Skip to main content
SpringerLink
Log in

Differentiation of Central Slovenian and Moscow populations of Rana temporaria frogs using peptide biomarkers of temporins family

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Skin secretion represents the only means of defense for the majority of frog species. That phenomenon is based on the fact that the main components of the secretion are peptides demonstrating greatly varying types of bioactivity. They fulfill regulatory functions, fight microorganisms and may be even helpful against predators. These peptides are considered to be rather promising pharmaceuticals of future generation as according to the present knowledge microorganisms are unlikely to develop resistance to them. Mass spectrometry sequencing of these peptides is the most efficient first step of their study providing reliably their primary structures, i.e., amino acids sequence and S-S bond motif. Besides discovering new bioactive peptides, mass spectrometry appears to be an efficient tool of taxonomy studies, allowing for distinguishing not only between closely related species, but also between populations of the same species. Application of several tandem mass spectrometry tools (CID, HCD, ETD, EThcD) available with Orbitrap mass analyzer allowed us to obtain full sequence of about 60 peptides in the secretion of Slovenian population of brown ranid frog Rana temporaria. The problem of sequence inside C-terminal cycle formed by two Cys and differentiation of isomeric Leu and Ile residues was done in top-down mode without any derivatization steps. Besides general biomarkers of Rana temporaria species, Central Slovenian population of Rana temporaria demonstrates six novel temporins and one brevinin 1, which may be treated as biomarkers of that population.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Kastin AJ, editor. Handbook of biologically active peptides. second ed. Boston: Academic Press; 2013.

    Google Scholar 

  2. Bowie JH, Separovic F, Tyler MJ. Host-defense peptides of Australian anurans. Part 2. Structure, activity, mechanism of action, and evolutionary significance. Peptides. 2012;37(1):174–88. https://doi.org/10.1016/j.peptides.2012.06.017.

    Article  CAS  PubMed  Google Scholar 

  3. Conlon JM, Kolodziejek J, Nowotny N. Antimicrobial peptides from ranid frogs: taxonomic and phylogenetic markers and a potential source of new therapeutic agents. Biochim Biophys Acta. 2004;1696(1):1–14. https://doi.org/10.1016/j.bbapap.2003.09.004.

    Article  CAS  PubMed  Google Scholar 

  4. Samgina TY, Gorshkov VA, Artemenko KA, Vorontsov EA, Klykov OV, Ogourtsov SV, et al. LC-MS/MS with 2D mass mapping of skin secretions’ peptides as a reliable tool for interspecies identification inside Rana esculenta complex. Peptides. 2012;34(2):296–302. https://doi.org/10.1016/j.peptides.2012.02.017.

    Article  CAS  PubMed  Google Scholar 

  5. Xu X, Lai R. The chemistry and biological activities of peptides from amphibian skin secretions. Chem Rev. 2015;115(4):1760–846. https://doi.org/10.1021/cr4006704.

    Article  CAS  PubMed  Google Scholar 

  6. AmphibiaWeb 2020. (online) Avaliable at: https://amphibiaweb.org.(Accessed 31 Oct 2020).

  7. Conlon JM. Reflections on a systematic nomenclature for antimicrobial peptides from the skins of frogs of the family Ranidae. Peptides. 2008;29(10):1815–9. https://doi.org/10.1016/j.peptides.2008.05.029.

    Article  CAS  PubMed  Google Scholar 

  8. Wang G. Bioinformatic analysis of 1000 amphibian antimicrobial peptides uncovers multiple length-dependent correlations for peptide design and prediction. Antibiotics (Basel). 2020;9(8):491. https://doi.org/10.3390/antibiotics9080491.

    Article  CAS  PubMed Central  Google Scholar 

  9. Conlon JM, Mechkarska M, Lukic ML, Flatt PR. Potential therapeutic applications of multifunctional host-defense peptides from frog skin as anti-cancer, anti-viral, immunomodulatory, and anti-diabetic agents. Peptides. 2014;57:67–77. https://doi.org/10.1016/j.peptides.2014.04.019.

    Article  CAS  PubMed  Google Scholar 

  10. Mansour SC, Pena OM, Hancock REW. Host defense peptides: front-line immunomodulators. Trends Immunol. 2014;35(9):443–50. https://doi.org/10.1016/j.it.2014.07.004.

    Article  CAS  PubMed  Google Scholar 

  11. Hancock REW, Haney E, Gill EE. The immunology of host defence peptides: beyond antimicrobial activity. Nat Rev Immunol. 2016;16(5):321–34. https://doi.org/10.1038/nri.2016.29.

    Article  CAS  PubMed  Google Scholar 

  12. Mwangi J, Hao X, Lai R, Zhang ZY. Antimicrobial peptides: new hope in the war against multidrug resistance. Zool Res. 2019;40(6):488–505. https://doi.org/10.24272/j.issn.2095-8137.2019.062.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Anastasi A, Erspamer V, Bertaccini G. Occurence of bradykinin in the skin of rana temporaria. Comp Biochem Physiol. 1965;14:43–52. https://doi.org/10.1016/0010-406x(65)90007-1.

    Article  CAS  PubMed  Google Scholar 

  14. Simmaco M, Mignogna G, Canofeni S, Miele R, Mangoni ML, Barra D. Temporins, antimicrobial peptides from the European red frog Rana temporaria. Eur J Biochem. 1996;242(3):788–92. https://doi.org/10.1111/j.1432-1033.1996.0788r.x.

    Article  CAS  PubMed  Google Scholar 

  15. Conlon JM, Aronsson U. Multiple bradykinin-related peptides from the skin of the frog, Rana temporaria. Peptides. 1997;18(3):361–5. https://doi.org/10.1016/s0196-9781(96)00339-7.

    Article  CAS  PubMed  Google Scholar 

  16. Simmaco M, Mignogna G, Barra D. Antimicrobial peptides from amphibian skin: what do they tell us? Biopolymers. 1998;47(6):435–50. https://doi.org/10.1002/(sici)1097-0282(1998)47:6%3C435::aid-bip3%3E3.0.co;2-8.

    Article  CAS  PubMed  Google Scholar 

  17. Samgina TY, Gorshkov VA, Vorontsov YEA, Lebedev AT, Artemenko KA, Ogourtsov SV, et al. Mass spectral study of the skin peptide of brown frog Rana temporaria from Zvenigorod population. J Anal Chem. 2011;66:1353–60. https://doi.org/10.1134/S1061934811140152 Original Russian paper in Mass-spektrometria. 2011;8(1):7-14.

    Article  CAS  Google Scholar 

  18. Samgina TY, Vorontsov EA, Gorshkov VA, Hakalehto E, Hanninen O, Zubarev RA, et al. Composition and Antimicrobial activity of the skin peptidome of Russian brown frog Rana temporaria. J Proteome Res. 2012;11(12):6213–22. https://doi.org/10.1021/pr300890m.

    Article  CAS  PubMed  Google Scholar 

  19. Mangoni ML, Rinaldi AC, DiGiulio A, Mignogna G, Bozzi A, Barra D, et al. Structure-function relationships of temporins, small antimicrobial peptides from amphibian skin. Eur J Biochem. 2000;267(5):1447–54. https://doi.org/10.1046/j.1432-1327.2000.01143.x.

    Article  CAS  PubMed  Google Scholar 

  20. Mangoni ML. Temporins, anti-infective peptides with expanding properties. Cell Mol Life Sci. 2006;63:1060–9. https://doi.org/10.1007/s00018-005-5536-y.

    Article  CAS  PubMed  Google Scholar 

  21. Mangoni ML, Grazia AD, Cappiello F, Casciaro B, Luca V. Naturally occurring peptides from Rana temporaria: antimicrobial properties and more. Curr Top Med Chem. 2016;16(1):54–64. https://doi.org/10.2174/1568026615666150703121403.

    Article  CAS  PubMed  Google Scholar 

  22. Musale V, Casciaro B, Mangoni ML, Abdel-Wahab YHA, Flatt PR, Conlon JM. Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti-diabetic agents. J Pept Sci. 2018;24:2. https://doi.org/10.1002/psc.3065.

    Article  CAS  Google Scholar 

  23. Romero SM, Cardillo AB, Martínez Ceron MC, Camperi SA, Giudicessi SL. Temporins: an approach of potential pharmaceutic candidates. Surg Infect (Larchmt). 2020;21(4):309–22. https://doi.org/10.1089/sur.2019.266.

    Article  PubMed  Google Scholar 

  24. Mangoni ML, Saugar JM, Dellisanti M, Barra D, Simmaco M, Rivas L. Temporins, small antimicrobial peptides with leishmanicidal activity. J Biol Chem. 2005;280(2):984–90. https://doi.org/10.1074/jbc.m410795200.

    Article  CAS  PubMed  Google Scholar 

  25. Chen Q, Wade D, Kurosaka K, Wang ZY, Oppenheim JJ, Yang D. Temporin A and related frog antimicrobial peptides use formyl peptide receptor-like 1 as a receptor to chemoattract phagocytes. J Immunol. 2004;173(4):2652–9. https://doi.org/10.4049/jimmunol.173.4.2652.

    Article  CAS  PubMed  Google Scholar 

  26. Apponyi MA, Pukala TL, Brinkworth CS, Maselli VM, Bowie JH, Tyler MJ, et al. Host-defence peptides of Australian anurans: structure, mechanism of action and evolutionary significance. Peptides. 2004;25(6):1035–54. https://doi.org/10.1016/j.peptides.2004.03.006.

    Article  CAS  PubMed  Google Scholar 

  27. Samgina TY, Artemenko KA, Bergquist J, Trebse P, Torkar G, Tolpina MD, et al. Differentiation of frogs from two populations belonging to the Pelophylax esculentus complex by LC-MS/MS comparison of their skin peptidomes. Anal Bioanal Chem. 2017;409(7):1951–61. https://doi.org/10.1007/s00216-016-0143-3.

    Article  CAS  PubMed  Google Scholar 

  28. Durban J, Juárez P, Angulo Y, Lomonte B, Flores-Diaz M, Alape-Girón A, et al. Profiling the venom gland transcriptomes of Costa Rican snakes by 454 pyrosequencing. BMC Genomics. 2011;12:259. https://doi.org/10.1186/1471-2164-12-259.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Tan CH, Tan KY, Fung SY, Tan NH. Venom-gland transcriptome and venom proteome of the Malaysian king cobra (Ophiophagus hannah). BMC Genomics. 2015;16:687. https://doi.org/10.1186/s12864-015-1828-2.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Samgina TY, Tolpina MD, Hakalehto E, Artemenko KA, Bergquist J, Lebedev AT. Proteolytic degradation and deactivation of amphibian skin peptides obtained by electrical stimulation of their dorsal glands. Anal Bioanal Chem. 2016;408(14):3761–8. https://doi.org/10.1007/s00216-016-9462-7.

    Article  CAS  PubMed  Google Scholar 

  31. Samgina TY, Artemenko KA, Gorshkov VA, Lebedev AT, Nielsen ML, Savistski ML, et al. Electrospray ionization tandem mass spectrometry sequencing of novel skin peptides from Ranid frogs containing disulfide bridges. Eur J Mass Spectrom (Chichester). 2007;13(2):155–63. https://doi.org/10.1255/ejms.867.

    Article  CAS  Google Scholar 

  32. Samgina TY, Artemenko KA, Gorshkov VA, Ogurtsov SV, Zubarev RA, Lebedev AT. Mass spectrometric study of peptides secreted by the skin glands of the brown frog Rana arvalis from the Moscow region. Rapid Commun Mass Spectrom. 2009;23(9):1241–8. https://doi.org/10.1002/rcm.3994.

    Article  CAS  PubMed  Google Scholar 

  33. Lebedev AT, Vasileva ID, Samgina TY. FT-MS in the de novo top-down sequencing of natural nontryptic peptides. Mass Spectrom. Rev. 2021. https://doi.org/10.1002/mas.21678.

  34. Samgina TY, Tolpina MD, Trebse P, Torkar G, Artemenko KA, Bergquist J, et al. LTQ Orbitrap Velos in routine de novo sequencing of non-tryptic skin peptides from the frog Rana latastei with traditional and reliable manual spectra interpretation. Rapid Commun Mass Spectrom. 2016;30(2):265–76. https://doi.org/10.1002/rcm.7436.

    Article  PubMed  Google Scholar 

  35. Samgina TY, Vorontsov EA, Gorshkov VA, Artemenko KA, Zubarev RA, Ytterberg JA, et al. Collision-induced dissociation fragmentation inside disulfide C-terminal loops of natural non-tryptic peptides. J Am Soc Mass Spectrom. 2013;24(7):1037–44. https://doi.org/10.1007/s13361-013-0632-y.

    Article  CAS  PubMed  Google Scholar 

  36. Samgina TY, Vorontsov EA, Gorshkov VA, Artemenko KA, Zubarev RA, Lebedev AT. Mass spectrometric de novo sequencing of natural non-tryptic peptides: comparing peculiarities of collision-induced dissociation (CID) and high energy collision dissociation (HCD). Rapid Commun Mass Spectrom. 2014;28(23):2595–604. https://doi.org/10.1002/rcm.7049.

    Article  CAS  PubMed  Google Scholar 

  37. Harrison AG, Young AB, Bleiholder C, Suhai S, Paizs B. Scrambling of sequence information in collision-induces dissociation of peptides. J Am Chem Soc. 2006;128(32):10364–5. https://doi.org/10.1021/ja062440h.

    Article  CAS  PubMed  Google Scholar 

  38. Samgina TY, Kovalev SV, Gorshkov VA, Artemenko KA, Poljakov NB, Lebedev AT. N-terminal tagging strategy for de novo sequencing of short peptides by ESI-MS/MS and MALDI-MS. J Am Soc Mass Spectrom. 2010;21:104–11. https://doi.org/10.1016/j.jasms.2009.09.008.

    Article  CAS  PubMed  Google Scholar 

  39. Samgina TY, Tolpina MD, Surin AK, Kovalev SV, Bosch RA, Alonso IP, et al. Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban treefrog Osteopilus septentrionalis. Rapid Commun Mass Spectrom. 2021;35(7):e9061. https://doi.org/10.1002/rcm.9061.

    Article  CAS  PubMed  Google Scholar 

  40. Samgina TY, Artemenko KA, Gorshkov VA, Poljakov NB, Lebedev AT. Oxidation versus carboxamidomethylation of S-S bond in ranid frog peptides: pro and contra for de novo MALDI-MS sequencing. J Am Soc Mass Spectrom. 2008;19(4):479–87. https://doi.org/10.1016/j.jasms.2007.12.010.

    Article  CAS  PubMed  Google Scholar 

  41. Frese CK, Altelaar AF, van den Toorn H, Nolting D, Griep-Raming J, Heck AJ, et al. Toward full peptide sequence coverage by dual fragmentation combining electron-transfer and higherenergy collision dissociation tandem mass spectrometry. Anal Chem. 2012;84(22):9668–73. https://doi.org/10.1021/ac3025366.

    Article  CAS  PubMed  Google Scholar 

  42. Lebedev AT, Damoc E, Makarov AA, Samgina TY. Discrimination of leucine and isoleucine in peptides sequencing with Orbitrap Fusion Mass Spectrometer. Anal Chem. 2014;86(14):7017–22. https://doi.org/10.1021/ac501200h.

    Article  CAS  PubMed  Google Scholar 

  43. Zhokhov SS, Kovalyov SV, Samgina TY, Lebedev AT. An EThcD-based method for discrimination of leucine and isoleucine residues in tryptic peptides. J Am Soc Mass Spectrom. 2017;28(8):1600–11. https://doi.org/10.1007/s13361-017-1674-3.

    Article  CAS  PubMed  Google Scholar 

  44. Barra D, Simmaco M, Boman HG. Gene-encoded peptide antibiotics and innate immunity. Do ‘animalcules’ have defence budgets? FEBS Lett. 1998;430(1-2):130–4. https://doi.org/10.1016/s0014-5793(98)00494-3.

    Article  CAS  PubMed  Google Scholar 

  45. Goraya J, Knoop FC, Conlon JM. Ranatuerin 1T: an antimicrobial peptide isolated from the skin of the frog. Rana temporaria. Peptides. 1999;20(2):159–63. https://doi.org/10.1016/s0196-9781(98)00174-0.

    Article  CAS  PubMed  Google Scholar 

  46. Mechkarska M, Kolodziejek J, Musale V, Coquet L, Leprince J, Jouenne T, et al. Peptidomic analysis of the host-defense peptides in skin secretions of Rana graeca provides insight into phylogenetic relationships among Eurasian Rana species. Comp Biochem Physiol Part D Genomics Proteomics. 2019;29:228–34. https://doi.org/10.1016/j.cbd.2018.12.006.

    Article  CAS  PubMed  Google Scholar 

  47. Xi X, Li B, Kwok HF, Chen T. A review on Bradykinin-related peptides isolated from amphibian skin secretion. Toxins. 2015;7(3):951–70. https://doi.org/10.3390/toxins7030951.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kӧnig E, Bininda-Emonds ORP, Shaw C. The diversity and evolution of anuran skin peptides. Peptides. 2015;63:96–117. https://doi.org/10.1016/j.peptides.2014.11.003.

    Article  CAS  Google Scholar 

  49. Couture R, Harrisson M, Vianna RM, Cloutier F. Kinin receptors in pain and inflammation. Eur J Pharmacol. 2001;429(1-3):161–76. https://doi.org/10.1016/s0014-2999(01)01318-8.

    Article  CAS  PubMed  Google Scholar 

  50. Schroeder C, Beug H, Müller-Esterl W. Cloning and functional characterization of the ornithokinin receptor. Recognition of the major kinin receptor antagonist, HOE140, as a full agonist. J Biol Chem. 1997;272(19):12475–81. https://doi.org/10.1074/jbc.272.19.12475.

    Article  CAS  PubMed  Google Scholar 

  51. Samgina TY, Gorshkov VA, Vorontsov YA, Artemenko KA, Zubarev RA, Lebedev AT. Mass spectrometric study of bradykinin-related peptides (BRPs) from the skin secretion of Russian ranid frogs. Rapid Commun Mass Spectrom. 2011;25(7):933–40. https://doi.org/10.1002/rcm.4948.

    Article  CAS  PubMed  Google Scholar 

  52. Artemenko KA, Zubarev RA, Samgina TY, Lebedev AT, Savitski MM. Two dimensional mass mapping as a general method of data representation in comprehensive analysis of complex molecular mixtures. Anal Chem. 2009;81(10):3738–45. https://doi.org/10.1021/ac802532j.

    Article  CAS  PubMed  Google Scholar 

  53. Rinaldi AC, Mangoni ML, Rufo A, Luzi C, Barra D, Zhao H, et al. Temporin L: antimicrobial, haemolytic and cytotoxic activities, and effects on membrane permeabilization in lipid vesicles. Biochem. J. 2002;368(Pt 1):91–100. https://doi.org/10.1042/bj20020806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Kim JB, Iwamuro S, Knoop FC, Conlon JM. Antimicrobial peptides from the skin of the Japanese mountain brown frog, Rana ornativentris. J Pept Res. 2001;58(5):349–56. https://doi.org/10.1034/j.1399-3011.2001.00947.x.

    Article  CAS  PubMed  Google Scholar 

  55. Bachem Peptide Calculator (online) Avaliable at: https://www.bachem.com/knowledge-center/peptide-calculator/ (Accessed 26 Mar 2021)

  56. Proteus Structure Prediction Server 2.0 (online) Avaliable at: http://www.proteus2.ca/proteus2/ (Accessed 23 Mar 2021)

Download references

Acknowledgements

The authors would like to acknowledge the Core Facility Center “Arktika” of Northern (Arctic) Federal University for providing instrumentation required for the experiments.

Funding

This research was supported by the Russian Science Foundation grant (no. 21-73-20105).

Author information

Authors and Affiliations

  1. Department of Organic Chemistry, Lomonosov Moscow State University, 119991, Moscow, Russia

    T. Yu. Samgina, I. D. Vasileva, S. V. Kovalev & A. T. Lebedev

  2. University of Ljubljana Faculty of Health Sciences, Zdravstvena pot 5, 1000, Ljubljana, Slovenia

    P. Trebse

  3. Department for Biology, Chemistry and Home Economics, University of Ljubljana Faculty of Education, Kardeljeva ploščad 16, 1000, Ljubljana, Slovenia

    G. Torkar

  4. Pushchino Branch, Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Prospekt Nauki 6, Pushchino, Moscow, 142290, Russia

    A. K. Surin

  5. Department of Medicinal Biochemistry and Biophysics, Division of Molecular Biometry, Karolinska Institutet, 17177, Stockholm, Sweden

    R. A. Zubarev

  6. Department of Pharmacological & Technological Chemistry, I.M. Sechenov First Moscow State Medical University, Moscow, 119146, Russia

    R. A. Zubarev

Authors
  1. T. Yu. Samgina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. I. D. Vasileva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S. V. Kovalev
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. Trebse
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Torkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. K. Surin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. R. A. Zubarev
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. A. T. Lebedev
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to T. Yu. Samgina or A. T. Lebedev.

Ethics declarations

Ethics approval

All of the experiments with amphibians were carried out according to the rules published in Appendix A of the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (ETS No. 123), accepted in Strasbourg, 15 June 2006, and according to the Russian law GOST33219–2014, developed on the basis of the convention mentioned above by the Euro-Asian Council for Standardization, Metrology and Certification (EASC).

Conflict of interest

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Samgina, T.Y., Vasileva, I.D., Kovalev, S.V. et al. Differentiation of Central Slovenian and Moscow populations of Rana temporaria frogs using peptide biomarkers of temporins family. Anal Bioanal Chem 413, 5333–5347 (2021). https://doi.org/10.1007/s00216-021-03506-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-021-03506-1

Keywords

Search

Navigation