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Mitochondrial repair effects of oxygen treatment on Alzheimer’s disease model mice revealed by quantitative proteomics Methodology 1. Animals Triple-transgenic AD mice [B6; 129-Psen1tm1Mpm Tg (APPSwe, tauP301L) 1Lfa/Mmjax] purchased from The Jackson Laboratory were divided into an oxygen-treated (OT) group (N = 12) and a control (Ctrl) group (N = 9). The oxygen treatment methods are described in detail elsewhere. 2. Mitochondrion isolation The hippocampus was homogenized with isolation buffer (225 mM sucrose, 75 mM mannitol, 1 mM EGTA, and 5 mM HEPES; pH 7.4) by Potter homogenization. Large cellular debris and nuclei were pelleted by centrifugation for 3 min at 1300 ×<i>g</i>. The resulting supernatant was centrifuged for 10 min at 21,000 ×<i>g</i> and the pellet was re-suspended in 1.5 mL of a 15% (v/v) Percoll (Sigma) solution. A stock Percoll solution was prepared as a mixture of Percoll and isolation buffer. A 1-mL aliquot of 40% Percoll solution was placed into a 4.0-mL polycarbonate tube and a neat layer of 1.5 mL of 24% Percoll was placed over the 40% Percoll solution aliquot. The re-suspended pellet was placed gently onto the Percoll gradient column. Separation was performed by centrifugation at 30,700 ×<i>g</i> for 50 min. After centrifugation, the upper band comprised of myelin and synaptosomes was removed carefully, and then the lower band containing mitochondria was collected. 3. Protein sample preparation and iTRAQ labeling To isolate mitochondria, we employed a method developed originally for use with lens tissues that was modified slightly for preparation of hippocampal mitochondria. Briefly, the mitochondria were lysed in ice-cold lysis buffer (KeyGEN, China) with an ultrasonic crusher (Branson Ultrasonics, USA). Subsequent protein digestion was performed by filter-aided sample preparation. After digestion, the tryptic peptides were labeled with iTRAQ reagents (AB SCIEX, USA) in accordance with the manufacturer’s protocol. Samples from the OT group were labeled with reagents 113 and 114; Ctrl group samples were labeled with reagents 115 and 116. 4. LC-MS/MS analysis LC-MS/MS analysis was performed with a nanoLC trap column (ChromXP C18) and a TripleTOF 5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON). Samples were loaded into a ChromXP C18 (3 μm, 120 Å) nanoLC trap column. Online chromatography separation was performed in a EksigentnanoLC-Ultra™ 2D System (AB SCIEX, Concord, ON). The scan scope (m/z) for TOF-MS is 350–1500 and that for MS/MS is 400–1250. The MS/MS data were analyzed for protein identification with ProteinPilot Software v.4.5 (AB Sciex Inc., USA). Gene Ontology (GO) categorization and protein-protein interaction were performed with the PANTHER and STRING databases, respectively.