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.
Highlights
The toxic secretions of venomous animals have long been a source of biologically active molecules, yet remain a promising source of new drugs [1, 2]
One major advantage of top-down proteomics (TDPs) lies in the ability to provide complete molecular specificity of an intact protein to determine its “proteoforms”, the set of molecules that arise from combinations of genetic variation and post-translational modifications (PTMs) [13]
The topdown methodology is divided into two major sections: denaturing top-down and native top-down, with the latter employing native GELFrEE and native MS/MS [28]
Summary
Venom Samples—Ophiophagus hannah (king cobra) venom milked from two individuals original from Malaysia was purchased from the Kentucky Reptile Zoo, and was pooled by zoo staff. Data Analysis—Intact protein mass and fragment ions were searched against protein sequences and candidate proteoforms from the suborder Serpente, including sequences, features and modifications for the king cobra [36]. This was accomplished by downloading a UniprotKB flatfile on June 10, 2014 and the search database was created using the Database Manager application within ProSightPC v3.0 (Thermo Fisher Scientific). Proteins were identified from fragmentation spectra using ProSightPC v3.0 (Thermo Fisher Scientific) and were searched against the Serpente database with 331,349 candidate proteoforms in the same ProSight Warehouse described above. The bottom-up analysis was performed using a single digestion that was analyzed by LC-MS/MS in triplicate (MS technical replicates) to reduce under-sampling associated with the DDA method
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