Fields, L.; Qin, J.; Ibarra, A. E.; Selby, K. G.; Gao, T.; Dang, T. C.; Lu, H.; Li, L.

Endogenous peptides are critical regulators of signaling and immunity but remain difficult to characterize in organisms with incomplete genomic annotation. We developed a hybrid discovery platform that integrates transformer-based de novo sequencing (Casanovo), neuropeptide-focused database searching (EndoGenius), and empirical false discovery rate estimation via NovoBoard. This pipeline enables confident identification of endogenous peptides while expanding coverage beyond conventional database-only or de novo-only approaches. Applied to neuroendocrine tissues from Callinectes sapidus and Cancer borealis, the workflow revealed numerous high-abundance novel peptides and provided structural and genomic support for their biological relevance. Notably, we report the first histone-2A-derived antimicrobial peptide in the C. sapidus and characterize naturally occurring sequence variants. We also identified unexpected peptide homologies between crustaceans and Rattus norvegicus, enabling annotation of conserved housekeeping proteins in sparsely annotated genomes. This hybrid platform establishes a scalable, open-source strategy for advancing neuropeptidomics and endogenous peptide discovery in emerging model organisms.