| Research Abstract |
Topological mass spectrometry analysis is a methodology to screen for novel neuropeptides that combines the techniques of MALDI-TOF MS, molecular cloning and quadrupole orthogonal acceleration time-of-flight (Q-TOF) MS. In the present study, we applied this strategy to the identification of neuropeptides involved in the regulation of water balance in the crayfish, Procambarus clarkii, specifically through anal respiration. Firstly, we performed direct MALDI-TOF MS analysis on slices of the 6^<th> abdominal ganglion, which has been known to contain some neurons controlling the movement of anal or hindgut. The data indicated the presence of orcokinin, its related peptide, crustacean-SIFamide, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). In addition to such already known neuropeptides, mass numbers of 1270 and 1330 were observed, implying the presence of the unknown neuropeptides. Extraction and purification was then performed by using their mass numbers a
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s a marker. Q-TOF MS/MS analysis revealed that the peptide with a mass number of 1270 Da has the C-terminal sequence of L/I-Asp-His-Val-Phe-L/I-Arg-Phe-amide, which is homological to myosuppressin. Subsequently, molecular biological approach was used, revealing that the precursor proteins both of myosuppressin-like peptide (MSLP) and CCAP contain only one copy each of the neuropeptide sequence. Their surrounding sequences are-KRQDLDHVFLRFGRSQ(Stop) and -KRPFCNAFTGCGKKR-, respectively. This information and their mass numbers could confirm their structures as pEDLDHVFLRFamide for MSLP and PFCNAFTGCa (C-C : disulfide bonding) for CCAP, respectively. Moreover, in situ hybridization (ISH) analysis suggested that posterior median cell cluster, which has been known to control hindgut directly, produce not only orcokinin but possibly also MSLP. The neuropeptides thus identified were synthesized and analyzed by bioassays on the movements of anus and the hindgut, and also on anal intake of water. Together, the neuropeptides seemed to have their respective effects by different sites and/or modes of action. Less
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