A Comprehensive Proteomic Study of the Skin Secretions of the Frog Lithobates spectabilis

Abstract

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.

Novel Kazal-type proteinase inhibitors from the skin secretion of the Splendid leaf frog, Cruziohyla calcarifer

“…Among the processes that could be involved in their control are spatial and temporal regulation, zymogen activation, peptidase degradation, and macromolecular peptidase inhibitors. However, these mechanism have not been proven in amphibians up to now [5] , [6] , [7] , [8] , [9] , [10] , [11] , [12] .…”

Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus

“…Demesa-Balderrama et al [27] and Cavalcante et al [28] conducted proteomics studies with Lithobates spectabilis and Dermatonotus muelleri skin secretion after electrophoresis and classical in-gel digestion. However, such approaches are purely analytical and rely on protein separation by electrophoresis, not allowing (typically) protein recovery and/or subsequent biological/biochemical assays.…”

“…We employed the ion-exchange batch processing protocol [21] to obtain soluble protein fractions from D. melanostictus parotoid macrogland secretion. Following this methodology, we identified by proteomics proteins already described in other studies, such as alcohol dehydrogenase [NADP(+)], calmodulin, galectin, histone H2B and prostaglandin reductase 1 [18, 27-29].…”

“…Demesa-Balderrama et al [27] performed an in-gel digestion and de novo peptide sequencing to study proteins from Lithobates spectabilis skin secretion. The authors found 111 de novo peptides, identifying 15 proteins (E-value < 0.077).…”

Protein Composition and Biomedical Potential of the Skin Secretion of Hylarana erythraea (Schlegel, 1837) (Anura: Ranidae) from Langkawi Archipelago, Kedah, Peninsular Malaysia

“…The in-solution digestion method was followed Demesa-Balderrama et al ( 2016 ) and Proaño-Bolaños et al ( 2017 ), with some modifications. 100 µL of each sample was re-solubilised in 100 µL of 6 M urea, 100 mM Tris–HCl buffer, pH 7.8.…”