Affinity selection mass spectrometry (AS-MS) for identification of acetylcholinesterase ligands in Strychnos peckii B.L. Rob

Introduction Strychnos L. species are traditionally used worldwide and are known to biosynthesize a wide range of structurally diverse alkaloids. Several of these compounds have been reported to interact with targets in the central nervous system, including acetylcholinesterase (AChE). Given its use in paralytic preparations, an aqueous extract of S. peckii was investigated for prospecting acetylcholinesterase ligands using affinity selection mass spectrometry (AS-MS), with the enzyme immobilized on magnetic particles. Methods For the AS-MS assay, 200 µg of eelAChE was immobilized on 10 mg of amino-functionalized magnetic beads via Schiff-base chemistry using glutaraldehyde as a linker. Control samples were prepared under identical conditions, with enzyme inactivation achieved using 100% MeOH (60 min). Enzymatic activity was monitored by choline formation. The workflow was optimized using galantamine as the reference ligand across four stages (incubation, magnetic separation, ligand dissociation, and LC-HRMS identification), establishing an affinity ratio (AR) cut-off of 1.2. Dissociated ligands were analyzed by LC-HRMS and processed in MZmine for ion alignment, deconvolution, and peak-area calculation. Results and Discussion Metabolites with AR > 1.2 were classified as AChE ligands and annotated based on MS/MS fragmentation data, interpretation of the molecular network, in silico tools, and manual curation guided by biosynthetic pathway analysis. The identified ligands were lyaloside (1) (AR, 3.73), 10-hydroxygeissoschizol (2) (AR, 3.69), alkaloid A (3) (AR, 2.51), 5-carboxystrictosidine (4) (AR, 2.05), corynantheol (5) (AR, 2.04), dolichantoside/isodolichantoside (6) (AR, 1.90), alkaloid B (7) (AR, 1.68), (strictosidine (AR, 1.66) (8), corynantheidal (9) (AR, 1.63), desoxycordifoline (10) (AR, 1.57), kaempferol-3- O -rhamnoside (11) (AR, 1.36), kaempferol (12) (AR, 1.35), alkaloid C (13) (AR, 1.29), and harman-3-carboxylic acid (14) (AR, 1.27). Conclusion Overall, the ligands were enriched in monoterpene indole alkaloids and related scaffolds, demonstrating that AS-MS is a rapid, label-free strategy to prioritize enzyme binding metabolites from complex natural matrices and expand the AChE interacting chemical space of S. peckii .