MetaboLights

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identification was performed using a targeted lipidomics approach. Individual lipid species were assigned based on predefined compound-specific precursor-to-product ion transitions (MRM transitions) together with their expected chromatographic retention times. Chromatographic peak assignment and integration were performed using Agilent MassHunter Quantitative Analysis software version 10.1 (Agilent Technologies).No untargeted feature annotation or database-based metabolite identification was performed.MetaboLightsPublicLiquid Chromatography MS - positive - reverse-phaseChromatographic separation was performed using an Agilent 1290 series HPLC system (Agilent Technologies) equipped with a ZORBAX Eclipse Plus C18 column (2.1 × 100 mm, 1.8 µm; Agilent Technologies). The column temperature was maintained at 60°C, and the injection volume was 1 µL.The mobile phases consisted of 50% water, 30% acetonitrile and 20% isopropanol (v/v/v) containing 10 mM ammonium formate (mobile phase A), and 1% water, 9% acetonitrile and 90% isopropanol (v/v/v) containing 10 mM ammonium formate (mobile phase B).The gradient started at 10% B with a flow rate of 0.4 mL/min and increased to 45% B over 2.7 min, 53% B over 0.1 min, 65% B over 6.2 min, 89% B over 0.1 min, 92% B over 1.9 min, and 100% B over 0.1 min. Mobile phase B was then maintained at 100% for 2.3 min. For re-equilibration, B was decreased from 100% to 10% over 0.1 min and maintained for 0.9 min. The flow rate was then increased to 0.6 mL/min for 1 min and subsequently returned to 0.4 mL/min over 0.1 min. The system was maintained at 10% B for a further 0.9 min at 0.4 mL/min. The total chromatographic cycle time was 16.5 min.Targeted lipidomics in Alexander's disease cellular models.Mariona JoveUniversity of Lleidablankblood plasmaU-87MG cellmass spectrometry assayCell pellets were resuspended in 10 µL of water containing BHT and sonicated for 20 min in a bath sonicator at 4°C. A 10 µL aliquot of the resulting homogenate was mixed with 100 µL of butanol:methanol (1:1, v/v) containing 10 mM ammonium formate and a mixture of internal standards (ISTDs). Samples were thoroughly vortexed and sonicated for 1 h at room temperature. Following extraction, samples were centrifuged at 14,000 × g for 10 min at 20°C, and the supernatants were transferred to sample vials fitted with glass inserts for LC-MS analysis.Pooled quality control (QC) samples were prepared and included throughout the analytical sequence to monitor analytical performance. A processing blank was also included to assess background contributions. Internal standards were added to the extraction solvent and were present in all samples for normalization and quantification.blankHomo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS15503Mariona Jove. University of Lleida. mariona.jove@udl.cat.Raw LC-MS data were processed using Agilent MassHunter Quantitative Analysis software version 10.1 (Agilent Technologies). Chromatographic peaks were assigned to individual lipid species based on predefined precursor-to-product ion transitions and retention times, and peak areas were integrated using MassHunter.During data preprocessing, lipid species with peak areas below 500 were excluded from further analysis. In addition, lipid signals detected in the processing blank at levels greater than 20% of the corresponding signal in the pooled quality control (QC) samples were removed to minimize background contributions.Analytical stability was assessed using the relative standard deviation (RSD) of the internal standards (ISTDs) across all samples, which was below 20%.Quantification was based on the ratio between the peak area of each lipid species and that of its corresponding internal standard, and data were expressed as nmol/mL. The complete dataset of lipid intensities was subsequently processed using the SERRF online platform for signal normalization. For triglycerides and cholesterol esters, SERRF-normalized signals were divided by the median signal of the corresponding internal standard to calculate concentrations.Mutationmariona.jove@udl.catBiological replicates of U-87 MG astrocytoma cells, including non-transfected cells and cells stably expressing GFP-tagged wild-type (wt) GFAP or the GFAP variants R239C, R239G, R79C, and E373K, were grown in 100 mm culture dishes under the conditions described above. Twelve biological replicates were collected for each experimental condition. An additional independent validation experiment was performed for GFP-GFAP wt and GFP-GFAP R239C cells, with six biological replicates per condition.For sample collection, all procedures were performed independently for each biological replicate and on ice. Cells were washed twice with cold PBS and gently scraped from each dish in 2 mL PBS. Cell suspensions were centrifuged at 2,380 × g for 5 min at 4°C, and the supernatants were carefully discarded. The resulting cell pellets were immediately snap-frozen on dry ice and stored at −80°C until lipid extraction and analysis.Metabolomicsblanktargeted analysisstudy reference materialsolvent blanklong term referenceHomo sapiensLipidomicstargeted metabolite profilingAgilent 1290 Infinity HPLCsample preparation blankexperimental sampleGlial Fibrillary Acidic Proteinblood plasmaAlexander diseaseU-87MG cellAgilent 6495 Triple Quadrupoleblanktargeted analysisstudy reference materialsolvent blanklong term referenceHomo sapiensLipidomicstargeted metabolite profilingAgilent 1290 Infinity HPLCsample preparation blankexperimental sampleGlial Fibrillary Acidic Proteinblood plasmaAlexander diseaseU-87MG cellAgilent 6495 Triple QuadrupoleMass spectrometric analysis was performed using an Agilent 6495 triple quadrupole LC-MS system (Agilent Technologies) equipped with an Agilent Jet Stream electrospray ionization (AJS-ESI) source. Data were acquired in both positive and negative ionization modes using dynamic scheduled multiple reaction monitoring (MRM), with compound-specific precursor-to-product ion transitions.Source parameters were as follows for positive and negative ionization modes, respectively: gas temperature, 150°C for both modes; drying gas flow, 17 L/min for both modes; nebulizer pressure, 20 psi for both modes; sheath gas temperature, 200°C for both modes; and sheath gas flow, 10 L/min for both modes. The capillary voltage was set to 3500 V in positive mode and 4000 V in negative mode, while the nozzle voltage was set to 1000 V in positive mode and 500 V in negative mode.falseTargeted lipidomics in Alexander's disease cellular modelsHigh-throughput targeted lipidomic data obtained from biological replicates (n=12) of four cellular models of Alexander disease using U-87 MG non-transfected and GFP-GFAP expressing cells (WT, R239C, R239G, R79C, and E373K variants).2026-09-282026-08-30MTBLS15503