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Parallel Peptidome and Transcriptome Analyses of Amphibian Skin Secretions Using Archived Frozen Acid-Solvated Samples

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Abstract

Amphibian skin secretions are unique sources of bioactive peptides and their donor species are currently rapidly disappearing from the biosphere. Here, we report that both peptides and polyadenylated mRNAs from skin granular glands remain amenable to study in samples of stimulated skin secretions following their storage in 0.1 % aqueous trifluoroacetic acid at −20 °C for many years. Frozen acidified solutions of toad (Bombina variegata) skin secretions, stored for 12 years, were thawed and samples removed for direct reverse phase HPLC fractionation. Additional samples were removed, snap frozen and lyophilised for construction of cDNA libraries following polyadenylated mRNA capture using magnetic oligo-dT beads and reverse transcription. Using the bombesin and bradykinin peptides found in bombinid toad skin as models, individual variant peptides of each type were located in reverse phase HPLC fractions and their corresponding biosynthetic precursor-encoding mRNA transcripts were cloned from the cDNA library using a RACE PCR strategy. This study illustrates unequivocally that both amphibian skin secretion peptides and their biosynthetic precursor-encoding polyadenylated mRNAs are stable in frozen acid-solvated skin secretion samples for considerable periods of time—a finding that may have fundamental implications in the study of archived materials but also in the wider field of molecular biology.

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Authors and Affiliations

  1. Natural Drug Discovery Group, School of Pharmacy, Queen’s University Belfast, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, UK

    Bing Bai, Hui Wang, Mei Zhou, Yang Yu, Sijia Ding, Tianbao Chen, Lei Wang & Chris Shaw

  2. Tangshan Gongren Hospital, Hebei United University, Tangshan City, 063000, Hebei Province, China

    Yingqi Zhang

Authors
  1. Bing Bai
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  2. Yingqi Zhang
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  3. Hui Wang
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  4. Mei Zhou
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  5. Yang Yu
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  6. Sijia Ding
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  7. Tianbao Chen
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  8. Lei Wang
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  9. Chris Shaw
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Corresponding authors

Correspondence to Lei Wang or Chris Shaw.

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Bing Bai and Yingqi Zhang contributed equally to this study.

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Bai, B., Zhang, Y., Wang, H. et al. Parallel Peptidome and Transcriptome Analyses of Amphibian Skin Secretions Using Archived Frozen Acid-Solvated Samples. Mol Biotechnol 54, 187–197 (2013). https://doi.org/10.1007/s12033-012-9551-6

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  • DOI: https://doi.org/10.1007/s12033-012-9551-6

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