MAGE-TAB Version	1.1
Comment[MetaboBank accession]	MTBKS47
Study Title	A novel method for single-grain-based metabolic profiling of Arabidopsis seed
Study Description	Introduction: In plant metabolomics, metabolite contents are often normalized by sample weight. However, accurate weighing of very small samples, such as individual Arabidopsis thaliana seeds (approximately 20 microgram), is difficult, which may lead to irreproducible results.; Objectives: We aimed to establish alternative normalization methods for seed-grain-based comparative metabolomics of A. thaliana.; Methods: Arabidopsis thaliana seeds were assumed to have a prolate spheroid shape. Using a microscope image of each seed, the lengths of major and minor axes were measured by fitting a projected 2-dimensional shape of each seed as an ellipse. Metabolic profiles of individual diploid or tetraploid A. thaliana seeds were measured by our highly sensitive protocol ("widely targeted metabolomics") that uses liquid chromatography coupled with tandem quadrupole mass spectrometry. Mass spectrometric analysis of 1 microliter of solution extract identified more than 100 metabolites. The data were normalized by various seed-size measures, including seed volume (single-grain-based analysis). For comparison, metabolites were extracted from 4 mg of diploid and tetraploid A. thaliana seeds and their metabolic profiles were analyzed by normalization of weight (weight-based analysis).; Results: A small number of metabolites showed statistically significant differences in the single-grain-based analysis compared to weight-based analysis. A total of 17 metabolites showed statistically different accumulation between ploidy types with similar fold changes in both analyses.; Conclusion: Seed-size measures obtained by microscopic imaging were useful for data normalization. Single-grain-based analysis enables evaluation of metabolism of each seed and elucidates the metabolic profiles of precious bioresources by using small amounts of samples.

Experimental Design	genotype design
Experimental Factor Name	ploidy	preparation
Experimental Factor Type	ploidy	preparation

Person Last Name	Takahashi	Fukushima	Hirai yokota	Sawada
Person First Name	Mikiko	Atsushi	Masami	Yuji
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 Center for Sustainable Resource Science	RIKEN Center for Sustainable Resource Science
Person Roles	submitter	submitter	submitter	submitter

PubMed ID
Publication DOI	10.1007/s11306-017-1211-1

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	Diploid and autotetraploid plants were maintained at 23 degree C under continuous illumination (ca. 60 micromol/sec/m^2) on rockwool supplied with 0.2 g/L HYPONeX solution. To harvest mature seeds for metabolomic analysis, 1-week-old plants grown on agar-solidified, half-strength Murashige and Skoog medium were transferred to soil [PRO-MIX (Premier Horticulture, Canada): vermiculite = 2:1] and grown under 16 h light/8 h dark with a nutrient solution reported as a control medium. For biological replicates, seeds were independently collected from 12 individuals for each genotype	A visually normal seed was selected from each of the 12 individual plants under a microscope. After the estimation of seed volume as described above, each seed was placed in a separate well of a 96-well plate using a Vacuseed (Polaris Instruments Ltd, Cambridge, U.K.) with a 3-mm zirconia bead. The metabolites were extracted using 500 microliter extraction solvent (80% methanol, 0.1% formic acid, 16.8 nmol/L lidocain, and 105 nmol/L 10-camphorsulfonic acid as internal standards) with a Multi-beads Shocker (Shake Master NEO, Bio Medical Science) at 1000 rpm for 10 min. After centrifugation, 250 microliter of the extract was transferred to a new plate, dried, dissolved in 250 microliter of ultra-pure water, and filtered using Whatman UNIFILTER plates 384 (GE Healthcare). The sample preparation process was automatically performed by a liquid handling system (Microlab Star Plus, Hamilton). 1 microliter of the solution extract (final concentration =ca. 40 microgram/mL) prepared from a seed (ca. 20 microgram in the case of diploid) was subjected to widely targeted metabolomics using LC-QqQ-MS (UPLC-TQS, Waters)16.8 nmol/L lidocain, and 105 nmol/L 10-camphorsulfonic acid	Column name: ACQUITY UPLC HSS T3 Column, 100 angstrom, 1.8 micrometer, 1 mm X 50 mm (Waters); Column temperature: 30.0 degree C; Column type: ODS; Flow gradient: Gradient condition: 99.9% A/0.1% B at 0 min, 99.9% A/0.1% B at 0.25 min, 91% A/9% B at 0.4 min, 83% A/17% B at 0.8 min, 0.1% A/99.9% B at 1.9 min, 0.1% A/99.9% B at 2.1 min, 99.9% A/0.1% B at 2.11 min, 99.9% A/0.1% B at 2.7 min; Flow rate: 0.24 ml/min; Instrument: UPLC-TQS, Waters; Solvent: A: 0.1% V/V formic acid in water, B: 0.1% V/V formic acid in acetonitrile;	Desolvation temperature: 600C; Fragmentation method: CID; Instrument: UPLC-TQS, Waters; Ion isolation: ESI; Ion mode: positive/negative; Ion source: LC; Ion source temperature: 150C; Ion spray voltage: 0.5kV(pos)/0.8kV(neg); Mass analyser: QqQ;	data not submitted	data not submitted
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	MTBKS47.sdrf.txt

Comment[Study type]	untargeted metabolite profiling
Comment[Experiment type]	liquid chromatography-mass spectrometry	quadrupole mass spectrometer
Comment[Submission type]	LC-MS
Comment[BioProject]	PRJDB15389
Comment[Related study]	RPMM:RPMM0049	Metabolonote:SE125
Comment[Contributor]
Comment[Submission Date]	2019-10-05
Comment[Last Update Date]	2025-01-31
