SE143:/S1/M1/D1

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Sample Set Information

ID SE143
Title Application of a metabolomic method combining one-dimensional and two-dimensional gas chromatography-time-of-flight/mass spectrometry to metabolic phenotyping of natural variants in rice.
Description We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC × GC) gas chromatography-time-of-flight (TOF)–mass spectrometry. This method was applied to the metabolic phenotyping of natural variants in rice for the 68 world rice core collection (WRC) and two other varieties. Ten metabolites were selected as metabolite representatives, and the selected ion current of each metabolite peak obtained from both techniques were statistically compared. Our method of combining 1D- and GC × GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs.
Authors Kusano M, Fukushima A, Kobayashi M, Hayashi N, Jonsson P, Moritz T, Ebana K, Saito K.
Reference J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Aug;855(1):71-9. Epub 2007 May 16.
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Sample Information

ID S1
Title rice
Organism - Scientific Name Oryza sativa
Organism - ID NCBI taxonomy 4530
Compound - ID
Compound - Source
Preparation Twenty-five rice seeds for each of the 68 WRC cultivars and var. Dahonggu and Pokkari were sown on April 19, 2005, at a rice field in NIAS, Tsukuba, Japan. Seeds were harvested independently for each cultivar after 40 days, starting from the day on which the first panicle of rice was observed. The seeds were threshed from the panicles manually and then collected by each cultivar, after they were dried at 30 °C for three days. All seeds in the husks were stored at 5 °C under dark conditions until analysis.
Sample Preparation Details ID
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Analytical Method Information

ID M1
Title 1D-GC-TOF/MS
Method Details ID MS1
Sample Amount 1 μL
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Analytical Method Details Information

ID MS1
Title One-dimensional gas chromatography-time-of-flight/mass spectrometry (1D-GC-TOF/MS) analysis
Instrument GC:Agilent 6890N gas chromatograph (Agilent Technologies, Wilmingston, USA)
MS:Pegasus III TOF mass spectrometer (LECO, St. Joseph, MI, USA)
Instrument Type
Ionization EI
Ion Mode Positive
Description One hundred milligrams of each material was extracted with extraction buffer [methanol/chloroform/water (3:1:1, v/v/v)] at a concentration of 100 mg/ml and containing 10 stable isotope reference compounds. Each isotope compound was adjusted to a final concentration of 15 ng/μl for each 1-μl injection. After centrifugation, 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).

One microliter of each sample was injected in the splitless mode by an CTC CombiPAL autosampler (CTC analytics, Zwingen, 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. Four replicates were subjected to 1D-GC-TOF/MS analysis. Helium was used as the carrier gas at a constant flow rate of 1 ml/min. The temperature program for metabolome analysis started with a 2-min isothermal step at 80 °C and this was followed by temperature ramping at 30 °C to a final temperature of 320 °C, which was maintained for 3.5 min. Data acquisition was performed on a Pegasus III TOF mass spectrometer (LECO, St. Joseph, MI, USA) with an acquisition rate of 30 spectra/s 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). 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, using the method of Kopka et al. The normalized responses are peak areas corrected using the sample weight and a constant amount of the representative internal standard compound ([13C4]-hexadecanoic acid).

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Data Analysis Information

ID D1
Title Data Processing
Data Analysis Details ID DS1
Recommended decimal places of m/z
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Data Analysis Details Information

ID DS1
Title Mass spectral data processing
Description To compare the response of the metabolite peaks with the same algorithm, the Leco ChromaTOF optimized for Pegasus 4D software version 2.32 (Leco, St. Joseph, MI, USA) was used. Data, including baseline correction, peak deconvolution, and peak annotation for 1D- and GC × GC-TOF/MS were processed. In parallel, non-processed MS data from the 1D-GC-TOF/MS analysis were exported in the NetCDF format to MATLAB software 6.5 (Mathworks, Natick, MA, USA), where all data-pretreatment procedures such as data normalization, baseline correction, and the subsequent data treatments were performed using custom scripts to perform multivariate statistical analysis for metabolite phenotype clustering. The resolved MS spectra obtained from the custom scripts were matched against reference mass spectra by using the National Institute of Standards and Technology (NIST) mass spectral search program for the NIST/EPA/NIH mass spectral library (version 2.0) and our custom mass spectral search software written in JAVA (http://www.metabolome.jp/). Two mass spectral libraries – an in-house metabolite library in PRIMe (Platform for RIKEN Metabolomics, http://prime.psc.riken.jp) and the library in the Golm Metabolome Database (GMD) at CSB.DB – were used for the collection of mass spectra obtained by analysis. The extracted MS spectra were finally identified or annotated according to their RI and comparison with the reference mass spectra in the libraries.
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