Journal
MOLECULAR & CELLULAR PROTEOMICS
Volume 15, Issue 7, Pages 2423-2434Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/mcp.M115.056523
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Funding
- W. M. Keck Foundation
- FAPERJ from the government of Rio de Janeiro State - Brazil [100.039/2014]
- Swiss National Science Foundation under Early Postdoc Mobility fellowship
- National Science Foundation Graduate Research Fellowship [2014171659]
- NIH GM [067193]
- Office for Research at Northwestern University
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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 (similar to 130 kDa) and the multichain toxin cobra venom factor (similar to 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.
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