Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry
Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide’s analgesic effect with very low side effects.
KeywordsDisulfide bridges Mass spectrometry Venom peptides Protein structure
Matrix-assisted laser desorption ionization
MALDI mass spectrometry
Electrospray ionization mass spectrometry
Nanoliquid chromatography tandem mass spectrometry
- E. coli
Quadrupole-time of flight
Ultra performance liquid chromatography
High-performance liquid chromatography
Tandem mass spectrometry
The authors thank the members of the lab for fruitful discussions.
KLW was supported by the ASPIRE Graduate Student Fellowship through Clarkson University’s CUPO Office. MM was supported by the Fulbright Senior Postdoctoral Fellowship awarded by the Romania-USA Fulbright Commission to MM (guest) and CCD (host). MVG’s work was supported through REDE Fapemig (CBB-RED-00006-14), CNPq (471070/2012-2), Capes Toxinology (AUX-PE 1444/2011), and Fapemig (PPM-00482-15).
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