Proteomic Analysis of Motor Cortex

All proteomic analyses were performed according to protocols previously described elsewhere [24 (link), 25 (link)]. Nine animals per group were euthanized and used in the proteomic approach. The samples of motor cortex from two animals were pooled, and all the procedures were carried out in triplicate. Briefly, the proteomic consists of protein extraction by lysis buffer. Then, the samples were reduced, alkylated, and finally digested by trypsin and desalted by C18 spin column (Pierce, Thermo Fisher, USA). Afterward, the samples were resuspended in the solution containing 12 μL of alcohol dehydrogenase standard (1 pmol/μL) + 108 μL of 3% acetonitrile and 0.1% formic acid.
The reading and identification of the peptides were performed on a nanoAcquity UPLC-Xevo QTof MS system (Waters Corporation, Wilmslow, UK), which were interpreted by Protein Lynx Global Server (PLGS) software applying the Monte-Carlo algorithm. After comparing the experimental groups, it was considered p < 0.05 for downregulated proteins and 1 − p > 0.95 for upregulated proteins. It was used the Rattus norvegicus proteome downloaded from Uniprot. After, the proteins identified were analyzed by a bioinformatic approach using Cytoscape 3.6.1 (Java®) with ClueGO plugin [26 (link)].