Identification of GYRKPPFNGSIFamide (crustacean-SIFamide) in the crayfish Procambarus clarkii by topological mass spectrometry analysis.

A new concept relating to the purification protocol for biological proteins and peptides has been designed as "topological mass spectrometry analysis," in combination with MALDI-TOF MS using slices of tissues, chromatographic purification from the extract of tissues, molecular cloning for the determination of the precursor structure, and capillary LC-MS/MS analysis for elucidation of its posttranslational modifications. In an actual application, we identified an alpha-amidated neuropeptide from the red swamp crayfish (Procambarus clarkii) brain. Initially, an MS number of around m/z 1382 was found by the direct MALDI-TOF MS analysis with slices of the accessory lobe of the brain. After two steps of reversed-phase HPLC separation with brain extract, the structure of a 1381 Da peptide was sequenced to the GYRKPPFNGSIFamide (named crustacean-SIFamide). Subsequently, the cDNA has been characterized and encodes a 76 amino acid precursor protein that contains a signal sequence, one copy of GYRKPPFNGSIFG and one additional peptide. The RT-PCR analysis implied that the mRNA of the neuropeptide was expressed throughout the nervous system of the crayfish. Furthermore, immunostaining demonstrated that the neuropeptide is distributed in the olfactory lobe, accessory lobe, olfactory globular tract, and olfactory lobe cells. In addition, database searches revealed that there are homologous sequences of the AYRKPPFNGSIFamide in the genome library of fruitfly Drosophila melanogaster and AYRKPPFNGSLFamide isolated from the grey fleshfly Neobellieria bullata, and GYRKPPFNGSIFamide isolated from the giant tiger prawn Penaeus monodon. These results suggested that the neuropeptide family might be widely distributed in arthropods and plays a significant role in the nervous system.