Lysosomal multi-omics reveals altered sphingolipid catabolism as driver of lysosomal dysfunction in the aging brain.
Description
Total lipid extracts of mouse brain lysosomes were prepared from 3month, 12month, 18month, and 24month old mice. The lipid composition of these extracts were analyzed by untargeted lipidomics using liquid chromatography coupled to high-resolution tandem mass spectrometry.
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Total lipid extracts were prepared using a modified MTBE lipid extraction protocol [1]. The extracts were analyzed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-MS/MS) [1-2]. The LC-MS/MS analyses were performed on an Agilent 1290 Infinity LC coupled to an Agilent 6560 Quadrupole Time-of-Flight (Q-TOF) mass spectrometer. The separation was achieved using an C18 CSH (1.7 µm; 2.1 x 100 mm) column (Waters, Milford, MA). Mobile phase A was 10 mM ammonium formate with 0.1% formic acid in water/acetonitrile (40:60, v/v) and mobile phase B was 10 mM ammonium formate with 0.1% formic acid in acetonitrile/isopropanol (10:90, v/v). The gradient was ramped from 40 % to 43 % B in 1 min, ramped to 50 % in 0.1 min, ramped to 54 % B in 4.9 minutes, ramped to 70 % in 0.1 min, and ramped to 99 % B in 2.9 min. The gradient was returned to initial conditions in 0.5 min and held for 1.6 min for column equilibration. The flow rate was 0.4 mL/min. The column was maintained at 55 °C and the auto-sampler was kept at 5 °C. A 2 µL injection was used for all samples. The MS parameters were as follows: extended dynamic range, 2 GHz; gas temperature, 200°C; gas flow, 10 L/min; nebulizer, 50 psi; sheath gas temperature, 300°C; sheath gas flow, 12 L/min; VCap, 3.5kV (+), 3.0kV (–); nozzle voltage, 250V; reference mass m/z 121.0509, m/z 1221.9906 (+), m/z 119.0363, m/z 980.0164 (–); MS and MS/MS Range m/z 100–1700; acquisition rate, 3 spectra/s; isolation, narrow (~ 1.3 m/z); collision energy 20 eV (+), 25 eV (–); max precursors/cycle, 3; precursor abundance-based scan speed, 25,000 counts/spectrum; ms/ms threshold, 5,000 counts and 0.001%; active exclusion enabled yes; purity, stringency 70 %, cut off 0 %; isotope model, common organic molecules; static exclusion ranges, m/z 40 to 151 (+,–). LC-MS/MS data was analyzed via Agilent’s Lipid Annotator (v 1.0). The default settings for feature finding and identification parameters were used. The LC-MS data from the MS1 workflow were processed using Agilent’s MassHunter Profinder (v 10.0). Batch targeted feature extraction using default parameters and the PCDL file created from Lipid Annotator were used for feature extraction. The processed data generated from Profinder which included peak area and lipid identification was exported into MetaboAnalyst 5.0 [4] for multivariate analysis. Univariate analysis was done using Prism 6 (GraphPad, La Jolla, CA). 1. Oleson, B.J., et al., Early life changes in histone landscape protect against age-associated amyloid toxicities through HSF-1-dependent regulation of lipid metabolism. Nat Aging 2024 Vol. 4 Issue 1 Pages 48-61. 2. Abdel-Megied AM, et. al. Characterization of the cellular lipid composition during SARS-CoV-2 infection. Anal Bioanal Chem. 2023 Sep;415(22):5269-5279. doi: 10.1007/s00216-023-04825-1. 3. Pang, Z., et al., MetaboAnalyst 5.0: narrowing the gap between raw spectra and functional insights. Nucleic Acids Res, 2021. 49(W1): p. W388-w396.