Supplemental Data S1-S5 for "A Signal Peptide-based Strategy for Mining Endogenous Secretory Peptidome of Skin from a Single Frog, Odorrana grahami"

We developed a pipeline that integrates transcriptomics and peptidomics-based screenings to discover the endogenous secretory peptidome from the skin of a single Odorrana grahami specimen, a frog species without prior genomic or transcriptomic data. We generated the first transcriptome of O. grahami and constructed a search protein set (SPS) by incorporating all possible ORF_shift proteins into a filtered protein set (FPS), which improved the identification rate of putative endogenous secretory proteins by 61.6%. We discovered 107 endogenous secretory peptides (ESPs) belonging to 14 families, of which eight families were new to the genus Odorrana, as well as three others that were new to this particular species; moreover, 62 peptides were novel. We also revealed the diversity and conservation of ESPs in the Odorrana skin, as well as the convergent evolution of two peptides across different families. We found variation in the peptide composition of the frog skin active peptide (FSAP) family between the individuals we studied and those previously studied. Therefore, we suggested that these peptides should be considered within the context of the pan-transcriptome and pan-peptidome. In addition, we detected 16 ESPs and 126 corresponding truncated peptides at the protein level. We recommend that a database precursor ion search accuracy of 3 ppm is reasonable for data obtained from modern high-precision mass spectrometers. Our results demonstrate the efficiency of our experimental approach in identifying ESPs in the amphibian skin, as well as its potential application to other species or tissues.