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

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.