Nerve growth factor on hippocampal mitochondria in VD rats: TMT Proteomic analysis of Data
We used Sprague-Dawley rats to establish VD model, and used the proteomic method based on relative quantification (iTRAQ) to identify the differentially expressed proteins in hippocampus mitochondria. A total of 33 differentially expressed proteins were identified between the VD rats and the VD rats treated with nerve growth factor groups. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis were done after that.
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Adult male Sprague-Dawley rats (220±20g; No. 00010751; Experimental Animal Center, Wuhan First Hospital, Wuhan, Hubei Province, China; Laboratory animal facility license number: 00014834) were kept in the laboratory with temperature of 22±2 ℃ and humidity of 65±2%, 4 rats per cage. Animals were fed in a 12 h light/dark cycle and were given food and water at will. Mitochondrial preparation Executed rats were stripped of hippocampal brain tissue, rinsed with frozen saline, wiped dry with filter paper, weighed and put into a small beaker. Add a small amount of homogenate medium to cut brain tissue as soon as possible, and pour it into glass homogenizer. Rotate and grind the crushing rod up and down to make the tissue fully crushed. The homogenate was poured into the 2 ml microcentrifuge tube. After the centrifuge (4000 rpm/min, 15min), the supernatant was collected to a new tube. The preparation of mitochondria was carried out according to the procedure of the mitochondrial extraction kit (Pierce, USA). Total mitochondrial cell protein extraction Samples were taken out under freezing condition and frozen on ice. The samples were added 10% SDS (Final concentration 1 %) and protease inhibitor PMSF (7.5 mM) to lyses for 30 minutes, and mixed every 5 minutes for 10 seconds. Protein supernatant was centrifuged at 12000g for 8 mins. BCA quantitative analysis was used, and then detected by SDS-PAGE. Protein was measured using the BCA method. TMT Sample Preparation Take 100 mg of above protein sample and supplement the volume with lysate to 100 µl. The final concentration of 10 mM TCEP was added and the reaction time was 60 min at 37 ℃. Add the iodoacetamide (40 mM) at room temperature for 40 minutes. Pre-cooled acetone (6:1 w/w) was added to each tube, and precipitated at -20℃ for 4 hours. The Protein samples were centrifuged at 10000g for 20 mins and then the precipitations were collected. The precipitations were dissolved with 100 µl TEAB (100 mM). Finally, the trypsin solution (1:50 w/w) was added for enzymatic hydrolysis at 37 ℃ overnight. The above samples were labeled using an TMT reagent 10-plex protein quantitation kit (Thermo Fisher, USA) as follows: The sham-operate control were labeled with TMT10-130N, TMT10-130C and TMT10-131, and the VD samples were labeled with TMT10-128C, TMT10-129N and TMT10-129C, respectively; the VD rats treated with NGF samples were labeled with TMT10-126, TMT10-127N and TMT10-128N, respectively.