Proteomic and peptidomic characterization of Rhaebo guttatus (Anura: Bufonidae) skin secretion

Rhaebo guttatus is a South American frog species from the Bufonidae family. Its defense against predators is primarily attributed to small molecules such as steroids, alkaloids, and biogenic amines. However, there is a lack of studies focusing on the proteins and peptides in its venom. This study aimed to analyze these biomolecules and investigate their biological significance. Using nano liquid chromatography coupled to high-resolution mass spectrometry (nanoLC-ESI-q-ToF), we performed shotgun proteomics and peptidomics analyses. The proteomic profile of R. guttatus showed similarities to those of other frog species, including proteins associated with muscle contraction, enzymes involved in oxidative stress-likely reflecting the skin's constant exposure to solar radiation and reactive oxygen species-and immune-related proteins such as galectin and annexin. These proteins appear to play a role in maintaining amphibian homeostasis, as suggested by previous studies. Peptidomic analysis revealed that some natural peptides may originate from the degradation of proteins present in the venom secretion. Interestingly, certain classes of proteins identified in the peptidomics were not detected in the proteomic dataset, underscoring the complementary nature of these omics approaches for unraveling venom molecular diversity. Among the peptides identified, we propose that some derive from BASP1, a protein typically associated with the brain. In this context, we hypothesize that these peptides originate from neuromuscular junctions and may participate in axonal regeneration and synaptogenesis processes. BASP1 is also recognized as a transcriptional suppressor of various tumors, suggesting potential pharmacological applications. Additionally, several de novo peptides with ALC >90 % were identified. In silico analyses, including prediction and molecular modeling, suggested antimicrobial activity for some of these peptides, paving the way for future studies with biotechnological and therapeutic interests.