Analytical workflow for rapid screening and purification of bioactives from venom proteomes

Abstract

Animal venoms are important sources for finding new pharmaceutical lead molecules. We used an analytical platform for initial rapid screening and identification of bioactive compounds from these venoms followed by fast and straightforward LC-MS only guided purification to obtain bioactives for further chemical and biological studies. The analytical platform consists of a nano-LC separation coupled post-column to high-resolution mass spectrometry and parallel on-line bioaffinity profiling for the acetylcholine binding protein (AChBP) in a chip based fluorescent enhancement based bioassay. AChBP is a stable structural homologue of the extracellular ligand binding domain of the α7-nicotinic acetylcholine receptor (α7-nAChR). This receptor is an extensively studied medicinal target, previously associated with epilepsy, Alzheimer's, schizophrenia and anxiety. The workflow is demonstrated with the venom of the Naja mossambica mossambica. Two medium affinity AChBP ligands were found. After subsequent LC-MS guided purification of the respective venom peptides, the purified peptides were sequenced and confirmed as Cytotoxin 1 and 2. These peptides were not reported before to have affinity for the AChBP. The purified peptides can be used for further biological studies.

Keywords: (E)-3-(3-(4-diethylamino-2-hydroxybenzylidene)-3,4,5,6-tetrahydropyridin-2-yl)pyridine; ACN; AChBP; CNS; DAHBA; EDA; EICs; ELISA BR; ESI; HPLC; HRS; IT TOF; LC; Liquid chromatography; Ls-AChBP; Lymnaea stagnalis acetylcholine binding protein; Lymnaea stagnalis acetylcholine binding protein (Ls-AChBP); MALDI-TOF/TOF; MS; Nano-liquid chromatography–mass spectrometry (nano-LC–MS); Nicotinic acetylcholine receptor; On-line microfluidics; PLA2; Q TOF; Snake venom proteome screening; TFA; TFX3; TIC; Venom purification; acetonitrile; acetylcholine binding protein; central nervous system; effect directed analysis; electrospray ionization; enzyme-linked immunosorbent assay blocking reagent; extracted ion chromatograms; high-performance liquid chromatography; high-resolution screening; i.d.; internal diameter; ion-trap-time-of-flight; mass spectrometry; matrix assisted laser desorption ionization with tandem time of flight detection; phospholipase A2; quadrupole-time-of-flight; three-finger toxin; total ion chromatogram; trifluoroacetic acid; α7-nAChR; α7-nicotinic acetylcholine receptor.