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Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra
黑杨的莽草酸羟基肉桂转移酶(HCT)活性分析

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Abstract

Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building blocks differ in their degree of methoxylation and their biosynthetic pathway is catalyzed by more than 10 enzymes. HCT plays a crucial role by channeling the phenylpropanoids towards the production of coniferyl and sinapyl alcohols. Interestingly, HCT has been reported to be implicated in the pathway both upstream and downstream of the 3-hydroxylation of the aromatic ring of p-coumaroyl shikimate (Figure 1) (Hoffmann et al., 2003; Hoffmann et al., 2004; Vanholme et al., 2013b). These features highlight the importance of developing an assay to reliably measure HCT activity in planta. Here, we describe a UPLC-MS-based method for the analysis of HCT activity in xylem total protein extracts of Populus nigra, which can be adapted to other woody and herbaceous plant species. The protocol was initially described in Vanholme et al. (2013a).

Keywords: enzyme activity(酶活性), HCT(HCT), Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase(羟基肉桂酰基-CoA莽草酸/ quinate羟基肉桂酰基转移酶), BAHD acyltransferase(BAHD酰基转移酶), p-coumaroyl shikimate(对香豆酰基莽草酸)


Figure 1. The two enzymatic reactions of the phenylpropanoid pathway catalyzed by HCT. HCT (AEN02914) converts p-coumaroyl-CoA into p-coumaroyl shikimic acid (first HCT-reaction), which is converted to caffeoyl shikimic acid by C3H (coumarate 3-hydroxylase). HCT converts it further to caffeoyl-CoA (second HCT-reaction).

Materials and Reagents

  1. Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (Biosolve, catalog number: 77-86-1 )
  2. Dithiothreitol (DTT) (AG Scientific, catalog number: C-1029 )
  3. Polyvinylpolypyrrolidone (PVPP) (Sigma-Aldrich, catalog number: P-6755 )
  4. Glycerol (Sigma-Aldrich, catalog number: G-7893 )
  5. Complete Mini Protease Inhibitor Cocktail Tablets (Roche, catalog number: 0 4693159001 )
  6. Bio-Rad Protein Assay (Bio-Rad Laboratories, catalog number: 500-0006 )
  7. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A-7906 )
  8. p-coumaroyl-CoA (TransMIT, catalog number: C030 )
  9. Shikimic acid (Sigma-Aldrich, catalog number: S-5375 )
  10. Caffeoyl shikimate (AnalytiCon Discovery GmbH, catalog number: NP-00058 )
  11. Coenzyme-A hydrate (CoA) (Sigma-Aldrich, catalog number: A-3164 )
  12. Acetonitrile ULC/MS grade (Biosolve, catalog number: BIO-012041 )
  13. Formic acid ULC/MS grade (Biosolve, catalog number: BIO-06914131 )
  14. Liquid chromatography (LC) sample vials (Waters, catalog numbers: 186002639 and 2639531020
  15. Ice
  16. Liquid nitrogen
  17. Protein Extraction Buffer (see Recipes)
  18. Reaction Mix (see Recipes)

Equipment

  1. Nunc 96-well microplate without lid and flat bottom wells (Thermo Fisher Scientific, catalog number: 269787 )
  2. Safe-Lock tubes 2.0 ml (Eppendorf, catalog number: 3706 )
  3. Stainless steel surgical scalpel blades No.22 (Swann-Morton, catalog number: 0 308 )
  4. Mortar (150 x 70 mm; 700 ml) (The Morgan Advanced Materials Company plc, catalog number: 12906-6a )
  5. Pestle (150 x 36 mm) (Haldenwanger, catalog number: 12906-2 )
  6. Balance (Mettler Toledo, model: XP-105 Delta Range )
  7. Vortex (IKA, model: MS2 Minishaker L002050 )
  8. Temperature controlled benchtop microcentrifuge (Eppendorf, model: 5417R )
  9. Temperature controlled microplate spectrophotometer (Molecular Devices, model: Spectra Max 250 )
  10. Thermoblock (Eppendorf Thermomixer Compact) or water bath
  11. UPLC-MS system (In our case: Waters Acquity UPLC system (WATERS) connected to a Thermo LTQ XL mass spectrometer (Thermo Fisher Scientific) or a Synapt HDMS Q-Tof (WATERS). Chromatographic separation was performed on a Waters Acquity BEH RP C18 (2.1 mm x 150 mm, 1.7 μm) column (WATERS))
  12. Ultrafreezer
  13. Optional: Qubit 2.0 Fluorometer (Invitrogen)

Software

  1. XCalibur 2.0 software (Thermo Fisher Scientific, Waltham) was used to acquire, analyze and manage mass spectrometry information

Procedure

  1. Protein extraction
    1. Collect 15-cm stem segments derived from the base of 1-m tall sprouts emerging from the trunk of a poplar tree using secateurs. If 1-year old trees are used, segments of the main stem can be used.
    2. Immediately submerge the fresh plant tissue into liquid nitrogen and store the samples at -80 °C until further use.
    3. Incubate the frozen stem segments on ice for a few minutes and make a small incision in the bark on one end of the segment. The bark can be easily pealed from the underlying tissue. Some part of the cambium can stick to the debarked stem, but can be easily removed by gently scrubbing the stem with a scalpel. Scrape the xylem tissue using a scalpel, making sure the scraped tissue is immediately collected in a precooled mortar (use liquid nitrogen to keep the material frozen during sampling).
    4. Grind the scraped xylem tissue to a fine powder using a mortar and pestle.
    5. For xylem total protein extraction, weigh ~100 mg of ground xylem tissue in a precooled 2-ml tube and add 1 ml of ice-cold protein extraction buffer. The composition of the protein extraction buffer is given as Recipe 1, at the end of the protocol.
    6. Vortex thoroughly and incubate on ice for 1 h, inverting the tubes every 5-10 min to prevent precipitation.
    7. Centrifuge at 20,000 x g for 10 min at 4 °C and transfer the supernatant to a new precooled tube. Keep all samples on ice.

  2. Protein quantification using the Bradford method (Bradford, 1976)
    1. Prepare the BSA standards for the calibration curve as follows:
      Volume of BSA (100 μg/ml)
      Volume of Water
      BSA Concentration
      0 μl
      240 μl
      0 μg/ml
      6 μl
      234 μl
      2.5 μg/ml
      12 μl
      228 μl
      5 μg/ml
      18 μl
      222 μl
      7.5 μg/ml
      24 μl
      216 μl
      10 μg/ml
      27 μl
      213 μl
      11.25 μg/ml
      30 μl
      210 μl
      12.5 μg/ml

    2. For the calibration curve, add 240 μl of each standard (above) to a separate well of a 96-well flat bottom microplate. All measurements are performed in triplicate.
    3. For the quantification of xylem total protein samples, add 210 μl of water in each well and 30 μl of the protein extract dilutions in triplicate. Prepare the dilutions using the protein extraction buffer, ranging from 5x to 100x diluted (depending on the amount of plant material used for the extraction).
    4. Add 60 μl of Bio-Rad Protein Assay reagent to each well and incubate at room temperature for 5 min.
    5. Read absorbance at 595 nm (A595) on a plate spectrophotometer.
      For the calculation of protein concentrations, generate a standard curve by plotting the BSA concentration (X-axis) versus A595 (Y-axis). After obtaining the trend line, use its corresponding equation and the absorbance of the protein sample to resolve the unknown concentration. The correlation coefficient of the trend line should be close to 1.00 (preferentially above 0.97) and all measured absorbances of the protein samples should fall into the linear range.
      Notes:
      1. Figure 2 shows an example of protein quantification using the Bradford method.
      2. As alternative to Bradford, Qubit (Invitrogen) can be used according manufacturer’s instructions for accurate and efficient quantification of protein concentrations.


        Figure 2. Total protein quantification with Bradford assay. 1Normalized absorbance is calculate by subtracting the absorbance value of the blank from the value obtained for each sample. 2Divide this value by the volume of protein extract used in the assay to calculate the final concentration (in this case, we used 2 μl).

  3. HCT activity assay
    HCT activity is measured by the conversion of p-coumaroyl-CoA and shikimate into p-coumaroyl shikimate (Figure 1).
    1. Prior to the preparation of enzymatic reactions, boil an aliquot of the xylem protein extract for 10 min, because the boiled protein extract will be used as negative control.
    2. The reaction mix is prepared in 1.5-ml tubes and contains 100 mM Tris-HCl pH 7, 1 mM DTT, 100 μM p-coumaroyl-CoA, 100 μM shikimic acid and 10 μg xylem protein extract (Recipe 2).
    3. Start the reaction by adding the corresponding volume of the protein extract. Use the same amount of protein of the boiled extract as negative control.
    4. Incubate at 30 °C for 30 min.
      Note: For an in-depth analysis, different time-points can be used.
    5. Terminate the reaction by boiling the samples for 5 min.
      Note: After boiling, place the reaction tubes on ice for at least 5 min, followed by a fast spin. This brings the droplets at the lid, caused by evaporation and condensation, back into the main sample, avoiding a change in the final product concentration.
    6. Transfer the total reaction volume (40 μl) to a LC sample vial for analysis of reaction products.

  4. Product identification and quantification
    10 μl of the aqueous phase is subjected to reversed phase LC-MS and LC-MS (Hoffmann et al., 2003). Here, the specific conditions can differ depending on the equipment used. We present our in house conditions optimized to identify and quantify p-coumaroyl shikimate on a Waters Acquity UPLC system connected to a Thermo LTQ XL mass spectrometer. Chromatographic separation is performed on an Acquity BEH C18 column (2.1 mm x 150 mm, 1.7 μm) using a gradient elution.
    1. The mobile phase is composed of water containing 1% acetonitrile and 0.1% formic acid (Buffer A) and acetonitrile containing 1% water and 0.1% formic acid (Buffer B). The column temperature is maintained at 40 °C and the autosampler temperature at 10 °C.
    2. A flow rate of 350 μl/min is applied during the gradient elution initializing at time 0 min 5% (B), time 30 min 50% (B), time 33 min 100% (B).
    3. The eluent is subsequently directed to the mass spectrometer, via electrospray ionization (ESI) in negative mode.
    4. MS source parameters are as follows: capillary temperature 300 °C, capillary voltage –24 V, source voltage 3.5 V, source current 100 μA, sheath gas flow 30, aux gas flow 20, sweep gas flow 5. The mass range is set between m/z 95-500.
    5. The reaction product, p-coumaroyl shikimate, is characterized based on m/z 319, retention times 8.13 min (trans isomer) 9.64 min (cis isomer), and fragmentation spectra (Figure 3). Compound concentrations were quantified based on curve fitting and peak area integration of the extracted ion chromatograms for m/z 319 using XCalibur's QuanBrowser.


      Figure 3. MS2 fragmentation spectra of p-coumaroyl shikimate (m/z 319)
      Note: For accurate mass measurements of p-coumaroyl shikimate, a Synapt HDMS Q-Tof mass spectrometer can be used (Vanholme et al., 2013b).

Recipes

  1. Protein Extraction Buffer
    Prepare the protein extraction buffer as follows:
    Stock Solution
    Volume/Amount
    Final Concentration
    100 mM Tris-HCl pH 7.5
    2 ml
    20 mM
    100 mM DTT
    1 ml
    10 mM
    100% Glycerol
    1.5 ml
    15%
    PVPP
    100 mg
    1%
    7x Complete Mini Protease Inhibitor
    1.33 ml
    1x
    Water To
    10 ml

    Mix thoroughly.
    Notes:
    1. Dissolve one tablet of Complete Mini Protease Inhibitor in 1.5 ml water to prepare the 7x stock solution.
    2. Since PVPP is insoluble, it may be necessary to cut the end of the pipet tip to add the protein extraction buffer to the ground xylem material.
  2. Reaction Mix
    Prepare the enzymatic reaction mix as follows:
    Stock Solution
    Volume
    Final Concentration
    500 mM Tris-HCl pH 7.0
    8 μl
    100 mM
    20 mM DTT
    2 μl
    1 mM
    2 mM p-coumaroyl-CoA
    2 μl
    100 μM
    2 mM shikimic acid
    2 μl
    100 μM
    Xylem protein extract
    x μl
    10 μg
    Water To
    40 μl


Acknowledgments

The protocol was briefly described by Vanholme and coworkers in Vanholme et al. (2013a). We gratefully acknowledge funding through the European Commission’s Directorate-General for Research within the 7th Framework Program (FP7/2007-2013) under the grant agreement N° 211917 (ENERGYPOPLAR), N° 211868 (NOVELTREE) and N° 311804 (MULTIBIOPRO), the Hercules program of Ghent University for the Synapt Q-Tof (grant no. AUGE/014) and the Multidisciplinary Research Partnership ‘Biotechnology for a Sustainable Economy’ (01MRB510W) of Ghent University. RV is indebted to the Research Foundation-Flanders for a postdoctoral fellowship.

References

  1. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
  2. Hoffmann, L., Maury, S., Martz, F., Geoffroy, P. and Legrand, M. (2003). Purification, cloning, and properties of an acyltransferase controlling shikimate and quinate ester intermediates in phenylpropanoid metabolism. J Biol Chem 278(1): 95-103.
  3. Hoffmann, L., Besseau, S., Geoffroy, P., Ritzenthaler, C., Meyer, D., Lapierre, C., Pollet, B. and Legrand, M. (2004). Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell 16(6): 1446-1465.
  4. Vanholme, B., Cesarino, I., Goeminne, G., Kim, H., Marroni, F., Van Acker, R., Vanholme, R., Morreel, K., Ivens, B., Pinosio, S., Morgante, M., Ralph, J., Bastien, C. and Boerjan, W. (2013a). Breeding with rare defective alleles (BRDA): a natural Populus nigra HCT mutant with modified lignin as a case study. New Phytol 198(3): 765-776.
  5. Vanholme, R., Cesarino, I., Rataj, K., Xiao, Y., Sundin, L., Goeminne, G., Kim, H., Cross, J., Morreel, K., Araujo, P., Welsh, L., Haustraete, J., McClellan, C., Vanholme, B., Ralph, J., Simpson, G.G., Halpin, C. and Boerjan, W. (2013b). Caffeoyl Shikimate Esterase (CSE) Is an Enzyme in the Lignin Biosynthetic Pathway. Science DOI: 10.1126/science.1241602.

简介

木质素是沉积在二次增厚的植物细胞壁中的复杂酚类聚合物。聚合物主要衍生自当偶联时产生对羟基苯基,愈创木基和紫丁香基单元(分别为H,G和S单元)的三种主要单独的木质素:p - 香豆酰基,松柏基和芥子醇进入聚合物。结构单元的甲氧基化程度不同,它们的生物合成途径被超过10种酶催化。 HCT通过将苯丙素类导向松柏醇和芥子醇的生产来发挥关键作用。有趣的是,据报道,HCT涉及p - 香豆酰基莽草酸的芳环的3-羟基化的上游和下游途径(图1)(Hoffmann等, 2003; Hoffmann等人,2004; Vanholme等人,2013b)。这些特征突出了开发在植物中可靠地测量HCT活性的测定法的重要性。在这里,我们描述了基于UPLC-MS的方法,用于分析黑曲霉的木质部总蛋白提取物中的HCT活性,其可以适用于其他木本和草本植物物种。该方案最初在Vanholme等人(2013a)中描述。

关键字:酶活性, HCT, 羟基肉桂酰基-CoA莽草酸/ quinate羟基肉桂酰基转移酶, BAHD酰基转移酶, 对香豆酰基莽草酸


图1.由HCT催化的苯丙素途径的两个酶反应。HCT(AEN02914)将p' - 香豆酰基-CoA转化为p' - 香豆酰基 莽草酸(第一HCT-反应),其通过C3H(香豆酸3-羟化酶)转化为咖啡酰基莽草酸。 HCT将其进一步转化为咖啡酰辅酶A(第二HCT反应)。

材料和试剂

  1. Tris碱(2-氨基-2-羟甲基 - 丙烷-1,3-二醇)(Biosolve,目录号:77-86-1)
  2. 二硫苏糖醇(DTT)(AG Scientific,目录号:C-1029)
  3. 聚乙烯聚吡咯烷酮(PVPP)(Sigma-Aldrich,目录号:P-6755)
  4. 甘油(Sigma-Aldrich,目录号:G-7893)
  5. 完全微型蛋白酶抑制剂混合片(Roche,目录号:04693159001)
  6. Bio-Rad蛋白质测定(Bio-Rad Laboratories,目录号:500-0006)
  7. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A-7906)
  8. p - 香豆酰基-CoA(TransMIT,目录号:C030)
  9. 莽草酸(Sigma-Aldrich,目录号:S-5375)
  10. Caffeoyl shikimate(AnalytiCon Discovery GmbH,目录号:NP-00058)
  11. 辅酶A水合物(CoA)(Sigma-Aldrich,目录号:A-3164)
  12. 乙腈ULC/MS级(Biosolve,目录号:BIO-012041)
  13. 甲酸ULC/MS级(Biosolve,目录号:BIO-06914131)
  14. 液相色谱(LC)样品小瓶(Waters,目录号:186002639和2639531020)
  15. 液氮
  16. 蛋白质提取缓冲液(参见配方)
  17. 反应混合物(参见配方)

设备

  1. Nunc 96孔微孔板(无盖和平底孔)(Thermo Fisher Scientific,目录号:269787)
  2. 安全锁管2.0ml(Eppendorf,目录号:3706)
  3. 不锈钢外科手术刀刀片22号(Swann-Morton,目录号:0308)
  4. 砂浆(150×70mm; 700ml)(The Morgan Advanced Materials Company plc,目录号:12906-6a)
  5. 杵(150×36mm)(Haldenwanger,目录号:12906-2)
  6. 平衡(Mettler Toledo,型号:XP-105 Delta Range)
  7. Vortex(IKA,型号:MS2 Minishaker L002050)
  8. 温度控制的台式微量离心机(Eppendorf,型号:5417R)
  9. 温度控制微孔板分光光度计(Molecular Devices,型号:Spectra Max 250)
  10. Thermoblock(Eppendorf Thermomixer Compact)或水浴
  11. UPLC-MS系统(在我们的情况下:连接到Thermo LTQ XL质谱仪(Thermo Fisher Scientific)或Synapt HDMS Q-Tof(WATERS)的Waters Acquity UPLC系统(WATERS))色谱分离在Waters Acquity BEH RP C18(2.1mm×150mm,1.7μm)柱(WATERS))
  12. Ultrafreezer
  13. 可选:Qubit 2.0荧光计(Invitrogen)

软件

  1. 使用XCalibur 2.0软件(Thermo Fisher Scientific,Waltham)获取,分析和管理质谱信息

程序

  1. 蛋白质提取
    1. 收集15厘米茎段,从1米高的芽出芽从杨树的树干使用剪枝的基地。如果使用1年树,可以使用主茎的部分
    2. 立即将新鲜植物组织浸没在液氮中,并将样品储存在-80℃直到进一步使用
    3. 在冰上孵育冻结的茎段几分钟,并在树干的一端在树皮上做一个小切口。树皮可以容易地从下面的组织剥离。形成层的一些部分可以粘附到剥皮的茎,但可以通过用手术刀轻轻地擦洗茎容易地去除。使用手术刀刮取木质部组织,确保刮取的组织立即收集在预冷的砂浆中(使用 液氮保持材料在采样期间冻结)
    4. 使用研钵和研杵将刮取的木质部组织研磨成细粉
    5. 对于木质部总蛋白提取,在预冷的2ml管中称重〜100mg研磨的木质部组织并加入1ml冰冷的蛋白提取缓冲液。 蛋白质提取缓冲液的组成在方案1结束时以配方1给出
    6. 彻底涡旋并在冰上孵育1小时,每5-10分钟倒转管以防止沉淀
    7. 在4℃下以20,000×g离心10分钟,并将上清液转移到新的预冷却的管中。 保持所有样品在冰上。

  2. 使用Bradford方法的蛋白质定量(Bradford,1976)
    1. 准备校准曲线的BSA标准如下:
      BSA(100μg/ml)的体积
      水量
      BSA浓度
      0微升
      240微升
      0μg/ml
      6微升
      234微升
      2.5μg/ml
      12微升
      228微升
      5μg/ml
      18微升
      222微升
      7.5μg/ml
      24微升
      216微升
      10μg/ml
      27微升
      213微升
      11.25μg/ml
      30微升
      210微升
      12.5μg/ml

    2. 对于校准曲线,将240μl的每种标准品(上文)加入96孔平底微量培养板的单独孔中。所有测量一式三份
    3. 对于木质部总蛋白质样品的定量,在每孔中加入210μl水,并且一式三份地加入30μl蛋白质提取物稀释液。使用蛋白质提取缓冲液制备稀释液,稀释范围为5x至100x(取决于用于提取的植物材料的量)。
    4. 每孔加入60μlBio-Rad Protein Assay试剂,室温孵育5分钟
    5. 在板分光光度计上读取595nm处的吸光度(A 595)。
      对于蛋白质浓度的计算,通过绘制BSA浓度(X轴)对A 595(Y轴)产生标​​准曲线。获得趋势线后,使用其对应的方程和蛋白质样品的吸光度来分辨未知浓度。趋势线的相关系数应该接近1.00(优选地大于0.97),并且蛋白质样品的所有测量的吸光度应当落入线性范围。
      注意:
      1. 图2显示了使用Bradford方法的蛋白质定量的实例。
      2. 作为Bradford的替代品,可以根据制造商的说明书使用Qubit(Invitrogen),以准确和有效地定量蛋白质浓度。


        图2.使用Bradford测定的总蛋白质定量 1 通过从每个样品获得的值中减去空白的吸光度值计算标准化的吸光度。 2 将此值除以测定中使用的蛋白质提取物的体积,以计算最终浓度(在这种情况下,我们使用2μl)。

  3. HCT活性测定
    通过将对 - 香豆酰基-CoA和莽草酸转化为对 - 香豆酰基莽草酸(图1)来测量HCT活性。
    1. 在制备酶反应之前,将木质部蛋白提取物的等分试样煮沸10分钟,因为煮沸的蛋白提取物将用作阴性对照。
    2. 反应混合物在1.5ml管中制备,并含有100mM Tris-HCl pH 7,1mM DTT,100μM对香豆酰基-CoA,100μM莽草酸和10μg木质部蛋白提取物(配方2)。
    3. 通过加入相应体积的蛋白质提取物开始反应。使用相同量的煮沸提取物的蛋白质作为阴性对照
    4. 在30℃孵育30分钟。
      注意:为了进行深入分析,可以使用不同的时间点。
    5. 通过煮沸样品5分钟终止反应。
      注意:煮沸后,将反应管置于冰上至少5分钟,然后快速旋转。这使得由蒸发和冷凝引起的盖子上的液滴回到主样品中,避免了最终产物浓度的变化。
    6. 将总反应体积(40μl)转移到LC样品瓶中用于分析反应产物
  4. 产品识别和量化
    将10μl水相进行反相LC-MS和LC-MS(Hoffmann等人,2003)。这里,具体条件可以根据使用的设备而不同。我们提供我们的内部条件优化以在连接到Thermo LTQ XL质谱仪的Waters Acquity UPLC系统上鉴定和定量 - 香豆酰基莽草酸。使用梯度洗脱在Acquity BEH C18柱(2.1mm×150mm,1.7μm)上进行色谱分离。
    1. 流动相由含有1%乙腈和0.1%甲酸的水(缓冲液A)和含有1%水和0.1%甲酸的乙腈(缓冲液B)组成。柱温度保持在40℃,自动进样器温度保持在10℃
    2. 在时间0min 5%(B),时间30min 50%(B),时间33min 100%(B)的梯度洗脱初始化期间施加350μl/min的流速。
    3. 随后将洗脱液通过电喷雾电离(ESI)以负模式引导至质谱仪。
    4. MS源参数如下:毛细管温度300℃,毛细管电压-24V,源电压3.5V,源电流100μA,鞘气流30,辅助气流20,吹扫气流5.质量范围设定在 m /z 95-500。
    5. 反应产物对香豆酰基莽草酸的特征在于m/z 319,保留时间8.13分钟(反式异构体)9.64分钟(顺式异构体)和碎裂光谱(图3)。基于使用XCalibur's QuanBrowser的m/z 319的提取离子色谱图的曲线拟合和峰面积积分来定量化合物浓度。


      图3.对香豆酰基莽草酸(m/z 319)的MS 2 为了准确测量对香豆酰基莽草酸的质量,可以使用Synapt HDMS Q-Tof质谱仪(Vanholme et al。,2013b)。 。

食谱

  1. 蛋白提取缓冲液
    准备蛋白提取缓冲液如下:
    库存解决方案
    量/ 金额
    最终 浓度 n
    100mM Tris-HCl pH7.5 2 ml
    20 mM
    100 mM DTT
    1 ml
    10 mM
    100%甘油
    1.5 ml
    15%
    PVPP
    100 mg
    1%
    7x完全微型蛋白酶抑制剂 1.33 ml
    1x
    水到
    10 ml

    彻底混合。
    注意:
    1. 将一片完全微型蛋白酶抑制剂溶解在1.5ml水中,制备7x储备溶液。
    2. 因为PVPP是不溶的,所以可能需要切割移液管吸头的末端以将蛋白质提取缓冲液添加到研磨的木质部材料中。
  2. 反应混合物
    准备酶反应混合物如下:
    库存解决方案

    最终集中
    500mM Tris-HCl pH7.0
    8微升
    100 mM
    20 mM DTT
    2微升
    1 mM
    2mM p-coumaroyl-CoA
    2微升
    100μM
    2mM莽草酸 2微升
    100μM
    木质部蛋白提取物
    x微博
    10μg
    水到
    40微升


致谢

该方案由Vanholme及其同事在Vanholme等人(2013a)中简要描述。 我们衷心感谢通过欧盟委员会第七框架内研究总司的资助 授予协议N°211917(ENERGYPOPLAR),N°211868(NOVELTREE)和N°311804(MULTIBIOPRO)的程序(FP7/2007-2013),根特大学的Hercules程序Synapt Q-Tof(授予号AUGE/014)和多学科研究伙伴关系"根特大学的可持续经济生物技术"(01MRB510W)。 RV负责研究基金会佛兰德斯的博士后研究。

参考文献

  1. Bradford,M.M。(1976)。 利用蛋白质染料结合原理的快速灵敏的微克量蛋白定量方法。 Anal Biochem 72:248-254。
  2. Hoffmann,L.,Maury,S.,Martz,F.,Geoffroy,P。和Legrand,M。(2003)。 酰基转移酶的纯化,克隆和性质,其在苯丙素代谢中控制莽草酸和奎尼酸酯中间体。 a> J Biol Chem 278(1):95-103
  3. Hoffmann,L.,Besseau,S.,Geoffroy,P.,Ritzenthaler,C.,Meyer,D.,Lapierre,C.,Pollet,B.and Legrand,M。(2004)。 羟基肉桂酰辅酶的沉默莽草酸/quinate hydroxycinnamoyltransferase影响苯丙素生物合成。 植物细胞 16(6):1446-1465
  4. Vanholme,B.,Cesarino,I.,Goeminne,G.,Kim,H.,Marroni,F.,Van Acker,R.,Vanholme,R.,Morreel,K.,Ivens,B.,Pinosio, ,Morgante,M.,Ralph,J.,Bastien,C.and Boerjan,W。(2013a)。 具有罕见缺陷型等位基因(BRDA)的育种:天然黑猩猩 HCT 具有修饰的木质素的突变体作为案例研究。 New Phytol 198(3):765-776。
  5. Vanholme,R.,Cesarino,I.,Rataj,K.,Xiao,Y.,Sundin,L.,Goeminne,G.,Kim,H.,Cross,J.,Morreel,K.,Araujo, Welsh,L.,Haustraete,J.,McClellan,C.,Vanholme,B.,Ralph,J.,Simpson,GG,Halpin,C。和Boerjan, Caffeoyl莽草酸酯酶(CSE)是木质素生物合成中的酶 ce DOI :10.1126/science.1241602。

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引用:Cesarino, I., Vanholme, R., Goeminne, G., Vanholme, B. and Boerjan, W. (2013). Shikimate Hydroxycinnamoyl Transferase (HCT) Activity Assays in Populus nigra. Bio-protocol 3(22): e978. DOI: 10.21769/BioProtoc.978.
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