Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of N-glycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools.

Pedal peptide (PP) and orcokinin (OK) are related neuropeptides that were discovered in protostomian invertebrates (mollusks, arthropods). However, analysis of genome/transcriptome sequence data has revealed that PP/OK‐type neuropeptides also occur in a deuterostomian phylum—the echinoderms. Furthermore, a PP/OK‐type neuropeptide (starfish myorelaxant peptide, SMP) was recently identified as a muscle relaxant in the starfish Patiria pectinifera. Here mass spectrometry was used to identify five neuropeptides (ArPPLN1a‐e) derived from the SMP precursor (PP‐like neuropeptide precursor 1; ArPPLNP1) in the starfish Asterias rubens. Analysis of the expression of ArPPLNP1 and neuropeptides derived from this precursor in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed a widespread pattern of expression, with labeled cells and/or processes present in the radial nerve cords, circumoral nerve ring, digestive system (e.g., cardiac stomach) and body wall‐associated muscles (e.g., apical muscle) and appendages (e.g., tube feet and papulae). Furthermore, our data provide the first evidence that neuropeptides are present in the lateral motor nerves and in nerve processes innervating interossicular muscles. In vitro pharmacological tests with SMP (ArPPLN1b) revealed that it causes dose‐dependent relaxation of apical muscle, tube foot and cardiac stomach preparations from A. rubens. Collectively, these anatomical and pharmacological data indicate that neuropeptides derived from ArPPLNP1 act as inhibitory neuromuscular transmitters in starfish, which contrasts with the myoexcitatory actions of PP/OK‐type neuropeptides in protostomian invertebrates. Thus, the divergence of deuterostomes and protostomes may have been accompanied by an inhibitory–excitatory transition in the roles of PP/OK‐type neuropeptides as regulators of muscle activity.

Gamete maturation and spawning in starfish is triggered by a gonad‐stimulating substance (GSS), which is present in extracts of the radial nerve cords. Purification of GSS from the starfish Patiria pectinifera identified GSS as a relaxin‐like polypeptide, which is now known as relaxin‐like gonad‐stimulating peptide (RGP). Cells expressing RGP in the radial nerve cord of P. pectinifera have been visualized, but the presence of RGP‐expressing cells in other parts of the starfish body has not been investigated. Here we addressed this issue in the starfish Asterias rubens. An A. rubens RGP (AruRGP) precursor cDNA was sequenced and the A chain and B chain that form AruRGP were detected in A. rubens radial nerve cord extracts using mass spectrometry. Comparison of the bioactivity of AruRGP and P. pectinifera RGP (PpeRGP) revealed that both polypeptides induce oocyte maturation and ovulation in A. rubens ovarian fragments, but AruRGP is more potent than PpeRGP. Analysis of the expression of AruRGP in A. rubens using mRNA in situ hybridization revealed cells expressing RGP in the radial nerve cords, circumoral nerve ring, and tube feet. Furthermore, a band of RGP‐expressing cells was identified in the body wall epithelium lining the cavity that surrounds the sensory terminal tentacle and optic cushion at the tips of the arms. Discovery of these RGP‐expressing cells closely associated with sensory organs in the arm tips is an important finding because these cells are candidate physiological mediators for hormonal control of starfish spawning in response to environmental cues. J. Comp. Neurol. 525:1599–1617, 2017. © 2016 Wiley Periodicals, Inc.

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

Calcitonin (CT) is a peptide hormone released by the thyroid gland that regulates blood Ca2+ levels in mammals. The CT gene is alternatively spliced, with one transcript encoding CT and another transcript encoding the CT-like neuropeptide calcitonin-gene related peptide (α-CGRP), which is a powerful vasodilator. Other CT-related peptides in vertebrates include adrenomedullin, amylin, and intermedin, which also act as smooth muscle relaxants. The evolutionary origin of CT-type peptides has been traced to the bilaterian common ancestor of protostomes and deuterostomes and a CT-like peptide (DH31) has been identified as a diuretic hormone in some insect species. However, little is known about the physiological roles of CT-type peptides in other invertebrates. Here we characterized a CT-type neuropeptide in a deuterostomian invertebrate—the starfish Asterias rubens (Phylum Echinodermata). A CT-type precursor cDNA (ArCTP) was sequenced and the predicted structure of the peptide (ArCT) derived from ArCTP was confirmed using mass spectrometry. The distribution of ArCTP mRNA and the ArCT peptide was investigated using in situ hybridization and immunohistochemistry, respectively, revealing stained cells/processes in the nervous system, digestive system, and muscular organs, including the apical muscle and tube feet. Investigation of the effects of synthetic ArCT on in vitro preparations of the apical muscle and tube feet revealed that it acts as a relaxant, causing dose-dependent reversal of acetylcholine-induced contraction. Furthermore, a muscle relaxant present in whole-animal extracts of another starfish species, Patiria pectinifera, was identified as an ortholog of ArCT and named PpCT. Consistent with the expression pattern of ArCTP in A. rubens, RT-qPCR revealed that in P. pectinifera the PpCT precursor transcript is more abundant in the radial nerve cords than in other tissues/organs analyzed. In conclusion, our findings indicate that the physiological action of CT-related peptides as muscle relaxants in vertebrates may reflect an evolutionarily ancient role of CT-type neuropeptides that can be traced back to the common ancestor of deuterostomes.