Characterization and Quantification of AST-C Peptides in Homarus americanus Using Mass Spectrometry

Neuropeptides are small signaling molecules found throughout the nervous system that influence animal behavior. Using the American lobster, Homarus americanus, as a model system, this research focused on an allatostatin type-C (AST-C) peptide, pQIRYHQCYFNPISCF (disulfide bond between underlined cysteine residues), and a structurally similar crustacean peptide, SYWKQCAFNAVSCFamide. These neuropeptides influence cardiac muscle contraction patterns and stomatogastric nervous system activity in the lobster. To understand their roles, this study sought to develop a method to quantify peptides in the pericardial organ (PO) and other crustacean tissues. Overall analysis involved microdissection to isolate tissues, tissue extraction, extract purification and concentration, and analysis by chip-based nano-electrospray ionization-liquid chromatography-mass spectrometry (nanoESI-LC-MS). In the present study, pQIRYHQCYFNPISCF was identified in the PO. To quantify target peptides, internal standards were tested as recovery and calibration references. However, experiments with pQIRYHQCYFNPISCF and other peptides showed evidence of adsorptive losses during sample preparation and analysis, with improvements in recovery resulting from the use of isopropanol-prewashed polypropylene vials. Preliminary results also suggested that introducing polyethylene glycol (PEG) in solution reduced adsorptive losses for hydrophobic peptides, but may have compromised hydrophilic peptide detection. Future directions include characterizing other sources of analyte loss and developing techniques to recover these signals. Since both target peptides as detected in the lobster are post-translationally modified, other directions include identifying modified and unmodified forms of these peptides in H. americanus. Ultimately, quantifying AST-C peptides and viii identifying their modified and unmodified forms will help explain how neuropeptides regulate behavior within the lobster and more complex systems.