Online ligand screening for cytochrome C from herbal medicines through “four-in-one” measurement

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC–energy-resolved-affinity MS (PCI-LC–ER-AMS) to achieve “four-in-one” monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC–ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through “four-in-one” measurement.