Proteomics Of Optic Ganglion From The Sepia Esculenta And The Octopus Vulgaris

Sepia esculenta and Octopus vulgaris are both mollusk animals belonging to class Cephalopoda. The nervous system of Cephalopoda is relatively advanced, and the ganglion of Cephalopoda is easy to anatomise and separate, making it ideal for carrying out neuroscience researches. In this paper, the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was chosen to separate the total proteins of optic ganglion from Sepia esculenta. Five analytical approaches such as the precipitation separation of TCA/acetone, the buffer extraction, the column chromatography, the lyolysis buffer, and the lyolysis-ultracentrifugation were compared to extract and separate the protein from the optic ganglion of Sepia esculenta respectively. We indicated the lyolysis-ultracentrifugation was the best method for separating the proteome of optic ganglion from Sepia esculenta. With the optimization of other aspects such as the covering range of carrier amopholytes, the loading quantity of first dimension (IEF) and the separate gel's concentration of second dimension (SDS-PAGE), we established a stable 2D-PAGE sample preparation method, with high resolution and perfect repetition, adapting to the proteome research in the nervous system of Sepia esculenta and Octopus vulgaris.Using the optimized method of 2D-PAGE, 2D-PAGE maps of optic and cerebral ganglion from Sepia esculenta were obtained. Peptide mass fingerprint (PMF) and database search were used to identify the proteins on 2D-PAGE gel, and then we established the anatomic maps of optic ganglion and cerebral ganglion of Sepia esculenta primarily. From the maps, we can found the protein spots of cerebral ganglion were much more than that of optic ganglion. The experiment results showed that the two high score protein, found both in optic and cerebral ganglion, were mitochondrial malate dehydrogenase precursor (pre-MDH) and SNAP-type proteins. Also, there were several protein spots with relative high score, such as elongation factor G, tubulin and actin .We chose differential proteomics to search for the differential proteins of optic ganglion from Octopus vulgaris treated with methanol. Gas-chromatography (GC) was used to prove the existence of methanol in the optic ganglion, and to calculate the concentration of that methanol. Using the optimized method of 2D-PAGE, we established the differential proteome between the methanol-induced optic ganglion and normal ganglion. Basing on the 2D-PAGE maps, we found that total significant 18 protein changed evidently, 6 were down-regulated, 7 were up-regulated, 2 were disappear and 3 were increment. All of these differential protein spots were identified by peptide mass fingerprinting (PMF) and database search. The identified results indicated that two up-regulated protein, beta-tubulin and beta-actin, were the most significant differential protein. Besides, there were five other protein which had obtained relative high score. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Succinyl-CoA synthetase (SCS) alpha subunit were up-regulate; putative ABC transporter was down-regulated; the other two, Alcohol dehydrogenase (ADH) and Acyl-CoA dehydrogenase long-chain specific (LACD) were increment. We suggested the disorders of beta-tubulin and beta-actin would result in the ablepsia of Octopus vulgaris. With the aid of LOCtree, the subcellular locations of differential proteins were predicted, and most of the differential proteins were mitochondrial proteins adding up to 38.89%. This showed that mitochondria had been seriously destructed by methanol toxicity and we suggested some protective compounds for mitochondria may help to cure methanol poisoning. With comparison and analysis, we found that those differential proteins composed a complex net reflecting the structural and functional disorders in the methanol-induced nerve cells. These can help us know further about the methanol's toxic mechanism to nervous system, the responses and the tolerant mechanism of the organism in methanol poisoning, and the therapy for methanol poisoning.The proteomics research of the nervous system of Cephalopoda using proteome analytical techniques and the establishment of the methanol-induced differential proteome expressed by the ganglion cells have not yet been discribed in detail and will be of great importance for the research related.