MAGE-TAB Version	1.1
Comment[MetaboBank accession]	MTBKS89
Study Title	Medicago truncatula shoot metabolite analysis using stable isotopes
Study Description	Metabolites in the Medicago truncatula shoot are investigated by using stable isotope labelings. This analysis was performed to estimate the elemental composition with high reliability using the number of stable isotopes incorporated in the compound as an index and to add information to discuss the biosynthetic pathway. The Medicago seedlings were labeled with 13C, 15N, 18O, and 34S, respectively in the same condition. Following peak picking by using PowerFT, the metabolite list was prepared from unlabeled data and blank data. The isotopic peaks were searched and listed using ShiftedIonsFinder. Moreover, the flavonoid-like peaks were also searched using ShiftedIonsFinder.

Experimental Design	species design
Experimental Factor Name
Experimental Factor Type

Person Last Name	Ara
Person First Name	Takeshi
Person Mid Initials
Person Affiliation	Kazusa DNA Research Institute
Person Roles	submitter

PubMed ID	29780292
Publication DOI	DOI: 10.5511/plantbiotechnology.14.0609c

Protocol Name	Sample collection 1	Sample collection 2	Sample collection 3	Sample collection 4	Sample collection 5	Sample collection 6	Extraction	Chromatography	Mass spectrometry	Data processing	Metabolite identification
Protocol Type	Sample collection	Sample collection	Sample collection	Sample collection	Sample collection	Sample collection	Extraction	Chromatography	Mass spectrometry	Data processing	Metabolite identification
Protocol Description	M. truncatula, Jemalong A17, were sown on an agar medium which prepared in a 500ml sealed glass bottle. The medium contained 1mM potassium phosphate monobasic, 30microM boric acid, 11microM manganese(II) chloride, 1microM zinc chloride, 1mM potassium chloride, 1mM molybdic acid (sodium salt), 3.2microM cupric chloride, 84nM cobalt chloride, 1mM calcium chloride, 50microM Fe-ethylenediaminetetraacetic acid (EDTA), 1mM magnesium sulfate, 10mM ammonium nitrate, 10mM potassium nitrate, and 0.5% glucose. The plants were grown for five weeks under the following conditions: temperature, 25/25 degree C (day/night); day/night rhythm, 18h/6h. During cultivation, the air was exchanged daily. The shoot (aerial part) and root were separated with a scalpel and they were immediately frozen using liquid nitrogen. After lyophilization, the sample was stored at room temperature.	M. truncatula, Jemalong A17, were sown on an agar medium which prepared in a 500ml sealed glass bottle. The medium contained 1mM potassium phosphate monobasic, 30microM boric acid, 11microM manganese(II) chloride, 1microM zinc chloride, 1mM potassium chloride, 1mM molybdic acid (sodium salt), 3.2microM cupric chloride, 84nM cobalt chloride, 1mM calcium chloride, 50microM Fe-ethylenediaminetetraacetic acid (EDTA), 1mM magnesium sulfate, 10mM ammonium nitrate, 10mM potassium nitrate, and 0.5% 13C6-glucose(ISOTEC). The plants were grown for five weeks under the following conditions: temperature, 25/25 degree C (day/night); day/night rhythm, 18h/6h. During cultivation, the 13C-labeled air (388ppm 13C-CO2 and balance air) was exchanged daily. The shoot (aerial part) and root were separated with a scalpel and they were immediately frozen using liquid nitrogen. After lyophilization, the sample was stored at room temperature.	M. truncatula, Jemalong A17, were sown on an agar medium which prepared in a 500ml sealed glass bottle. The medium contained 1mM potassium phosphate monobasic, 30uM boric acid, 11microM manganese(II) chloride, 1microM zinc chloride, 1mM potassium chloride, 1mM molybdic acid (sodium salt), 3.2microM cupric chloride, 84nM cobalt chloride, 1mM calcium chloride, 50uM Fe-ethylenediaminetetraacetic acid (EDTA), 1mM magnesium sulfate, 10mM 15N-ammonium nitrate (SI Science), 10mM 15N-potassium nitrate (SI Science), and 0.5% glucose. The plants were grown for five weeks under the following conditions: temperature, 25/25 degree C (day/night); day/night rhythm, 18h/6h. During cultivation, the air was exchanged daily. The shoot (aerial part) and root were separated with a scalpel and they were immediately frozen using liquid nitrogen. After lyophilization, the sample was stored at room temperature.	M. truncatula, Jemalong A17, were sown on an agar medium which prepared in a 500ml sealed glass bottle. The medium contained 1mM potassium phosphate monobasic, 30microM boric acid, 11microuM manganese(II) chloride, 1microM zinc chloride, 1mM potassium chloride, 1mM molybdic acid (sodium salt), 3.2microM cupric chloride, 84nM cobalt chloride, 1mM calcium chloride, 50uM Fe-ethylenediaminetetraacetic acid (EDTA), 1mM magnesium sulfate, 10mM ammonium nitrate, 10mM potassium nitrate, and 0.5% glucose. The plants were grown for five weeks under the following conditions: temperature, 25/25 degree C (day/night); day/night rhythm, 18h/6h. During cultivation, the 18O-labeled air (21.7% 18O-O2, 0.0394% CO2, and balance N2) was exchanged daily. The shoot (aerial part) and root were separated with a scalpel and they were immediately frozen using liquid nitrogen. After lyophilization, the sample was stored at room temperature.	M. truncatula, Jemalong A17, were sown on an agar medium which prepared in a 500ml sealed glass bottle. The medium contained 1mM potassium phosphate monobasic, 30uM boric acid, 11microM manganese(II) chloride, 1microM zinc chloride, 1mM potassium chloride, 1mM molybdic acid (sodium salt), 3.2microM cupric chloride, 84nM cobalt chloride, 1mM calcium chloride, 50microM Fe-ethylenediaminetetraacetic acid (EDTA), 1mM 34S-magnesium sulfate (SI Science), 10mM ammonium nitrate, 10mM potassium nitrate, and 0.5% glucose. The plants were grown for five weeks under the following conditions: temperature, 25/25 degree C (day/night); day/night rhythm, 18h/6h. During cultivation, the air was exchanged daily. The shoot (aerial part) and root were separated with a scalpel and they were immediately frozen using liquid nitrogen. After lyophilization, the sample was stored at room temperature.	80% methanol was prepared from methanol (Optima LC/MS, Fisher Chemical) and water (Optima LC/MS, Fisher Chemical).	The metabolites were extracted with 80% methanol as follows: 1) Measure about 10mg of plant sample in 1.5ml microtube. 2) Ground the sample in the tube with pestle. 3) Add 20 times the volume of cool 80% methanol and mixed well. 4) Incubate it on ice for 30min and then centrifuge it at 15,000/g for 20 min at 4 degree C. 5) Collect the supernatants in a new glass vial. 6) Repeat steps 3 to 5 once. 7) Combine all the supernatants in same glass vial. 8) Clean up the solution with the disc filter (millex-LG, 0.20 micrometre, MILLIPORE).	Instrument: Agilent 1200 HPLC (Agilent Technologies) HPLC equipped with photodiode array detector; Comment: A 10 microL sample is injected into HPLC. Elute monitoring by PDA equipped with Agilent1200 HPLC in 2 nm step.; Max wave length for elute monitering: 950.0 nm; Min wave length for elute monitering: 190.0 nm; Column name: TSKgel ODS-100V (4.6 x 250mm, 5 micrometre; TOSOH); Column temperature: 40.0 degree C; Elute monitor: photodiode array detector; Flow gradient: Gradient: (B);3 to 97% (0.0 to 45.0min), 97% (45.1 to 50.0min), 3% (50.1 to 57.0min); Flow rate: 0.5 ml/min; Solvent: A; 0.1% formic acid aq. B; ACN (addition 0.1% formic acid fc.)	Instrument: LTQ-Orbitrap XL (Thermo Fisher Scientific) Hybrid linear ion trap (LTQ)/orbitrap mass spectrometer; MS scan setting: Orbitrap-MS conditions: Filter 1: FTMS + c norm !corona !pi res=60000 o(100.0-1500.0); 2: ITMS + c norm !corona !pi Dep MS/MS Most intense ion from (1); 3: ITMS + c norm !corona !pi Dep MS/MS 2nd most intense ion from (1); 4: ITMS + c norm !corona !pi Dep MS/MS 3rd most intense ion from (1); 5: ITMS + c norm !corona !pi Dep MS/MS 4th most intense ion from (1); 6: ITMS + c norm !corona !pi Dep MS/MS 5th most intense ion from (1)., Rejected mass=235.00000, 376.00000, 609.00000, 810.00000, 1123.00000.; Data processing software: Xcalibur (Thermo Fisher Scientific K.K); Ion isolation: ESI; Ion mode: Positive; Mass analyser: LTQ-Orbitrap; Resolution: 60000; Scan mass range: m/z 100.0-1500.0	data not submitted	data not submitted
Protocol Parameters							Post extraction;Derivatization	Chromatography instrument;Autosampler model;Column model;Column type;Guard column;Detector;Signal range;Resolution	Scan polarity;Scan m/z range;Instrument;Ion source;Mass analyzer
Protocol Hardware								Agilent 1200 HPLC (Agilent Technologies) HPLC equipped with photodiode array detector	LTQ-Orbitrap XL (Thermo Fisher Scientific) Hybrid linear ion trap (LTQ)/orbitrap mass spectrometer;
Protocol Software									Xcalibur (Thermo Fisher Scientific K.K)

Public Release Date	2020-10-05

Term Source Name	Metabolonote
Term Source File	http://metabolonote.kazusa.or.jp
Term Source Version

SDRF File	MTBKS89.sdrf.txt

Comment[Study type]	untargeted metabolite profiling
Comment[Experiment type]	liquid chromatography-mass spectrometry	orbitrap
Comment[Submission type]	LC-DAD-MS
Comment[BioProject]	PRJDB14177
Comment[Related study]	Metabolonote:SE37
Comment[Contributor]	Hideki Nagasaki 1, Sachiko Osawa 1, Hideki Hirakawa, 1 Kota Kera 1, Yoshiyuki Ogata 2, Yoshiki Nagashima 1, Norimoto Shimada 1,3, Nozomu Sakurai 1. Daisuke Shibata 1, Hideyuki Suzuki 1 (1:Kazusa DNA Research Institute, 2:Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 3:Tokiwa Phytochemical Co.,Ltd.)
Comment[Submission Date]	2019-10-05
Comment[Last Update Date]	2024-06-14