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Metabolite Profiling of Mature Arabidopsis thaliana Seeds Using Gas Chromatography-Mass Spectrometry (GC-MS)
采用气相色谱质谱仪(GC-MS)分析成熟拟南芥种子代谢谱   

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参见作者原研究论文

本实验方案简略版
Plant Physiology
Nov 2014

 

Abstract

Metabolite profiling using gas chromatography-mass spectrometry (GC-MS) permits the annotation and quantification of a relatively wide variety of metabolites, covering a wide range of biochemical groups of metabolites. Lisec et al. (2006) established a method for GC-MS profiling in plants. Based on this protocol, we provide here a detailed GC-MS-based metabolite profiling protocol to identify compounds belonging to several biochemical groups in the primary metabolism of mature Arabidopsis thaliana seeds (Cohen et al., 2014). The protocol uses methoxyamine hydrochloride and N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) as derivatization reagents, as previous studies indicated these are the most appropriate compounds for profiling of plant metabolites. The protocol is relatively rapid, delivers reproducible results, and can be employed to profile metabolites of many other types of plant tissues with only minor modifications. In this context, developing seeds can serve as an excellent system for studying metabolic regulation, since during their development, a massive synthesis of reserve compounds occurs controlled under tight transcriptional regulation and associated with temporally distinct metabolic switches.

Materials and Reagents

  1. 2 ml microfuge safe seal lock-cap tubes (SARSTEDT, catalog number: 72-695-500 )
  2. 1.5 ml microfuge safe seal lock-cap tubes (SARSTEDT, catalog number: 72-690-001 )
  3. Mature dry Arabidopsis thaliana (ecotype Colombia-0) seeds
  4. Liquid nitrogen
  5. HPLC-grade methanol (Merck Millipore, catalog number: 67-56-1 )
  6. HPLC-grade chloroform (Merck Millipore, catalog number: 67-66-3 )
  7. HPLC-grade water (Avantor Performance Materials, J.T. Baker, catalog number: 7732-18-5 )
  8. Pyridine (Merck Millipore, catalog number: 110-86-1 )
  9. DL-norleucine internal standard (Sigma-Aldrich, catalog number: N1398 )
  10. Methoxyamine hydrochloride (Sigma-Aldrich, catalog number: 226904 )
  11. N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) (Sigma-Aldrich, catalog number: 24589-78-4 )
  12. n-alkanes mixture (see Recipes)
    n-Dodecane (C12) (Sigma-Aldrich, catalog number: 44010 )
    n-Pentadecane (C15) (Sigma-Aldrich, catalog number: 76509 )
    n-Octadecane (C18) (Sigma-Aldrich, catalog number: 74691 )
    n-Nonadecane (C19) (Sigma-Aldrich, catalog number: 74158 )
    n-Docosane (C22) (Sigma-Aldrich, catalog number: 43942 )
    n-Octacosane (C28) (Sigma-Aldrich, catalog number: 74684 )
    n-Dotriacontane (C32) (Sigma-Aldrich, catalog number: 44253 )
    n-Hexatriacontane (C36) (Sigma-Aldrich, catalog number: 52919 )
  13. Pre-cooled (4 °C) extraction buffer containing DL-norleucine internal standard solution (see Recipes)
  14. Methoxyamine hydrochloride derivatization reagent (see Recipes)
  15. n-alkanes time standards mix solution (see Recipes)
  16. N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) derivatization reagent + pre-mixed n-alkanes time standards (see Recipes)

Equipment

  1. Vacuum desiccator
  2. Liquid nitrogen dewar
  3. Table-top centrifuge (able to reach 20,817 x g at 4 °C) (Eppendorf)
  4. Table-top vortex (Benchmark Scientific Inc.)
  5. CentriVap benchtop centrifugal vacuum concentrator (Labconco)
  6. Benchtop multi-Therm heat-shaker (Benchmark Scientific Inc.)
  7. GC-MS system (Agilent Technologies, model: 7890A ) coupled with a mass selective detector and a Gerstel multipurpose sampler MPS2S (or any other GC-MS system)
  8. DB-5ms capillary column (30 m, 0.25 mm i.d., and 0.25 μm thickness) (or any other column suitable for primary metabolic profiles) (Agilent Technologies, catalog number: 122-5532G )
  9. VF-5ms capillary column (30 m + 10 m EZ-guard, 0.25 mm i.d., and 0.25 μm thicknesses) or any other column suitable for primary metabolic profiles (Agilent Technologies, catalog number: CP9013 )
  10. 10 mm Certified Clear Screw Thread Kit: 2 ml Thermo ScientificTM NationalTM 10 mm wide opening screw thread vials and inserts manufactured from clear glass + PTFE/silicone septa (Thermo Fisher Scientific, catalog number: CERT4010-91 ).
  11. 200 μl MicroSert glass inset (31 x 5 mm) (Thermo Fisher Scientific, catalog number: C4012-465 )
  12. 1 ml clear glass high recovery reaction vials with attached 13-425 open top phenolic screw-cap + PTFE/silicone septa (WHEATON, catalog number: W986294NG )
  13. 20 ml pre-assembled EPA vials + PTFE screw-caps (Thermo Fisher Scientific, catalog number: B7800-20 )

Procedure

  1. Extraction of metabolites from Arabidopsis thaliana seeds (Figures 1A1-1A8)
    1. Weight 20 mg of mature dry Arabidopsis thaliana seeds and place them into 2 ml lock-cap microfuge tubes (Figure 1A1). Seeds were harvested at full maturity at the end of desiccation period. Following collection, the seeds were allowed to fully-dry under vacuum desiccation for another 3 d. Please see Note 1 for seed stock, desiccation processes and replicate recommendations.
    2. Insert small metal grinding balls into each tube and place tubes in liquid nitrogen (Figure 1A2).
    3. Insert tubes into a 4 °C pre-cooled ball mill plastic adaptors and immediately place the adaptors in the ball mill. Homogenize samples for 4 min at 24 Hz and transfer the samples back into the liquid nitrogen (Figure 1A3).
    4. Add 1 ml of 4 °C pre-cooled extraction buffer containing the DL-norleucine internal standard (see Recipes) and vortex vigorously (Figure 1A4). Please see Note 2 for safety recommendations.
    5. Centrifuge samples on a 4 °C pre-cooled table-top centrifuge for 10 min at 20,817 x g. This step permits the separation of polar supernatant (methanol/water) and non-polar (chloroform) phases (Figure 1A5).
    6. Collect the supernatant into new 2 ml lock-cap microfuge tubes and add 300 μl of HPLC-grade chloroform and 300 μl of HPLC-grade water. Vortex vigorously each sample for 10 sec to allow water and chloroform thoroughly mixed, and centrifuge samples on a 4 °C pre-cooled table-top centrifuge for 10 min at 20,817 x g (Figure 1A6).
    7. Collect 300 μl aliquots from the supernatant into new 1.5 ml lock-cap microfuge tubes (Figure 1A7).
    8. At this stage, the 300 μl aliquots can be immediately transferred into -80 °C for future analyses without being derivatized (Figure 1A81). In case derivatization takes place, dry 300 μl aliquots using a CentriVap benchtop centrifugal vacuum concentrator for 5-6 h at 25 °C (Figure 1A82).


      Figure 1. Workflow of extraction metabolites from Arabidopsis thaliana seeds

  2. Derivatization using methoxyamine hydrochloride and N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) reagents (Figures 2B1-2B5)
    1. Methoxymate the dry aliquots by re-suspending the pellet in 40 μl of methoxyamine hydrochloride solution (see Recipe 2) (Figure 2B1).
      Note: Methoxyamine hydrochloride reagent is extremely toxic, thus, derivatization process must be handled with the highest care under the fume hood.
    2. Transfer the whole amount of samples into reaction vials (Figure 2B2).
    3. Incubate reaction vials for 2 h at 37 °C in order to protect carbonyl moieties (Figure 2B3).
    4. Trimethylsilylate acidic protons by adding 70 μl of N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) containing pre-mixed n-alkanes time standards (see Recipes) and incubate reaction vials for 30 min at 37 °C (Figure 2B4). The addition of n-alkanes allows the determination of retention time indices.
      Note: MSTFA reagent is extremely toxic, thus, derivatization process must be handled with the highest care under the fume hood.
    5. Cool reaction vials in room temperature and transfer the whole amount of samples (approximately 110 μl) into inserts placed in GC-MS running vials (Figure 2B5).


      Figure 2. Workflow of derivatization processes

  3. GC-MS analysis (Figure 3C)
    1. Analyses were carried out on a GC-MS system (Agilent, model: 7890A) coupled with a mass selective detector (Agilent, model: 5975c) and a Gerstel multipurpose sampler MPS2S (Figure 3C). The analysis was performed under the conditions described in Table 1, whereby the DB-5ms capillary column (together with a duraguard, DG) were used to quantify soluble and protein-bound amino acids, and the VF-5ms capillary column was used to quantify sugars, sugar acids, sugar alcohols, tricarboxylic acid (TCA) cycle intermediates and organic acids. The VF-5ms capillary column was used as it can reach a higher temperature, thus, allowing better separation of metabolites within chromatographs.



      Figure 3. Workflow of GC-MS running and Data analysis

      Table 1. GC-MS metabolite profiling conditions


  4. Data analysis (Figure 3D)
    1. Peak finding, peak integration and retention time correction were performed with the Agilent GC/MSD Productivity ChemStation package (http://www.agilent.com).
    2. The corresponding mass spectra and retention time indices were compared with standard substances and commercially available electron mass spectrum libraries available from the National Institute of Standards and Technology (http://www.nist.gov/) and Max Planck Institute for Plant Physiology, Golm, Germany (http://www.mpimp-golm.mpg.de/).
    3. Integrated peaks of mass (m/z) fragments were normalized against the DL-norleucine internal standard signal. The relative peaks of the DL-norleucine internal standard should be of similar height/area among all samples (see representative ion chromatograms appear in Figure 4).
    4. Following internal standard normalization, anomalous values were excluded from the metabolite dataset based on the calculated relative peak area of every metabolite. Representative total-ion-chromatograms (TIC) appear in Figure 5 shows differential peak areas from overlaid chromatograms from seeds of wild-type Arabidopsis thaliana (Colombia-0) against two transgenic lines.


      Figure 4. Representative ion chromatograms of wild-type Arabidopsis thaliana seeds (ecotype Colombia-0) and seeds from two transgenic lines. Ion chromatograms (large square) show similar peaks underlying the characteristic 158 m/z mass fragment (small square) of the DL-norleucine internal standard.


      Figure 5. Representative total-ion-count (TIC) chromatograms of wild-type Arabidopsis thaliana seeds (ecotype Colombia-0) and seeds from two transgenic lines. Differences are shown in various peaks between the wild-type and transgenic lines.

Notes

  1. In the case of Arabidopsis, mature dry seeds can be collected from fully-matured plants at the end of desiccation, usually 4-6 weeks after germination on soil (depending on irrigation light and humidity growing conditions). WT ecotypes and mutant lines can be ordered by several different public seed collections such as the SALK Institute for Biological Studies (http://www.salk.edu/) or RIKEN (http://www.riken.jp/en/). The availability of Arabidopsis T-DNA mutant lines can be checked in the websites of T-DNA Express: Arabidopsis Gene Mapping Tool (http://signal.salk.edu/cgi-bin/tdnaexpress) or The Arabidopsis Information Resource (https://www.arabidopsis.org/). We recommend desiccating seeds under vacuum as desiccation under heat in an oven might induce too harsh conditions triggering some metabolic changes. We recommend using at least 5 biological replicates from each genotype/treatment. Apart from biological replicates, at least one blank sample containing no biological material should be prepared and subjected to all extraction and derivatization protocols.
  2. Extraction buffer and both methoxyamine hydrochloride and MSTFA derivatization reagents are extremely toxic, thus, apart for seeds sampling and weighting procedures, all extraction and derivatization processes must be performed with extreme care under fume hood.

Recipes

  1. Pre-cooled (4 °C) extraction buffer containing DL-norleucine internal standard solution
    1. Prepare a 2.5:1:1 mixture of HPLC-grade methanol, HPLC-grade chloroform and HPLC-grade water (v:v:v). The extraction buffer must be prepared freshly before the extraction.
    2. For preparation of DL-norleucine internal standard solution, dissolve 2 mg of DL-norleucine in 1 ml of HPLC-grade water and vortex vigorously. Mix 7 μl of internal standard per 1 ml of extraction buffer.
    3. Internal standard tube should be stored at -20 °C when unused. The internal standard allows the normalization of peaks in case of material loss during extraction procedures.
  2. Methoxyamine hydrochloride derivatization reagent
    1. Dissolve 20 mg methoxyamine hydrochloride in 1 ml of pyridine at room temperature.
    2. This reagent needs to be prepared freshly before the experiment.
  3. n-alkanes time standards mix solution
    1. Dissolve 2 μl or 2 mg of soluble or solid n-alkane, respectively, in 1 ml of pyridine in separate 20 ml glass vials with Teflon screw-caps (Table 2).
    2. For the preparation of n-alkanes time standards mix solution, transfer 500 μl from each C12 to C32 n-alkane solutions and 1,000 μl from the C36 n-alkane solution (as the C36 peak can be observed better at this concentration) into a new 20 ml glass vial and vortex vigorously (Table 2).
    3. All separate n-alkane vials and n-alkanes time standards mix solution vial should be stored at -20 °C when unused.

      Table 2. Preparation of n-alkanes time standards mix solution


  4. N-methyl-N-trimethylsilyltriflouroacetamide (MSTFA) derivatization reagent + pre-mixed n-alkanes time standards
    Calculate the amount of MSTFA needed for the analysis (70 μl per sample) and add 7 μl per 70 μl MSTFA of pre-mixed n-alkanes time standards mix solution directly into the MSTFA mixture. n-alkanes time standards mix solution should be gently warmed up at 70 °C prior addition into the MSTFA mixture. 
    Note: This reagent needs to be prepared freshly before the experiment. For example: an experiment of 50 samples would require 3.5 ml of MSTFA (70 μl x 50 samples) mixed with 350 μl of pre-warmed n-alkanes time standards mix solution (7 μl x 50 samples).

Acknowledgments

This is a detailed protocol of the analyses performed in Cohen et al. (2014). Both are fundamentally based on Lisec et al. (2006) with modifications made to profile Arabidopsis thaliana seeds. We would like to acknowledge the Israel Science Foundation for supporting this research (ISF grants #231-09 and #1004/15).

References

  1. Cohen, H., Israeli, H., Matityahu, I. and Amir, R. (2014). Seed-specific expression of a feedback-insensitive form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis stimulates metabolic and transcriptomic responses associated with desiccation stress. Plant Physiol 166(3): 1575-1592.
  2. Lisec, J., Schauer, N., Kopka, J., Willmitzer, L. and Fernie A.R. (2006). Gas chromatography mass spectrometry-based metabolite profiling in plants. Nat Protoc 1(1):387-396.

简介

使用气相色谱 - 质谱(GC-MS)的代谢物分析允许注释和量化相对多种代谢物,涵盖广泛的代谢物的生物化学组。 Lisec等人。 (2006)建立了一种在植物中进行GC-MS分析的方法。基于该方案,我们在这里提供详细的基于GC-MS的代谢物分析方案以鉴定属于成熟拟南芥种子的主要代谢中的几个生物化学基团的化合物(Cohen等人。,2014)。该方案使用甲氧基胺盐酸盐和N,N-甲基-N,N-三甲基甲硅烷基三氟乙酰胺(MSTFA)作为衍生化试剂,如先前的研究所表明的,这些是用于分析植物代谢物的最合适的化合物。该方案相对快速,提供可重复的结果,并且可以用于简化许多其它类型的植物组织的代谢物,只有很少的修改。在这种情况下,开发种子可以作为研究代谢调节的优秀系统,因为在它们的开发期间,储备化合物的大规模合成在严格的转录调节下发生控制并且与时间上不同的代谢开关相关。

材料和试剂

  1. 2 ml microfuge安全密封锁帽管(SARSTEDT,目录号:72-695-500)
  2. 1.5 ml microfuge安全密封锁帽管(SARSTEDT,目录号:72-690-001)
  3. 成熟干种拟南芥(哥伦比亚生态型-0)种子
  4. 液氮
  5. HPLC级甲醇(Merck Millipore,目录号:67-56-1)
  6. HPLC级氯仿(Merck Millipore,目录号:67-66-3)
  7. HPLC级水(Avantor Performance Materials,J.T.Baker,目录号:7732-18-5)
  8. 吡啶(Merck Millipore,目录号:110-86-1)
  9. DL-正亮氨酸内标(Sigma-Aldrich,目录号:N1398)
  10. 甲氧基胺盐酸盐(Sigma-Aldrich,目录号:226904)
  11. N,N-三甲基甲硅烷基三氟乙酰胺(MSTFA)(Sigma-Aldrich,目录号:24589-78-4)
  12. n - 烷烃混合物(参见配方)
    十二烷(C12)(Sigma-Aldrich,目录号:44010) - 十五烷(C15)(Sigma-Aldrich,目录号:76509)
    正十八烷(C18)(Sigma-Aldrich,目录号:74691)
    - 十九烷(C19)(Sigma-Aldrich,目录号:74158)
    - 二十二烷(C22)(Sigma-Aldrich,目录号:43942)
    (C28)(Sigma-Aldrich,目录号:74684)
    三十二烷(C32)(Sigma-Aldrich,目录号:44253)
    n - 高三十烷(C36)(Sigma-Aldrich,目录号:52919)
  13. 含有DL-正亮氨酸内标溶液(见配方)的预冷(4℃)提取缓冲液
  14. 甲氧胺盐酸盐衍生试剂(参见配方)
  15. n - 烷烃时间标准混合解决方案(参见配方)
  16. N-甲基-N,N-三甲基甲硅烷基三氟乙酰胺(MSTFA)衍生化试剂+预混合的正 - 烷烃时间标准(参见配方)。

设备

  1. 真空干燥器
  2. 液氮杜瓦瓶
  3. 台式离心机(能够在4℃达到20,817×g)(Eppendorf)
  4. 桌面涡流(Benchmark Scientific Inc.)
  5. CentriVap台式离心真空浓缩器(Labconco)
  6. 台式多Therm热摇床(Benchmark Scientific Inc.)
  7. 与质量选择检测器和Gerstel多用途取样器MPS2S(或任何其他GC-MS系统)相连的GC-MS系统(Agilent Technologies,型号:7890A)
  8. DB-5ms毛细管柱(30m,0.25mm i.d.和0.25μm厚度)(或任何其他适用于初级代谢图谱的柱)(Agilent Technologies,目录号:122-5532G)
  9. VF-5ms毛细管柱(30m + 10m EZ-guard,0.25mm i.d.和0.25μm厚度)或适用于初级代谢图谱的任何其它柱(Agilent Technologies,目录号:CP9013)
  10. 10 mm认证的清除螺纹螺纹套件:2毫升Thermo Scientific 国家 TM 10毫米宽开口螺纹小瓶和由透明玻璃+ PTFE /硅胶隔片制造的插入件(Thermo Fisher科学,目录号:CERT4010-91)。
  11. 200μlMicroSert玻璃插入物(31×5mm)(Thermo Fisher Scientific,目录号:C4012-465)
  12. 1 ml透明玻璃高回收反应瓶,附有13-425开口酚醛螺旋盖+ PTFE /硅胶隔片(WHEATON,目录号:W986294NG)
  13. 20ml预装配的EPA小瓶+ PTFE螺旋盖(Thermo Fisher Scientific,目录号:B7800-20)

程序

  1. 从拟南芥种子中提取代谢物(图1A1-1A8)
    1. 重量20mg成熟干种拟南芥种子,并将其放入2ml锁帽微量离心管中(图1A1)。在干燥期结束时完全成熟收获种子。收集后,使种子在真空干燥下完全干燥另外3天。请参见注1,了解种子原料,干燥过程和重复建议。
    2. 将小金属研磨球插入每个管中,并将管置于液氮中(图1A2)
    3. 将管插入4°C预冷球磨塑料适配器,并立即将适配器放置在球磨机中。在24Hz下均匀化样品4分钟,并将样品转移回液氮(图1A3)。
    4. 加入1ml含有DL-正亮氨酸内标(见Recipes)的4℃预冷却的提取缓冲液并剧烈涡旋(图1A4)。有关安全建议,请参阅注释2
    5. 在4℃预冷却的台式离心机上在20,817×g离心样品10分钟。该步骤允许分离极性上清液(甲醇/水)和非极性(氯仿)相(图1A5)。
    6. 收集上清液到新的2毫升锁帽微量离心管,加入300微升的HPLC级氯仿和300微升的HPLC级水。大力涡旋每个样品10秒,以使水和氯仿充分混合,并在4℃预冷却的台式离心机上在20,817×g离心10分钟(图1A6)。
    7. 从上清液收集300微升等分试样到新的1.5毫升锁帽微量离心管(图1A7)。
    8. 在这个阶段,300μl等分试样可以立即转移到-80℃用于未衍生化的分析(图1A8 1)。在发生衍生化的情况下,使用CentriVap台式离心真空浓缩器在25℃下干燥300μl等分试样5-6小时(图1A8 2)。


      图1.拟南芥种子提取代谢物的工作流程

  2. 使用甲氧胺盐酸盐和N,N-甲基-N'-三甲基甲硅烷基三氟乙酰胺(MSTFA)试剂(图2B1-2B5)进行衍生化
    1. 通过将沉淀重悬于40μl甲氧胺盐酸盐溶液中(见配方2)(图2B1),将干燥的等分试样甲氧基化。
      注意:甲氧基胺盐酸盐试剂是极毒性的,因此,衍生化过程必须在通风橱中以最高的谨慎处理。
    2. 将全部样品转移到反应瓶中(图2B2)。
    3. 孵育反应瓶在37℃下2小时,以保护羰基部分(图2B3)。
    4. 通过加入70μl含有预混合的正 - 烷烃时间标准的N,N-甲基-N,N-三甲基甲硅烷基三氟乙酰胺(MSTFA)来处理三甲基甲硅烷基化物酸性质子Recipes)并在37℃下孵育反应小瓶30分钟(图2B4)。添加n n - 烷烃允许确定保留时间指数。
      注意:MSTFA试剂毒性极大,因此,必须在通风橱内最高度谨慎地处理衍生化过程。
    5. 在室温下冷却反应瓶,将全部样品(约110μl)转移到置于GC-MS运行瓶中的插入物中(图2B5)。


      图2.衍生过程的工作流程

  3. GC-MS分析(图3C)
    1. 在与质量选择性检测器(Agilent,型号:5975c)和Gerstel多用途采样器MPS2S(图3C)偶联的GC-MS系统(Agilent,型号:7890A)上进行分析。在表1所述的条件下进行分析,由此使用DB-5ms毛细管柱(与duraguard,DG一起)来量化可溶性和蛋白质结合的氨基酸,并且使用VF-5ms毛细管柱来定量糖,糖酸,糖醇,三羧酸(TCA)循环中间体和有机酸。使用VF-5ms毛细管柱,因为它可以达到更高的温度,从而允许在色谱仪中更好地分离代谢物。



      图3. GC-MS运行和数据分析的工作流程

      表1. GC-MS代谢物分析条件


  4. 数据分析(图3D)
    1. 使用Agilent GC/MSD Productivity ChemStation软件包进行峰检测,峰积分和保留时间校正( http ://www.agilent.com )。
    2. 将相应的质谱和保留时间指数与可从国家标准和技术研究所获得的标准物质和市售电子质谱库进行比较( http://www.nist.gov/)和马克斯普朗克植物生理学研究所,德国( http://www.mpimp-golm.mpg.de/)。
    3. 将整合的质量峰(m/z)片段针对DL-正亮氨酸内标信号进行标准化。 DL-正亮氨酸内标的相对峰在所有样品中应具有相似的高度/面积(参见图4中代表性的离子色谱图)。
    4. 在内标标准化之后,基于计算的每种代谢物的相对峰面积,从代谢物数据集中排除异常值。图5中显示的代表性总离子色谱(TIC)显示来自野生型拟南芥(Colombia-0)的种子与两个转基因品系的重叠色谱图的差异峰面积。


      图4.野生型拟南芥种子(生态型哥伦比亚-0)和来自两个转基因品系的种子的代表性离子色谱图。 离子色谱图(大正方形)显示DL-正亮氨酸内标的特征性158m/z 质量片段(小正方形) />

      图5.野生型拟南芥种子(生态型哥伦比亚-0)和来自两个转基因品系的种子的代表性总离子计数(TIC)色谱图。显示了差异在野生型和转基因品系之间的各种峰

笔记

  1. 在拟南芥的情况下,可以在干燥结束时,通常在土壤上发芽后4-6周(取决于灌溉光和湿度生长条件)从完全成熟的植物收集成熟干种子。 WT生态型和突变体系可以通过若干不同的公共种子收集物进行排序,例如SALK生物研究所( http://www.riken.jp/en/)。可以在T-DNA Express:Arabidopsis基因映射工具( http://signal.salk.edu/cgi-bin/tdnaexpress )或拟南芥信息资源( https://www.arabidopsis.org/) )。我们建议在真空下干燥种子,在烘箱中加热干燥可能会诱发过于苛刻的条件,引发一些代谢变化。我们建议每个基因型/治疗至少使用5个生物学重复。除了生物复制品,至少应制备一个不含生物材料的空白样品,并进行所有提取和衍生化方案。
  2. 提取缓冲液和甲氧胺盐酸盐和MSTFA衍生化试剂都是极其有毒的,因此,除了种子取样和加重程序外,所有的提取和衍生化过程必须在通风橱下非常小心地进行。

食谱

  1. 含有DL-正亮氨酸内标溶液的预冷(4℃)提取缓冲液
    1. 制备HPLC级甲醇,HPLC级氯仿和HPLC级水(v:v:v)的2.5:1:1混合物。提取缓冲液必须在提取前新鲜制备。
    2. 为制备DL-正亮氨酸内标溶液,将2mg DL-正亮氨酸溶于1ml HPLC级水中并剧烈涡旋。混合7μl内标每1毫升提取缓冲液。
    3. 内部标准管在未使用时应储存在-20°C。内部标准允许在萃取过程中材料损失的情况下峰的归一化
  2. 甲氧胺盐酸盐衍生化试剂
    1. 在室温下将20mg甲氧基胺盐酸盐溶于1ml吡啶中
    2. 该试剂需要在实验前新鲜配制
  3. n - 烷烃时间标准混合解决方案
    1. 在具有Teflon螺旋盖的单独的20ml玻璃小瓶中(表2),将2μl或2mg的可溶性或固体n-烷烃分别溶解在1ml吡啶中。
    2. 为了制备 n' - 烷烃时间标准混合溶液,将500μl从各C 12至C 32正链烷烃溶液转移至500μl,并从C 36 em- (在该浓度下可以更好地观察到C 36峰)加入新的20ml玻璃瓶中并剧烈涡旋(表2)。
    3. 所有单独的 n - 烷烃瓶和 n -alkanes时间标准混合溶液瓶在未使用时应储存在-20°C。

      表2.准备 - 烷烃时间标准混合解决方案


  4. N-甲基-N,N-三甲基甲硅烷基三氟乙酰胺(MSTFA)衍生化试剂+预混合的正 - 烷烃时间标准
    计算分析所需的MSTFA的量(每个样品70μl),并将7μl每70μlMSTFA预混合的正 - 烷烃时间标准混合溶液直接加入MSTFA混合物中。 n - 烷烃时间标准混合溶液应在70℃下轻轻预热,然后加入MSTFA混合物。
    注意:该试剂需要在实验前新鲜配制。例如:50个样品的实验需要3.5ml的MSTFA(70μl×50个样品)与350μl的预热的正烷烃时间标准混合溶液(7μl×50个样品)混合。

致谢

这是在Cohen等人中进行的分析的详细方案。 (2014年)。两者基本上都是基于Lisec 。 (2006),其中对拟南芥种子进行了修饰。我们要感谢以色列科学基金会支持这项研究(ISF拨款#231-09和#1004/15)。

参考文献

  1. Cohen,H.,Israel,H.,Matityahu,I.和Amir,R。(2014)。  拟南芥中CYSTATHIONINE-γ-SYNTHASE的反馈不敏感形式的种子特异性表达刺激与干燥胁迫相关的代谢和转录组反应。 植物生理学 166(3):1575-1592
  2. Lisec,J.,Schauer,N.,Kopka,J.,Willmitzer,L。和Fernie A.R. (2006)。  基于气相色谱质谱法的代谢物分析在植物中。 Nat Protoc 1(1):387-396
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cohen, H., Matityahu, I. and Amir, R. (2016). Metabolite Profiling of Mature Arabidopsis thaliana Seeds Using Gas Chromatography-Mass Spectrometry (GC-MS). Bio-protocol 6(21): e1981. DOI: 10.21769/BioProtoc.1981.
  2. Cohen, H., Israeli, H., Matityahu, I. and Amir, R. (2014). Seed-specific expression of a feedback-insensitive form of CYSTATHIONINE-γ-SYNTHASE in Arabidopsis stimulates metabolic and transcriptomic responses associated with desiccation stress. Plant Physiol 166(3): 1575-1592. 
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