A combined strategy of neuropeptide predictions and tandem mass spectrometry identifies evolutionarily conserved ancient neuropeptides in the sea anemone Nematostella vectensis

Abstract

Neuropeptides are a class of bioactive peptides and are responsible for various physiological processes including metabolism, development and reproduction. Although accumulated genome and transcriptome data have reported a number of neuropeptide candidates, it still remains difficult to obtain a comprehensive view of neuropeptide repertoires due to their small and variable nature. Neuropeptide prediction tools usually work only for those peptides for which sequentially related homologs have previously been identified. Recent peptidomics technology has enabled systematic structural identification of neuropeptides by using the combination of liquid chromatography and tandem mass spectrometry. However, obtaining reliable identifications of endogenous peptides is still difficult using a conventional tandem mass spectrometry-based peptide identification approach using protein database because a large search space has to be scanned due to the absence of a cleavage enzyme specification. We developed a pipeline consisting of the prediction of in silico cleaved endogenous neuropeptides followed by peptide-spectrum matching enabling highly sensitive and reliable neuropeptide identification. This approach effectively reduces the search space of peptide-spectrum matching, and thus increases search sensitivity. To identify neuropeptides in Nematostella vectensis, a basal eumetazoan having one of the most primitive nervous systems, we scanned the Nematostella protein database for sequences displaying structural hallmarks of metazoan neuropeptides, including C/N-terminal structures and modifications. Peptide-spectrum matching was performed against the in silico cleaved peptides and successfully identified dozens of neuropeptides at high confidence. The identification of Nematostella neuropeptides structurally related the tachykinin, GnRH/AKH, neuromedin-U/pyrokinin peptide families indicate that these peptides already originated in the eumetazoan ancestor of all animal species, most likely concomitantly with the development of a nervous system.