SE229:/S032/M01

Sample Set Information

ID	SE229
Title	LC-MS based untargeted metabolome analysis of Oryza
Description	Compounds in the Oryza species were analyzed using liquid chromatography-mass spectrometry (LC-MS) in a untargeted manner. The same analytical conditions are applied to all samples. Therefore, the compound peaks can be compared to each other by the mass values, the retention time of the LC, and the CID mass spectrum.
Authors	Yutaka Sato (National Institute of Genetics), Nozomu Sakurai (National Institute of Genetics, Kazusa DNA Research Institute, email: sakurai (at) kazusa.or.jp)
Reference
Comment

Sample Information

ID	S032
Title	Rice seed, O. rufipogon, W1102 (No.16-2)
Organism - Scientific Name	Oryza rufipogon
Organism - ID	NCBI taxonomy: 4529
Compound - ID
Compound - Source
Preparation	The sample was provided by the Oryzabase sponsored by NBRP.
Sample Preparation Details ID	SS1
Comment

Sample Preparation Details Information

ID	SS1
Title	Sample preparation
Description	The harvested sample was stored at low temperature. The sample was ground to a fine powder using a homogenizer Multi-beads shocker MB3200(S) (Yasui Kikai Corp., Osaka, Japan) with 3,000 rpm for 15 min and stored at -80 degree C until use.
Comment_of_details

Analytical Method Information

ID	M01
Title	LC-QTOF-MS, ESI, Positive
Method Details ID	MS01
Sample Amount	0.25 mg / 2 ul injection
Comment

Analytical Method Details Information

ID	MS01
Title	LC-Q-Tof-MS, ESI, Positive
Instrument	Nexera X2 (Shimadzu), Compact (Bruker Daltonics)
Instrument Type	LC-QTOF-MS
Ionization	ESI
Ion Mode	Positive
Description	- Compound Extraction Compounds in the sample were extracted by 75%(v/v) methanol containing 1 uM 7-hydroxy-5-methylflavone as an internal control (IS). The sample in methanol in a 2 mL tube was homogenized using a zirconia bead (5 mm diameter) and Mixer Mill MM 400 (Verder Scientific, Co., Ltd.) at 25 Hz for 2 min, twice. A homogenate was centrifuged at 17,400 x g, 5 min at 4 degree C. A supernatant, or if separated into two liquid layers, the methanol layer, was applied to a C 18 silica column (MonoSpin C18, GL Sciences Inc.) to remove highly hydrophobic contaminants. The filtrate was passed through a polytetrafluoroethylene (PTFE) filter (pore size 0.2 um, Millipore) and used for LC-MS analyses. - Liquid chromatography (LC)-mass spectrometry (MS) analysis Nexera X2 system (Shimadzu Corporation) and Compact system (Bruker Japan K.K.) were used. An aliquot (2 uL) of the methanol extract was applied to an InertSustain AQ-C18 column (3 um x 1.5 mm x 50 mm, GL Sciences) connected after a guard column (InertSustain AQ-C18 Cartridge Guard Column, 3 um x 1.5 mm x 10 mm, GL Sciences), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile (Solvent B). The gradient program was as follows: 2% B (0 min), 2% B (1.5 min), 98% B (13.5 min), 98% B (17 min), 2% B (17.01 min), and 2% B (18.5 min). The flow rate was set at 0.2 mL/min. The column oven temperature was set to 40 degree C. The compounds separated by the LC were detected using the mass spectrometer under the conditions below: Ionization, Electrospray Ionization (ESI); Polarity, Positive; Scan rate, 2 Hz; Mass scan range, 50-1200; End plate offset, 500 V; Capillary voltage, 4000 V; Nebulizer gas (N2) pressure, 2.5 bar; Dry gas (N2) flow, 8.0 L/min; Dry gas temperature, 200 degree C; Transfer Funnel1 RF, 200.0 Vpp; Funnel2 RF, 200.0 Vpp; In-source CID Energy, 0.0 eV; Hexapole RF, 50.0 Vpp; Quadrupole Ion Energy, 3.0 eV; Low mass m/z, 55.00; Collision energy, 10.0 eV; Collision RF, 450.0 Vpp; Transfer time, 80.0 us; and PrePulse storage, 3.0 us. The data-dependent MS/MS spectra were obtained with the conditions below: Isolation width, 3-15 Da; Collision energy 35 eV; Precursor ion number, 5; Active Exclusion, on; Exclude, after 3 spectra; Release, after 0.3 min; Reconsider precursor, on; and if current intens. / previous intens., 2.0. For mass calibration, 1 mM sodium formate in 50% (v/v) 2-propanol was injected directly into the MS at 38.50–40.50 min of LC separation with a flow rate 0.1 mL/min. The eluent at 0-3 min was wasted. The raw data were obtained by Hystar software (ver.3.2 SR4, Bruker Daltonik, GmbH).
Comment_of_details	[column] InertSustain AQ-C18 (1.5 x 50 mm, 3 micrometer; GL Sciences) [gradient] Solvent A: water containing 0.1% v/v formic acid; Solvent B: acetonitrile; Gradient 2% B (0 min), 2% B (1.5 min), 98% B (13.5 min), 98% B (17 min), 2% B (17.01 min), and 2% B (18.5 min) [total separation time] 18.5 min