SE143:/S1/M2/D1

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.
Comment

PubMed

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
Comment

Analytical Method Information

ID	M2
Title	GC × GC-TOF/MS
Method Details ID	MS2
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS2
Title	Two-dimensional gas chromatography-time-of-flight/mass spectrometry (GC × GC-TOF/MS)
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). The GC × GC-TOF/MS system consisted of the Agilent 6890N gas chromatograph and Pegasus 4D TOF-MS (Leco, St. Joseph, MI, USA). An Rtx-5 Sil MS column containing 5% diphenyl–95% dimethylpolysiloxane as the stationary phase was used as the first column (I.D., 30 m × 0.25 mm and film thickness, 0.25 μl) and an Rtx-50 column containing 50% methyl–50% phenyl polysiloxane was used as the second column (I.D., 1.0 m × 0.18 mm and film thickness, 0.20 μl). Both columns were purchased from Restek (Bellefonte, USA). They were connected using a universal Press-Tight® connector (Restek). A thermal modulator and the first and second ovens were independently controlled by Leco ChromaTOF optimized for Pegasus 4D software version 2.32 (Leco, St. Joseph, MI, USA). The second column was housed within the second oven. A 1-μl portion of each extract was injected in the splitless mode; three replicate injections were made for each biological sample. The temperature of the GC inlet and transfer line was set at 250 °C. The first column was maintained at 70 °C for 2 min and then increased at the rate of 15 °C/min to 320 °C, and this temperature was maintained for 4 min. The second column was initially set at 80 °C and the temperature program followed was the same as that used for the first column. The modulator was operated at 30 °C for a 3.0-s period; this temperature was higher than that of the first GC oven. The carrier gas (helium) was maintained at a constant flow rate of 1 ml/min. Signals within the mass range from m/z 60 to 600 were collected at a rate of 100 spectra/s after 273 s of solvent delay. The linear retention time (RTcompound x) was calculated using the RT value of each compound on the first (1st RTcompound x) and second dimensions (2nd RTcompound x) as mentioned below: The alkane mixtures used in GC × GC-TOF/MS analysis were analyzed to determine the RI. The algorithm was applied in the Leco ChromaTOF software to calculate the RI. The normalized response was then calculated as described by Kopka et al.
Comment_of_details

Data Analysis Information

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

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.
Comment_of_details