Identification of neuropeptides in gastropod mollusks. - Classical and brand-new approaches –

For the understanding of the neural regulation of homeostasis, reproduction and behaviors, elucidation of structure and function of neuropeptides is inevitable. A classical technique for peptide purification is the separation of peptidic extract from nervous tissue by the high-performance liquid chromatography, which was followed by screening of fractions with biological or immunological assays. Although this approach requires relatively large amount of tissues for extraction and the screening is time- consuming, we have identified more than 15 kinds of bioactive neuropeptides in the nervous tissue of a marine snail, Thais clavigera. The purified peptides include TEP (Thais excitatory peptide)-1/-2, FRFamide, WWamide and others. The molecular cloning of precursor for each neuropeptide demonstrated that structurally related peptides are aligned in tandem on most of the precursor proteins. To identify the peptides on the precursors, we conducted the de novo sequencing of peptides with the nanoLC-Orbitrap-MS/MS analysis. It identified most of the neuropeptides found on the precursor proteins in the extract from 50 of Thais ganglia. Thus, this technique is suitable for the comprehensive identification of peptides from relatively small amount of tissues. However, several peptides that had been identified by the classical technique, such as APGWamide and leucokinin, were not identified, suggesting the limitation of this brand-new technique.