Inhibition of autophagy-lysosomal function exacerbates microglial and monocyte lipid metabolism reprograming and dysfunction after brain injury.
Description
Targeted lipidomics for mouse brain lysosomes with sham and TBI treatment. Dataset also includes lipidomic anaylsis of microglia and monocytes.
Files
Steps to reproduce
Total lipid extracts from the lysosomes were prepared using methyl tert butyl ether (MTBE) lipid extraction protocol with slight modifications as described previously (Mehrabani-Tabari, 2025, 40345661). Briefly, 400 µL of cold methanol and 10 µL of internal standard (EquiSPLASH) were added to each sample. The sample was incubated at 4°C, 650 rpm shaking for 15 min. Next, 500 µL of cold MTBE was added followed by incubation at 4°C for 1 h with 650 rpm shaking. Cold water (500 µL) was added slowly, and the resulting extract was maintained 4°C, 650 rpm shaking for 15 min. Phase separation was completed by centrifugation at 8,000 g for 8 min at 4 °C. The upper, organic phase was removed and set aside on ice. The bottom, aqueous phase was re-extracted with 200 µL of MTBE followed by 15 min incubation at 4 °C with 650 rpm shaking. Phase separation was completed by centrifugation at 8,000 g for 8 min at 4 °C. The upper, organic phase was removed and combined with a previous organic extract. The latter was dried under a steady stream of nitrogen at 30 °C. The recovered lipids were reconstituted in 100 µL of acetonitrile:isopropanol:water (1:2:1, v/v/v). Total lipid extracts were analyzed by liquid chromatography coupled to targeted tandem mass spectrometry (LC-MS/MS). The LC-MS/MS analyses were performed on an Ultimate 3000 Ultra High-Performance Liquid Chromatograph coupled to a Thermo TSQ Altis Tandem Quadrupole Mass Spectrometer (Thermo Scientific, San Jose, CA). LC-MS/MS methodology was adapted from the literature (Medina, 2023, 36716250) and previous publication (Mehrabai-Tabari, 2025, Gouda, 2024, 39128713). The separation was achieved using an ACQUITY Amide BEH column (1.7 µm; 2.1 x 100 mm) column (Waters, Milford, MA) maintained at 45 °C. Mobile phase compositions for solvents A and B consisted of ACN/H2O (95:5, v/v) and (50:50, v/v) respectively, with 10 mM ammonium acetate The gradient profile had a flow rate of 0.6 mL min−1 and ramped from 0.1 to 20% B in 2 min, from 20 to 80% B in 3 min, dropped from 80 to 0.1% B in 0.1 min, and held 0.1% B for 2.9 min. Total chromatographic run time was 8.0 min. The injection volume was 2 μL. The auto-sampler was maintained at 7 °C. Electrospray ionization was achieved using either negative or positive mode. Mass spectrometry detection was done using selective reaction monitoring where predetermined precursor to product ion transitions were used. ESI source parameters were set as follows: voltage 3500 V in positive mode and −2500 V in negative mode, sheath gas (Arb) = 60, aux gas (Arb) = 15, sweep gas (Arb) = 1 and ion transfer tube temperature of 380 °C. Nitrogen was used as the nebulizer and argon as collision gas (1.5 mTor). The vaporizer temperature was set to 350 °C. LC-MS/MS data was acquired using Thermo’s Xcalibur software and data processing was achieved using Xcalibur 4.2 and TraceFinder 5.1. Additional data analysis was done using Prism 6 (GraphPad, La Jolla, CA) and MetaboAnalyst.
Institutions
- University of Maryland BaltimoreMD, Baltimore