SE204:/S17/M112

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Sample Set Information

ID SE204
Title Untargeted metabolome analysis of tobacco (BY-2) cells using LC-MS / LC-MSを用いたタバコ(BY-2)培養細胞のメタボローム解析
Description Untargeted metabolome analyses of tobacco (BY-2) cell cultures using liquid chromatography-mass spectrometry (LC-MS).

The information on the peaks detected in this study was available at the Plant Metabolome Repository website (http://metabolites.in/plants).


液体クロマトグラフィー-質量分析(LC)を用いたタバコ(BY-2)培養細胞のノンターゲットメタボローム解析です。

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) から公開されています。

Authors Sakurai N1,2, Suda K1, Masumoto H1, Kugou 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、桝本寛1、久郷和人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 S17
Title Medium, 7 days
Organism - Scientific Name
Organism - ID
Compound - ID
Compound - Source
Preparation
Sample Preparation Details ID SS1
Comment

Sample Preparation Details Information

ID SS1
Title Preparation of the samples
Description The tobacco BY-2 cells were provided by the RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. The cells were cultured according to a general protocol. The cells or culture media were sampled after 0, 3, or 7 days after inoculation. The samples were immediately frozen in liquid nitrogen, and stored at -80C. The cell sample was homogenated using mortar and pestle under liquid nitrogen into a fine powder and stored at -80C until use.
Comment_of_details

Analytical Method Information

ID M112
Title ESI Negative (Method 1)
Method Details ID MS11
Sample Amount 5 mg FW / 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 containing 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 for 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 analyses.

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 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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