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Quantification of Low Molecular Weight Thiols in Arabidopsis
拟南芥中低分子量硫醇的定量测定   

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Abstract

Low-molecular-weight (LMW) thiols are a class of highly reactive compounds due to their thiol moiety. They play important roles in the maintenance of cellular redox homeostasis, detoxification, and development. Monobromobimane (mBBr) is weakly fluorescent but selectively reacts with thiols to yield highly fluorescent thioethers (mBSR) products, which is especially useful for the quantification of LMW thiols. The stable mBSR products can be separated by high-performance liquid chromatography (HPLC) equipped with a fluorescent detector. The main cellular LMW thiols are L-cysteine, gamma-glutamylcysteine, and glutathione (GSH). The following protocol describes the extraction and quantification of L-cysteine, gamma-glutamylcysteine, and glutathione from Arabidopsis tissues as reported (Xiang and Oliver, 1998; Zhao et al., 2014; Wang et al., 2015) with minor revision. Modifications may be required if the HPLC system or the C18 column is different.

Keywords: Cysteine(半胱氨酸), Glutathione(谷胱甘肽), Arabidopsis(拟南芥), HPLC(高效液相色谱法), Fluorescent(荧光)

Materials and Reagents

  1. 1.5 ml microcentrifuge tube
  2. 0.2 μm nylon filter (Sigma-Aldrich, catalog number: Z259969 )
  3. 100 ml glass syringe
  4. Pipette tip
  5. Fresh Arabidopsis thaliana tissues (any plant part you want to test, 50-100 mg is sufficient)
  6. L-cysteine (Sigma-Aldrich, catalog number: 778672 )
  7. Gamma-glutamylcysteine (γ-Glu-Cys) (Sigma-Aldrich, catalog number: G0903 )
  8. L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  9. Double distilled water (supplied by School of Life Sciences, University of Science and Technology of China)
  10. Double distilled water
  11. Hydrochloric Acid (HCl) (Sigma-Aldrich, catalog number: 435570 ) (see Recipes)
  12. 2-(N-Morpholino)ethanesulfonic acid hydrate (MES) (Sigma-Aldrich catalog number: M8250 ) (see Recipes)
  13. Ethylene diaminetetraacetic acid disodium salt dihydrate (EDTA.2Na.2H2O) (Sangon Biotech, catalog number: A100105 ) (see Recipes)
  14. Monobromobimane (mBBr) (Sigma-Aldrich, catalog number: 69898 ) (see Recipes)
  15. Trifluoroacetic acid (TFA) (Sigma-Aldrich, catalog number: 302031 ) (see Recipes)
  16. Acetonitrile (OCEANPAK, catalog number: Ac00030281 ) (see Recipes)

Equipment

  1. Analytic balance (Mettler-Toledo International Inc., model: ML104 )
  2. Mortar and Pestle
  3. Refrigerated microcentrifuge (Thermo Fisher Scientific, Eppendorf, model: 5424R )
  4. 37 °C degree incubator (Shanghai Jinghong Laboratory Instrument, model: GNP-9080 )
  5. Reverse-phase C18 column (5 µm, 110A, 150 x 4.6 mm) (Phenomenex, model: Gemini C18 column ) or equivalent, guard column (Phenomenex, SecurityGuard Standard, model: AJ0-7597 )
  6. HPLC equipment (Agilent Technologies, model: 1200 series )
    1. Pump (Agilent Technologies, model: G1312A )
    2. Sampler (Agilent Technologies, model: G1328A )
    3. Column heater (Agilent Technologies, model: G1316A )
    4. FLD detector (Agilent Technologies, model: G1321A )
  7. 200 μl pipette
  8. pH meter (Thermo Fisher Scientific, Mettler Toledo, model: FE20–FiveEasy PlusTM )
  9. Ultrasonic cleaner (Shanghai Sonxi Ultrasonic Instrument, model: DS-2510DT )

Software

  1. Suitable data collection and processing software (Agilent Technologies, model: 1200 ChemStation)
  2. Standard curve plotting (Microsoft Excel)

Procedure

  1. Preparation of samples and standards
    1. Fresh tissues are sampled into a 1.5 ml microcentrifuge tube and weighed with an analytical balance. 50-100 mg is sufficient for each biological sample.
    2. Samples are ground in the microcentrifuge tube with a mortar and a pestle with 2 volumes of 0.15 M HCl added by 200 μl pipette (for example, 100 mg tissue needs 200 μl).
    3. The homogenate is centrifuged at 12,000 x g, 4 °C, for 15 min and the supernatant is transferred into a new 1.5 ml microcentrifuge tube. This step can be repeated if necessary to remove insoluble substances.
    4. Prepare 10 mM stock solutions of L-cysteine, gamma-glutamylcysteine, and glutathione, and then prepare the following standards: 0, 50, 100, 200, 500, 1,000 μM of L-cysteine, gamma-glutamylcysteine, and glutathione by diluting the stocks with 0.15 M HCl.
    5. 100 μl of the supernatant from step A3 (or standards) is transferred to 1.5 ml microcentrifuge tubes containing 2 μl of 0.5 M EDTA, 2.6 μl of 300 mM mBBr in acetonitrile, and 100 μl of 1.75 M MES (pH 7.4). The mixture was incubated in the dark at 37 °C for 1 h to allow the derivatization reaction to completed.
    6. The mixture is centrifuged as in step A3 for 5 min before quantification.

  2. Quantification by HPLC (refer to Agilent 1200 HPLC ChemStation Operation)
    Select “Edit entire method” from “Method” menu and set parameters according to the following text. For quantification, 50 μl sample from step A6 is injected into the sample chamber of the HPLC system and separated using a reverse-phase C18 column and a flow rate of 0.8 ml/min on an Agilent 1200 HPLC system. Solvents A and B are used to elute the fluorescent derivatives with the gradient shown in the table below. The fluorescent derivatives (mBSR) are detected using fluorescence detector with excitation wavelength at 260 nm and emission at wavelength 474 nm.

    Table 1. HPLC elution program
    Time (min)
    A (%)
    B (%)
    Rate of flow (ml/min)
    0.0
    90
    10
    0.8
    0.3
    85
    15
    0.8
    14.0
    80
    20
    0.8
    15.0
    0
    100
    0.8
    19.0
    0
    100
    0.8
    20.0
    90
    10
    0.8
    24.0
    90
    10
    0.8

    1. Analyze standards and samples using the HPLC program above with at least 3 replicates.
    2. Peak areas are integrated using the ChemStation software.
      Select “Data analysis” from the “View” menu to enter the picture data analysis.
      Select “Load signal” from the “File” menu to select your data file.
      Select “Integrate” from the “Integration” menu, and then the data is integrated.

  3. Calculations
    1. Prepare standard curves by plotting the concentration (μM) of the standards (Y-axis) vs the peak areas (X-axis) and add a trendline.
    2. Determine the peak area for each sample and determine concentration using the trendline. Concentration (μM) = a * peak area + b (a and b are already calculated in the trendline).

Recipes

  1. 0.15 M HCl (stored at room temperature)
    1.27 ml HCl (37%)
    100 ml double distilled water
    Filtered through 0.2 μm nylon filter using 100 ml syringe
  2. 1.75 M MES (pH 7.4) (stored in 4 °C)
    3.41 g MES
    10 ml double distilled water
    Adjust pH to 7.4 using NaOH and filter through 0.2 μm nylon filter using 100 ml syringe
  3. 0.5 M EDTA.2Na.2H2O (stored in room temperature)
    18.6 g EDTA.2Na.2H2O
    Dissolved in double distilled water, pH adjusted to 8.0 with NaOH, and volume brought to100 ml
    Filtered through 0.2 μm nylon filter using 100 ml syringe
  4. 300 mM monobromobimane (stored in -20 °C)
    0.025 g monobromobimane
    307.3 μl acetonitrile
    12,000 x g, 4 °C, 30min to remove insoluble substances
  5. Solvent A: 0.1% (v/v) trifluoroacetic acid (HPLC grade) (freshly prepared)
    1 ml trifluoroacetic acid
    999 ml double distilled water
    Filtered through 0.2 μm nylon filter using 100 ml syringe
  6. Solvent B: 90% (v/v) acetonitrile (HPLC grade) (freshly prepared)
    900 ml 100% acetonitrile
    100 ml 0.1% (v/v) trifluoroacetic acid
    Filtered through 0.2 μm nylon filter using 100 ml syringe
    Solvent A and Solvent B need degas in the ultrasonic cleaner for 30 min with loose lid
  7. 10 mM L-cysteine (stored in -20 °C)
    0.0606 g L-cysteine
    50 ml 0.15 M HCl
  8. 10 mM gamma-glutamylcysteine (stored in -20 °C)
    0.1251 g gamma-glutamylcysteine
    50 ml 0.15 M HCl
  9. 10 mM glutathione (stored in -20 °C)
    0.1537 g glutathione
    50 ml 0.15 M HCl
  10. Standards (stored in -20 °C)

    0 μM
    50 μM
    100 μM
    200 μM
    500 μM
    1,000 μM
    10 mM
    0 μl
    5 μl
    10 μl
    20 μl
    50 μl
    100 μl
    0.15 M HCl
    1,000 μl
    995 μl
    990 μl
    980 μl
    950 μl
    900 μl

Acknowledgments

This protocol was modified from previous work described by Fahey and Newton (1987).

References

  1. Fahey, R. C. and Newton, G. L. (1987). Determination of low-molecular-weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Methods Enzymol 143: 85-96.
  2. Wang, Z., Mao, J. L., Zhao, Y. J., Li, C. Y. and Xiang, C. B. (2015). L-Cysteine inhibits root elongation through auxin/PLETHORA and SCR/SHR pathway in Arabidopsis thaliana. J Integr Plant Biol 57(2): 186-197.
  3. Xiang, C. and Oliver, D. J. (1998). Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis. Plant Cell 10(9): 1539-1550.
  4. Zhao, Q., Wu, Y., Gao, L., Ma, J., Li, C. Y. and Xiang, C. B. (2014). Sulfur nutrient availability regulates root elongation by affecting root indole-3-acetic acid levels and the stem cell niche. J Integr Plant Biol 56(12): 1151-1163.

简介

低分子量(LMW)硫醇是一类高反应性化合物,由于它们的硫醇部分。 它们在维持细胞氧化还原稳态,解毒和发育中起重要作用。 单溴溴烷(mBBr)是弱荧光的,但选择性地与硫醇反应产生高荧光硫醚(mBSR)产物,其对于定量LMW硫醇特别有用。 稳定的mBSR产物可以通过配备有荧光检测器的高效液相色谱(HPLC)分离。 主要的细胞LMW硫醇是L-半胱氨酸,γ-谷氨酰半胱氨酸和谷胱甘肽(GSH)。 以下方案描述了从所报道的拟南芥组织中提取和定量L-半胱氨酸,γ-谷氨酰半胱氨酸和谷胱甘肽(Xiang和Oliver,1998; Zhao等人, 2014; Wang ,,2015)。 如果HPLC系统或C18柱不同,可能需要进行修改。

关键字:半胱氨酸, 谷胱甘肽, 拟南芥, 高效液相色谱法, 荧光

材料和试剂

  1. 1.5 ml微量离心管
  2. 0.2μM尼龙过滤器(Sigma-Aldrich,目录号:Z259969)
  3. 100毫升玻璃注射器
  4. 移液管头
  5. 新鲜拟南芥组织(要测试的任何植物部分,50-100mg就足够了)
  6. L-半胱氨酸(Sigma-Aldrich,目录号:778672)
  7. γ-谷氨酰半胱氨酸(γ-Glu-Cys)(Sigma-Aldrich,目录号:G0903)
  8. L谷胱甘肽还原(Sigma-Aldrich,目录号:G4251)
  9. 双蒸水(由中国科学技术大学生命科学学院提供)
  10. 双蒸水
  11. 盐酸(HCl)(Sigma-Aldrich,目录号:435570)(参见Recipes)
  12. 2-(N-吗啉代)乙磺酸水合物(MES)(Sigma-Aldrich目录号:M8250)(参见配方)
  13. 乙二胺四乙酸二钠盐二水合物(EDTA 2Na 2H O)(Sangon Biotech,目录号:A100105) br />
  14. 单溴甲烷(mBBr)(Sigma-Aldrich,目录号:69898)(参见配方)
  15. 三氟乙酸(TFA)(Sigma-Aldrich,目录号:302031)(参见Recipes)
  16. 乙腈(OCEANPAK,目录号:Ac00030281)(参见配方)

设备

  1. 分析天平(Mettler-Toledo International Inc.,型号:ML104)
  2. 砂浆和杵
  3. 冷冻微量离心机(Thermo Fisher Scientific,Eppendorf,型号:5424R)
  4. 37℃培养箱(上海景洪实验室仪器,型号:GNP-9080)

  5. 反相C18柱(5μm,110A,150 x 4.6 mm)(Phenomenex,型号:Gemini C18柱)或同等级,保护柱(Phenomenex,SecurityGuard Standard,型号:AJ0-7597)
  6. HPLC设备(Agilent Technologies,型号:1200系列)
    1. 泵(Agilent Technologies,型号:G1312A)
    2. 采样器(Agilent Technologies,型号:G1328A)
    3. 柱加热器(Agilent Technologies,型号:G1316A)
    4. FLD检测器(Agilent Technologies,型号:G1321A)
  7. 200μl移液器
  8. pH计(Thermo Fisher Scientific,Mettler Toledo,型号:FE20-FiveEasy Plus TM
  9. 超声波清洗机(上海松士超声波仪器,型号:DS-2510DT)

软件

  1. 合适的数据收集和处理软件(Agilent Technologies,型号:1200化学工作站)
  2. 标准曲线绘图(Microsoft Excel)

程序

  1. 样品和标准品的制备
    1. 将新鲜组织取样到1.5ml微量离心管中并称重 ?与分析天平。 50-100mg对于每种生物学是足够的 ?样品
    2. 样品在微量离心管中用a研磨 研钵和杵用2体积的0.15M HCl通过200μl移液管加入 ?(例如,100毫克组织需要200微升)
    3. 匀浆 在12,000×g,4℃下离心15分钟,并且上清液 转移到新的1.5ml微量离心管中。这一步可以 如果需要重复,以除去不溶性物质。
    4. 准备10 ?mM L-半胱氨酸,γ-谷氨酰半胱氨酸的储备溶液 谷胱甘肽,然后制备以下标准:0,50,100,200, 500μM,1000μM的L-半胱氨酸,γ-谷氨酰半胱氨酸和谷胱甘肽 用0.15M HCl稀释储备液。
    5. 100μl上清液 从步骤A3(或标准品)转移至1.5ml微量离心机 含有2μl0.5M EDTA,2.6μl300mM mBBr的管, 乙腈和100μl的1.75M MES(pH 7.4)。混合物 在黑暗中在37℃孵育1小时以允许衍生化 反应完成。
    6. 将混合物如步骤A3中离心5分钟,然后定量

  2. HPLC定量(参见Agilent 1200 HPLC化学工作站操作)
    从"方法"菜单中选择"编辑整个方法",并根据以下文本设置参数。为了定量,将来自步骤A6的50μl样品注入HPLC系统的样品室中,并使用反相C18柱并在Agilent 1200 HPLC系统上以0.8ml/min的流速分离。溶剂A和B用于以如下表所示的梯度洗脱荧光衍生物。使用荧光检测器检测荧光衍生物(mBSR),激发波长为260nm,发射波长为474nm。

    表1. HPLC洗脱程序
    时间(分)
    A(%)
    B(%)
    流速(ml/min)
    0.0
    90
    10
    0.8
    0.3
    85
    15
    0.8
    14.0
    80
    20
    0.8
    15.0
    0
    100
    0.8
    19.0
    0
    100
    0.8
    20.0
    90
    10
    0.8
    24.0
    90
    10
    0.8

    1. 使用上述HPLC程序分析标准品和样品,至少重复3次。
    2. 使用ChemStation软件对峰面积进行积分。
      从"查看"菜单中选择"数据分析",进入图像数据分析。
      从"文件"菜单中选择"加载信号"以选择数据文件。
      从"积分"菜单中选择"积分",然后积分数据。

  3. 计算
    1. 通过绘制标准曲线的浓度(μM) 标准(Y轴)峰值区域(X轴)并添加趋势线。
    2. 确定每个样品的峰面积并确定浓度 使用趋势线。浓度(μM)= a *峰面积+ b(a和b是 ?已在趋势线中计算)。

食谱

  1. 0.15 M HCl(在室温下储存)
    1.27ml HCl(37%) 100ml双蒸水
    使用100ml注射器通过0.2μM尼龙过滤器过滤
  2. 1.75 M MES(pH 7.4)(储存在4℃) 3.41克MES
    10ml双蒸水
    使用NaOH调节pH至7.4,并使用100ml注射器通过0.2μM尼龙过滤器过滤
  3. 0.5M EDTA 2Na 2H 2 O(在室温下储存)
    18.6g EDTA。 2Na 2H 2 O 溶解在双蒸水中,用NaOH调节pH至8.0,体积达到100ml,
    使用100ml注射器通过0.2μM尼龙过滤器过滤
  4. 300mM单溴甲烷(储存在-20℃)
    0.025g单溴甲烷 307.3μl乙腈
    12,000×g ,4℃,30分钟以除去不溶物质
  5. 溶剂A:0.1%(v/v)三氟乙酸(HPLC级)(新鲜制备) 1ml三氟乙酸 999 ml双蒸水
    使用100ml注射器通过0.2μM尼龙过滤器过滤
  6. 溶剂B:90%(v/v)乙腈(HPLC级)(新鲜制备) 900ml 100%乙腈 100ml 0.1%(v/v)三氟乙酸 使用100ml注射器通过0.2μM尼龙过滤器过滤 溶剂A和溶剂B需要在超声波清洗器中脱气30分钟,具有松开的盖子
  7. 10mM L-半胱氨酸(储存在-20℃) 0.0606g L-半胱氨酸 50ml 0.15M HCl
  8. 10mMγ-谷氨酰半胱氨酸(保存在-20℃) 0.1251gγ-谷氨酰半胱氨酸 50ml 0.15M HCl
  9. 10mM谷胱甘肽(保存在-20℃) 0.1537克谷胱甘肽 50ml 0.15M HCl
  10. 标准(储存于-20°C)

    0μM
    50μM
    100μM
    200μM
    500μM
    1,000μM
    10 mM
    0微升
    5微升
    10微升
    20微升
    50微升
    100微升
    0.15 M HCl
    1000微升
    995微升
    990微升
    980μl
    950微升
    900μl

致谢

该协议从Fahey和Newton(1987)描述的先前工作修改。

参考文献

  1. Fahey,R.C。和Newton,G.L。(1987)。 使用单溴甲烷荧光标记和高效液相色谱法测定低分子量硫醇。 Methods Enzymol 143:85-96。
  2. Wang,Z.,Mao,J.L.,Zhao,Y.J.,Li,C.Y.and Xiang,C.B。(2015)。 L-半胱氨酸通过植物拟南芥中的生长素/PLETHORA和SCR/SHR途径抑制根伸长。 J Integr Plant Biol 57(2):186-197。
  3. Xiang,C。和Oliver,D.J。(1998)。 谷胱甘肽代谢基因协调应答拟南芥中的重金属和茉莉酸。 植物细胞 10(9):1539-1550。
  4. Zhao,Q.,Wu,Y.,Gao,L.,Ma,J.,Li,C.Y.and Xiang,C.B。(2014)。 硫营养素可??用性通过影响根吲哚-3-乙酸水平和干细胞生态位来调节根伸长。 Integral Plant Biol 56(12):1151-1163。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Miao, Z., Wang, Z. and Xiang, C. (2016). Quantification of Low Molecular Weight Thiols in Arabidopsis. Bio-protocol 6(1): e1704. DOI: 10.21769/BioProtoc.1704.
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