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species were identified by targeted multiple reaction monitoring (MRM), in which each lipid was detected using a predefined precursor-to-product ion transition characteristic of its lipid class, in positive and/or negative ionisation mode. Class-specific stable-isotope-labelled internal standards were used to confirm identity and for normalisation. Peak detection and integration were performed in SCIEX OS software (Version 1.4, AB Sciex, Framingham, MA, USA).MetaboLightsPublicLiquid Chromatography MS - alternating - hilicLiquid Chromatography MS - alternating - reverse-phaseChromatographic separation was performed on an Agilent 1290 Infinity II liquid chromatography system coupled to the mass spectrometer. Three separations were used. (1) MTBE extract on a Waters BEH HILIC column (2.1 x 100 mm, 1.7 um) at 45 C, with mobile phase A: water/acetonitrile (5:95, v/v) with 10 mM ammonium acetate, and mobile phase B: water/acetonitrile (1:1, v/v) with 10 mM ammonium acetate and 0.01% ammonia solution (28%). (2) MTBE extract on a Phenomenex Kinetex C18 column (2.1 x 100 mm, 2.6 um), with mobile phase A: water/acetonitrile/methanol (1:1:1, v/v/v) with 7 mM ammonium acetate, and mobile phase B: isopropanol with 7 mM ammonium acetate. (3) IPA extract on an Agilent Zorbax Eclipse Plus C18 column (2.1 x 100 mm, 1.8 um) at 50 C, with mobile phase A: water/acetonitrile/methanol (1:1:1, v/v/v) with 0.1% formic acid and 1 mM ammonium acetate, and mobile phase B: isopropanol/acetonitrile (90:10, v/v) with 0.05% formic acid and 10 mM ammonium acetate. All samples were analysed in randomised order.Cholesterol maintains the degradative capacity of lysosomes during clearance and recycling of dysfunctional mitochondria.Haoning YangNanyang Technological University, Lee Kong Chian School of Medicinekeratinocytemass spectrometry assayTwo parallel lipid extractions were performed. For the MTBE extraction (HILIC and Kinetex C18 streams): 225 uL of -20 C pre-cooled methanol containing mixed internal standards was added to the cell samples and vortexed for 10 s; 750 uL of -20 C pre-cooled methyl tert-butyl ether (MTBE) was then added and the mixture centrifuged at 20,000 x g for 10 min at 4 C. The upper organic layer was collected, dried, and reconstituted in 80 uL dichloromethane/methanol (1:1) for LC-MS analysis. For the IPA extraction (EC C18 stream): 500 uL of -20 C pre-cooled isopropanol (IPA) containing mixed internal standards was added to the cell samples and vortexed for 10 s; 88 uL water was added and the mixture centrifuged at 20,000 x g for 10 min at 4 C. The upper layer was collected, dried, and reconstituted in 36 uL dichloromethane/methanol (1:1) with 0.1% formic acid for LC-MS analysis. Quality control (QC) samples were prepared by pooling aliquots from all samples, and six QC injections were analysed within each batch to monitor method reliability and instrument stability.Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15181Yasunori Saheki. Lee Kong Chian School of Medicine, Nanyang Technological University; Institute of Resource Development and Analysis, Kumamoto University. yasunori.saheki@ntu.edu.sg.Haoning Yang. Lee Kong Chian School of Medicine, Nanyang Technological University. yanghaoning0815@gmail.com.Chromatographic peaks of the detected lipid species were integrated using SCIEX OS software (Version 1.4, AB Sciex, Framingham, MA, USA). The raw peak areas of each lipid species were normalised to the corresponding internal standard for each lipid class to correct for instrument drift.Treatmentyanghaoning0815@gmail.comN/TERT-1 wild-type immortalised human keratinocytes were cultured and treated with either ethanol (vehicle control) or a combination of oligomycin and antimycin A (O/A) to induce mitochondrial dysfunction. For each treatment group, three independent biological replicates were prepared. Cells were harvested for lipid extraction. Quality control (QC) samples were prepared from a separate pooled batch of wild-type N/TERT-1 cells and analysed alongside the study samples within each batch to monitor method reliability and instrument stability.Metabolomicstargeted analysiskeratinocyteliquid chromatography-mass spectrometryAgilent 1290 Infinity II UHPLCHomo sapiensLipidomicsAB SCIEX QTRAP 6500targeted metabolite profilingexperimental sampletargeted analysiskeratinocyteliquid chromatography-mass spectrometryAgilent 1290 Infinity II UHPLCHomo sapiensLipidomicsAB SCIEX QTRAP 6500targeted metabolite profilingexperimental sampleSamples were analysed on an AB Sciex QTRAP 6500 quadrupole-linear ion trap mass spectrometer (SCIEX, Framingham, MA, USA) equipped with an electrospray ionisation (ESI) source, operated in multiple reaction monitoring (MRM) mode in both positive and negative ionisation modes. Instrument parameters were: curtain gas (CUR) 40 psi; collision gas (CAD) medium; ionspray voltage +/-4500 V; source temperature 350 C; nebuliser gas (GS1) and drying gas (GS2) both 55 psi; entrance potential (EP) +/-10 V; and collision cell exit potential (CXP) 10 V. For each treatment group (ethanol control and O/A), data were obtained from three independent biological replicates.falseCholesterol maintains the degradative capacity of lysosomes during clearance and recycling of dysfunctional mitochondriaEfficient clearance and recycling of dysfunctional mitochondria through the robust catabolic activity of lysosomes is essential for cellular health. However, how membrane lipids contribute to maintaining the degradative capacity of lysosomes remains poorly understood. Here, we show that cholesterol plays a critical role in preserving the functional integrity of degradative lysosomes. Clearance of damaged mitochondria by degradative lysosomes is tightly coupled with the acute accumulation of phosphatidylinositol 4-phosphate (PI4P) on the lysosomal surface via PI4KIIα activity. This PI4P accumulation activates oxysterol-binding protein (OSBP)-mediated cholesterol transport from the endoplasmic reticulum (ER) to lysosomal membranes. The resulting efflux of cholesterol from the ER activates sterol regulatory element-binding protein 2 (SREBP-2), enhancing cholesterol production. Sustained cholesterol accumulation on lysosomal membranes maintains lysosomal acidity and membrane integrity for efficient mitochondrial degradation. This degradation process then leads to the release of free fatty acids and their recycling and storage through the formation of DGAT1-dependent lipid droplets. These findings uncover a key phosphoinositide-regulated cholesterol transport pathway that promotes the clearance and recycling of dysfunctional mitochondria, a process whose impairment is closely linked to neurodegeneration.2026-07-302026-07-29MTBLS15181