Mass spectral characterization of peptide transmitters/hormones in the nervous system and neuroendocrine organs of the American lobster Homarus americanus

Abstract

The American lobster Homarus americanus is a decapod crustacean with both high economic and scientific importance. To facilitate physiological investigations of peptide transmitter/hormone function in this species, we have used matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS) to elucidate the peptidome present in its nervous system and neuroendocrine organs. In total, 84 peptides were identified, including 27 previously known H. americanus peptides (e.g., VYRKPPFNGSIFamide [Val(1)-SIFamide]), 23 peptides characterized previously from other decapods, but new to the American lobster (e.g., pQTFQYSRGWTNamide [Arg(7)-corazonin]), and 34 new peptides de novo sequenced/detected for the first time in this study. Of particular note are a novel B-type allatostatin (TNWNKFQGSWamide) and several novel FMRFamide-related peptides, including an unsulfated analog of sulfakinin (GGGEYDDYGHLRFamide), two myosuppressins (QDLDHVFLRFamide and pQDLDHVFLRFamide), and a collection of short neuropeptide F isoforms (e.g., DTSTPALRLRFamide and FEPSLRLRFamide). Our data also include the first detection of multiple tachykinin-related peptides in a non-brachyuran decapod, as well as the identification of potential individual-specific variants of orcokinin and orcomyotropin-related peptide. Taken collectively, our results not only expand greatly the number of known H. americanus neuropeptides, but also provide a framework for future studies on the physiological roles played by these molecules in this commercially and scientifically important species.

Figure 1

Direct tissue peptide profiling of H. americanus brain by MALDI FTMS. Signals correspond to the protonated molecular ions, [M+H]⁺, where M is the molecular weight of each peptide. Peptide identity was assigned based on accurate mass measurement. Novel peptides are indicated by stars with open stars indicating known peptides in other decapods but new to H. americanus and filled stars showing new peptides reported for the first time in this study.

Figure 2

Collision-induced dissociation spectrum of a de novo sequenced B-type allatostatin, TNWNKFQGSWamide. The sequence-specific b-type and y-type fragment ions and immonium ion characteristic of tryptophan are labeled, with derived amino acid sequence listed above the fragmentation spectrum.

Figure 3

Collision-induced dissociation spectra of three de novo sequenced FMRFamide-related peptides QDLDHVFLRFamide, DTSTPALRLRFamide, and SGRNFLRFamide. All precursor ions are doubly charged. The sequence-specific b-type and y-type fragment ions and immonium ions are labeled.

Figure 4

Collision-induced dissociation spectrum of a de novo sequenced orcokinin-precursor related peptide VYGPRDIANLY. The identities of isoleucine and leucine are determined based on knowledge of orcokinin prepro-hormone sequence (Dickinson et al., 2007a).