Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry

• Published in: Journal of the American Society for Mass Spectrometry Vol. 29; no. 5; pp. 827 - 841
• Main Authors: Wormwood, Kelly L., Ngounou Wetie, Armand Gatien, Gomez, Marcus Vinicius, Ju, Yue, Kowalski, Paul, Mihasan, Marius, Darie, Costel C.
• Format: Journal Article
• Language: English
• Published: New York Springer US 01-05-2018; Springer Nature B.V
• Subjects: Amino Acid Sequence; Analgesics; Analgesics - chemistry; Analytical Chemistry; Animals; Bioinformatics; Biotechnology; Chemistry; Chemistry and Materials Science; Cysteine - analysis; Disulfides - analysis; Focus: 29th Sanibel Conference; Ions; Linkages; Mass spectrometry; Organic Chemistry; Pain; Peptides; Peptides - chemistry; Peptidomics: Bridging the Gap Between Proteomics and Metabolomics by MS: Research Article; Pharmacology; Proteomics; Scientific imaging; Side effects; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy; Spider Venoms - chemistry; Spiders - chemistry; Structural analysis; Tandem Mass Spectrometry - methods; Trypsin; Venom peptides; Mass spectrometry; Protein structure; Disulfide bridges
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1007/s13361-018-1904-3

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.
Graphical Abstract
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