Background

Endogenous secretory peptides (ESPs) play crucial roles in amphibian skin, yet their identification remains challenging in species lacking genomic data. This study developed an innovative pipeline integrating transcriptomics and peptidomics to identify ESPs in the skin of Odorrana grahami. This approach enhances endogenous secretory protein prediction accuracy by rescuing candidates erroneously discarded during SignalP-based screening. Such false negatives typically result from inaccurate annotation of N-terminal start sites within 5′-UTRs by protein prediction tools like TransDecoder.

Results

Our approach enhanced potential endogenous secretory protein identification rates by 61.6%, discovering 107 putative ESPs (16 validated at the protein level). Among these, 74 ESPs are newly reported in O. grahami (including 62 novel peptides). These ESPs span 14 known families (11 newly reported in O. grahami, 8 of which are first reported within the genus Odorrana). The frog skin active peptide (FSAP) family (n = 83)—comprising the largest subset of ESPs identified in this study—showed unexpected diversity between our studied individual and previously reported individuals within the population. Collectively, O. grahami (n = 226) and Odorrana andersonii (n = 205) currently hold the highest documented counts of FSAP family peptides in amphibians. Phylogenetic analysis delineated five FSAP clades (A–E) containing 18 clustered groups, with the hypervariable clade D harboring diverse non-AMPs and tachykinin-convergent peptides. GO and KEGG pathway analyses indicated that ESPs in O. grahami skin are predominantly immunity-related.

Conclusions

This study highlights underestimated FSAP family peptide diversity in Odorrana and provides an adaptable framework for ESP discovery across taxa.