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

  • Kelly L. Wormwood
  • Armand Gatien Ngounou Wetie
  • Marcus Vinicius Gomez
  • Yue Ju
  • Paul Kowalski
  • Marius Mihasan
  • Costel C. Darie
Focus: 29th Sanibel Conference, Peptidomics: Bridging the Gap Between Proteomics and Metabolomics by MS: Research Article

Abstract

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

Keywords

Disulfide bridges Mass spectrometry Venom peptides Protein structure 

Abbreviations:

MALDI

Matrix-assisted laser desorption ionization

MS

Mass spectrometry

MALDI-MS

MALDI mass spectrometry

ESI

Electrospray ionization

ESI-MS

Electrospray ionization mass spectrometry

nanoLC

Nanoliquid chromatography

nanoLC-MS/MS

Nanoliquid chromatography tandem mass spectrometry

DTNB

5,5-Dithio-bis-(2-nitrobenzoic acid)

E. coli

Escherichia coli

FA

Formic acid

ACN

Acetonitrile

Q-TOF

Quadrupole-time of flight

UPLC

Ultra performance liquid chromatography

HPLC

High-performance liquid chromatography

MS/MS

Tandem mass spectrometry

HCAA

α-Cyano-4-hydroxycinnamic acid

TFA

Trifluoroacetic acid

TOPA

3,4,6-Trihydroxy-phenylalanine

Cys

Cysteine

Notes

Acknowledgements

The authors thank the members of the lab for fruitful discussions.

Supplementary material

13361_2018_1904_MOESM1_ESM.pptx (351 kb)
ESM 1 Supplemental Figure 1 A) MS spectra of the undigested Phα1β peptide demonstrating of the presence of the fully disulfide linked peptide by m/z 862.9 (7+), 1006.6 (6+), 1207.7 (5+) and 1509.1 (4+), which correspond to the mass of the Phα1β peptide minus 12 Da and B) The MS/MS fragmentation for each of the previously mentioned precursor ions demonstrating the inability for the peaks to fragment sufficiently enough to assign disulfide arrangements. Supplemental Figure 2 Identification of Cys7-8 disulfide linked. A) MSMS fragmentation of the precursor ion (inset) with m/z 863.37(2+) corresponds to the mass of the peptide DNQCYCPPGSSLGIFK with cysteines disulfide linked (minus 2 Da). B) MSMS fragmentation of the precursor ion (inset) with m/z 879.33(2+) corresponds to the mass of the peptide DNQCYCPPGSSLGIFK with cysteines disulfide linked (minus 2 Da), and modified by 32 Da. C) MSMS fragmentation of the precursor ion (inset) with m/z 895.82(2+) corresponds to the mass of the peptide DNQCYCPPGSSLGIFK with cysteines disulfide linked (minus 2 Da), and modified by 64 Da. The spectra from A, B & C resulted from an AspN digestion. Supplemental Figure 3 Confirmation of the Cys7-8 and Cys9-10 disulfide linkage by MSMS fragmentation of the precursor ion (inset) with m/z 1044.88 (2+). This corresponds to the peptide DNQCYCPPGSSLGIFKCSCA with Cys 7-to Cys 10 disulfide linked (minus 4 Da). Supplemental Figure 4 Confirmation of all disulfide linkages present in the peptide by MALDI-MS analysis of the Phα1β peptide. A) MS spectrum of the intact peptide and the peptide fragments; B) LIFT MS/MS spectrum of the intact peptide. These experiments suggest that all Cys 1 to Cys 12, Cys 1 to Cys 8 and Cys 9 to Cys 12 are disulfide linked (A). These experiments also suggest that Cys 3 to Cys 12, Cys 9 to Cys 12 and Cys 11-12 are also disulfide linked. Supplemental Figure 5 Zoom into the 6 kDa region of MALDI-MS spectra for the peaks that correspond to the intact recombinant (A) and native (B) Phα1β peptide, which demonstrates that all cysteine residues are disulfide linked. Supplemental Figure 6 MALDI-MS spectra for the peaks that correspond to the intact and truncated recombinant (A) and native (B) Phα1β peptide, as analyzed in linear (A) and reflective (B) positive mode. Supplemental Figure 7 MALDI-MS/MS fragmentation of the recombinant (A) and native (B) Phα1β peptides. In both spectra, we identified two fragments with m/z 1233.26(1+) and 2133.82(1+) (A) or 1233.56(1+) and 2134.02(1+) (B), which corresponded to peptides FKCSCAHANKY and CIPRGEICTDDCECCGCDNQ, each with their cysteines intramolecularly disulfide linked. (PPTX 351 kb)

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Copyright information

© American Society for Mass Spectrometry 2018

Authors and Affiliations

  • Kelly L. Wormwood
    • 1
  • Armand Gatien Ngounou Wetie
    • 1
  • Marcus Vinicius Gomez
    • 2
  • Yue Ju
    • 3
  • Paul Kowalski
    • 3
  • Marius Mihasan
    • 1
    • 4
  • Costel C. Darie
    • 1
  1. 1.Biochemistry & Proteomics Group, Department of Chemistry & Biomolecular ScienceClarkson UniversityPotsdamUSA
  2. 2.Institute of Education and Research Santa Casa Belo Horizonte-Laboratory of ToxinsBelo HorizonteBrazil
  3. 3.Bruker DaltonicsBillericaUSA
  4. 4.Department of BiologyAlexandru Ioan Cuza University of IasiIasiRomania

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