Project description:Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric L-amino acid oxidase (LAAO, ~130 kDa) and the multi-chain toxin cobra venom factor (~147 kDa). The LAAO complex exhibited two clusters of multi-proteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new anti-venoms or other biotherapeutics.
Project description:Complementary bottom-up MS/MS analyses contributed to complete a locus-resolved venom phenotype map for O. hannah, the world's longest venomous snake and a species of medical concern across its wide distribution range in forests from India through Southeast Asia. Its venom composition convincingly explains the main neurotoxic effects of human envenoming caused by king cobra bite. The integration of efficient chromatographic separation of the venom components, and locus-resolved toxin identification through top-down and bottom-up MS/MS-based species-specific database searching, promises a bright future to the field of venom research.