SE198:/S12/M161

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Sample Set Information

ID SE198
Title Untargeted metabolome analysis of 15N- or 34S-labeled plants (lettuce) / 15Nまたは34S全標識した植物のLC-MSメタボローム解析(レタス)
Description Untargeted metabolome analyses of lettuce were performed using liquid chromatography – mass spectrometry (LC-MS). This data acquisition aims at an improvement of peak annotation by using plant samples that were fully labeled with stable isotope 15N or 34S.

Two lettuce varieties, Leaf Lettuce Green and King Crown were used.

The information of the peaks detected in Leaf Lettuce Green was available at the Plant Metabolome Repository website (http://metabolites.in/plants).


15Nおよび34Sで全標識した植物試料を用いることで、液体クロマトグラフィー-質量分析(LC)で検出される化合物ピークの推定を向上させることを目的として、ノンターゲットメタボローム解析を行った。

植物試料として、二種類のレタス(リーフレタスグリーンおよびキングクラウン)を用いた。

リーフレタスグリーンで検出されたピークは、植物メタボロームレポジトリ (http://metabolites.in/plants) から公開されている。

Authors Sakurai N1,2, Suda K1, Akimoto N1, Hoshi K1, Osawa S1, Ikeda C1, Ozawa K1, Yamada M1, Muneto R1, Shibata D1 (1 Kazusa DNA Research Institute, 2 National Institute of Genetics), Contact: sakurai AT nig.ac.jp (replace AT with @)


櫻井望1,2、須田邦裕1、秋元奈弓1、星久美1、大澤祥子1、池田千晶1、小澤馨史1、山田学1、宗藤玲子1、柴田大輔1(1 かずさDNA研究所, 2 国立遺伝学研究所)

Reference
Comment

Sample Information

ID S12
Title Grand Rapids (Leaf Lettuce Green) (Control), freeze dried
Organism - Scientific Name Lactuca sativa L. var crispa
Organism - ID NCBI taxonomy: 466611
Compound - ID
Compound - Source
Preparation
Sample Preparation Details ID SS1
Comment

Sample Preparation Details Information

ID SS1
Title Preparation of the samples
Description Seeds of lettuce “Grand Rapids (Leaf Lettuce Green)” (Lactuca sativa L. var crispa) and “King Crown” (Lactuca sativa L. var capitata) were purchased from SAKATA SEED CORPORATION (Kanagawa, Japan). The seeds were germinated on vermiculite as a support medium in pots. The plants were grown in a growth chamber (12 h light/12 hr dark, 22 C) for 33 days and then on a cultivation shelf (16 hr light/ 8 hr dark, 20-28 C). The leaves were harvested 77 and 83 days after germination for Leaf Lettuce Green and King Crown, respectively. The plants were fertilized using a solution containing 3 mM KNO3, 1 mM Ca(NO3)2, 0.5 mM MgSO4, 0.5 mM NH4H2PO4, 0.05 mM Fe(III)-ethylenediamine-N,N,N’,N’-tetraacetic acid (EDTA), 49 uM H3BO3, 9 uM MnSO4, 0.8 uM ZnSO4, 0.3 uM CuSO4, and 0.1 uM Na2MoO4. For the stable isotope labeling, KNO3, Ca(NO3)2, and NH4H2PO4 were replaced with their 15N labeled chemicals (99.2-99.8 Atom%) and MgSO4 was replaced with its 34S labeled chemical (98 Atom%). labeled chemicals were purchased from Shoko Science Co., Ltd. (Yokohama, Japan). The plants were fertilized properly using the same amount of the solution in one time for control, 15N-labeled, and 34S-labeled plants. In total, 2,530 and 3,040 mL of the solution was used for growing three individuals of Leaf Lettuce Green and King Crown, respectively. The shoot of three individuals for each treatment was harvested, mixed, weighed, and homogenated using mortar and pestle under liquid nitrogen into a fine powder. The sample was stored at -80 C until use. A part of the powdered sample was freeze-dried and stored at room temperature.
Comment_of_details

Analytical Method Information

ID M161
Title ESI Negative (Method 6)
Method Details ID MS11
Sample Amount 0.3 mg DW / 20 uL injection
Comment


Analytical Method Details Information

ID MS11
Title LC-FT-MS, ESI, Negative
Instrument Agilent1100 HPLC (Agilent), LTQ-FT (Thermo Fisher Scientific)
Instrument Type LC-FTICR-MS
Ionization ESI
Ion Mode Negative
Description Metabolite extraction

300 mg of powdered sample was mixed with 900 uL of 100% methanol solution, and 18 mg freeze-dried sample was mixed with 1200 uL of 75% methanol solution in 2 mL tube. Both 100% and 75% methanol solution contain 25 uM of 7-hydroxy-5-methylflavone as internal standard (IS). The sample was homogenized using Mixer Mill MM 300 (QIAGEN) at 25 Hz, for 2 min, twice. The homogenate was centrifuged under 17,400 x g, 5 min at 4 C. A supernatant was passed through a Polytetrafluoroethylene (PTFE) filter (pore size 0.2 um, Millipore), and the filtrate was applied to a C18 silica column (MonoSpin C18, GL Science) to remove highly-hydrophobic contaminants. The filtrate passed through the C18 column was used for LC-MS analyzes.

The same extraction procedure with 75% methanol was performed without a sample to prepare mock samples and used as negative controls.


Liquid chromatography (LC)-mass spectrometry (MS) analysis


Agilent 1100 system (Agilent) and Finnigan LTQ-FT (Thermo Fisher Scientific) were used. Aliquots (5 uL) of the methanol extract were applied to a TSK-gel ODS-100V column (4.6 x 250 mm, 5 um, TOSO), and separated by water containing 0.1%(v/v) formic acid (Solvent A) and acetonitrile containing 0.1%(v/v) formic acid (Solvent B). The gradient program was as follows: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min). The flow rate was set at 0.25 mL/min for 0-100 min, and 0.5 mL/min for 100.1-107 min.


The conditions of mass spectrometry were as below:


- Method 1: This method is used for measuring the accurate mass of the peaks and obtaining MS/MS spectra. [Full scan] resolution, 100,000 by FT; m/z range, 100-1500 [MS2 scan] data-dependent scan by IT for the most intense 5 ions in the full scan. [Dynamic exclusion setting for MS2 scan] repeat count, 3; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; exclusion duration, 20 s.


- Method 2: Same as Method 1 except below: [Dynamic exclusion setting for MS2 scan] exclusion duration, 30 s.


- Method 3: Same as Method 1 except below: [Dynamic exclusion setting for MS2 scan] repeat count, 2; exclusion duration, 30 s.


- Method 4: Same as Method 1 except below: [Dynamic exclusion setting for MS2 scan] off.


- Method 5: This method is used for obtaining MS3 spectra. [Full scan] resolution, 12,500 by FT; m/z range, 100-1500. [MS2 scan] data-dependent scan by IT for the most intense 5 ions in the full scan. [Dynamic exclusion setting for MS2 scan] same as Method 1. [MS3 scan] data-dependent scan by IT for the most intense 2 ions in the MS2 scan


- Method 6: This method is used for obtaining MS2 spectra in high-resolution: [Full scan] resolution, 12,500 by FT; m/z range, 100-1500. [MS2 scan] data-dependent scan by FT for the most intense 5 ions in the full scan. resolution, 12,500. [Dynamic exclusion setting for MS2 scan] repeat count, 1; repeat duration, 30 s; exclusion list size, 500; margin, 10 ppm; exclusion duration, 40 s.


- Method 7: Same as Method 5 except below: [Dynamic exclusion setting for MS2 scan] same as Method 2


ESI: electrospray ionization, FT: Fourier transformation ion cyclotron resonance type mass spectrometry, IT: ion trap type mass spectrometry.


The mass analyses with electrospray ionization (ESI) in positive or negative modes were performed. The combinations of the Methods 1-7 and ionization polarity are different between the samples, but, at least one method for accurate mass measurement (Method 1-4) was applied for each sample. The raw data were acquired using Xcalibur software (Thermo Fisher Scientific).

Comment_of_details [column] TSK-gel ODS-100V (4.6 x 250 mm, 5 micrometer; TOSOH)

[gradient] Solvent A: water containing 0.1% v/v formic acid; Solvent B: acetonitrile containing 0.1% v/v formic acid; Gradient: 3% B (0 min), 97% B (90 min), 97% B (100 min), 3% B (100.1 min), and 3% B (107 min)

[total separation time] 107 min

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