title: Mapping Proteoforms and Protein Complexes From King Cobra Venom Using Both Denaturing and Native Top-down Proteomics.
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 (∼130 kDa) and the multichain toxin cobra venom factor (∼147 kDa). The l-amino acid oxidase complex exhibited two clusters of multiproteoform 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 antivenoms or other biotherapeutics.
authors.affiliation: From 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.
authors.forename: Rafael D
authors.identifier: 0000-0002-0349-235X
authors.initials: RD
authors.lastname: Melani
authors.affiliation: §Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, 60208.
authors.forename: Owen S
authors.initials: OS
authors.lastname: Skinner
authors.affiliation: §Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, 60208.
authors.forename: Luca
authors.initials: L
authors.lastname: Fornelli
authors.affiliation: From the ‡Proteomics Unit, Rio de Janeiro Proteomics Network, Departamento de Bioquímica. Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, 21941-909, Brazil; n-kelleher@northwestern.edu gilberto@iq.ufrj.br.
authors.forename: Gilberto B
authors.initials: GB
authors.lastname: Domont
authors.affiliation: §Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, 60208.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Compton
authors.affiliation: §Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, 60208 n-kelleher@northwestern.edu gilberto@iq.ufrj.br.
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D004546:Elapid Venoms / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017815:Elapidae / Q000378:metabolism*; D050830:L-Amino Acid Oxidase / Q000302:isolation & purification; D011489:Protein Denaturation; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D004546:Elapid Venoms; C016310:cobra venom factor; D050830:L-Amino Acid Oxidase