Disulfide C-terminal loop fragments derived from AMPs and the presence of peptidases
have been previously reported in the skin secretions of different amphibians. However, there are only a few studies on the
identification of enzymes in frog skin secretion based on the primary structure of these proteins. Similarly, little data exist
regarding the identification of disulfide C-terminal loops at large scale. Therefore, a comprehensive study on this issue
certainly could bring in much more information for understanding this molecular process and its biochemical consequences.
Thus, the aim of this work was to characterize the presence of disulfide C-terminal loop fragments of AMPs and
identify the proteins and probable enzymes present in the completely unknown secretion contents of the frog Lithobates
spectabilis. For this purpose, high-resolution mass spectrometry was applied to analyze the skin secretions processed by
two different protocols: (1) using a cocktail of enzymatic inhibitors and 2) without any protease inhibitors, maintaining the
solution for 2 hours at 10°C. Results from procedure-1, revealed 122 molecular masses, whereas procedure-2 permitted
253 different molecular masses to be identified. Fifty-nine peptides including 22 disulfide C-terminal loop-containing
peptides were obtained following procedure-2. Polyacrylamide gel electrophoresis separation, tryptic digestion and LCMS/
MS were used for “de novo” sequencing of 111 different peptides and the unequivocal identification of fifteen proteins
including at least three different peptidases. Additionally, it was possible to fully sequence eight peptides, including
a ranatuerin-related peptide identified here as Spectabilin, that was subsequently chemically synthesized and showed high
antibacterial, antiparasitic and cytotoxic activities.