DOI: 10.5061/dryad.nn5qc
title: Data from: Age- and diet-associated metabolome remodeling characterizes the aging process driven by damage accumulation

description: Aging is thought to be associated with increased molecular damage, but
 representative markers vary across conditions and organisms, making it
 difficult to assess properties of cumulative damage throughout lifespan.
 We used nontargeted metabolite profiling to follow age-associated
 trajectories of &gt;15,000 metabolites in Drosophila subjected to
 control and lifespan-extending diets. We find that aging is associated
 with increased metabolite diversity and low-abundance molecules,
 suggesting they include cumulative damage. Remarkably, the number of
 detected compounds leveled-off in late-life, and this pattern associated
 with survivorship. Fourteen-percent of metabolites showed age-associated
 changes, which decelerated in late-life and long-lived flies. In contrast,
 known metabolites changed in abundance similarly to nontargeted
 metabolites and transcripts, but did not increase in diversity. Targeted
 profiling also revealed slower metabolism and accumulation of
 lifespan-limiting molecules. Thus, aging is characterized by gradual
 metabolome remodeling, and condition- and advanced age-associated
 deceleration of this remodeling is linked to mortality and molecular
 damage.
description: LC_MS peak
 intensitiesDrosophila 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.