DOI: 10.5061/dryad.nn5qc/1
title: LC_MS peak intensities

description: Drosophila Lifespan Extension and Sampling: Progenies used in aging assays and metabolite profiling experiments were prepared by mating wild-type animals of Canton-S background, which were backcrossed for seven generations.  Aging progeny was grown on rich media (flystocks.bio.indiana.edu) at regular density.  Newly eclosing F1 adult males were collected for three days, mixed and distributed within vials containing respective food for sampling at predetermined ages.  The recipes for preparation of the defined diet have been described (Troen et al., 2007).  Between 30-50 flies were sampled at each age, rapidly frozen in liquid nitrogen and stored at -80 °C.  Once all samples were collected they were immediately processed for LC-MS profiling.  Metabolite Profiling: Three LC-MS methods were used to measure polar metabolites and lipids in whole fly homogenates.  Conditions for the analysis were set using a panel of routinely analyzed 293 standards. Polar and lipid-associated species were extracted from 7 and 2 flies, respectively, in three separate replicates which were ran in randomized order. All data were acquired using an LC-MS system comprised of a Nexera X2 U-HPLC (Shimadzu, Marlborough, MA) and a Q Exactive hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific; Waltham, MA).  Hydrophilic interaction liquid chromatography (HILIC) analyses of water soluble metabolites in the positive ionization mode were carried out by extracting 10 µL homogenate using 90 µL of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA).  The samples were centrifuged (10 min, 9,000 x g, 4 °C) and the supernatants were injected directly onto a 150 x 2 mm Atlantis HILIC column (Waters; Milford, MA).  The column was eluted isocratically at a flow rate of 250 µL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 min followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 min.  The electrospray ionization voltage was 3.5 kV and data were acquired using full scan analysis over m/z 70-800 at 70,000 resolution and a 3 Hz data acquisition rate.  Negative ion mode analyses of polar metabolites were achieved using a HILIC method under basic conditions.  Briefly, 30 µL homogenate was extracted using 120 µL of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA).  The samples were centrifuged (10 min, 9,000 x g, 4 °C) and the supernatants were injected directly onto a 150 x 2.0 mm Luna NH2 column (Phenomenex; Torrance, CA) that was eluted at a flow rate of 400 µL/min with initial conditions of 10% mobile phase A (20 mM ammonium acetate and 20 mM ammonium hydroxide in water) and 90% mobile phase B (10 mM ammonium hydroxide in 75:25 v/v acetonitrile/methanol) followed by a 10 min linear gradient to 100% mobile phase A.  MS full scan data were acquired over m/z 70-800.  The ionization source voltage is -3.0 kV and the source temperature is 325 °C.  Lipids were extracted from 10 µL of homogenate using 190 µL of isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids; Alabaster, AL).  After centrifugation, supernatants were injected directly onto a 150 x 3.0 mm Prosphere HP C4 column (Grace, Columbia, MD).  The column was eluted isocratically with 80% mobile phase A (95:5:0.1 vol/vol/vol 10 mM ammonium acetate/methanol/acetic acid) for 2 min followed by a linear gradient to 80% mobile-phase B (99.9:0.1 vol/vol methanol/acetic acid) over 1 min, a linear gradient to 100% mobile phase B over 12 min, then 10 min at 100% mobile-phase B.  Full scan MS analyses (m/z 400-1000) were carried out in the positive ion mode using full scan analysis at 70,000 resolution and 3 Hz data acquisition rate.  All raw data were processed using Progenesis CoMet software (version 2.0, NonLinear Dynamics) for feature alignment, signal detection, and signal integration.  Signal peak areas were converted into numerical intensity values and normalized to internal standards added to each sample and to total signal at each time point.