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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 M03
Method Details ID MS03
Sample Amount 14μg

Analytical Method Details Information

Instrument Agilent CE capillary electrophoresis system,Agilent G3250AA LC/MSD TOF system,Agilent 1100 series binary HPLC pump,G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit
Instrument Type
Ionization ESI
Ion Mode Positive and negative
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 for CE-MS> Each sample was extracted in 200 volumes of methanol containing 8μM of two reference compounds (methionine sulfone for cation and camphor 10-sulfonic acid for anion analyses) using a Retsch mixer mill MM310 at a frequency of 27 Hz for 1 min. The extracts were then centrifuged at 20,400 × g for 3 min at 4 °C. Five hundred-μl aliquot of the supernatant was transferred into a tube. Five hundred μl of chloroform and 200 μl of water was added into the tube to perform liquid-liquid distribution. The upper layer was evaporated for 30 min at 45°C by a centrifugal concentrator to obtain two layers. For removing high-molecular-weight compounds such as oligo-sugars, the upper layer was centrifugally filtered through a Millipore 5-kDa cutoff filter at 9,100 g for 120 min at 4°C. The filtrate was dried for 120 min by a centrifugal concentrator. The residue was dissolved into 20 μl of water containing 200 μM of internal standards (3-aminopyrrolidine for cation and trimesic acid for anion analyses) that were used for compensation of migration time in the peak annotation step.

<CE-TOF MS instruments>

All CE-TOFMS experiments were performed using an Agilent CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany), an Agilent G3250AA LC/MSD TOF system (Agilent Technologies, Palo Alto, CA), an Agilent 1100 series binary HPLC pump, and the G1603A Agilent CE-MS adapter and G1607A Agilent CE-ESI-MS sprayer kit. The G2201AA Agilent ChemStation software for CE and the Analyst QS software for TOFMS were used.

<Separation column and electrolytes>

Separations were carried out using a fused silica capillary (50 μm i.d. x 100 cm total length) filled with 1 M formic acid for cation analyses or with 20 mM ammonium formate (pH 10.0) for anion analyses as the electrolyte. The capillary temperature was maintained at 20 °C.

<Sample injection>

The sample solutions were injected at 50 mbar for 15 sec (15 nL). The sample tray was cooled below 4 °C.

<Separation parameters>

Prior to each run the capillary was flushed with electrolyte for 5 min. The applied voltage for separation was set at 30 kV. Fifty percent (v/v) methanol/water containing 0.5 μM reserpine was delivered as the sheath liquid at 10 μL/min.


ESI-TOFMS was conducted in the positive ion mode for cation analyses or in the negative ion mode for anion analyses, and the capillary voltage was set at 4 kV.

<Dry gas condition>

A flow rate of heated dry nitrogen gas (heater temperature 300 °C) was maintained at 10 psig.

<Voltage settings in TOF/MS>

The fragmentor, skimmer, and Oct RFV voltage were set at 110V, 50V, and 160V for cation analyses or at 120V, 60V, and 220V for anion analyses, respectively.

<Mass calibration>

Automatic recalibration of each acquired spectrum was performed using reference masses of reference standards. The methanol dimer ion ([2M+H]+, m/z = 65.0597) and reserpine ([M+H]+, m/z = 609.2806) for cation analyses or the formic acid dimer ion ([2M-H]-, m/z = 91.0037) and reserpine ([M-H]-, m/z = 607.2661) for anion analyses provided the lock mass for exact mass measurements.

<Mass data acquirement>

Exact mass data were acqu ired at a rate of 1.5 cycles/sec over a 50-1000 m/z range.

<Quality control>

In an every single sequence analysis (maximum 36 samples) on our CE-MS system, we analyzed the standard compound mixture at the first and the end of sample analyses. The detected peak area of standard compound mixture was checked in point of reproducible sensitivity. Standard compound mixture composed of major detectable metabolites including amino acids and organic acids, and this mixture was newly prepared at least once a half year. In all analyses in this study, there were no differences in the sensitivity of standard compounds mixture.


Data Analysis Information

ID D03
Title In-house software
Data Analysis Details ID DS03
Recommended decimal places of m/z Default

Data Analysis Details Information

Title In-house software
Description An original data file (.wiff) was converted to an unique binary file (.kiff) using in-house software (nondisclosure). Peak picking and alignment were performed using the another in-house software (nondisclosure), peaks were picked and aligned among samples automatically. By contrast with the detected m/z and migration time values of standard compounds including internal standards, peaks were annotated automatically using the same software. For normalization, the individual area of the detected peaks was divided by the peak area of the internal reference standards. Based on the calibration curves for standard compounds, peak area values were converted into values corresponding to amounts.
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