SE53:/MS02

Sample Set Information

ID	SE53
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
Comment

The original paper is available at PLOS ONE.

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

Analytical Method Details Information

ID	MS02
Title	LC-TOF/MS
Instrument	Waters Acquity UPLC system and Waters Q-Tof Premier
Instrument Type	UPLC-QTOF-MS
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 LC-MS> Five mg DW per 150 μl of extraction medium (methanol/water [2:5 v/v] with reference compounds [0.5 mg l-1 flavonol-2'-sulfonic acid and 1.0 mg l-1 ampicilin]) each sample was used for the extraction of plant material using a Retsch mixer mill MM310 at a frequency of 20 Hz for 5 min at 4°C. After centrifugation for 10 min at 15,000 × g, the supernatant was transferred into a 2 ml tube. Thirty volumes of methanol were added to the tube and then extracted again using the mixer mill at a frequency of 20 Hz for 5 min at 4°C. After centrifugation for 10 min at 15,000 × g, the resulting supernatant was transferred into the tube. Two hundred-μl aliquot of the extracts was filtered using an Oasis® HLB -μelusion plate (30 μm, Waters Co., Massachusetts, USA). The extracts were evaporated to dryness in an SPD2010 SpeedVac® concentrator from ThermoSavant (Thermo electron corporation, Waltham, MA, USA). The extracts were dissolved by 160 μl of 20% aqueous methanol containing 0.5 mg l-1 lidocaine and d-camphor sulfonic acid. <LC-TOF/MS conditions> After filtration of the extracts (Ultrafree-MC, 0.2 μm pore size; Millipore), the sample extracts (5 μl) were analyzed using an LC-MS system equipped with an electrospray ionization (ESI) interface (HPLC, Waters Acquity UPLC system; MS, Waters Q-Tof Premier). The analytical conditions were as follows. HPLC: column, Acquity bridged ethyl hybrid (BEH) C18 (pore size 1.7 μ m, length 2.0 × 100 mm, Waters); solvent system, acetonitrile (0.1% formic acid):water (0.1% formic acid); gradient program, 1 : 99 v/v at 0 min, 1 : 99 v/v at 0.1 min, 99.5 : 0.5 at 15.5 min, 99.5 : 0.5 at 17.0 min, 1 : 99 v/v at 17.1 min and 1 : 99 at 20 min; flow rate, 0.3 ml min=1; temperature, 38°C; MS detection: capillary voltage, +3.0 keV; cone voltage, 23 V for positive mode and 35 V for negative mode; source temperature, 120°C; desolvation temperature, 450°C; cone gas flow, 50 l h=1; desolvation gas fiow, 800 l/ h; collision energy, 2 V for positive mode and 5 V for negative mode ; detection mode, scan (m/z 100-2000; dwell time 0.45 sec; interscan delay 0.05 sec, centroid). The scans were repeated for 19.5 min in a single run. The data were recorded using MassLynx version 4.1 software (Waters).
Comment_of_details