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Feb 2017

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Analysis of Xyloglucan Composition in Arabidopsis Leaves
拟南芥叶中木葡聚糖组分分析   

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Abstract

Xyloglucan is one of the main components of the primary cell wall in most species of plants. This protocol describes a method to analyze the composition of the enzyme-accessible and enzyme-inaccessible fractions of xyloglucan in the model species Arabidopsis thaliana. It is based on digestion with an endoglucanase that attacks unsubstituted glucose residues in the backbone. The identities and relative amounts of released xyloglucan fragments are then determined using MALDI-TOF mass spectrometry.

Keywords: Xyloglucan (木葡聚糖), Arabidopsis (拟南芥), Cell wall (细胞壁), Hemicellulose (半纤维素), Primary wall (初生壁), MALDI-TOF (MALDI-TOF)

Background

In many flowering plants xyloglucan is a major component of primary cell walls, where it plays an important role in growth regulation. Sequential extraction protocols offer a way of separating distinct xyloglucan domains (Pauly et al., 1999). Some xyloglucan appears to be trapped inside cellulose microfibrils while another fraction is bound to their surface through hydrogen bonding. The rest of the xyloglucan, possibly the majority of it, occupies the space between microfibrils (Park and Cosgrove, 2015). Part of this later xyloglucan can be extracted through direct endoglucanase digestion of cell wall material. Much of the remaining xyloglucan can then be released through alkaline treatment. Studies of mutants deficient in xyloglucan exoglycosidases have shown that these enzymes, together with Xyloglucan Endotransglycosylases/Hydrolases, act mostly on the enzyme-accessible fraction (Sampedro et al., 2010; Günl and Pauly, 2011; Günl et al., 2011; Sampedro et al., 2012; Sampedro et al., 2017). Digestion of Arabidopsis xyloglucan with endoglucanases that attack unsubstituted glucose residues results in a mixture of three and four-glucose subunits that can be quickly and easily analyzed through MALDI-TOF mass spectrometry (Lerouxel et al., 2002; Günl et al., 2010). The area of the ion peaks can be then used to quantify the abundance of the different fragment, although there is evidence of significant differences in response factors (Tuomivaara et al., 2015). This protocol incorporates some changes from our previous versions, such as the use of SDHB (Super-DHB) matrix, which reduces the noise, and the addition of NaCl during extraction to prevent the formation of potassium adducts.

Materials and Reagents

  1. Pipette tips (2 μl, 200 μl, 1,000 μl)
  2. 1.5 ml microcentrifuge tubes
  3. Centrifugal filters, modified PES, 10K (VWR, catalog number: 82031-348 )
  4. 200 μl PCR tubes
  5. Mature Arabidopsis leaves (2 or 3 leaves)
  6. Liquid nitrogen
  7. Ethanol (Merck, catalog number: 1.00983 )
  8. Type II purified water
  9. Acetone (Sigma-Aldrich, catalog number: 179973 )
  10. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  11. Cellulase suspension from Trichoderma longibrachiatum (Megazyme, catalog number: E-CELTR )
  12. Pyridine (VWR, catalog number: 27199.292 )
  13. Thimerosal (Sigma-Aldrich, catalog number: T5125 )
  14. Sodium hydroxide (NaOH) (Merck, catalog number: 106469 )
  15. Acetic acid (AppliChem, catalog number: 141008.1611 )
  16. 2,5-Dihydroxybenzoic acid (Sigma-Aldrich, catalog number: 39319 )
  17. 2-Hydroxy-5-methoxybenzoic acid (Sigma-Aldrich, catalog number: 146188 )
  18. Acetonitrile (Merck, catalog number: 1.00029 )
  19. Xyloglucan oligosaccharide mixture (Megazyme, catalog number: O-XGHON )
  20. Digestion buffer (see Recipes)
  21. Super-DHB (SDHB) matrix (see Recipes)

Equipment

  1. Variable volume single channel manual pipettes (0.2 to 2 μl, 2 to 20 μl, 10 to 100 μl, 100 to 1,000 μl)
  2. Pellet pestles (Sigma-Aldrich, catalog number: Z359947 )
  3. Bench pillar drill, 250 W
  4. Insulation foam block
  5. Polycarbonate cover
  6. Dry block heater
  7. Vortexer
  8. Microcentrifuge (Eppendorf, model: 5424 )
  9. SpeedVac System (Thermo Fisher Scientific, Thermo ScientificTM, model: SavantTM SC210 P1 )
  10. Ultrasonic bath
  11. Orbital shaker
  12. MALDI target plate (Bruker, model: MTP 384 ground steel TF, catalog number: 8209519 )
  13. Mass spectrometer (Bruker, model: UltraFlex III MALDI-TOF/TOF )

Software

  1. Flex Analysis Version 3.0 (Bruker)

Procedure

  1. Cell wall extraction
    1. Collect 100 mg of mature Arabidopsis leaves (2 or 3 leaves) in a microcentrifuge tube.
      Note: We usually collect fully expanded leaves with no signs of senescence from 4 to 5 weeks old plants grown in 16-h days at 22 °C/18 °C light/dark temperature.
    2. Grind for a few seconds with a pestle mounted on a bench pillar drill at about 600 rpm, keeping the sample submerged in liquid N2 (see Figure 1). Do not add liquid N2 to the sample. Pestles should be changed between samples.


      Figure 1. Grinding setup. The bottom half of a plastic 60 ml container was inserted in a block of insulation foam and covered with a perforated piece of polycarbonate. A channel was added to facilitate addition of liquid N2.

    3. Add 1 ml of 80% ethanol. Vortex for 5 sec and heat for 10 min at 50 °C in a dry block heater. Centrifuge for 5 min at 3,000 x g and discard the supernatant.
    4. Add 1 ml of 100% ethanol to the precipitate. Vortex for 5 sec and keep at room temperature for 10 min. Centrifuge for 5 min at 3,000 x g and discard the supernatant.
    5. Add 1 ml of acetone to the precipitate. Vortex for 5 sec and keep at room temperature for 10 min. Centrifuge for 5 min at 3,000 x g and discard the supernatant.
    6. Dry samples in a Speed-Vac for 5 min approximately.
      Note: We try to avoid complete drying to facilitate resuspension. Samples can also be air-dried. Dried samples can be stored at -20 °C for later processing.

  2. Extraction of enzyme-accessible xyloglucan
    1. Add 1 ml of 100 mM NaCl to dried wall residue and resuspend in an ultrasonic bath for at least 20 min. Vortex the sample for 5 sec several times during resuspension. Centrifuge for 3 min at 18,000 x g and discard the supernatant.
    2. Add 1 ml of water to cell wall residue, vortex for 5 sec and incubate in an ultrasonic bath for at least 5 min. Centrifuge for 3 min at 18,000 x g and discard the supernatant. Repeat for a total of two times.
    3. Add 350 μl of digestion buffer (see Recipes).
      Note: Complete resuspension is not necessary as it will be finished during overnight incubation.
    4. Pipette a volume of cellulase suspension that contains 6 units of enzyme per sample into a centrifugal filter. Volume will vary depending on enzyme batch. Add 450 μl of digestion buffer. Centrifuge for 5 min at 14,000 x g and discard the filtrate.
    5. Add 450 μl of digestion buffer. Centrifuge for 5 min at 14,000 x g and discard the filtrate. Repeat for a total of two times.
    6. Add 50 μl of digestion buffer per sample to the retentate and pipette to a clean centrifuge tube.
      Note: Steps B4 to B6 can be carried out while cell wall residue is resuspended.
    7. Pipette 50 μl of the enzyme solution obtained in step B6 to each of the resuspended cell wall samples from step B3.
    8. Incubate overnight in an orbital shaker at 37 °C and 150 rpm.
    9. The next day wash one centrifugal filter per sample by adding 500 μl of water, centrifuging for 3 min at 14,000 x g and removing the filtrate. Repeat this process for a total of 3 times.
      Note: This step is necessary to remove traces of a polymer, which will otherwise interfere with the MALDI-TOF analysis.
    10. Centrifuge the cell wall digestion for 5 min at 18,000 x g.
    11. Pipette the supernatant to a pre-washed centrifugal filter. Centrifuge for 10 min at 14,000 x g.
    12. Discard the filter and dry the filtrate on a Speed-Vac.
      Note: Some residue is usually visible at the bottom of the tube.
    13. Store at -20 °C.

  3. Extraction of non-accessible xyloglucan
    1. Wash the cell wall residue from the accessible xyloglucan extraction (precipitate from step B10) by adding 1 ml of water, centrifuging for 5 min at 18,000 x g and discarding the supernatant. Repeat for a total of two times.
    2. Add 900 μl of 17% NaOH (w/v) and incubate overnight in an orbital shaker at 37 °C.
    3. Add 300 μl of acetic acid and centrifuge for 5 min at 18,000 x g.
    4. Pipette 500 μl of the supernatant onto a centrifugal filter, centrifuge at 14,000 x g for 10-15 min and discard the filtrate. Add additional supernatant and repeat until all of it has been filtered.
    5. Add 500 μl of digestion buffer to the filter, centrifuge at 14,000 x g for 15-20 min and discard the filtrate. Repeat for a total of two times.
    6. Add 350 μl of digestion buffer to the filter and pipette the retentate to a new microcentrifuge tube.
    7. Follow the procedure for extraction of enzyme-accessible xyloglucan starting in step B4.

  4. MALDI-TOF analysis
    1. Resuspend dried xyloglucan digestion (step B13) in 20 μl of 10 mM NaCl by repeated pipetting. Centrifuge at 18,000 x g for 2 min.
    2. Pipette 2 μl from the top of the solution into a clean 200 μl PCR tube and add 6 μl of SDHB solution (see Recipes). Pipette 2 μl of the mixture onto the MALDI target plate.
    3. To prepare a calibration spot mix 2 μl of a 2 mM solution of xyloglucan oligosaccharides (Megazyme) with 6 μl of SDHB solution. Pipette 2 μl of the mixture onto the MALDI target plate.
    4. Air dry at room temperature for approximately 30 min.
      Note: A dissecting microscope can be used to check the samples. Some crystals should be visible on the plate (Figure 2).


      Figure 2. Examples of crystallization on the MALDI plate. Although the sample on the left is cleaner both produced usable spectra. Bars = 1 mm.

    5. Load the target plate onto a MALDI-TOF/TOF mass spectrometer.
    6. Operate the mass spectrometer in reflectron and positive mode, at an accelerating voltage of 25 kV with a PIE (Pulsed Ion Extraction) of 10 nsec. Set matrix suppression at 450 Da. Average a total of 600 laser shots delivered in 3 sets of 200 shots (at 100 Hz) to 3 different locations.
    7. Calibrate the mass range using the calibration spot. The three main peaks correspond to the following sodium adducts: XXXG (C39H66033Na1, 1085.3384 Da), XXLG/XLXG (C45H76038Na1, 1247.3912) and XLLG (C51H86043Na1, 1409.4440). These abbreviations correspond to the standard xyloglucan oligosaccharide nomenclature (Tuomivaara et al., 2015).
    8. Compile mass spectra for each sample by plotting the mass over charge (m/z) ratio (x-axis) of all detected ion species against their measured intensities (y-axis) using Flex Analysis Version 3.0 (Bruker).

Data analysis

  1. Using Flex Analysis label peaks using SNAP (Sophisticated Numerical Annotation Procedure) with signal to noise threshold set at 3. The program will calculate the area of the peaks from the isotopic envelope for each m/z (Figure 3). Export the data to Excel.


    Figure 3. Screenshot of Flex Analysis showing a list of identified peaks with their areas, and a detail of the 1597 m/z peak

  2. Manually select the peaks with an m/z corresponding to xyloglucan fragments. Those present in wild-type xyloglucan are shown in Table 1. Sum the total area of the peaks and calculate the proportion of the total area that corresponds to each of the identified xyloglucan fragments. Average at least three independent extractions. Student’s t-test or other statistical methods can be used to compare the proportions of xyloglucan fragments in different genotypes. Extraction of non-accessible xyloglucan results in loss of acetyl groups and only non-acetylated fragments will be observed. Changes that are more evident in accessible xyloglucan, compared to non-accessible xyloglucan, are likely caused by xyloglucan metabolism in the wall.

    Table 1. Structure and m/z values of xyloglucan fragments present in wild-type Columbia leaves. Structure abbreviations correspond to the standard xyloglucan oligosaccharide nomenclature, with acetylation indicated by underlined symbols (Tuomivaara et al., 2015). Adduct composition is indicated as the number of hexoses (H), deoxyhexoses (DH), pentoses (P), acetyl groups (Ac) and sodium atoms (Na). The m/z values of other oligosaccharide adducts can be calculated using the following formula: 162 x H + 146 x DH + 132 x P + 42 x Ac + 41.

Notes

An internal standard, such as malto-oligosaccharides or cello-oligosaccharides can be added to the SDHB solution for absolute quantification. Washes with water before extraction will remove a small amount of xyloglucan (Günl and Pauly, 2011). The endoglucanase used in this protocol appears to cut in front of every single unsubstituted glucose. Other endoglucanase with different specificities can produce a different set of fragments when analyzing unusual xyloglucan (Günl et al., 2011). This method cannot separate fragments of identical mass, such as XLXG and XXLG, although in some cases MALDI-TOF/TOF can be used on the same samples to identify the fragments (Sampedro et al., 2010; Sampedro et al., 2012; Sampedro et al., 2017).

Recipes

  1. Digestion buffer
    36.6 μl acetic acid
    29 μl pyridine
    20 mg thimerosal
    100 ml of water
    Store at 4 °C
  2. SDHB matrix
    9 mg ml-1 2,5-dihydroxybenzoic acid
    1 mg ml-1 2-hydroxy-5-methoxybenzoic
    70% acetonitrile
    30% water
    Store at -20 °C for one month

Acknowledgments

This protocol was briefly described in Sampedro et al. (2017). This work was supported by the Ministerio de Economía y Competividad (grant No. BIO2012-40032-C03-01) and the Xunta de Galicia (grant No. PGIDIT10PXIB200305PR).

References

  1. Günl, M., Gille, S. and Pauly, M. (2010). OLIgo mass profiling (OLIMP) of extracellular polysaccharides. J Vis Exp (40).
  2. Günl, M., Neumetzler, L., Kraemer, F., de Souza, A., Schultink, A., Pena, M., York, W. S. and Pauly, M. (2011). AXY8 encodes an α-fucosidase, underscoring the importance of apoplastic metabolism on the fine structure of Arabidopsis cell wall polysaccharides. Plant Cell 23(11): 4025-4040.
  3. Günl, M. and Pauly, M. (2011). AXY3 encodes a α-xylosidase that impacts the structure and accessibility of the hemicellulose xyloglucan in Arabidopsis plant cell walls. Planta 233(4): 707-719.
  4. Lerouxel, O., Choo, T. S., Seveno, M., Usadel, B., Faye, L., Lerouge, P. and Pauly, M. (2002). Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting. Plant Physiol 130(4): 1754-1763.
  5. Park, Y. B. and Cosgrove, D. J. (2015). Xyloglucan and its interactions with other components of the growing cell wall. Plant Cell Physiol 56(2): 180-194.
  6. Pauly, M., Albersheim, P., Darvill, A. and York, W. S. (1999). Molecular domains of the cellulose/xyloglucan network in the cell walls of higher plants. Plant J 20(6): 629-639.
  7. Sampedro, J., Gianzo, C., Iglesias, N., Guitian, E., Revilla, G. and Zarra, I. (2012). AtBGAL10 is the main xyloglucan β-galactosidase in Arabidopsis, and its absence results in unusual xyloglucan subunits and growth defects. Plant Physiol 158(3): 1146-1157.
  8. Sampedro, J., Pardo, B., Gianzo, C., Guitian, E., Revilla, G. and Zarra, I. (2010). Lack of α-xylosidase activity in Arabidopsis alters xyloglucan composition and results in growth defects. Plant Physiol 154(3): 1105-1115.
  9. Sampedro, J., Valdivia, E. R., Fraga, P., Iglesias, N., Revilla, G. and Zarra, I. (2017). Soluble and membrane-bound β-glucosidases are involved in trimming the xyloglucan backbone. Plant Physiol 173(2): 1017-1030.
  10. Tuomivaara, S. T., Yaoi, K., O'Neill, M. A. and York, W. S. (2015). Generation and structural validation of a library of diverse xyloglucan-derived oligosaccharides, including an update on xyloglucan nomenclature. Carbohydr Res 402: 56-66.

简介

木葡聚糖是大多数植物种类中主要细胞壁的主要成分之一。 该方案描述了一种分析拟南芥拟南芥中木葡聚糖的可酶酶和不可酶酶级分的组成的方法。 它是基于内切葡聚糖酶的消化,其攻击骨架中的未取代的葡萄糖残基。 然后使用MALDI-TOF质谱法测定释放的木葡聚糖片段的身份和相对量。
【背景】在许多开花植物中,木葡聚糖是主要细胞壁的主要成分,其在生长调节中起重要作用。顺序提取方案提供了分离不同木葡聚糖结构域的方法(Pauly et al。,1999)。一些木葡聚糖似乎被捕获在纤维素微原纤维内,而另一部分通过氢键结合到它们的表面。木葡聚糖的其余部分(可能大多数)占据了微原纤维之间的距离(Park和Cosgrove,2015)。可以通过细胞壁材料的直接内切葡聚糖酶消化来提取部分后来的木葡聚糖。然后可以通过碱处理释放大部分剩余的木葡聚糖。缺乏木葡聚糖外切糖苷酶的突变体的研究表明,这些酶与葡聚糖内切葡糖基酶/水解酶一起主要作用于酶易接近部分(Sampedro等人,2010;Günl和Pauly,2011;Günl ,2011; Sampedro等人,2012; Sampedro等人,2017)。消化拟南芥木葡聚糖与内切葡聚糖酶攻击未经取代的葡萄糖残基导致三个和四个葡萄糖亚基的混合物,可以通过MALDI-TOF质谱快速和容易地分析(Lerouxel等人, ,2002;Günl等人,2010)。然后可以使用离子峰的面积来量化不同片段的丰度,尽管有应答因素有显着差异的证据(Tuomivaara等人,2015)。该协议包含了我们以前版本的一些改变,例如使用降低噪音的SDHB(Super-DHB)矩阵,以及在提取期间加入NaCl以防止形成钾加合物。

关键字:木葡聚糖, 拟南芥, 细胞壁, 半纤维素, 初生壁, MALDI-TOF

材料和试剂

  1. 移液头(2μl,200μl,1,000μl)
  2. 1.5 ml微量离心管
  3. 离心过滤器,改良PES,10K(VWR,目录号:82031-348)
  4. 200μlPCR管
  5. 成熟的拟南芥叶子(2或3叶)
  6. 液氮
  7. 乙醇(Merck,目录号:1.00983)
  8. II型净化水
  9. 丙酮(Sigma-Aldrich,目录号:179973)
  10. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  11. 木霉木霉纤维素酶悬浮液(Megazyme,目录号:E-CELTR)
  12. 吡啶(VWR,目录号:27199.292)
  13. 硫柳汞(Sigma-Aldrich,目录号:T5125)
  14. 氢氧化钠(NaOH)(Merck,目录号:106469)
  15. 乙酸(AppliChem,目录号:141008.1611)
  16. 2,5-二羟基苯甲酸(Sigma-Aldrich,目录号:39319)
  17. 2-羟基-5-甲氧基苯甲酸(Sigma-Aldrich,目录号:146188)
  18. 乙腈(Merck,目录号:1.00029)
  19. 葡聚糖寡糖混合物(Megazyme,目录号:O-XGHON)
  20. 消化缓冲液(参见食谱)
  21. 超级DHB(SDHB)矩阵(见配方)

设备

  1. 可变体积单通道手动移液器(0.2至2μl,2至20μl,10至100μl,100至1,000μl)
  2. 颗粒杵(Sigma-Aldrich,目录号:Z359947)
  3. 台架钻,250瓦
  4. 绝缘泡沫块
  5. 聚碳酸酯盖
  6. 干块加热器
  7. Vortexer
  8. 微量离心机(Eppendorf,型号:5424)
  9. SpeedVac系统(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Savant TM SC210 P1)
  10. 超声波浴
  11. 轨道摇床
  12. MALDI靶板(Bruker,型号:MTP 384 Ground steel TF,目录号:8209519)
  13. 质谱仪(Bruker,型号:UltraFlex III MALDI-TOF / TOF)

软件

  1. Flex Analysis 3.0版(Bruker)

程序

  1. 细胞壁提取
    1. 在微量离心管中收集100毫克成熟的拟南芥叶(2或3叶)。
      注意:我们通常在22°C / 18°C光/黑暗温度下,在16小时内从4至5周龄的植物中收集完全扩张的叶子,没有衰老迹象。
    2. 用约600转/分钟的台式钻头上的研杵研磨数秒钟,保持样品浸没在液体N 2(见图1)。不要向样品中加入液氮N 2。应在样品之间更换杵。


      图1.研磨设置将塑料60毫升容器的下半部分插入绝缘泡沫块中,并覆盖有多孔聚碳酸酯。加入通道以便于添加液体N 2。

    3. 加入1毫升80%乙醇。旋转5秒,并在50℃下在干燥块加热器中加热10分钟。以3,000×g离心5分钟,弃去上清液
    4. 向沉淀物中加入1ml 100%乙醇。涡旋5秒,并在室温下保持10分钟。以3,000×g离心5分钟,弃去上清液
    5. 向沉淀物中加入1毫升丙酮。涡旋5秒,并在室温下保持10分钟。以3,000×g离心5分钟,弃去上清液
    6. 干燥样品在Speed-Vac中大约5分钟。
      注意:我们尽量避免完全干燥以促进再悬浮。样品也可以风干。干燥样品可以储存在-20°C进行后续处理。

  2. 提取可酶切的木葡聚糖
    1. 向干燥的壁残留物中加入1ml 100mM NaCl,并重悬于超声浴中至少20分钟。在再悬浮过程中将样品涡旋5秒钟。以18,000×g离心3分钟,弃去上清液。
    2. 向细胞壁残渣中加入1ml水,旋转5秒,并在超声波浴中孵育至少5分钟。以18,000×g离心3分钟,弃去上清液。重复两次。
    3. 加入350μl消化缓冲液(参见食谱)。
      注意:完全重新悬浮是不必要的,因为它将在夜间孵化完成。
    4. 移取一定量的纤维素酶悬浮液,每个样品含有6单位酶到离心过滤器中。体积将根据酶批次而变化。加入450μl消化缓冲液。离心5分钟,放置14,000 x g,然后弃去滤液。
    5. 加入450μl消化缓冲液。以14,000xg离心5分钟并弃去滤液。重复两次。
    6. 将50μl每个样品的消化缓冲液加入到渗余物中并移液至干净的离心管 注意:步骤B4至B6可以在细胞壁残留物重悬时进行。
    7. 将50μl步骤B6中得到的酶溶液移至步骤B3的每个重悬细胞壁样品。
    8. 在37°C和150 rpm的轨道振荡器中孵育过夜
    9. 第二天,通过加入500μl水洗涤每个样品的一个离心过滤器,以14,000xg离心3分钟并除去滤液。重复此过程总共3次。
      注意:此步骤是去除痕量的聚合物,否则会影响MALDI-TOF分析。
    10. 以18,000 x g离心细胞壁消化5分钟。
    11. 将上清液移至预先冲洗的离心过滤器。以14,000 x g离心10分钟。
    12. 丢弃过滤器并在Speed-Vac上干燥滤液。
      注意:有些残留物通常在管底部可见。
    13. 储存于-20°C。

  3. 提取不可接触的木葡聚糖
    1. 通过加入1ml水,从可接近的木葡聚糖提取物(步骤B10的沉淀)洗涤细胞壁残留物,以18,000×g离心5分钟并弃去上清液。重复两次。
    2. 加入900μl17%NaOH(w / v),并在37°C的轨道振荡器中孵育过夜。
    3. 加入300μl乙酸,并以18,000 x g离心5 min
    4. 将500μl上清液吸取到离心过滤器上,以14,000 x g离心10-15分钟,弃去滤液。添加额外的上清液并重复,直到所有的上清液都被过滤
    5. 向滤器中加入500μl消化缓冲液,以14,000 x g离心15-20分钟,弃去滤液。重复两次。
    6. 将350μl消化缓冲液加入过滤器,并将渗余液移至新的微量离心管
    7. 按照步骤B4中提取可酶切的木葡聚糖的步骤。

  4. MALDI-TOF分析
    1. 通过重复移取将干燥的木葡聚糖消化(步骤B13)重悬于20μl10mM NaCl中。以18,000 x g离心2分钟。
    2. 将2μl从溶液顶部吸取到干净的200μlPCR管中,加入6μlSDHB溶液(参见食谱)。移取2μl混合物到MALDI靶板上。
    3. 准备2μl2mM木葡聚糖寡糖(Megazyme)溶液与6μlSDHB溶液的校准点混合物。将2μl混合物吸入MALDI靶板上
    4. 在室温下空气干燥大约30分钟 注意:解剖显微镜可用于检查样品。板上应有一些晶体(图2)。


      图2. MALDI板上结晶的实例。尽管左侧的样品更清洁,但两者均可生成可用的光谱。酒吧= 1毫米。

    5. 将目标板装载到MALDI-TOF / TOF质谱仪上。
    6. 以10ns的PIE(脉冲离子提取)在25kV的加速电压下以反射和正模式操作质谱仪。在450 Da处设置矩阵抑制。平均共有600张激光照片,以3套200张(100 Hz)传送到3个不同的地点。
    7. 使用校准点校准质量范围。三个主峰对应于以下钠加合物:XXXG(C39 66 33 1 1, 1085.3384Da),XXLG / XLXG(C H 38 1 1,127.3912)和XLLG C H 86 1 1 1> 1,1409.4440)。这些缩写对应于标准的木葡聚寡糖命名法(Tuomivaara et al。,2015)。
    8. 使用Flex Analysis 3.0版(通过绘制所有检测到的离子物质的电荷质量与其测量的强度(y轴))的质量(x轴),绘制每个样品的质谱图(布鲁克)

数据分析

  1. 使用具有信号噪声阈值设置为3的SNAP(复杂数值注释步骤)的Flex Analysis标签峰。程序将从每个m / z的同位素包络计算峰的面积(图3 )。将数据导出到Excel。


    图3. Flex Analysis的屏幕截图显示了具有其区域的已识别峰的列表,以及1597
    />
  2. 手动选择对应于木葡聚糖片段的m / z 的峰。存在于野生型木葡聚糖中的那些显示在表1中。总和峰的总面积并计算对应于每个鉴定的木葡聚糖片段的总面积的比例。平均至少三次独立提取。可以使用Student's t检验或其他统计方法来比较不同基因型中木葡聚糖片段的比例。不可接近的木葡聚糖的提取导致乙酰基的损失,并且仅观察到非乙酰化的片段。与不可接触的木葡聚糖相比,在可及的木葡聚糖中更明显的变化可能是由于木葡聚糖在墙壁中的代谢引起的。

    表1.野生型哥伦比亚叶中存在的木葡聚糖片段的结构和m / z 值。 结构缩写对应于标准的木葡聚糖寡糖命名法,乙酰化由下划线的符号表示(Tuomivaara等人,2015)。加合物组成指示为己糖(H),脱氧己糖(DH),戊糖(P),乙酰基(Ac)和钠原子(Na)的数目。其他寡糖加合物的m / z值可以使用下式计算:162×H + 146×DH + 132×P + 42×Ac + 41.

笔记

可以向SDHB溶液中添加诸如麦芽低聚糖或低聚糖的内标,用于绝对定量。提取前用水冲洗少量木葡聚糖(Günl和Pauly,2011)。本协议中使用的内切葡聚糖酶似乎在每个单一未取代的葡萄糖前面切割。当分析不寻常的木葡聚糖时,具有不同特异性的其他内切葡聚糖酶可以产生不同的片段(Günlet al。,2011)。该方法不能分离相同质量的片段,例如XLXG和XXLG,尽管在某些情况下,可以将MALDI-TOF / TOF用于相同的样品以鉴定片段(Sampedro等人,2010; Sampedro等人,2012; Sampedro等人,2017)。

食谱

  1. 消化缓冲液
    36.6μl乙酸
    29μl吡啶
    20毫克硫柳汞
    100ml水
    储存于4°C
  2. SDHB矩阵
    9mg ml 2,5-二羟基苯甲酸
    1mg ml 2-羟基-5-甲氧基苯甲酸 70%乙腈 30%水
    在-20°C储存一个月

致谢

这个协议在Sampedro等人(2017)中简要描述。这项工作得到经济部长(授权号BIO2012-40032-C03-01)和Xunta de Galicia(授权号PGIDIT10PXIB200305PR)的支持。

参考

  1. Günl,M.,Gille,S.和Pauly,M。(2010)。 细胞外多糖的OLIgo质谱分析(OLIMP)。 Vis Vis < / em>(40)。
  2. Günl,M.,Neumetzler,L.,Kraemer,F.,de Souza,A.,Schultink,A.,Pena,M.,York,W.S。和Pauly,M。(2011)。 AXY8编码α-岩藻糖苷酶,强调了重组代谢对细胞结构的重要性拟南芥细胞壁多糖。植物细胞23(11):4025-4040。
  3. Günl,M。和Pauly,M。(2011)。 AXY3 编码影响结构和可达性的α-木糖苷酶拟南芥植物细胞壁中的半纤维素木葡聚糖。植物233(4):707-719。
  4. Lerouxel,O.,Choo,T.S.,Seveno,M.,Usadel,B.,Faye,L.,Lerouge,P。和Pauly,M。(2002)。 通过酶促低聚糖指纹图谱对植物细胞壁突变体的快速结构表型。 Physiol 130(4):1754-1763。
  5. Park,Y.B。和Cosgrove,D.J。(2015)。 木葡聚糖及其与日益增长的细胞壁的其他成分的相互作用。 Cell Physiol 56(2):180-194。
  6. Pauly,M.,Albersheim,P.,Darvill,A.and York,W.S。(1999)。 高等植物细胞壁中纤维素/木葡聚糖网络的分子结构域。植物J 20(6):629-639。
  7. Sampedro,J.,Gianzo,C.,Iglesias,N.,Guitian,E.,Revilla,G。和Zarra,I。(2012)。 AtBGAL10是拟南芥中主要的木葡聚糖β-半乳糖苷酶,其缺失导致不寻常的木葡聚糖亚基和生长缺陷。植物生理学158(3):1146-1157。
  8. Sampedro,J.,Pardo,B.,Gianzo,C.,Guitian,E.,Revilla,G。和Zarra,I.(2010)。 拟南芥中缺乏α-木糖苷酶活性改变木葡聚糖组成并产生生长缺陷。植物生理学154(3):1105-1115。
  9. Sampedro,J.,Valdivia,E.R.,Fraga,P.,Iglesias,N.,Revilla,G。和Zarra,I.(2017)。 可溶性和膜结合的β-葡糖苷酶参与修剪木葡聚糖骨架。植物生理学173(2):1017-1030。
  10. Tuomivaara,S.T.,Yaoi,K.,O'Neill,M.A.and York,W.S。(2015)。 不同木葡聚糖衍生的寡糖的文库的生成和结构验证,包括木葡聚糖命名法的更新。
    Carbohydr Res 402:56-66。
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Copyright: © 2017 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. Sampedro, J., Gianzo, C., Guitián, E., Revilla, G. and Zarra, I. (2017). Analysis of Xyloglucan Composition in Arabidopsis Leaves. Bio-protocol 7(19): e2569. DOI: 10.21769/BioProtoc.2569.
  2. Sampedro, J., Valdivia, E. R., Fraga, P., Iglesias, N., Revilla, G. and Zarra, I. (2017). Soluble and membrane-bound β-glucosidases are involved in trimming the xyloglucan backbone. Plant Physiol 173(2): 1017-1030.
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