DOI: 10.6084/m9.figshare.28765562
title: Adult <i>Drosophila</i> Aqueous Metabolomic datasets for normal and high-sugar diets

description: <b>Metabolomics sample preparation:</b>Aqueous metabolites for targeted LC-MS profiling of whole flies and hemolymph samples were extracted using a protein precipitation methods described by the Seattle Northwest Metabolomics Research Center (NW-MRC) [1,2,3]- Refs below.<i>Whole fly Samples:</i> Whole adult male flies were frozen in liquid nitrogen after 7 or 14 days on a normal diet or HSD. Ten flies were used per biological sample, and 3 biological replicates were used for each diet and time point. Samples were first homogenized in 200 µL purified deionized water at 4 ˚C. Then 800 µL of cold methanol containing 124 µM 6C13-glucose and 25.9 µM 2C13-glutamate was added (reference internal standards were added to the samples to monitor sample prep). Afterward, samples were vortexed, stored for 30 minutes at -20 ˚C, sonicated in an ice bath for 10 minutes, centrifuged for 15 min at 14,000 rpm and 4 ˚C, and then 600 µL of supernatant was collected from each sample. Lastly, recovered supernatants were dried on a SpeedVac and reconstituted in 0.5 mL of LC-matching solvent containing 17.8 µM 2C13-tyrosine and 39.2 3C13-lactate (reference internal standards were added to the reconstituting solvent to monitor LC-MS performance). Samples were transferred into LC vials and placed into a temperature-controlled autosampler for LC-MS analysis.<b>Metabolomics:</b>Targeted LC-MS metabolite analysis was performed on a duplex-LC-MS system composed of two Shimadzu UPLC pumps, CTC Analytics PAL HTC-xt temperature-controlled auto-sampler, and AB Sciex 6500+ Triple Quadrupole MS equipped with ESI ionization source. UPLC pumps were connected to the auto-sampler in parallel and could perform two chromatography separations independently from each other. Each sample was injected twice on two identical analytical columns (Waters XBridge BEH Amide XP), where separations were performed in hydrophilic interaction liquid chromatography (HILIC) mode. While one column was performing separation and MS data acquisition in ESI+ ionization mode, the other column was getting equilibrated for sample injection, chromatography separation, and MS data acquisition in ESI- mode. Each chromatography separation was 18 minutes (total analysis time per sample was 36 minutes). MS data acquisition was performed in multiple-reaction-monitoring (MRM) mode. LC-MS system was controlled using AB Sciex Analyst 1.6.3 software. Measured MS peaks were integrated using AB Sciex MultiQuant 3.0.3 software. Up to 158 metabolites (plus 4 spiked standards) were measured across the fly samples study set, and up to 148 metabolites (plus 34 SILISs) were measured in the hemolymph sample set. For the hemolymph set, absolute concentrations of 30 metabolites were determined. In addition to the two study samples, two sets of quality control (QC) samples were used to monitor the assay performance and data reproducibility. One QC [QC(I)] was a pooled human serum sample used to monitor system performance, and the other QC [QC(S)] was a pooled study sample. This QC was used to monitor data reproducibility. Each QC sample was injected per every 10 study samples. The median CV for the fly set was 2.8%, while for the hemolymph was 11.1%. References:<br><i> </i><i>Kurup, K., Matyi, S., Giles, C.B., Wren, J.D., Jones, K., Ericsson, A., Raftery, D., Wang, L., Promislow, D., Richardson, A., and Unnikrishnan, A. (2021). Calorie restriction prevents age-related changes in the intestinal microbiota. Aging (Albany NY) </i><i>13</i><i>, 6298-6329. 10.18632/aging.202753.</i><i> </i><i>Meador, J.P., Bettcher, L.F., Ellenberger, M.C., and Senn, T.D. (2020). Metabolomic profiling for juvenile Chinook salmon exposed to contaminants of emerging concern. Sci Total Environ </i><i>747</i><i>, 141097. 10.1016/j.scitotenv.2020.141097.</i><i> </i><i>Hanson, A.J., Banks, W.A., Bettcher, L.F., Pepin, R., Raftery, D., and Craft, S. (2019). Cerebrospinal fluid lipidomics: effects of an intravenous triglyceride infusion and apoE status. Metabolomics </i><i>16</i><i>, 6. 10.1007/s11306-019-1627-x.</i>