0 Journal Article
%T A Four-Parameter LC–MS Strategy for Reliable Ligand Screening of Cytochrome c in Herbal Medicines
%A Omar Zayed
%A Leila Ibrahim
%A Ahmed Mansour
%J Specialty Journal of Pharmacognosy, Phytochemistry, and Biotechnology
%@ 3062-441X
%D 2024
%V 4
%N 2
%R 10.51847/shtboZjldY
%P 151-171
%X Online affinity-based ligand screening utilizing liquid chromatography (LC) integrated with various detection systems is a prevalent strategy for isolating bioactive constituents from herbal medicines (HMs). Nonetheless, the occurrence of false-positive results remains a widespread challenge due to inadequate data documentation regarding the formation and stability of the ligand-protein complex. To address this limitation and enhance the reliability of identified hits, this study developed a post-column infusion liquid chromatography–energy-resolved-affinity mass spectrometry (PCI-LC–ER-AMS) platform. This configuration enables a comprehensive "four-in-one" analytical monitoring approach, tracking: 1) the reduction in response for potential ligands; 2) the reduction in protein response; 3) ions associated with the formed ligand-protein complexes; and 4) the dissociation strength of the ligand-protein binding. As a proof-of-concept application, ligand fishing targeting Cytochrome C (Cyt C) within complex herbal matrices was performed. To validate the utility of the method, a simulated sample composed of twelve predefined ligands alongside two negative controls was subjected to LC separation and mixed with Cyt C prior to quadrupole time-of-flight mass spectrometry (Qtof-MS) analysis. In comparison to control assays lacking either Cyt C or the ligands, all twelve target ligands—and notably none of the negative controls—demonstrated reduced response intensities. These decreases were perfectly aligned with twelve negative peaks detected in the Cyt C ion current chromatogram at the corresponding retention times. Furthermore, sequential ions representing each distinct ligand-Cyt C complex were successfully recorded. Through the generation of breakdown curves, the optimal collision energy (OCE) required to disrupt the non-covalent interactions was found to be positively correlated with binding affinity. The optimized platform was subsequently applied to evaluate two herbal medicines, namely Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata. The screening successfully isolated 24 compounds exclusively from SR; among these, flavonoid glycosides demonstrated elevated OCE values and superior binding strengths relative to their aglycone counterparts. Subsequent affinity measurements and cell-based assays confirmed the pronounced interactions between Cyt C and each of the discovered components. In summary, PCI-LC–ER-AMS provides an effective, high-confidence online ligand screening methodology for Cyt C in herbal remedies via multi-dimensional "four-in-one" analytical verification.
%U https://galaxypub.co/article/a-four-parameter-lcms-strategy-for-reliable-ligand-screening-of-cytochrome-c-in-herbal-medicines-ds8iyxexfizc9by