Manual mass spectrometry de novo sequencing of the anionic host defense peptides of the Cuban Treefrog Osteopilus septentrionalis

Abstract

Rationale Host defense peptides accumulated in the skin glands of the animals constitute the basis of the adaptive and immune system of amphibians. The peptidome of the Cuban frog Osteopilus septentrionalis was established using tandem mass spectrometry as the best analytical tool to elucidate the sequence of these peptides. Methods Manual interpretation of complementary collision‐induced dissociation (CID), higher energy collision‐induced dissociation (HCD), and electron transfer dissociation (ETD) tandem mass spectra recorded with an Orbitrap Elite mass spectrometer in liquid chromatography/mass spectrometry (LC/MS) mode was used to sequence the peptide components of the frog skin secretion, obtained by mild electrostimulation. Results Although the vast majority of amphibian peptides discovered so far are cationic, surprisingly only anionic peptides were identified in the skin secretion of the Cuban frog Osteopilus septentrionalis. Mass spectrometry allowed the sequences to be established of 16 representatives of new peptide families: septenins 1 and septenins 2. The highest sequence coverage when dealing with these anionic peptides was obtained with CID normalized collision energy 35 and HCD normalized collision energy 28. Conclusions Mirror‐symmetrical peptides are sequenced using N‐terminal acetylation. Acetylated Ser is reliably distinguished from isomeric Glu by the loss of ketene from b‐ions containing the corresponding residue. Calculations of the physicochemical and structural properties of the discovered anionic septenins 1 and 2 allowed the mechanism of their interaction with microbe cells to be postulated.

Mass Spectrometry Differentiation between Rana arvalis Populations Based on Their Skin Peptidome Composition

“…Small protonated peptides often lose H 2 O/NH 3 molecules, leading to informative but rather complicated spectra with long series of y n 0 ions. 9 Figure 3 illustrates HCD spectrum of m/z 694.928 2+ ion of temporin AV after elimination of an ammonia molecule [MH − NH 3 ] + . Mass accuracy in that case was 2.5 ppm.…”

“…Skin secretion released by the dorsal glands of amphibians in response to the external threat, irritation, or attack represents the main and often the only defensive weapon of amphibians, including Ranid frogs. Our scientific interests deal with the de novo sequencing of the amphibian skin secretion peptides by means of mass spectrometry. − The obtained results are summarized in the recent reviews. , Skin peptidomes of the European Ranid frogs, including Rana arvalis species, may contain the following features: (i) peptides with C-terminal disulfide 6–9 member cycles, called “rana box” (brevinins 1 and 2; esculentins 1 and 2; ranatuerins 1 and 2, and some other peptides’ families); (ii) amidation at the C-terminal temporins; (iii) melittin-related peptides (MRPs); (iv) bradykinin-related peptides (BRPs); (v) bombesins, and others. − Their activity usually leads to the death of pathogenic cells. Thus, disulfide-containing peptides, temporins, and MRPs are treated as cytolytics.…”

A Comprehensive Review of Patented Antimicrobial Peptides from Amphibian Anurans

“…During the last 30 years, the study of anuran skin secretion has been a topic of great interest due to its unique chemical properties and the associated biosynthetic pathways, which make it a powerful source of natural compounds with biological activities. − ,,,− The vast diversity of molecules found in anurans’ dermal glands can be classified into five main groups: (1) peptides and proteins, (2) alkaloids, (3) other biogenic amines, (4) steroid bufadienolides, and (5) volatile organic compounds. − ,,− ,,,− In recent years, our group has been working on the analysis of amphibian skins’ secretions using spectrometry for the study of toxins, volatile organic compounds, pigmentation, and AMPs’ diversity, − analyzing chemical diversity in terms of their ecological relevance. More specifically, we now have a more comprehensive understanding, suggesting that many defense substances can also be interpreted regarding host-symbiotic relationships .…”