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Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels
在二氨基荧光素(DAF)凝胶上检测植物样品中的蛋白质S-亚硝基硫醇(SNO)   

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Abstract

In plant cells, the analysis of protein S-nitrosothiols (SNOs) under physiological and adverse stress conditions is essential to understand the mechanisms of Nitric oxide (NO)-based signaling. We adapted a previously reported protocol for detecting protein SNOs in animal systems (King et al., 2005) for plant samples. Briefly, proteins from plant samples are separated via non-reducing SDS-PAGE, then the NO bound by S-nitrosylated proteins is released using UV light and, finally, the NO is detected using the fluorescent probe DAF-FM (Rodriguez-Ruiz et al., 2017). Thus, the approach presented here provides a relatively quick and economical procedure that can be used to compare protein SNOs content in plant samples and provide insight in NO-based signaling in plants.

Keywords: Nitric oxide(一氧化氮), S-nitrosothiols(S-亚硝基硫醇), S-nitrosation(S-亚硝化), S-nitrosylation(S-亚硝基化)

Background

Nitric oxide (NO) is a free radical which can interact with a diverse array of biomolecules including proteins, lipids, and nucleic acids. In the case of proteins, one of the most relevant post-translational modifications (PTMs) is the covalent attachment of an NO group to the thiol (-SH) side chain of cysteine (Cys) present in peptides or proteins. This modification generates a family of NO-derived molecules called S-nitrosothiols (SNOs) which are important compounds in both animal and plant systems (Foster et al., 2003; Lindermayr and Durner, 2009; Astier et al., 2011; Broniowska and Hogg, 2012). Although this PTM is often designated as S-nitrosylation, the more appropriate term is S-nitrosation. It is difficult to detect, quantify and identify protein SNOs in plant systems. While there are several techniques to detect SNOs such as chemiluminescence, the biotin switch method, mass spectrometry, fluorescence detection, and antibody detection (against S-nitrosocysteine) (Kettenhofen et al., 2007; Foster, 2012; Devarie-Baez et al., 2013; Diers et al., 2014; Barroso et al., 2016; Mioto et al., 2017) many of these techniques require tedious sample preparation procedures that are time consuming and require sophisticated, expensive equipment.

Materials and Reagents

  1. 10-cm-diameter polystyrene Petri dishes (Fisher Scientific, catalog number: 12654785 )
  2. Parafilm M All-Purpose Paraffin Wax Film (Bemis, catalog number: PM996 )
  3. Sweet green pepper fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain)
    Note: This company grows pepper plants in experimental glass-covered greenhouses under optimal conditions of light, temperature and humidity.
  4. Arabidopsis thaliana ecotype Columbia seeds (originally obtained from NASC, Nottingham Arabidopsis Stock Center)
  5. Ethanol (Fisher Scientific, catalog number: 10517694 )
  6. Commercial Bleach (20%)
  7. Murashige and Skoog medium (Sigma-Aldrich, catalog number: M5524 )
  8. Sucrose (Sigma-Aldrich, catalog number: 84097 )
  9. Phyto-agar (Sigma-Aldrich, catalog number: P8169-100G )
  10. Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, catalog number: 5000006 )
  11. Bovine serum albumin (BSA) Fraction V (Roche Diagnostics, Sigma-Aldrich, catalog number: 10735078001 )
  12. 4-20% Precast TGX Mini-Protean gel (Bio-Rad Laboratories, catalog number: 4561093 )
  13. Ascorbate (AsA) (Sigma-Aldrich, catalog number: A7631-25G )
  14. Copper(I) chloride (CuCl) (Sigma-Aldrich, catalog number: 651745-5G )
  15. N-ethylmaleimide (NEM) (Sigma-Aldrich, catalog number: E3876-5G )
  16. Dithiothreitol (DTT) (Roche Diagnostics, catalog number: 10708984001 )
  17. Reduced glutathione (GSH) (Sigma-Aldrich, catalog number: G4251-5G )
  18. β-Mercaptoethanol (ME) (Sigma-Aldrich, catalog number: M6250-10ML )
  19. Tris (AMRESCO, catalog number: 0497 )
  20. Ethylenediaminetetraacetic acid, disodium salt, dihydrate (Na2-EDTA) (Sigma-Aldrich, catalog number: E5134 )
  21. Triton X-100 (AMRESCO, catalog number: 0694 )
  22. Glycerol (AMRESCO, catalog number: E520 )
  23. Sodium dodecyl sulfate (SDS; electrophoresis grade)
  24. Bromophenol blue (Sigma-Aldrich, catalog number: B0126-25G )
  25. 3-Amino,4-aminomethyl-2’,7’-difluorescein (DAF-FM) (Sigma-Aldrich, catalog number: D2196 )
  26. Grinding buffer (see Recipes)
  27. Sample treatment buffer (2x) (see Recipes)
  28. Standard running buffer for SDS-PAGE containing 1 mM EDTA (see Recipes)
  29. Gel staining solution (see Recipes)

Equipment

  1. Set of Gilson micropipettes (Gilson, P10, P20 and P100)
  2. Plant growth cabinet (Panasonic Biomedical, model: MLR-352-PE )
  3. Porcelain mortar and pestle (VWR, catalog numbers: 410-0110 and 410-0120 , respectively)
  4. Refrigerated centrifuge Hettich Mikro 220R (Hettich Lab Technology, model: Mikro 220 R , catalog number: 2205)
  5. Vertical Slab gels Electrophoresis System (Bio-Rad Laboratories, model: Mini-PROTEAN®, catalog number: 1658003EDU )
  6. Standard UV-transilluminator (302-312 nm), used in molecular biology laboratory
  7. Molecular Imager PharosFX system (Bio-Rad Laboratories, model: PharosFXTM, catalog number: 1709460 )
    Note: This product has been discontinued.
  8. EvolutionTM 201 UV-visible spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: EvolutionTM 201 , catalog number: 912A0890)

Software

  1. ImageJ (free application available in https://imagej.net/)

Procedure

  1. Preparation of plant extracts
    1. Sweet green pepper (Capsicum annuum) fruits were provided by Syngenta Seeds S.A. (El Ejido, Spain) from plants grown in experimental greenhouses with optimal nutrients supplementation applied on rockwood as substrate.
    2. Surface-sterilize Arabidopsis thaliana ecotype Columbia seeds for 5 min in 70% ethanol containing 0.1% SDS. Then, place in sterile water containing 20% (v/v) commercial Bleach and 0.1% SDS for 20 min. Wash four times with sterile water. Grow seeds on Petri plates over commercial Murashige and Skoog medium (Sigma-Aldrich) at a pH of 5.5, containing 1% (w/v) sucrose and 0.8% (w/v) phyto-agar. Place the seedlings for 14 days at 16 h light, 22 °C/8 h dark, 18 °C, under a light intensity of 100 μE m-2 sec-1.
    3. Homogenize plant samples (between 0.1 to 0.5 g) in a mortar and pestle with Gridding buffer (see Recipes) in a ratio 1:1 (w/v) for pepper fruits and ratio 1:3 (w/v) for Arabidopsis. Perform these operations at 0-4 °C, for example using a container with ice (see Figure 1).


      Figure 1. Illustrative picture showing the Disposition of the mortar in the container with ice

    4. Centrifuge extracts at 27,000 x g at 4 °C for 20 min.
    5. Use the supernatants for the protein assays. Determine protein concentration with the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as the standard according with the ‘Microassay Procedure’ as outlined by the manufacturer.

  2. SDS-PAGE and S-nitrosothiols staining
    1. Pre-run SDS-PAGE using a precast 4-20% gradient TGX Mini-Protean gel (without samples) for 30 min at 30 mA per gel with standard running buffer containing 1 mM EDTA.
      Note: The gel is pre-run primarily to remove any potential traces of unpolymerized acrylamide.
    2. Prepare samples with sample treatment buffer (see Recipes) in ratio 1:1 (v/v). For each samples, 25 µg of protein is loaded per lane.
    3. Run the electrophoresis at 15 mA per gel. The electrophoresis should be stopped when the front line indicated by the bromophenol blue (used for tracing the migration of samples) is 1 cm from the end of the gel (usually for 45 to 60 min).
    4. Wash the gel with 20-25 ml ultrapure water containing 1 mM EDTA for 5 min.
    5. Coat the gel with Gel staining solution (28 µM DAF-FM) for 10 min at room temperature in the dark.
      Note: Cover with a piece of Parafilm of the same size and use a roller to gently smooth the staining solution over the gel such that there is a homogenous distribution of the solution.
    6. Expose the gel to UV light for 5 min.
    7. Take a picture with the fluor imager (PharosFMTM) according to the manufacturer’s instructions (excitation wavelength of 488 nm and emission wavelength of 530 nm) (see Figure 2).


      Figure 2. Detection and quantification of S-nitrosylated proteins in plant samples. A. Detection of endogenous S-nitrosylated (S-nitrosated) proteins (SNP) on DAF gels. Lane 1. Sweet green pepper fruits; Lane 2. Arabidopsis thaliana 14-day-old seedlings. Protein samples (25 µg) were separated by SDS-PAGE (gradient gels 4-20%) under non-reducing conditions. Molecular weight markers are indicated on the right. B. Densitometric scans of S-nitrosated proteins and its relative quantification (%) made by the ImageJ program

Data analysis

Quantification of the S-nitrosated protein bands can be done by densitometric analysis, for example, using the ImageJ program (see Figure 2B).

Notes

Any techniques could have false positives. Therefore, it is highly recommended to perform several internal controls to avoid potential artifacts in new plant samples. Before loading samples on DAF gels it is recommended that plant samples be pre-treated at 25 °C for 3 h with different chemicals capable of: A) decomposing SNOs, such as 20 mM ascorbate (AsA) and 0.1 mM CuCl; B) blocking free thiols, such as 5 mM N-ethylmaleimide (NEM); and C) reducing agents, such as 20 mM dithiothreitol (DTT), 20 mM reduced glutathione (GSH) and 100 mM β-mercaptoethanol (ME).

Recipes

  1. Grinding buffer
    50 mM Tris-HCl, pH 7.8
    0.1 mM EDTA
    0.2% (v/v) Triton X-100
    10% (v/v) glycerol
  2. Sample treatment buffer (2x)
    250 mM Tris-HCl, pH 6.8
    8% (w/v) SDS
    40% (w/v) glycerol
    0.006% (w/v) bromophenol blue
  3. Standard running buffer for SDS-PAGE containing 1 mM EDTA
    Running buffer: 0.375 mM Tris-HCl, pH 8.8
    1 mM EDTA
  4. Gel staining solution
    28 µM DAF-FM solution prepared in ultrapure water
    Note: Prepare before use and protected from the light.

Acknowledgments

MRR acknowledges an FPI contract (BES-2012-055904) from the Ministry of Economy and Competitiveness, Spain. This work has been supported by the ERDF co-financed grant AGL2015-65104-P from the Ministry of Economy and Competitiveness, Spain. The authors are also grateful for the previous work done by King et al. (2005) which has been of great value to adapt it to plant samples.

References

  1. Astier, J., Rasul, S., Koen, E., Manzoor, H., Besson-Bard, A., Lamotte, O., Jeandroz, S., Durner, J., Lindermayr, C. and Wendehenne, D. (2011). S-nitrosylation: an emerging post-translational protein modification in plants. Plant Sci 181(5): 527-533.
  2. Barroso, J. B., Valderrama, R., Carreras, A., Chaki, M., Begara-Morales, J. C., Sanchez-Calvo, B. and Corpas, F. J. (2016). Quantification and localization of S-nitrosothiols (SNOs) in higher plants. Methods Mol Biol 1424: 139-147.
  3. Broniowska, K. A. and Hogg, N. (2012). The chemical biology of S-nitrosothiols. Antioxid Redox Signal 17(7): 969-980.
  4. Devarie-Baez, N. O., Zhang, D., Li, S., Whorton, A. R. and Xian, M. (2013). Direct methods for detection of protein S-nitrosylation. Methods 62:171-176.
  5. Diers, A. R., Keszler, A. and Hogg, N. (2014). Detection of S-nitrosothiols. Biochim Biophys Acta 1840(2): 892-900.
  6. Foster, M. W. (2012). Methodologies for the characterization, identification and quantification of S-nitrosylated proteins. Biochim Biophys Acta 1820(6): 675-683.
  7. Foster, M. W., McMahon, T. J. and Stamler, J. S. (2003). S-nitrosylation in health and disease. Trends Mol Med 9(4): 160-168.
  8. Kettenhofen, N. J., Broniowska, K. A., Keszler, A., Zhang, Y. and Hogg, N. (2007). Proteomic methods for analysis of S-nitrosation. J Chromatogr B Analyt Technol Biomed Life Sci 851(1-2): 152-159.
  9. King, M., Gildemeister, O., Gaston, B. and Mannick, J. B. (2005). Assessment of S-nitrosothiols on diaminofluorescein gels. Anal Biochem 346(1): 69-76.
  10. Lindermayr, C. and Durner, J. (2009). S-Nitrosylation in plants: pattern and function. J Proteomics 73(1): 1-9.
  11. Mioto, P. T., Rodriguez-Ruiz, M., Mot, A. C., Zuccarelli, R., Corpas, F. J., Freschi, L. and Mercier, H. (2017). Alternative fluorimetric-based method to detect and compare total S-nitrosothiols in plants. Nitric Oxide 68: 7-13.
  12. Rodriguez-Ruiz, M., Mioto, P., Palma, J. M. and Corpas, F. J. (2017). S-nitrosoglutathione reductase (GSNOR) activity is down-regulated during pepper (Capsicum annuum L.) fruit ripening. Nitric Oxide 68: 51-55.

简介

在植物细胞中,生理和不利胁迫条件下的蛋白质S-亚硝基硫醇(SNO)的分析对于了解一氧化氮(NO)的信号传导机制至关重要。 我们调整了以前报告的用于检测动物系统中蛋白质SNO的方法(King等,2005),用于植物样品。 简言之,通过非还原性SDS-PAGE分离来自植物样品的蛋白质,然后使用UV光释放由S-亚硝基化蛋白质结合的NO,最后使用荧光探针DAF-FM检测NO(Rodriguez-Ruiz et 等等,2017)。 因此,本文提出的方法提供了相对快速和经济的方法,可用于比较植物样品中的蛋白质SNOs含量,并提供植物中基于NO的信号传导的洞察。
【背景】一氧化氮(NO)是一种自由基,可与各种生物分子阵列相互作用,包括蛋白质,脂质和核酸。在蛋白质的情况下,最相关的翻译后修饰(PTM)之一是NO基团与存在于肽或蛋白质中的半胱氨酸(Cys)的硫醇(-SH)侧链的共价连接。该修饰产生称为S-亚硝基硫醇(SNO)的家族,其是动物和植物系统中重要的化合物(Foster等人,2003; Lindermayr和Durner,2009; Astier等人,2011; Broniowska和Hogg,2012)。尽管该PTM通常被称为S-亚硝基化,但更合适的术语是S-亚硝基化。在植物系统中难以检测,定量和鉴定蛋白质SNO。虽然有几种检测SNO的技术,如化学发光,生物素转换法,质谱法,荧光检测和抗体检测(针对S-亚硝基半胱氨酸)(Kettenhofen et al。,2007; Foster,2012; Devarie-Baez et al。 ,2013; Diers等人,2014; Barroso等人,2016; Mioto等人,2017)许多这些技术需要繁琐的样品制备程序,其耗时且需要复杂且昂贵的设备。

关键字:一氧化氮, S-亚硝基硫醇, S-亚硝化, S-亚硝基化

材料和试剂

  1. 10厘米直径的聚苯乙烯培养皿(Fisher Scientific,目录号:12654785)
  2. Parafilm M多用途石蜡蜡(Bemis,目录号:PM996)
  3. 甜食青椒水果由先正达种子S.A.(El Ejido,西班牙)提供
    注意:该公司在最佳的光照,温度和湿度条件下,在实验玻璃覆盖的温室中种植胡椒植物。
  4. 拟南芥生态型哥伦比亚种子(最初来自NASC,诺丁汉拟南芥库存中心)
  5. 乙醇(Fisher Scientific,目录号:10517694)
  6. 商业漂白(20%)
  7. Murashige和Skoog培养基(Sigma-Aldrich,目录号:M5524)
  8. 蔗糖(Sigma-Aldrich,目录号:84097)
  9. 植物琼脂(Sigma-Aldrich,目录号:P8169-100G)
  10. Bio-Rad蛋白测定染料试剂(Bio-Rad Laboratories,目录号:5000006)
  11. 牛血清白蛋白(BSA)级分V(Roche Diagnostics,Sigma-Aldrich,目录号:10735078001)
  12. 4-20%预制TGX微型蛋白凝胶(Bio-Rad Laboratories,目录号:4561093)
  13. 抗坏血酸(AsA)(Sigma-Aldrich,目录号:A7631-25G)
  14. 氯化铜(I)(CuCl)(Sigma-Aldrich,目录号:651745-5G)
  15. N-马来酰亚胺(NEM)(Sigma-Aldrich,目录号:E3876-5G)
  16. 二硫苏糖醇(DTT)(Roche Diagnostics,目录号:10708984001)
  17. 还原型谷胱甘肽(GSH)(Sigma-Aldrich,目录号:G4251-5G)
  18. β-巯基乙醇(ME)(Sigma-Aldrich,目录号:M6250-10ML)
  19. Tris(AMRESCO,目录号:0497)
  20. 乙二胺四乙酸二钠盐,二水合物(Na 2 -EDTA)(Sigma-Aldrich,目录号:E5134)
  21. Triton X-100(AMRESCO,目录号:0694)
  22. 甘油(AMRESCO,目录号:E520)
  23. 十二烷基硫酸钠(SDS;电泳级)
  24. 溴苯酚蓝(Sigma-Aldrich,目录号:B0126-25G)
  25. 3-氨基-4-氨基甲基-2',7'-二氟荧光素(DAF-FM)(Sigma-Aldrich,目录号:D2196)
  26. 研磨缓冲液(参见食谱)
  27. 样品处理缓冲液(2x)(参见食谱)
  28. 含有1mM EDTA的SDS-PAGE的标准运行缓冲液(参见食谱)
  29. 凝胶染色溶液(参见食谱)

设备

  1. 一套Gilson微量移液器(Gilson,P10,P20和P100)
  2. 植物生长箱(Panasonic Biomedical,型号:MLR-352-PE)
  3. 瓷砂浆和杵(VWR,目录号:分别为410-0110和410-0120)
  4. 冷藏离心机Hettich Mikro 220R(Hettich Lab Technology,型号:Mikro 220 R,目录号:2205)
  5. 垂直平板凝胶电泳系统(Bio-Rad Laboratories,型号:Mini-PROTEAN ®,目录号:1658003EDU)
  6. 标准紫外 - 透射仪(302-312 nm),用于分子生物学实验室
  7. 分子成像仪PharosFX系统(Bio-Rad Laboratories,型号:PharosFX TM,目录号:1709460)
    注意:本产品已停产。
  8. 进行 TM 201紫外可见分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:Evolution TM, 201,目录号:912A0890) />

软件

  1. ImageJ( https://imagej.net/ 中提供的免费应用程序)

程序

  1. 植物提取物的制备
    1. 甜瓜青椒( Capsicum annuum )水果由先正达种子S.A.(El Ejido,西班牙)从在实验温室种植的植物提供,其中最佳营养补充施用于作为底物的木材上。
    2. 在70%含有0.1%SDS的乙醇中表达消毒拟南芥生态型Columbia种子5分钟。然后,放入含有20%(v / v)商业漂白剂和0.1%SDS的无菌水中20分钟。用无菌水洗涤四次。在含有1%(w / v)蔗糖和0.8%(w / v)植物琼脂的pH5.5下,在商业Murashige和Skoog培养基(Sigma-Aldrich)上培养Petri板上的种子。将幼苗放置16小时,光照时间为16小时,22℃/ 8小时黑暗,18℃,光强度为100微米/秒〜 。
    3. 在研钵和研杵与研钵缓冲液(参见食谱)中使植物样品(0.1至0.5g)以1:1比例(w / v)和胡椒果实比例1:3(w / v)拟南芥。在0-4°C进行这些操作,例如使用带冰的容器(见图1)

      图1.说明性图片显示冰箱中的砂浆的配置

    4. 离心机在4℃下以27,000×g离心提取20分钟
    5. 使用上清液进行蛋白质分析。用Bio-Rad蛋白质测定染料试剂(Bio-Rad Laboratories,Hercules,CA)测定蛋白质浓度,使用牛血清白蛋白作为标准,按照制造商概述的“微量测定法”(< br />
  2. SDS-PAGE和亚硝基硫醇染色
    1. 使用预先的4-20%梯度TGX Mini-Protean凝胶(无样品)进行SDS-PAGE预处理30分钟,每个凝胶30 mA,标准运行缓冲液含有1 mM EDTA。 注意:凝胶预先主要用于去除任何潜在痕量的未聚合丙烯酰胺。
    2. 以比例1:1(v / v)准备样品处理缓冲液(见配方)的样品。对于每个样品,每泳道加载25μg蛋白质
    3. 运行电泳每凝胶15 mA。当溴酚蓝(用于追踪样品的迁移)指示的前线距离凝胶末端1cm(通常为45〜60分钟)时,应停止电泳。
    4. 用含有1mM EDTA的20-25ml超纯水洗涤凝胶5分钟
    5. 将凝胶用凝胶染色溶液(28μMDAF-FM)在室温下黑暗涂覆10分钟。
      注意:盖上一块相同尺寸的石蜡膜,并使用滚筒将凝胶上的染色溶液轻轻平滑,使溶液均匀分布。
    6. 将凝胶暴露于紫外光下5分钟
    7. 根据制造商的说明书(激发波长488 nm,发射波长530 nm),使用荧光成像仪(PharosFM TM )拍照(见图2)。


      图2.植物样品中的S - 亚硝基化蛋白质的检测和定量。 :一种。在DAF凝胶上检测内源性的S - 亚硝基化(亚硝化)蛋白(SNP)。车道1.甜青椒水果; Lane 2。拟南芥 14日龄幼苗。在非还原条件下,通过SDS-PAGE(梯度凝胶4-20%)分离蛋白质样品(25μg)。分子量标记在右侧显示。 B.图像质量分数蛋白质的光密度扫描及其相对量化(%)ImageJ程序制作

数据分析

可通过光密度分析(例如使用ImageJ程序)(见图2B)进行定量鉴定。

笔记

任何技术都可能会有误报。因此,强烈建议执行多个内部控制,以避免新植物样品中的潜在伪像。在将样品加样到DAF凝胶之前,建议将植物样品在25℃预处理3小时,并使用不同的化学物质:A)分解SNO,如20mM抗坏血酸(AsA)和0.1mM CuCl; B)封闭游离硫醇,例如5mM N,N-乙基马来酰亚胺(NEM);和C)还原剂,例如20mM二硫苏糖醇(DTT),20mM还原型谷胱甘肽(GSH)和100mMβ-巯基乙醇(ME)。

食谱

  1. 研磨缓冲液
    50mM Tris-HCl,pH7.7
    0.1 mM EDTA
    0.2%(v / v)Triton X-100
    10%(v / v)甘油
  2. 样品处理缓冲液(2x)
    250mM Tris-HCl,pH6.8
    8%(w / v)SDS
    40%(w / v)甘油
    0.006%(w / v)溴酚蓝
  3. 含有1 mM EDTA的SDS-PAGE标准运行缓冲液 运行缓冲液:0.375mM Tris-HCl,pH8.8。
    1 mM EDTA
  4. 凝胶染色溶液
    28μMDAF-FM溶液用超纯水制备
    注意:使用前准备并保护灯光。

致谢

MRR承认西班牙经济与竞争力部的FPI合同(BES-2012-055904)。这项工作得到西班牙经济和竞争力部的经济和社会发展部的共同出资赠款AGL2015-65104-P的支持。作者还对以前的King等人的工作感激不尽。 (2005)已经很有价值地适应了植物样本。

参考

  1. Astier,J.,Rasul,S.,Koen,E.,Manzoor,H.,Besson-Bard,A.,Lamotte,O.,Jeandroz,S.,Durner,J.,Lindermayr,C.and Wendehenne,D 。(2011)。 一种新兴的植物中的翻译后蛋白质修饰。植物科学181(5):527-533。
  2. Barroso,JB,Valderrama,R.,Carreras,A.,Chaki,M.,Begara-Morales,JC,Sanchez-Calvo,B.and Corpas,FJ(2016)。&lt; a class =“ke-insertfile” href =“http://www.ncbi.nlm.nih.gov/pubmed/27094417”target =“_ blank”>在高等植物中的亚硝基硫醇(SNO)的定量和定位。 a>方法Mol Biol 1424:139-147。
  3. Broniowska,KA和Hogg,N。(2012)。抗氧化氧化还原信号 17(7):969-980。
  4. Devarie-Baez,N.O.,Zhang,D.,Li,S.,Whorton,A.R。和Xian,M。(2013)。 用于检测蛋白质的直接方法 -nitrosylation。 方法 62:171-176。
  5. Diers,AR,Keszler,A.和Hogg,N。(2014)。检测亚硝基硫醇。 Biochim Biophys Acta 1840(2):892-900。
  6. Foster,MW(2012)。表征方法,鉴定和定量的亚硝基化蛋白质。 Biochim Biophys Acta 1820(6):675-683。
  7. Foster,MW,McMahon,TJ和Stamler,JS(2003)。&nbsp; Trends Mol Med 9(4):160-168。
  8. Kettenhofen,NJ,Broniowska,KA,Keszler,A.,Zhang,Y.和Hogg,N。(2007)。用于分析S-亚硝化的蛋白质组学方法 Chromatogr B Analyt Technol Biomed Life Sci 851 (1-2):152-159。
  9. King,M.,Gildemeister,O.,Gaston,B.and Mannick,JB(2005)。&nbsp; 评估二氨基荧光素凝胶上的亚硝基硫醇。 ):69-76。
  10. Lindermayr,C.和Durner,J。(2009)。 植物中的亚硝酰化:模式和功能。蛋白质组学 73(1):1-9。
  11. Mioto,PT,Rodriguez-Ruiz,M.,Mot,AC,Zuccarelli,R.,Corpas,FJ,Freschi,L.and Mercier,H。(2017)。&lt; a class =“ke-insertfile”href = “http://www.ncbi.nlm.nih.gov/pubmed/28274830”target =“_ blank”>替代基于荧光的方法来检测和比较植物中的总氮 - 硝基硫醇。 a>一氧化氮 68:7-13。
  12. Rodriguez-Ruiz,M.,Mioto,P.,Palma,JM and Corpas,FJ(2017)。&nbsp; 在辣椒(辣椒)中果实成熟时,亚硝基谷胱甘肽还原酶(GSNOR)活性被下调。 a>一氧化氮 68:51-55。
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引用:Rodríguez-Ruiz, M., Mioto, P. T., Palma, J. M. and Corpas, F. J. (2017). Detection of Protein S-nitrosothiols (SNOs) in Plant Samples on Diaminofluorescein (DAF) Gels. Bio-protocol 7(18): e2559. DOI: 10.21769/BioProtoc.2559.
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