SE45:/MS01

Sample Set Information

ID	SE45
Title	Metabolomic Characterization of Knockout Mutants in Arabidopsis: Development of a Metabolite Profiling Database for Knockout Mutants in Arabidopsis
Description	Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/.
Authors	Atsushi Fukushima, Miyako Kusano, Ramon Francisco Mejia, Mami Iwasa, Makoto Kobayashi, Naomi Hayashi, Akiko Watanabe-Takahashi, Tomoko Narisawa, Takayuki Tohge, Manhoi Hur, Eve Syrkin Wurtele, Basil J. Nikolau, Kazuki Saito, RIKEN CSRS
Reference	Fukushima et al. (2014) Plant Physiology 165:948-961 (PMID: 24828308)
Comment	The raw data files are available at DropMet web site in PRIMe database, MetaboLights (Salek et al., 2013) (accession no. MTBLS47), and The MetabolomeExpress (Carroll et al., 2010).

PubMed

MeKO

The raw data files are available at DROP Met web site in PRIMe database of RIKEN.

Related data are deposited in MetaboLights.

Related data are deposited in MetabolomeExpress.

Analytical Method Details Information

ID	MS01
Title	GC-TOF/MS
Instrument	GC Agilent 6890N gas chromatograph / MS Pegasus IV TOF mass spectrometer
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	<Sample processing and extraction> Each sample was extracted at a concentration of 5 mg flesh weight (FW) of tissues per ml extraction medium (methanol / chloroform/water [3:1:1 v/v/v]) containing 10 stable isotope reference compounds: • [2H4]-succinic acid • [13C5,15N]-glutamic acid • [2H7]-cholesterol • [13C3]-myristic acid • [13C5]-proline • [13C12]-sucrose • [13C4]-hexadecanoic acid • [2H4]-1,4-butanediamine • [2H6]-2-hydoxybenzoic acid • [13C6]-glucose using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4°C. Each isotope compound was adjusted to a final concentration of 15 ng µl-1 for each 1-µl injection. After 5-min centrifugation at 15,100 × g, a 100 µl aliquot of the supernatant was drawn and transferred into a glass insert vial. The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo Electron Corporation, Waltham, MA, USA). For methoximation, 30 µl of methoxyamine hydrochloride (20 mg/ml in pyridine) was added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA with 1% TMCS at 37°C with shaking. For methoximation, 30 µl of methoxyamine hydrochloride (20 mg ml-1 in pyridine) were added to the sample. After 24 h of derivatization at room temperature, the sample was trimethylsilylated for 1 h using 30 µl of MSTFA at 37°C with shaking. After silylation 30 µl of n-heptane were added. All derivatization steps were performed in the vacuum glove box VSC-100 (Sanplatec, Japan) filled with 99.9995% (G3 grade) of dry nitrogen. <GC-TOF/MS conditions> Using the splitless mode by an CTC CombiPAL auto-sampler (CTC analytics, Zwin-gen, Switzerland), 1 µl of each sample was injected into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) featuring a 30 m x 0.25 mm inner diameter fused-silica capillary column with a chemically bound 0.25-µl film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA) for metabolome analysis. Helium was delivered as the carrier gas at a constant flow rate of 1 ml min-1. The temperature program for metabolome analysis started with a 2-min isothermal step at 80°C followed by temperature ramping of 30°C to a final temperature of 320°C, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200°C, respectively. Ions were generated with a 70-eV electron beam at an ionization current of 2.0 mA. Data acquisition was on a Pegasus IV TOF MS instrument (LECO, St. Joseph, MI, USA) at an acquisition rate of 30 spectra s-1 in the mass range of a mass-to-charge ratio of m/z = 60-800. The solvent delay was 237 sec. Alkane standard mixtures (C8-C20 and C21-C40) were purchased from Sigma-Aldrich (Tokyo, Japan) and used for calculating the retention index (RI). The normalized response for calculating the signal intensity of each metabolite from the mass-detector response was obtained with each selected ion current unique in each metabolite MS spectrum to normalize the peak response. For quality control, we injected methylstearate in every 6 samples. Quality control (QC) samples were prepared by mixing 100 μl of extracts of each wild-type sample. We run six QC samples per batch (30 samples in total).
Comment_of_details