High-resolution LC-MS/MS lipidomics characterization of wild-type and ATG7-deficient mouse renal proximal tubule cells

ABSTRACT: This study provides an untargeted lipidomics investigation of ATG7-deficient (cKO) mouse renal proximal tubule cells compared to wild-type (WT) controls to evaluate lipidomic alterations associated with autophagy deficiency. Lipids were extracted from cell culture wells using a single-phase isopropanol:methanol (1:1) extraction protocol at -80 °C, followed by protein precipitation and reconstitution in methanol:isopropanol (1:1). High-resolution LC-MS/MS data acquisition was performed on a Thermo Vanquish Horizon tandem LC system coupled to a Thermo Scientific Exploris 480 mass spectrometer equipped with a Heating Electrospray Ionization (HESI) source. Chromatographic separation was achieved via reverse-phase liquid chromatography using a Waters Premier BEH C18 column (50 mm × 2.1 mm, 1.7 µm) operating at 60 °C with a 7.5-minute segmented gradient. Data were acquired in Data-Dependent Acquisition (DDA) mode (Top 5) in both positive and negative ESI polarities in separate analytical runs across an m/z range of 150–2000. Raw mass spectrometry data files (.raw) were processed using Thermo Compound Discoverer (version 3.3). Structural lipid identification was performed by matching experimental spectra against the LipidBlast spectral library in compliance with Lipid Standards Initiative (LSI) guidelines, using strict accurate mass, retention time, and fragmentation criteria. Peak areas were median-normalized, log-transformed, and statistically evaluated to identify significantly altered lipid species between WT and ATG7-deficient cell groups.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse-phase, Liquid Chromatography MS - positive - reverse-phase

PROVIDER: MTBLS15861 | MetaboLights | 2026-09-30

REPOSITORIES: MetaboLights