Arabidopsis Metabolome Analysis Using Infusion ESI FT-ICR/MS
融合ESI FT-ICR/MS法分析拟南芥代谢组   

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Plant Molecular Biology
Jul 2014

 

Abstract

We made the method for Arabidopsis metabolome analysis based on direct-infusion Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) (IonSpec). This method was sufficiently applied to metabolic phenotyping of Arabidopsis. This method is simple in that after homogenizing samples, powdered samples are dissolved in extraction solvents (acetone and methanol) to 20% fresh weight/volume. Extracted sample solutions are dried and dissolved in 50% (v/v) acetonitrile. Mass analysis using FT-ICR/MS (IonSpec) is performed in positive and negative ionization operation modes. Mass spectra are acquired over the 100-1,000 m/z range and accumulated to improve the S/N ratio.

Keywords: Metabolome (代谢组学), Arabidopsis (拟南芥), Albino (白化), ESI FT-ICR/MS (ESI FT-ICR质谱)

Materials and Reagents

  1. 3-week-old Arabidopsis plants
  2. Liquid nitrogen
  3. Acetone (HPLC grade) (Wako Chemicals USA, catalog number: 014-08681 )
  4. Methanol (HPLC grade) (Wako Chemicals USA, catalog number: 134-14523 )
  5. Nitrogen gas
  6. EN1-16 (TAITEC, catalog number: 0076417-000 )
  7. Formic acid (HPLC grade) (Wako Chemicals USA, catalog number: 063-04192 )
  8. Lidocaine (anaesthetic) m/z 235.18104 (Wako Chemicals USA, catalog number: 120-02691 )
  9. Prochloraz (agricultural chemical) m/z 376.03863 (Wako Chemicals USA, catalog number: 164-25131 )
  10. Reserpine (alkaloid sedative drug) m/z 609.28121 (Wako Chemicals USA, catalog number: 184-00691 )
  11. Bombesin (peptide) m/z 810.41479 (Wako Chemicals USA, catalog number: 339-40861 )
  12. 28~30% ammonia solution (Wako Chemicals USA, catalog number: 016-03146 )
  13. Negative mode internal standards: 2.4-D (plant hormone) m/z 218.96157 (Wako Chemicals USA, catalog number: 040-18532 )
  14. Ampicillin (antibiotic) m/z 348.10180 (Wako Chemicals USA, catalog number: 017-20531 , CHAPS 8 detergent) m/z 613.388865 (Wako Chemicals USA, catalog number: 341-04721 )
  15. Tetra-N-acetylchitotetraose [(GluNAc)4] m/z 829.32023 (Tokyo Chemical Industry, catalog number: T2910 )
  16. 50% (v/v) acetonitrile (HPLC grade) (Wako Chemicals USA, catalog number: 018-19853 ) [use distilled water to dilute acetonitrile to 50%(v/v)]
  17. Acetic acid (Wako Chemicals USA, catalog number: 014-20063 )
  18. 28~30% ammonia solution (Wako Chemicals USA, catalog number: 016-03146 )
  19. Ultrapure water (recommended but not required)

Equipment

  1. An IonSpec Explorer FT-ICR/MS equipped with a 7-tesla actively shielded superconducting magnet (Agilent, Ionspec)
  2. Glass vial and teflon cap (GL Biochem, catalog number: 1030-46716 )
  3. Glass pipette
  4. 0.45 µm filters (PTFE) (ADVANTEC®, catalog number: 13HP045AN )
  5. Heat block

Procedure

  1. Preparation of samples for FT-ICR/MS analysis
    1. Homogenize samples with liquid nitrogen to create powder samples of whole plants using mortars and pestles.
    2. Dissolve in extraction solvents to 20% fresh weight/volume. Two extraction solvents, 100% acetone and 100% methanol, are used to elute various polar compounds.
    3. Filter extracted sample solutions through 0.45 µm filters (PTFE).
    4. Transfer filtered sample solutions to vials.
    5. Transfer 1 ml of sample solution to a separate vial. Place the sample solution into a draft chamber and apply nitrogen gas using EN1-16. At 40 °C solvents will evaporate using a heat block.
    6. Dissolve samples in 50% acetonitrile (0.1~1 ml depending on sample varieties) and store at -80 °C.
    7. Dilute samples 1:14 prior to ESI analysis.

  2. FT-ICR/MS analysis
    1. Perform Mass analysis using an IonSpec Explorer FT-ICR/MS in positive and negative ionization operation modes. Ions are generated from an ESI source with a fused silica needle of 0.005-inch i. d. (Oikawa et al., 2006). Samples are infused using a Harvard syringe pump mode 22 at a flow rate of 0.5 to 1.0 µl/min through a 100 µl Hamilton syringe. Set the potentials on the electrospray emitters to 3.0 kV and -0.3 kV for the positive and the negative electrosprays, respectively.
    2. For positive mode, add 99.5% formic acid to extracted sample solutions (step A7) at a final concentration of 0.1% (v/v). Positive mode internal standards: Lidocaine (anaesthetic) m/z 235.18104, Prochloraz (agricultural chemical) m/z 376.03863, Reserpine (alkaloid sedative drug) m/z 609.28121, Bombesin (peptide) m/z 810.41479.
    3. For negative mode, add 28~30% ammonia solution to extracted sample solutions at a final concentration of 0.1% (v/v).
      Negative mode internal standards: 2.4-D (plant hormone) m/z 218.96157, Ampicillin (antibiotic) m/z 348.10180, CHAPS 8 detergent) m/z 613.388865, (GluNAc)4 m/z 829.32023.
    4. Mass spectra are acquired over the 100-1,000 m/z range and accumulate to improve the S/N ratio. The time period for accumulation depends on the total ion concentration. Analyze peaks using the IonSpec Omega ver.8 software. Proofread m/z of each peak referencing the internal standards. Measure the product ion mass spectra of each sample three times.
    5. When ion peaks are detected at least twice out of three successive spectral scans, they are subjected to further data processing as ion signals from actual analytes.
    6. A total of four mass spectral peaks from two different extraction solvents (methanol and acetone) and two ionization operation modes (positive and negative) are aligned using our in-house Java program. (If you are interested in this in-house Java program, please contact Prof. Ohta.)

  3. Normalization of data
    Apply global normalization to data. To avoid zero division, missing values are filled with 105 as a background signal of FT-ICR/MS. Peak intensities are transformed using a logarithmic scale with a factor of 10. Four data matrices are used to apply global normalization.
    Global normalization calculation methods are as follows:
    1. Average intensity is calculated by dividing the total signal by the number of detected peaks in each spectrum.
    2. Average signal is calculated for all spectra in each elution and charge pair.
    3. Normalization factor is calculated for each spectrum by dividing the average intensity for each spectrum by total average intensity.
    4. Normalized intensity is calculated by multiplying the raw intensity in each spectrum by the previously calculated normalization factor. Empirical formulas are inferred by the accuracy of the FT-ICR/MS. Because the sample ions become adduct ions to attach protons and sodium ions, etc. on the ESI source, we assumed the following were involved in the detected peaks [M+H]+, [M+Na]+, [M+K]+, [M+H+methanol]+, [M+ammonium]+ in positive ionization mode and [M-H]- in negative ionization mode.
    5. Search for candidate compounds using KEGG (http://www.genome.jp/kegg/), NIST (http://webbook.nist.gov/chemistry/) and KNApSAck (Shinbo et al., 2006).

Notes

  1. In order to obtain reliably reproducible data, it is imperative that the plant growth environment be as uniform/consistent as possible. For example depending on the light environment the value of a plant chlorophyll fluorescence will fluctuate. The amount of light a plant receives when next to the side light on the growth incubator is completely different from the light it receives when on the center of the shelf. For this reason it is important to shuffle the location of growth plates, etc.
  2. It is important that FT-ICRMS maintains a steady vacuum. We must also be careful about the size of the sample cone on the end of the ESI spray nozzle. It is also important to make minor adjustments where necessary to the direction of ESI ionspray, in an effort to capture as many ions into ICR cells as possible.
  3. FT-ICRMS can have problems when the concentration of plant extraction samples is too high, or when other unwanted substances are included in samples. Regular baking and removal of ions from the chamber inner wall as well as cleaning of the ion source is required.

Acknowledgments

This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (Japan) [Grants-in-Aid for Scientific Research (No.17681022 to R.M.)].
FT-ICR/MS (IonSpec) machine belongs to Osaka Prefecture University, and for this project it was used according to directions as indicated by Professors Ohta and Oikawa of the same university. Data normalization techniques were established by Mr. Satou.
This protocol is modified and appended referencing the original, as featured in "Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins" (Satou et al., 2014).

References

  1. Oikawa, A., Nakamura, Y., Ogura, T., Kimura, A., Suzuki, H., Sakurai, N., Shinbo, Y., Shibata, D., Kanaya, S. and Ohta, D. (2006). Clarification of pathway-specific inhibition by Fourier transform ion cyclotron resonance/mass spectrometry-based metabolic phenotyping studies. Plant Physiol 142(2): 398-413.
  2. Satou, M., Enoki, H., Oikawa, A., Ohta, D., Saito, K., Hachiya, T., Sakakibara, H., Kusano, M., Fukushima, A., Saito, K., Kobayashi, M., Nagata, N., Myouga, F., Shinozaki, K. and Motohashi, R. (2014). Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins. Plant Mol Biol 85(4-5): 411-428.
  3. Shinbo, Y., Nakamura, Y., Altaf-Ul-Amin, M., Asahi, H., Kurokawa, K., Arita, M., Saito, K., Ohta, D., Shibata, D. and Kanaya, S. (2006). KNApSAcK: a comprehensive species-metabolite relationship database. Biotech Agric Forest 57: 165-181.

简介

我们基于直接输注傅立叶变换离子回旋共振质谱(FT-ICR/MS)(IonSpec)制备了拟南芥分析的方法。 该方法充分应用于拟南芥的代谢表型。 该方法的简单之处在于,在均质化样品后,将粉末样品溶解在提取溶剂(丙酮和甲醇)中至20%鲜重/体积。 将提取的样品溶液干燥并溶解在50%(v/v)乙腈中。 使用FT-ICR/MS(IonSpec)的质量分析在正和负电离操作模式中进行。 质谱在100-1,000m/z范围内获得并累积以提高S/N比。

关键字:代谢组学, 拟南芥, 白化, ESI FT-ICR质谱

材料和试剂

  1. 3周龄拟南芥植物
  2. 液氮
  3. 丙酮(HPLC级)(Wako Chemicals USA,目录号:014-08681)
  4. 甲醇(HPLC级)(Wako Chemicals USA,目录号:134-14523)
  5. 氮气
  6. EN1-16(TAITEC,目录号:0076417-000)
  7. 甲酸(HPLC级)(Wako Chemicals USA,目录号:063-04192)
  8. 利多卡因(麻醉剂)m/z 235.18104(Wako Chemicals USA,目录号:120-02691)
  9. 丙氯灵(农药)m/z 376.03863(Wako Chemicals USA,目录号:164-25131)
  10. 利血平(生物碱镇静药)m/z 609.28121(Wako Chemicals USA,目录号:184-00691)
  11. 铃蟾肽(肽)m/z 810.41479(Wako Chemicals USA,目录号:339-40861)
  12. 28〜30%氨溶液(Wako Chemicals USA,目录号:016-03146)
  13. 阴模内标:2.4-D(植物激素)m/z 218.96157(Wako Chemicals USA,目录号:040-18532)
  14. 氨苄青霉素(抗生素)m/z 348.10180(Wako Chemicals USA,目录号:017-20531,CHAPS 8去污剂)m/z 613.388865(Wako Chemicals USA,目录号:341-04721)
  15. 四-N,N-乙酰基壳四糖[(GluNAc)4] m/z 829.32023(Tokyo Chemical Industry,目录号:T2910)
  16. 50%(v/v)乙腈(HPLC级)(Wako Chemicals USA,目录号:018-19853)[使用蒸馏水稀释乙腈至50%(v /
  17. 乙酸(Wako Chemicals USA,目录号:014-20063)
  18. 28〜30%氨溶液(Wako Chemicals USA,目录号:016-03146)
  19. 超纯水(推荐但不需要)

设备

  1. 配有7特斯拉主动屏蔽超导磁体(Agilent,Ionspec)的IonSpec Explorer FT-ICR/MS。
  2. 玻璃瓶和特氟隆盖(GL Biochem,目录号:1030-46716)
  3. 玻璃吸管
  4. 0.45μm过滤器(PTFE)(ADVANTEC ,目录号:13HP045AN)
  5. 热块

程序

  1. 用于FT-ICR/MS分析的样品制备
    1. 用液氮匀化样品,用研钵和杵制成整株植物的粉末样品
    2. 溶于萃取溶剂至20%鲜重/体积。 二 萃取溶剂,100%丙酮和100%甲醇洗脱 各种极性化合物
    3. 通过0.45μm过滤器(PTFE)过滤提取的样品溶液
    4. 将过滤的样品溶液转移到小瓶中
    5. 转移1毫升样品溶液到一个单独的小瓶。 放置 样品溶液进入通风室并使用氮气 EN1-16。 在40°C时,溶剂会通过加热块蒸发。
    6. 将样品溶解在50%乙腈(0.1〜1ml,取决于样品品种),并存储在-80℃。
    7. 在ESI分析前稀释样品1:14。

  2. FT-ICR/MS分析
    1. 使用IonSpec Explorer FT-ICR/MS进行质谱分析 和负离子化操作模式。 离子由ESI产生 源用0.005英寸i的熔融石英针。 d。 (Oikawa等人, 2006)。 使用哈佛注射器泵模式22在a注入样品 流速通过100μlHamilton注射器为0.5至1.0μl/min。 组 电喷雾发射器的电势为3.0kV和-0.3kV 正和负电喷雾。
    2. 对于 正模式,向提取的样品溶液中加入99.5%甲酸(步骤   A7),终浓度为0.1%(v/v)。 正模式内部 标准:利多卡因(麻醉剂)m/z 235.18104,丙氯灵 (农业化学品)m/z 376.03863,利血平(生物碱镇静剂 药物)m/z 609.28121,铃蟾肽(肽)m/z 810.41479。
    3. 对于阴性模式,向萃取的样品溶液中加入28〜30%氨水溶液,最终浓度为0.1%(v/v)。
      阴性模式内标:2.4-D(植物激素)m/z 218.96157, 氨苄青霉素(抗生素)m/z 348.10180,CHAPS 8去垢剂)m/z 613.388865,   (GluNAc)4 m/z 829.32023
    4. 质谱通过 100-1,000m/z范围,并累积以提高S/N比。 时间 累积的时间取决于总离子浓度。 分析 峰使用IonSpec Omega ver.8软件。 每个峰的校对m/z   参考内部标准。 测量产物离子质量 每个样品的光谱三次。
    5. 当检测到离子峰时   在三个连续光谱扫描中至少两次,它们是 作为离子信号从实际进行进一步的数据处理 分析物。
    6. 共有四个质谱峰来自两个 不同的萃取溶剂(甲醇和丙酮)和两次离子化 操作模式(正和负)使用我们的内部对齐 Java程序。 (如果你对这个内部Java程序感兴趣, 请联系Ohta教授。)

  3. 数据规范化
    对数据应用全局规范化。 为了避免零分割,缺失值用10 5 填充作为FT-ICR/MS的背景信号。 使用10的因子的对数标度转换峰强度。使用四个数据矩阵应用全局正规化。
    全局归一化计算方法如下:
    1. 平均强度是通过将总信号除以每个光谱中检测到的峰的数目来计算的。
    2. 对每个洗脱和电荷对中的所有光谱计算平均信号。
    3. 通过除以每个光谱计算归一化因子   每个光谱的平均强度乘以总平均强度
    4. 归一化强度通过将原始强度乘以 每个光谱由先前计算的归一化因子。 经验公式通过FT-ICR/MS的准确性推断。 因为样品离子变为加合离子以附着质子和钠 离子,等。我们假设涉及到以下 检测到的峰[M + H] +,[M + Na] +,[M + K] +,[M + H + [M +铵] +,在正电离模式中,[M-H] - 为负 电离模式。
    5. 使用KEGG搜索候选化合物 ( http://www.genome.jp/kegg/),NIST( http://webbook.nist.gov/chemistry/) 和KNApSAck(Shinbo等人,2006)。

笔记

  1. 为了获得可靠的可再现数据,必须使植物生长环境尽可能均匀/一致。 例如,根据光环境,植物叶绿素荧光的值将波动。 植物在靠近生长培养箱上的侧灯时接收的光的量与在搁架的中心上接收的光完全不同。 由于这个原因,重要的是洗牌生长板的位置,等等。
  2. 重要的是FT-ICRMS保持稳定的真空。我们还必须小心ESI喷嘴末端的样品锥的尺寸。同样重要的是,在必要时对ESI离子喷射的方向进行微小调整,以尽可能多地捕获离子进入ICR细胞。
  3. 当植物提取样品的浓度过高时,或当样品中包含其他不需要的物质时,FT-ICRMS可能会有问题。需要定期烘焙并从室内壁除去离子以及清洁离子源。

致谢

这项工作得到教育,文化,体育,科学和技术部(日本)[科学研究助理(No.17681022至R.M.)]的支持。
FT-ICR/MS(IonSpec)机器属于大阪府立大学,对于该项目,根据同一所大学的Ohta教授和Oikawa教授指示使用。数据标准化技术由Satou先生建立 该协议被修改和附加参考原始,如"Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins"(Satou等人,2014)。

参考文献

  1. Oikawa,A.,Nakamura,Y.,Ogura,T.,Kimura,A.,Suzuki,H.,Sakurai,N.,Shinbo,Y.,Shibata,D.,Kanaya,S.and Ohta, 2006)。 通过傅里叶变换离子回旋共振/基于质谱法的代谢表型研究来澄清途径特异性抑制。 Plant Physiol 142(2):398-413。
  2. Satou,M.,Enoki,H.,Oikawa,A.,Ohta,D.,Saito,K.,Hachiya,T.,Sakakibara,H.,Kusano,M.,Fukushima,A.,Saito, Kobayashi,M.,Nagata,N.,Myouga,F.,Shinozaki,K.and Motohashi,R。(2014)。 拟南芥白色或转绿色突变体的转录组和代谢组的综合分析被破坏的核编码叶绿体蛋白。 Plant Mol Biol 85(4-5):411-428。
  3. Shinbo,Y.,Nakamura,Y.,Altaf-Ul-Amin,M.,Asahi,H.,Kurokawa,K.,Arita,M.,Saito,K.,Ohta,D.,Shibata,D.and Kanaya ,S.(2006)。 KNApSAcK:一个全面的物种 - 代谢物关系数据库。 Biotech Agric Forest 57:165-181。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Motohashi, R., Satou, M., Myouga, F., Oikawa, A. and Ohta, D. (2015). Arabidopsis Metabolome Analysis Using Infusion ESI FT-ICR/MS. Bio-protocol 5(9): e1463. DOI: 10.21769/BioProtoc.1463.
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