Peptidomics pp 145-157 | Cite as

Identification and Analysis of Bioactive Peptides in Amphibian Skin Secretions

  • J. Michael Conlon
  • Jérôme Leprince
Part of the Methods in Molecular Biology book series (MIMB, volume 615)


Skin secretions from anurans (frogs and toads), particularly those species belonging to the Hylidae and Ranidae families, are a rich source of biologically active peptides. Cytolytic peptides with broad-spectrum antimicrobial activities and highly variable amino acid sequences are often released into these secretions in high concentrations. Identification and characterization of these components can prove to be valuable in species identification, elucidation of evolutionary histories and phylogenetic relationships between species, and may lead to development of agents with potential for therapeutic application. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release in a procedure that does not appear to cause distress to the animals. The peptide components in the secretions are separated by reversed-phase HPLC on octadecylsilyl silica (C18) columns under standard conditions after partial purification on Sep-Pak cartridges. Individual peptides are identified by determination of their molecular masses by MALDI-TOF mass spectrometry and from their retention times. The use of mixtures of synthetic peptides of appropriate molecular mass as calibration standards enables mass determination to a high degree of precision.

Key words

Frog skin secretions antimicrobial peptide reversed-phase HPLC MALDI-TOF mass spectrometry 


  1. 1.
    Lazarus, L.H. and Attila, M. (1993) The toad, ugly and venomous, wears yet a precious jewel in his skin. Prog. Neurobiol. 41, 473–507.PubMedCrossRefGoogle Scholar
  2. 2.
    Nicolas, P. and Amiche, M. (2006) The dermaseptins, in Handbook of Biologically Active Peptides (Kastin, A.J., ed.), Elsevier, San Diego, CA, pp. 295–304.CrossRefGoogle Scholar
  3. 3.
    Apponyi, M.A., Pukala, T.L., Brinkworth, C.S., Maselli, V.M., Bowie, J.H., Tyler, M.J., et al. (2004) Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides 25, 1035–1054.PubMedCrossRefGoogle Scholar
  4. 4.
    Conlon, J.M., Kolodziejek, J., Nowotny, N. (2004) Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim. Biophys. Acta 1696, 1–14.PubMedGoogle Scholar
  5. 5.
    Nascimento, A.C.,Fontes, W., Sebben A. and Castro, M.S. (2003) Antimicrobial peptides from anurans skin secretions. Protein Pept. Lett. 10, 227–238.PubMedCrossRefGoogle Scholar
  6. 6.
    Vanhoye, D., Bruston, F., Nicolas, P. and Amiche, M. (2003) Antimicrobial peptides from hylid and ranin frogs originated from a 150-million-year-old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain. Eur. J. Biochem. 270, 2068–2081.PubMedCrossRefGoogle Scholar
  7. 7.
    Ohnuma, A., Conlon, J.M., Kawasaki, H. and Iwamuro S. (2006) Developmental and triiodothyronine-induced expression of genes encoding preprotemporins in the skin of Tago’s brown frog Rana tagoi. Gen. Comp. Endocrinol. 146, 242–250.PubMedCrossRefGoogle Scholar
  8. 8.
    Tyler, M.J., Stone, D.J. and Bowie, J.H. (1992) A novel method for the release and collection of dermal, glandular secretions from the skin of frogs. J. Pharmacol. Toxicol. Methods 28, 199–200.PubMedCrossRefGoogle Scholar
  9. 9.
    Nutkins, J.C. and Williams, D.H. (1989) Identification of highly acidic peptides from processing of the skin prepropeptides of Xenopus laevis. Eur. J. Biochem. 181, 97–102.PubMedCrossRefGoogle Scholar
  10. 10.
    Frost, D.R. (2008) Amphibian species of the world: an online reference. Version 5.2. Electronic database accessible at American Museum of Natural History, New York.
  11. 11.
    Davidson, C., Benard, M.F., Shaffer, H.B., Parker, J.M., O‘Leary, C., Conlon, J.M., et al. (2007) Effects of chytrid and carbaryl exposure on survival, growth and skin peptide defenses in foothill yellow-legged frogs. Environ. Sci. Technol. 41, 1771–1776.PubMedCrossRefGoogle Scholar
  12. 12.
    Conlon, J.M. (2007) Purification of naturally occurring peptides by reversed-phase HPLC. Nat. Protoc. 2, 191–197.PubMedCrossRefGoogle Scholar
  13. 13.
    Goraya, J., Wang, Y., Li, Z., O‘Flaherty, M., Knoop, F.C., Platz, J.E., et al. (2000) Peptides with antimicrobial activity from four different families isolated from the skins of the North American frogs Rana luteiventris, Rana berlandieri and Rana pipiens. Eur. J. Biochem. 267, 894–900.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • J. Michael Conlon
    • 1
  • Jérôme Leprince
    • 2
  1. 1.Department of Biochemistry, Faculty of Medicine and Health SciencesUnited Arab Emirates UniversityAl-AinUAE
  2. 2.European Institute for Peptide Research (IFRMP 23), INSERM U-413, UA CNRS, University of RouenMont-Saint-AignanFrance

Personalised recommendations