Mass spectrometry-based affinity capture of peptide ligands from complex venom mixtures

Animal venoms constitute a rich source of bioactive peptides targeting membrane receptors, including G protein-coupled receptors (GPCRs) and ligand-gated ion channels. However, identifying receptor-binding ligands directly from complex venom mixtures remains analytically challenging because venoms are highly diverse and conventional screening approaches have limited throughput. Here, we present a membrane-based affinity-capture workflow coupled to mass spectrometry to recover known receptor ligands and prioritize additional venom toxins for validation. The methodology combines native membrane preparations, label-free affinity capture, MALDI-TOF mass spectrometry, and LC-MS/MS-based proteomic identification within a single analytical workflow. Its performance was demonstrated using two pharmacologically distinct membrane receptor families: the human vasopressin type 2 receptor, a G protein-coupled receptor, and the muscle-type nicotinic acetylcholine receptor, a ligand-gated ion channel. The workflow recovered known ligands spanning endogenous peptides and disulfide-rich venom toxins and documented AVP-dependent changes in toxin recovery. Direct comparison of bound fractions obtained with nAChR-rich Torpedo membranes and control CHO membranes further identified toxins preferentially retained by each membrane preparation. By combining receptor-guided affinity selection with direct molecular identification, this strategy provides a versatile analytical platform for investigating ligand–receptor interactions and prioritizing bioactive peptides from highly complex natural mixtures. Beyond venom research, the approach is readily adaptable to other membrane receptors and molecular libraries for ligand discovery. Proteomic data are available via ProteomeXchange with identifier PXD084762.