Mitochondria-derived extracellular vesicles mediate Alzheimer’s disease pathology through transmitting microglial Ggt1
Description
Metabolomics analysis The mouse brains were dissected, collected in 1.5 ml Eppendorf tubes and weighed 20 mg on ice. 70% methanol containing internal standard was added to the tissue and shaken at 2,500 rpm for 5 min. The samples were incubated for 15 min at 4 °C, followed by a centrifugation step at 12,000 rpm for 10 min. The supernatant was stored at -20 °C for 30 min, followed by a centrifugation step at 12,000 rpm for 3 min. A 200 μL aliquots of supernatant were transferred for LC-MS analysis. All samples were analyzed with LC/MS methods. For HILIC (hydrophilic interaction chromatography), a Waters ACQUITY UPLC BEH HILIC Column (1.7 µm, 1 mm * 100 mm) was used. A gradient from A (20 mM ammonium formate, 10 % water, 10 % methanol, 80 % acetonitrile) to B (20 mM ammonium formate, 60% water, 40% acetonitrile) was used, employing a flow rate of 0.4 mL/min. The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) (ExionLC AD) and Tandem mass spectrometry (MS/MS). After the analysis by HILIC-MS/MS, the data acquisition was operated using the information-dependent acquisition (IDA) mode using Analyst TF 1.7.1 Software (Sciex, Concord, ON, Canada). For two-group analysis, differential metabolites were determined by VIP (VIP > 1) and P value (P value < 0.05, Student’s t test). VIP values were extracted from OPLS-DA result, which also contain score plots and permutation plots, were generated using R package MetaboAnalystR. The data were log transformed (log2) and mean centered before OPLS-DA. To avoid overfitting, a permutation test (200 permutations) was performed. Identified metabolites were annotated using KEGG Compound database (http://www.kegg.jp/kegg/compound/), annotated metabolites were then mapped to KEGG Pathway database (http://www.kegg.jp/kegg/pathway.html).
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Institutions
- Tongji UniversityShanghai, Shanghai