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.
Original language | English (US) |
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Pages (from-to) | 2423-2434 |
Number of pages | 12 |
Journal | Molecular and Cellular Proteomics |
Volume | 15 |
Issue number | 7 |
DOIs | |
State | Published - Jul 2016 |
Funding
This material is based upon work supported by FAPERJ research grant 100.039/2014 from the government of Rio de Janeiro State-Brazil for R.D.M., by the Swiss National Science Foundation under Early Postdoc Mobility fellowship for L.F., and by the National Science Foundation Graduate Research Fellowship under fellowship number 2014171659 for O.S.S. The work was also supported by NIH GM 067193, and the Office for Research at Northwestern University.
ASJC Scopus subject areas
- Analytical Chemistry
- Biochemistry
- Molecular Biology