Denaturing and native top-down venomics: Mapping proteoforms and protein complexes from king cobra venom

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF, LTQ Orbitrap Elite

ORGANISM(S): Ophiophagus Hannah (king Cobra) (naja Hannah)

TISSUE(S): Venom

SUBMITTER: RAFAEL MELANI  

LAB HEAD: Gilberto B Domont

PROVIDER: PXD003403 | Pride | 2016-05-18

REPOSITORIES: Pride