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Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.
Rafael D Melani1, Owen S Skinner2, Luca Fornelli2
1From the ‡Proteomics Unit, Rio de Janeiro Proteomics Network, Departamento de Bioquímica. Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-909, Brazil; §Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, 60208.
Top-down proteomics precisely characterized king cobra venom proteins and complexes, revealing toxin variations and modifications. This advanced method offers a clearer view of venom components for developing new antivenoms.
Area of Science:
- Proteomics
- Biochemistry
- Toxicology
Background:
- Bottom-up proteomics relies on computational inference to identify proteins from peptides.
- Top-down proteomics directly analyzes intact proteins, offering a more complete characterization.
- King cobra (Ophiophagus hannah) venom contains complex toxins critical for its lethality.
Purpose of the Study:
- To characterize whole proteins and protein complexes from Ophiophagus hannah venom using top-down proteomics.
- To gain a detailed understanding of toxin sequence variation, cleavage sites, and post-translational modifications (PTMs).
- To provide a precise molecular inventory of venom proteins for antivenom development.
Main Methods:
- Utilized GELFrEE (gel electrophoresis) and solution isoelectric focusing for protein fractionation.
- Employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analysis.
- Performed top-down proteomics in both native and denaturing modes to preserve non-covalent interactions.
Main Results:
- Identified 131 proteins and characterized 184 proteoforms from 14 toxin families.
- Provided detailed information on large venom glycoprotein complexes like l-amino acid oxidase and cobra venom factor.
- Revealed glycosylation patterns on the l-amino acid oxidase complex.
Conclusions:
- Top-down proteomics offers an unprecedented characterization of venom proteoforms and complexes.
- This methodology clarifies toxin variation and PTMs critical for venom function.
- A precise venom proteome inventory can advance snakebite research and biotherapeutic development.
