MetaboLights

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identification was conducted by matching acquired spectra against the LipidBlast spectral library. Spectral alignment and diagnostic fragments were confirmed through visual inspection of mirror plots (library vs. experimental data). Identification was validated according to Lipid Standards Initiative (LSI) guidelines. Lipids meeting the strict criteria for expected MS2 fragmentation patterns, mass accuracy threshold, and retention time (RT) alignment were retained in the final compound table.MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseLiquid chromatography was performed on a Thermo Vanquish Horizon LC system using a Waters Premier BEH C18 column (50 mm × 2.1 mm, 1.7 µm) maintained at 60°C. Injection volume was 2 µL for positive mode and 5 µL for negative mode, with a flow rate of 1.0 mL/min. A 7.5-minute segmented gradient was applied as follows: 0.0–1.0 min, 15–30% B; 1.0–1.25 min, 30–48% B; 1.25–5.0 min, 48–82% B; 5.5–5.75 min, 82–99% B; 5.75–6.0 min, 99% B (isocratic); 6.0–6.05 min, 99–15% B; 6.05–7.5 min, 15% B (isocratic re-equilibration).Autophagy regulator ATG7 links lipid metabolism to proximal tubule cell-fate decisions in health and disease.Mikhail KorzinkinInsilico Medicinekidneymass spectrometry assay Lipid extraction was performed following a previously described method54, with some modifications. A mixture of 1000 µL isopropanol: methanol (1:1) was added to each cell culture well. The plates were then incubated at -80 °C for 10 minutes. After incubation, the cells were scraped, and the resulting solution was transferred to Eppendorf tubes. The samples were continuously mixed in a thermomixer (Eppendorf) at 25 °C (950 rpm) for 30 minutes. Protein precipitation was achieved by centrifugation at 16,000 g for 10 minutes at 25 °C. The resulting single-phase supernatant was collected, dried under nitrogen (N2), and stored at -20°C until further analysis. Before analysis, the dried lipids were reconstituted in 100 µL methanol: isopropanol (1:1)Mus musculushttps://www.ebi.ac.uk/metabolights/MTBLS15861Alessandro Luciani. Institute of Physiology, University of Zurich. University of Zurich, 8057 Zurich, Switzerland. alessandro.luciani@physiol.uzh.ch.Mikhail Korzinkin. Insilico Medicine. mike@insilicomedicine.com.Martina Zanella. martina.zanella@fgcz.ethz.ch.Raw LC-MS dataset files (.raw) were processed using Thermo Compound Discoverer software (version 3.3). Peak areas were normalized across samples using median normalization. Processed peak areas were log-transformed, and statistical comparisons between experimental groups were performed using Student's t-test. Data visualization, including volcano plots and heatmaps, was performed in R using the ggplot2 and ComplexHeatmap packages.ATG7-KOmike@insilicomedicine.comLipids were extracted from cell culture wells by adding 1000 µL of pre-chilled isopropanol:methanol (1:1, v/v) to each well. Plates were incubated at -80°C for 10 minutes, after which cells were scraped and transferred to Eppendorf tubes. Samples were continuously mixed in a thermomixer at 25°C (950 rpm) for 30 minutes. Protein precipitation was achieved by centrifugation at 16,000 × g for 10 minutes at 25°C. The single-phase supernatant was collected, dried under a stream of nitrogen ($N_2$), and stored at -20°C. Prior to LC-MS analysis, dried lipid extracts were reconstituted in 100 µL of methanol:isopropanol (1:1, v/v).Metabolomicsultra-performance liquid chromatography-mass spectrometryLipidBlastMus musculusuntargeted analysisThermo Scientific Exploris 480LipidomicsCompound DiscovererThermo Vanquish Horizonkidneyuntargeted lipidomicsBiological Sample - MetabolomicsCell Cultureautophagiaultra-performance liquid chromatography-mass spectrometryLipidBlastMus musculusuntargeted analysisThermo Scientific Exploris 480LipidomicsCompound DiscovererThermo Vanquish Horizonkidneyuntargeted lipidomicsBiological Sample - MetabolomicsCell CultureautophagiaMass spectrometry was performed on a Thermo Scientific Exploris 480 mass spectrometer equipped with a Heated Electrospray Ionization (HESI) source operating in both positive and negative polarities in separate analytical runs. Source settings: sheath gas = 30, auxiliary gas = 13, spray voltage = 3800 V (positive mode) and -3500 V (negative mode), vaporizer temperature = 350°C, ion transfer tube temperature = 300°C, RF lens = 50%, with mild trapping enabled. Internal mass calibration was performed at the start of each run. Data were acquired in Data-Dependent Acquisition (DDA) mode (Top 5). MS1 parameters: resolution = 60,000, scan range = 150–2000 m/z, AGC target = 1e6, max injection time = 10 ms. MS2 parameters: isolation window = 1 m/z, stepped normalized collision energy (NCE) = 10, 30, and 340%, resolution = 15,000, AGC target = 1e5, max injection time = 10 ms.falseHigh-resolution LC-MS/MS lipidomics characterization of wild-type and ATG7-deficient mouse renal proximal tubule cellsThis 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.2026-09-302026-09-29MTBLS15861