Exploring the Sea Urchin Neuropeptide Landscape by Mass Spectrometry.

Neuropeptides are essential cell-to-cell signaling messengers and serve important regulatory roles in animals. Although remarkable progress has been made in peptide identification across the Metazoa, for some phyla such as Echinodermata, limited neuropeptides are known and even fewer have been verified on the protein level. We employed peptidomic approaches using bioinformatics and mass spectrometry (MS) to experimentally confirm 23 prohormones and to characterize a new prohormone in nervous system tissue from Strongylocentrotus purpuratus, the purple sea urchin. Ninety-three distinct peptides from known and novel prohormones were detected with MS from extracts of the radial nerves, many of which are reported or experimentally confirmed here for the first time, representing a large-scale study of neuropeptides from the phylum Echinodermata. Many of the identified peptides and their precursor proteins have low homology to known prohormones from other species/phyla and are unique to the sea urchin. By pairing bioinformatics with MS, the capacity to characterize novel peptides and annotate prohormone genes is enhanced. Graphical Abstract.

Urotensin II in invertebrates: from structure to function in Aplysia californica.

Neuropeptides are ancient signaling molecules that are involved in many aspects of organism homeostasis and function. Urotensin II (UII), a peptide with a range of hormonal functions, previously has been reported exclusively in vertebrates. Here, we provide the first direct evidence that UII-like peptides are also present in an invertebrate, specifically, the marine mollusk Aplysia californica. The presence of UII in the central nervous system (CNS) of Aplysia implies a more ancient gene lineage than vertebrates. Using representational difference analysis, we identified an mRNA of a protein precursor that encodes a predicted neuropeptide, we named Aplysia urotensin II (apUII), with a sequence and structural similarity to vertebrate UII. With in-situ hybridization and immunohistochemistry, we mapped the expression of apUII mRNA and its prohormone in the CNS and localized apUII-like immunoreactivity to buccal sensory neurons and cerebral A-cluster neurons. Mass spectrometry performed on individual isolated neurons, and tandem mass spectrometry on fractionated peptide extracts, allowed us to define the posttranslational processing of the apUII neuropeptide precursor and confirm the highly conserved cyclic nature of the mature neuropeptide apUII. Electrophysiological analysis of the central effects of a synthetic apUII suggests it plays a role in satiety and/or aversive signaling in feeding behaviors. Finding the homologue of vertebrate UII in the numerically small CNS of an invertebrate animal model is important for gaining insights into the molecular mechanisms and pathways mediating the bioactivity of UII in the higher metazoan.

Quantitative peptidomics reveal brain peptide signatures of behavior.

The honey bee genome predicts approximately 100 peptides from 36 prohormones, but the functions of many of these peptides are unknown. We used differential isotope labeling combined with mass spectrometric analysis to quantify approximately 50% of known bee brain peptides in the context of foraging, with 8 showing robust and dynamic regulation. Some showed differences in brain abundance as a function of experience; specifically, nectar and pollen collection led to quick changes in abundance. These changes were related to the act of food collection, not ingestion, because foragers bring food back to the hive for storage rather than eating it themselves. Other peptide differences in brain abundance were seen in bees that either flew to a nectar feeder or a pollen feeder, but did not yet collect any food. These differences likely reflect well-known predispositions of some bees to collect either nectar or pollen, but not both. Tachykinin, PBAN, and sNPF were among the peptides with the strongest changes in association with nectar and pollen foraging. These peptides are known to be involved in regulating food intake in solitary insects, suggesting an evolutionary connection between that behavior and social foraging. These results demonstrate that it is now possible to use quantitative peptidomics to help determine which brain peptides are bioactive and to elucidate their function in the regulation of behavior.

From the genome to the proteome: uncovering peptides in the Apis brain.

Neuropeptides, critical brain peptides that modulate animal behavior by affecting the activity of almost every neuronal circuit, are inherently difficult to predict directly from a nascent genome sequence because of extensive posttranslational processing. The combination of bioinformatics and proteomics allows unprecedented neuropeptide discovery from an unannotated genome. Within the Apis mellifera genome, we have inferred more than 200 neuropeptides and have confirmed the sequences of 100 peptides. This study lays the groundwork for future molecular studies of Apis neuropeptides with the identification of 36 genes, 33 of which were previously unreported.

monitoring activity-dependent peptide release from the CNS using single-bead solid-phase extraction and MALDI TOF MS detection.

To investigate dynamic peptidergic cell-cell communication, single micrometer-sized solid-phase extraction (SPE) beads were used to collect peptides from specific locations of well-characterized neurosecretory structures and even individual neuronal processes for off-line MALDI MS analyses. Peptide binding parameters of single SPE beads, including limits of collection, detection, and saturation capacity, were tested with 14C-labeled cytochrome c as well as with mixtures of multiple neuropeptides (bradykinin, Aplysia acidic peptide 1-20, and insulin). MALDI MS detection of secreted peptides was demonstrated in two well-characterized neurosecretory structures, the rat pituitary gland and single cultured Aplysia bag cell neurons. With cultured cells, precise placement of SPE beads allowed peptide collection from distinct neurites with spatial localization on the order of 200 microm, and SPE beads could be replaced within time frames that allowed analyte collection before and after cell stimulation paradigms. Comparison between pre- and poststimulation peptide profiles in both model systems allowed a directed strategy to determine which compounds were released with neuronal activity. Single SPE bead MALDI MS offers a novel approach to investigate peptide signaling that allows the detection and discovery of unknown intercellular signals secreted from a large variety of biological tissues.