MAGE-TAB Version 1.1 Comment[MetaboBank accession] MTBKS5 Study 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 Study Description We have developed a comprehensive method combining analytical techniques of one-dimensional (1D) and two-dimensional (GC x 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 x GC-TOF/MS is useful for the metabolic phenotyping of natural variants in rice for further studies in breeding programs. Experimental Design hardware variation design Experimental Factor Name cultivar Experimental Factor Type cultivar Person Last Name Takahashi Fukushima Kusano Person First Name Mikiko Atsushi Miyako Person Mid Initials Person Affiliation RIKEN Center for Sustainable Resource Science RIKEN Center for Sustainable Resource Science; Department of Agricultural and Life Science, Kyoto Prefectural University RIKEN Plant Science Center Person Roles submitter submitter submitter PubMed ID 17556050 Publication DOI Protocol Name Sample collection Extraction Chromatography Mass spectrometry Data processing Metabolite identification Protocol Type Sample collection Extraction Chromatography Mass spectrometry Data processing Metabolite identification Protocol Description 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 degreeC for three days. All seeds in the husks were stored at 5 degreeC under dark conditions until analysis. 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/microliter for each 1 microliter injection. After centrifugation, a 200 microliter 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).10 stable isotope reference compounds: [2H4]-succinic acid, [13C5,15N]-glutamic acid, [2H7]-cholesterol, [13C3]-myristic acid, [13C5]-proline, [13C12]-sucrose, [13C4]-hexadecanoic acid, [2H4]-1,4-butanediamine, [2H6]-2-hydoxybenzoic acid and [13C6]-glucose Column name: 30m x 25mm inner diameter fused-silica capillary column with a chemically bound 0.25 microliter film Rtx-5 Sil MS stationary phase (RESTEK, Bellefonte, USA); Column type: low polarity; Flow gas: Helium; Flow rate: 1.0 ml/min; Instrument: Agilent 6890N Gas Chromatograph (Agilent Technologies, Wilmingston, USA); Temperature gradient: Started with a 2 min isothermal step at 80 degree C and this was followed by temperature ramping at 30 degree C to a final temperature of 320 degree C, which was maintained for 3.5 min.; Acquisition rate: 30 spectra/s; Fragmentation method: EI; Instrument: Pegasus III TOF mass spectrometer (LECO, St. Joseph, MI, USA); Ion isolation: EI; Ion isolation energy: 70.0 eV; Ion mode: Positive; Ion source: GC; Mass analyser: TOF; Retenition index method: Alkane standard mixtures (C8-20 and C21-40) were used for calculating the retention index (RI).; Scan MZ range: m/z 60-800; 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 x 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 MSspectra were finally identified or annotated according to their RI and comparison with the reference mass spectra in the libraries. Protocol Parameters Post extraction;Derivatization Chromatography instrument;Autosampler model;Column model;Column type;Guard column Scan polarity;Scan m/z range;Instrument;Ion source;Mass analyzer Protocol Hardware Protocol Software Public Release Date 2020-10-05 Term Source Name RIKEN Plant Metabolome MetaDatabase (RPMM) Metabolonote Term Source File http://metabobank.riken.jp/pmm/db/plantMetabolomics http://metabolonote.kazusa.or.jp/ Term Source Version SDRF File MTBKS5.sdrf.txt Comment[Study type] untargeted metabolite profiling Comment[Experiment type] gas chromatography-mass spectrometry time-of-flight mass spectrometry Comment[Submission type] GC-MS Comment[BioProject] PRJDB15517 Comment[Related study] RPMM:RPMM0005 Metabolonote:SE143 Comment[Contributor] Comment[Submission Date] 2019-10-05 Comment[Last Update Date] 2026-01-29