A combined mass spectrometric and cDNA sequencing approach to the isolation and characterization of novel antimicrobial peptides from the skin secretions of Phyllomedusa hypochondrialis azurea

Alan Hunter Thompson, AJ Bjourson, David Francis Orr, Chris Shaw, Stephen McClean

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33 Citations (Scopus)

Abstract

Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent antimicrobial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea. Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. T-RACE reactions used primers designed for the highly conserved 5'-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 mu M when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli, Staphylococcus aureus and Micrococcus luteus. (c) 2007 Elsevier Inc. All rights reserved.
LanguageEnglish
Pages1331-1343
JournalPeptides
Volume28
Issue number7
DOIs
Publication statusPublished - Jul 2007

Fingerprint

Complementary DNA
Skin
Peptides
Micrococcus luteus
Microbial Sensitivity Tests
Amphibians
Post Translational Protein Processing
Gram-Negative Bacteria
Gene Library
Proteomics
Staphylococcus aureus
Escherichia coli
Anti-Bacterial Agents
Amino Acids
dermaseptin

Cite this

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title = "A combined mass spectrometric and cDNA sequencing approach to the isolation and characterization of novel antimicrobial peptides from the skin secretions of Phyllomedusa hypochondrialis azurea",
abstract = "Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent antimicrobial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea. Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. T-RACE reactions used primers designed for the highly conserved 5'-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 mu M when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli, Staphylococcus aureus and Micrococcus luteus. (c) 2007 Elsevier Inc. All rights reserved.",
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AU - Shaw, Chris

AU - McClean, Stephen

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AB - Studies conducted on amphibian skin secretions over the past 40 years have isolated and identified huge arrays of bioactive peptides, many of which have demonstrated potent antimicrobial activity. Such peptides are attracting increasing attention due to the growing problem of pathogenic microorganisms resistant to conventional antibiotics. The current study utilized a combined proteomic/genomic approach to facilitate the high throughput sequencing of five novel dermaseptins and four novel phylloseptins from the skin secretions of Phyllomedusa hypochondrialis azurea. Peptides were partially identified using Q-TOF MS/MS fragmentation and de novo sequencing, while a cDNA library was constructed from the lyophilized skin secretion. T-RACE reactions used primers designed for the highly conserved 5'-signal regions of previously deduced dermaseptin precursors. cDNA sequenced peptides were attributed to their respective fragmentation spectra to confirm the structure of the final processed peptides. Such an approach identified post-translational modifications in addition to deciphering isobaric amino acids. Several of the peptides were purified to homogeneity and displayed potent antimicrobial activity with minimum inhibitory concentrations starting at 0.4 mu M when tested against and range of Gram-positive and Gram-negative bacteria including Escherichia coli, Staphylococcus aureus and Micrococcus luteus. (c) 2007 Elsevier Inc. All rights reserved.

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