MetaboLights

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spectra files (.raw) were imported into Lipostar (Molecular Discovery). Peaks were selected using a signal threshold of 200 in MS and 100 in MS/MS. Peaks obtained in initial blank samples were subtracted from the experimental samples. M/z values of Leu-Enk were used for correction of peak m/z values, using reference values of 556.2771 in positive ion mode and 554.2615 in negative ion mode. M/z values of peaks were compared against the Lipidmaps Structural Database, for hits within 10ppm of the database value. Only identifications with at least 1 fragment within MS/MS, whose m/z matched that of the hypothetical fragmentations within 20ppm, were approved. Kendrick Mass Defect plots were then used to map the retention time for each class of lipid. Integrated peak areas for approved lipids were then exported for analysis. Peak intensities for each lipid were uploaded to Metaboanalyst for Partial Least Squares Discriminant Analysis (PLS-DA) of entire fly lipidomes after pareto scaling. Volcano plots of TAGs were generated in Prism using a minimum p-value of 0.05 and Log2FC > 0.5.MetaboLightsPublicLiquid Chromatography MS - negative - reverse-phaseLiquid Chromatography MS - positive - reverse-phaseSamples were analyzed using an ACQUITY UPLC I-Class PLUS System (Waters) using reverse phase liquid chromatography tandem mass spectrometry. Samples were injected using partial loop onto an ACQUITY Premier CSH C18 Column, 1.7 µm particle size, 2.1 x 100mm (55°C), and were eluted during an 18-minute gradient using a mixture of solvent A (60:39:1 acetonitrile:water:1M ammonium formate with 0.1% formic acid) and B (90:9:1 isopropanol:acetonitrile:1M ammonium formate with 0.1% formic acid); gradient programme consisted of Initial conditions 85 A:15 B, then 70 A:30 B at 2 minutes, 52 A: 48 B at 2.5 minutes, 18 A: 82 B at 11 minutes, 1 A: 99 B at 11.5 minutes until 15 minutes, where the gradient returned to 85 A: 15 B until 18 minutes to allow for column re-equilibration before the next sample was injected. Ten µL/min Leucine-Enkephalin (200 ng/ml) was infused once every minute as the lockspray. All solvents were Optima grade. Three different 'Assays' were used with different sample dilutions and loading volumes to allow less abundant, more polar lipids to be analysed. Assay 1: Triglyceride analysis in positive ion mode. The reconstituted samples were diluted 100-fold in Optima grade isopropanol (IPA) and 5 µL were loaded. MS data were collected for the full duration of the 18 min chromatographic run. Assay 2: Phospholipid analysis in positive ion mode. The reconstituted samples were used undiluted (i.e. 100 times more concentrated than in Assay 1) and 5 µL were loaded. After 9.5 mins of the chromatographic run, the flow was diverted to waste to prevent overloading the system with concentrated TGs and causing cross-contamination between runs. Assay 3: Phospholipid analysis in negative ion mode. The reconstituted samples were used undiluted (i.e. 100 times more concentrated than in Assay 1) and 10 µL were loaded. After 9.5 mins of the chromatographic run, the flow was diverted to waste to prevent overloading the system with concentrated TGs and causing cross-contamination between runs. Lipidomic analysis of Drosophila on different sugar feeding regimes +/- trametinib.Corinne Michelle SpickettAston UniversityWhole Organismmass spectrometry assay5 whole adult female flies were homogenised in 300 μL of methanol and 1 mL of Methyl tert-Butyl Ether (MTBE) using glass beads and a FastPrep-24 5G (MP Biomedicals). Samples were incubated for 1 hour at RT with continuous shaking before the addition of 200 μL of H2O to give a final volumetric ratio of approximately 10:3:2.5 MTBE:methanol:water. Samples were vortexed for 20 s and centrifuged for 10 min at 1000 g at 4°C. 1 mL of the upper lipid-containing phase was transferred to a glass collection vial. Lipid extracts were lypholised under nitrogen gas and stored at -80°C.Immediately before analysis, samples were reconstituted in 200µL Optima grade IPA. Dilutions were carried out as described in Chromatography.Drosophila melanogasterhttps://www.ebi.ac.uk/metabolights/MTBLS14468Corinne Michelle Spickett. Aston University. School of Biosciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK. c.m.spickett@aston.ac.uk.Not applicable - See next section as .raw files used. Treatmentc.m.spickett@aston.ac.ukAll experimental flies were reared from synchronised egg collections on standard sugar-yeast-agar (SYA) media consisting of 5% (w/v) granulated sugar (Tate & Lyle), 10% (w/v) brewer’s yeast (MP Biomedicals), 1.5% (w/v) agar (Merck) and preservatives 3% (v/v) nipagin (diluted from a 10% (w/v) stock solution of tegosept (Apex Bioresearch Products) prepared in 95% ethanol) and 0.3% (v/v) propionic acid (Thermo Fisher Scientific). Newly eclosed adult flies were transferred to fresh SYA food and allowed to mate for 24 hours before sorting by sex under carbon dioxide anaesthesia. Flies were transferred to experimental food vials at a density of 15 flies per vial containing SYA or high sugar SYA in which the sugar content of the media was increased to 40% (w/v). For drug treatments, trametinib (Insight Biotechnology) was added to the media to a final concentration of 15.6 µM (from a 62.4 mM stock solution dissolved in DMSO) maintaining a final DMSO concentration of 0.25% (v/v). For drug-free conditions, equivalent volumes of vehicle only (DMSO) were added to the media). Flies were maintained at 25°C in constant 60% humidity on a 22 h light:12 h dark cycle and were transferred to fresh vials every 2-3 days. Flies were snap frozen on liquid nitrogen at 10-days of age and stored at -80°C.MetabolomicsObesitySELECT SERIES Cyclic IMSWaters ACQUITY UPLC I-Class PLUS Systemmortality/aginguntargeted analysisLipidomicsdata-dependent acquisitionDrosophila melanogasterWhole Organismexperimental sampleObesitySELECT SERIES Cyclic IMSWaters ACQUITY UPLC I-Class PLUS Systemmortality/aginguntargeted analysisLipidomicsdata-dependent acquisitionDrosophila melanogasterWhole Organismexperimental sampleA SELECT SERIES Cyclic ion mobility separation mass spectrometry system (Waters) was used for the analysis of samples. In positive ion mode, capillary, cone and source offset voltages were set to 2V, 40V and 20V, respectively. Source temperature was 120 °C and desolvation temperature was 280 °C. Cone gas was set to 40 L/h and desolvation gas set to 600 L/h. Nebuliser gas was set to 6 bar, and reference capillary was set to 3 kV. In negative ion mode, capillary, cone and source offset voltages were set to 4V, 27V and 5V respectively. Source temperature was 120°C and desolvation temperature was 300°C. Cone gas, desolvation gas, nebuliser gas, and reference capillary settings were the same as for positive ion mode. The TOF was operated in V mode, with acquisition range 50-2000 m/z. Scan times of MS and MS/MS were 1 second and 0.1s, respectively. MS/MS was acquired using data-dependent acquisition, fragmenting the 5 most intense peaks with an intensity count of >1,000. A dynamic exclusion list was applied, preventing re-fragmentation of ions of the same m/z within 10 seconds. Fragmentation was performed using collision induced dissociation through a ramp of 25V to 45V, after quadrupole isolation using a low mass resolution of 4.7 and a high mass resolution of 15.0.falseLipidomic analysis of Drosophila on different sugar feeding regimes +/- trametinibConsumption of sugar-rich diets adversely affects health and reduces life expectancy through the excessive production and storage of triacylglycerides (TAGs) leading to metabolic dysfunction. Here, using Drosophila melanogaster, we identify the conserved Ras/MAPK signalling pathway as a key mediator of this response. Pharmacological inhibition of Ras/MAPK signalling with the MEK inhibitor, trametinib, was tested for its effect on phospholipid and triacylglycerol content of whole flies extracted by the MTBE method. Reverse phase LC-MSMS analysis was carried out on a Waters SELECT Cyclic IMS instrument operating in DDA mode.2026-09-022026-05-13MTBLS14468