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Sample Set Information

Title Covering chemical diversity of genetically-modified tomatoes using metabolomics for objective substantial equivalence assessment
Description We propose using multiple analytical platforms for the direct acquisition of an interpretable data set of estimable chemical diversity. As an example, we report an application of our multi-platform approach that assesses the substantial equivalence of tomatoes over-expressing the taste-modifying protein miraculin. In combination, the chosen platforms detected compounds that represent 86% of the estimated chemical diversity of the metabolites listed in the LycoCyc database. Following a proof-of-safety approach, we show that w92% had an acceptable range of variation while simultaneously indicating a reproducible transformation-related metabolic signature. We conclude that multi-platform metabolomics is an approach that is both sensitive and robust and that it constitutes a good starting point for characterizing genetically modified organisms.
Authors Miyako Kusano, Henning Redestig, Tadayoshi Hirai, Akira Oikawa, Fumio Matsuda, Atsushi Fukushima, Masanori Arita, Shin Watanabe, Megumu Yano, Kyoko Hiwasa-Tanase, Hiroshi Ezura, Kazuki Saito
Reference Kusano M et al. (2011) PLOS ONE 6: e16989

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

Sample Information

ID S01
Title Tomato
Organism - Scientific Name Solanum lycopersicum
Organism - ID NCBI taxonomy:4081
Compound - ID
Compound - Source
Preparation Solanum lycopersicum, L. cv. Moneymaker, Aichi-first, Ailsa Craig, MicroTom, M82, and Rutgers.

<Biosource species>
The two transgenic lines overexpressing milaculin gene (moneymaker background) were also used.



<Organ specification>

Green and red fruits. A three grade color scale (green, orange, and red) was employed to evaluate tomato color.

Sample Preparation Details ID SS01

Sample Preparation Details Information

Title Growth condition and Sampling
Description <Growth condition>

Seedlings of Solanum lycopersicum were potted in 1/2000 a Wagner pot containing compost soil (Kureha, Tokyo, Japan) for the soil experiment. Seeds were sown in 5 cm × 5 cm × 5 cm (height × length × width) rockwool cubes and grown in a hydroponics system (565 mg l-1 NO- 3 , 15.7 mg l-1 NH+4 , 202.2 mg l-1 PO- 3 , 218.4 mg l-1 K+, 19.9 mg l-1 Mg+2 , 95.0 mg l-1 Ca+2 and micronutrients) in an environmentally controlled growth room at 25 °C/20 °C (light/dark) and 600 ppm CO2 concentration with a light/dark cycle of 16 h/8 h for the hydroponic culture (HC) experiment. Seedlings were placed in a netted-greenhouse located at the Gene Research Center in University of Tsukuba

<Sampling and sampling date>

The fruits were harvested in spring (a pilot and HC experiments) and late summer (the soil experiment) in 2006, 2008, and 2009.

<Metabolism quenching method>

All samples were frozen within 30 s after sampling in liquid nitrogen. The frozen samples were lyophilized


Analytical Method Information

ID M01
Method Details ID MS01
Sample Amount An equivalent of 0.6 μg and 6 μg of the derivatized samples

Analytical Method Details Information

Instrument Agilent 6890N gas chromatograph (Agilent Technologies) and Pegasus IV TOF mass spectrometer (LECO)
Instrument Type
Ionization EI
Ion Mode Positive
Description <Sample processing and extraction>

The lyophilized sample in a 2 ml tube was frozen and then homogenized with a 5 mm of zirconia bead by a Mixer Mill (Retsch, Haan, Germany) at 20 Hz for 1 min. Five mg dry weight (DW) of the lyophilized samples were weighed for GC-MS and LC-MS analyses, while 25 mg DW of the samples for CE-MS analysis.

<Extraction and derivatization for GC-MS>
Each sample was extracted with a concentration of 2.5 mg DW 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,
[13C3]-myristic acid,
[13C4]-hexadecanoic acid,
[2H6]-2-hydoxybenzoic acid and

using a Retsch mixer mill MM310 at a frequency of 30 Hz for 3 min at 4℃. Each isotope compound was adjusted to a final concentration of 15 ng µl-1 for each 1-µl injection. After centrifugation for 5 min at 15,100 × g, a 200-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℃ with shaking. Thirty µl of n-heptane was added following silylation. All the 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>

One microliter of each sample was injected in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwin-gen, Switzerland) into an Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA) equipped with a 30 m × 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 used 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 ℃ and this was followed by temperature ramping at 30 ℃ to a final temperature of 320 ℃, which was maintained for 3.5 min. The transfer line and the ion source temperatures were 250 and 200 ℃, respectively. Ions were generated by a 70-eV electron beam at an ionization current of 2.0 mA. The acceleration voltage was turned on after a solvent delay of 273 s. Data acquisition was performed on a Pegasus IV TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra s-1 in the mass range of a mass-to-charge ratio of m/z = 60-800. Alkane standard mixtures (C8-C20 and C21-C40) were purchased from Sigma-Aldrich (Tokyo, Japan) and were used for calculating the retention index (RI) (Wagner et al. 2003; Schauer et al. 2005). The normalized response for the calculation of the signal intensity of each metabolite from the mass-detector response was obtained by each selected ion current that was unique in each metabolite MS spectrum to normalize the peak response. For quality control, we injected methylstearate in every 6 samples. Data was normalized using the CCMN algorithm (Redestig et al. 2009).


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Data Analysis Information

ID D01
Title Leco ChromaTOF, MATLAB, H-MCR, NIST mass spectral search program
Data Analysis Details ID DS01
Recommended decimal places of m/z Default

Data Analysis Details Information

Title Leco ChromaTOF, MATLAB, H-MCR, NIST mass spectral search program
Description Nonprocessed MS data from GC-TOF/MS analysis were exported in NetCDF format generated by chromatography processing and mass spectral deconvolutionsoftware, Leco ChromaTOF version 3.22 (LECO, St. Joseph, MI, USA) to MATLAB 6.5 (Mathworks, Natick, MA, USA), where all data pretreatment procedures, such as smoothing, alignment, time-window setting, and H-MCR, were carried out (Jonsson et al. 2006). The resolved MS spectra were matched against reference mass spectra using the NIST mass spectral search program for the NIST/EPA/NIH mass spectral library (version 2.0) and our custom software for peak annotation written in JAVA. Peaks were identified or annotated based on RIs and the reference mass spectra comparison to the Golm Metabolome Database (GMD) released from CSB.DB1 (Kopka et al. 2005)] and our in-house spectral library. The metabolites were identified by comparison with RIs from the library databases (GMD and our own library) and with those of authentic standards, and the metabolites were defined as annotated metabolites on comparison with mass spectra and RIs from these two libraries.

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