搜索

Rapid Determination of Cellulose, Neutral Sugars, and Uronic Acids from Plant Cell Walls by One-step Two-step Hydrolysis and HPAEC-PAD
采用一步两步式水解法和HPAEC-PAD快速测定植物细胞壁的纤维素、中性糖和糖醛酸   

评审
匿名评审
下载 PDF 引用 收藏 1 提问与回复 分享您的反馈 Cited by

本文章节

Abstract

The plant cell wall is primarily composed of the polysaccharides cellulose, hemicellulose and pectin. The structural and compositional complexity of these components are important for determining cell wall function during plant growth. Moreover, cell wall structure defines a number of functional properties of plant-derived biomass, such as rheological properties of foods and feedstock suitability for the production of cellulosic biofuels. A typical characterization of cell wall chemistry in the molecular biology lab consists of a mild acid hydrolysis for the quantification of hemicellulose and pectin-derived monomers and a separate analysis of cellulose by the Updegraff method. We have adopted a streamlined ‘one-step two-step’ hydrolysis protocol that allows for the simultaneous determination of cellulose content, neutral sugars, and uronic acids by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) of paired samples. In our work, this protocol has largely replaced Updegraff cellulose quantification and hydrolysis with 2 M TFA for the determination of matrix polysaccharide composition at the micro scale.

Keywords: Cellulose(纤维素), Cell Wall(细胞壁), Hemicellulose(半纤维素), HPAEC-PAD(离子色谱法)

Background

The protocol is based on paired analysis of samples hydrolyzed in 4% (w/v) sulfuric acid at 121 °C. One set of samples is first pretreated with 72% (w/w) sulfuric acid to swell cellulose and make it susceptible to dilute acid hydrolysis (Saeman hydrolysis in Figure 1; Saeman et al., 1945). The other set of samples are not subjected to this pretreatment, resulting in hydrolysis of non-crystalline matrix polysaccharides (Matrix hydrolysis in Figure 1). Comparison of the glucose recovered from each hydrolysis regime allows calculation of cellulose amount that is in good agreement with the more labor-intensive Updegraff (1969) protocol (Bauer and Ibáñez, 2014). In addition to glucose (Glc), other sugars derived from matrix polysaccharides can be quantified from the matrix hydrolysis samples (Gao et al., 2014). Thus, with relatively few manual manipulations, matrix monosaccharides and cellulose can be quantified from two hydrolysis samples and a total of four HPAEC-PAD experiments. Despite the number of chromatographic separations required by the protocol, the great reduction in ‘hands-on’ time required to prepare samples makes this technique well-suited to high throughput analyses. Although we have found that robust and reproducible analysis of rhamnose (Rha), arabinose (Ara), mannose (Man), and xylose (Xyl) requires multiple HPAEC-PAD runs, reports describing simultaneous quantification of all neutral and acidic sugars in a single run may allow further improvement of this protocol’s throughput (Zhang et al., 2012; Voiniciuc and Grünl, 2016). The steps of the protocol described here are outlined in Figure 1.


Figure 1. Protocol Overview. AIR = Alcohol insoluble residue.

Materials and Reagents

  1. 2 ml safe-lock microcentrifuge tubes (Eppendorf, catalog number: 022363352 )
  2. Aluminum foil
  3. 2 ml Sarstedt tubes with screw caps (SARSTEDT, catalog number: 72.694.007 )
  4. 2 ml screw cap autosampler vials (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: C4000-1W )*
  5. Autosampler vial caps with pre-slit septa (Phenomenex, catalog number: AR0-8977-13-B )*
  6. 5/32” Grinding Balls, 440C Stainless Steel, Treated (OPS Diagnostics, catalog number: GBSS 156-5000-01 )*
  7. Disposable anti-static polypropylene powder scoops (Cole-Parmer Instrument, catalog number: 06277-60 )
    Note: This product has been discontinued.
  8. Ethanol (Decon Labs, catalog number: V1016 )*
  9. Chloroform (Thermo Fisher Scientific, Fisher Scientific, catalog number: C607-4 )*
  10. Methanol (Thermo Fisher Scientific, Fisher Scientific, catalog number: A412P4 )*
  11. Acetone (Thermo Fisher Scientific, Fisher Scientific, catalog number: A184 )*
  12. 72% (w/w) sulfuric acid solution (RICCA Chemical, catalog number: R81916001A )*
  13. Ultrapure water (Milli-Q or equivalent)*
  14. 9 sugars:
    L-fucose (Fuc) (Sigma-Aldrich, catalog number: F2252 )*
    D-glucose (Glc) (Sigma-Aldrich, catalog number: G8270 )*
    D-galactose (Gal) (Sigma-Aldrich, catalog number: G0750 )*
    D-xylose (Xyl) (Sigma-Aldrich, catalog number: X1500 )*
    D-mannose (Man) (Sigma-Aldrich, catalog number: M8574 )*
    L-arabinose (Ara) (Sigma-Aldrich, catalog number: A3256 )*
    L-rhamnose (Rha) (Sigma-Aldrich, catalog number: W373011 )*
    D-galacturonic acid monohydrate (GalA) (Sigma-Aldrich, catalog number: 48280 )*
    D-glucuronic acid (GluA) (Sigma-Aldrich, catalog number: G5269 )*
  15. Sodium hydroxide solution (50%, w/w) (Thermo Fisher Scientific, Fisher Scientific, catalog number: SS254 )
  16. Sodium acetate, anhydrous (Sigma-Aldrich, catalog number: 71183 )
  17. Liquid nitrogen

*Note: These items can reliably be substituted with laboratory-grade equivalents from different vendors.

Equipment

  1. Freeze-dryer (Labconco, model: FreeZone 12 )* (optional)
  2. Magnet
  3. Aspirator
  4. Metal spatula
  5. Microcentrifuge (Eppendorf, model: 5417R )*
  6. Autoclave-compatible rack
  7. Autosampler
  8. Microbalance (Mettler Toledo, model: XS105 )*
  9. Ball mill (Retsch, model: MM 400 )*
  10. Dionex ICS-5000 HPAEC-PAD system, optionally equipped with an eluent generator (Thermo Fisher Scientific, Fisher Scientific, model: ICS-5000+ SYSTEM )
  11. CarboPac PA-20 Analytical column, 3 x 150 mm (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 060142 )
  12. CarboPac PA-20 Guard column, 3 x 30 mm (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 060144 )
  13. CarboPac PA-200 Analytical column, 3 x 250 mm (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 062896 )
  14. CarboPac PA-200 Guard column, 3 x 50 mm (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 062895 )

*Note: These items can reliably be substituted with laboratory-grade equivalents from different vendors.

Software

  1. Chromeleon 7 (Thermo Fisher Scientific)
  2. Microsoft Excel

Procedure

  1. Preparation of alcohol insoluble residue (AIR)
    1. Flash freeze < 300 mg fresh tissue in a 2 ml Eppendorf tube (Note 1). Homogenization and solvent extractions will be inefficient if more tissue is used. If plants are derived from agar-solidified plates, care must be taken to avoid taking any agar with the seedlings, as it will be hydrolyzed and quantified as Gal in later steps. If a freeze-dryer is not available, see Note 2 and skip to step A6.
    2. Leave the lid of the tube open, but cover the opening with a small piece of aluminum foil. Ensure the foil is vented by poking a small hole with a needle or pipet tip.
    3. Lyophilize frozen samples for 2-3 days.
    4. Add 3 steel balls to each tube and close the lids.
    5. Homogenize the dried samples for 2 min, shaking at 25 Hz at room temperature. Reverse the orientation of the tube holder and shake for an additional 2 min at 25 Hz.
    6. Ensure that a fine powder with no clumps is obtained before proceeding.
    7. Add 1.5 ml of 70% (v/v) ethanol to the tube and vortex. Remove the steel balls from the slurry using a magnet and discard them.
    8. Centrifuge at 20,000 x g for 10 min.
    9. Discard the supernatant using an aspirator that is compatible with organic solvents or pipet.
    10. Add 1.5 ml of 1:1 (v:v) chloroform:methanol. Disrupt the pellet with a small metal spatula and then thoroughly vortex the sample.
    11. Repeat steps A8-10 two more times for a total of 3 washes with chloroform:methanol.
    12. Centrifuge and remove the solvents as before (steps A8-9).
    13. Add 1.5 ml of acetone, disrupt the pellet, vortex, centrifuge, and discard the supernatant as before.
    14. Dry the final pellet either by using a vacuum centrifuge for at least 30 min or let air-dry overnight. The resulting material is the AIR (Note 3, Figure 2).


      Figure 2. AIR material after step A14

  2. One-step two-step hydrolysis
    1. For each AIR sample, accurately weigh ~1 mg of material into each of four 2 ml Sarstedt tubes on an analytical balance (Note 4). Avoid clumps of material. Record the exact weight of AIR in each tube. These four tubes are technical replicates that will be subjected to two different hydrolysis regimes as duplicates.
    2. Saeman hydrolysis samples
      1. To two tubes containing AIR, add 50 μl of 72% (w/w) sulfuric acid. Immediately cap the tubes, vortex vigorously, and centrifuge briefly to collect the sample in the bottom of the tube. Delayed or insufficient initial mixing can result in poor rehydration of the sample and incomplete hydrolysis.
      2. Incubate the tube at room temperature for 1 h. Vortex every 10 min or continuously with an Eppendorf Thermomixer.
      3. Add 1,400 μl water to each tube to adjust the sulfuric acid concentration to 4% (w/v). Vortex to mix.
    3. Matrix hydrolysis samples
      1. To the second set of two tubes, add 1,400 μl water first and then add 50 μl 72% (w/w) sulfuric acid to give a 4% (w/v) concentration of sulfuric acid. Cap the tubes and vortex.
    4. Recovery standards
      1. Prepare a standard mix consisting of 9 sugars (Fuc, Rha, Ara, Gal, Glc, Xyl, Man, GalA, and GluA), each at a concentration of 100 µg ml-1. It is recommended to prepare a concentrated stock of each sugar individually and then combine. Account for the extra mass of water if hydrated sugars are used. Prepare at least enough for use in this part of the protocol (1 ml) as well as for preparation of a standard curve in the next part of the protocol (138 μl). Sugar standards can be stored frozen once prepared.
      2. To account for sugar-specific losses during hydrolysis, a recovery standard is subjected to the same conditions as the matrix hydrolysis samples (Note 5). Pipet 500 μl of the standard mixture and 900 μl of water into a 2 ml Sarstedt tubes. Add 50 μl of 72% (w/w) sulfuric acid. Repeat with a second tube. Cap the tubes and vortex.
    5. Set all tubes except for one of the recovery standards in an autoclave-compatible rack and autoclave at 121 °C for 60 min. The second recovery standard tube is not autoclaved and serves as a control for calculating monosaccharide-specific correction factors.
    6. Cool samples to room temperature and centrifuge for 1 min at 20,000 x g to pellet any insoluble material.
    7. Dilute samples, recovery standard, and recovery standard control 100 fold by transferring 10 μl into an autosampler vial containing 990 μl of MilliQ water. Store samples at 4 °C for up to two weeks. Samples can be stored for a month or more if they are frozen at -20 °C. This dilution factor is convenient for AIR isolated from Arabidopsis seedlings, for more cellulose-rich AIR prepared from stems, wood, or other secondary tissues, a higher dilution factor should be used (200-500 fold).
    8. Standard curve samples
      1. Dilute standard mix from step B4 (9 sugars, 100 µg ml-1 each) to yield a standard curve between 0.05 µg ml-1 and 5 µg ml-1. Prepare this directly into 2 ml autosampler vials with pre-slit septa (Table 1). This range of concentrations generally covers the complete linear range of the instrument.
         
        Table 1. Preparation of standard curve samples

        *Note: Standard 8 is further diluted to prepare standards 1-4.

  3. HPAEC-PAD analysis
    1. HPAEC-PAD analysis #1 (All samples)
      1. Inject 25 μl of each standard, recovery standard, and sample onto a 3 x 150 mm CarboPac PA-20 analytical column equipped with a 3 x 50 mm guard column of the same material. Elute the compounds with 2 mM KOH at a flow rate of 0.4 ml min-1 for 30 min (Note 6). This run will resolve Fuc, Gal, Glc, Xyl, and Man. Rha and Ara are not consistently separated with these conditions, so they are quantified by a second analysis that is only necessary for the matrix hydrolysis samples.
      2. In our experience, retention times of peaks will shift earlier as more samples are run, and after ~50-100 samples it is necessary to perform a column flush with sodium acetate (see below, step C4).
    2. HPAEC-PAD analysis #2 (Matrix hydrolysis samples only)
      1. Inject 25 μl of each standard, recovery standard, and sample onto a 3 x 150 mm CarboPac PA-20 analytical column equipped with a 3 x 50 mm guard column of the same material. Elute the compounds with 18 mM KOH 0.4 ml min-1 for 30 min. This run will resolve Rha and Ara.
      2. A column flush may be required after ~50-100 samples are run (see below, step C4).
    3. HPAEC-PAD analysis #3 (Matrix hydrolysis samples only)
      1. Inject 25 μl of each standard, recovery standard, and sample onto a 3 x 150 mm CarboPac PA-200 analytical column equipped with a 3 x 50 mm guard column of the same material. Elute the compounds with a 10 min gradient of 50 mM to 200 mM sodium acetate in 100 mM NaOH at a flow rate of 0.4 ml min-1 (This is accomplished with a gradient of 5%-20% eluent B [100 mM NaOH, 1 M sodium acetate]. Eluent A consists of 100 mM NaOH and makes up the remaining volume). This run will resolve GalA and GluA.
    4. Column flushing procedure
      1. The performance of the CarboPac PA20 column degrades after running ~50-100 samples, leading to increasingly early retention times until peaks cannot be sufficiently resolved. This is remedied by flushing the column with 100 mM NaOH/1 M sodium acetate for 30 min followed by water for 30 min at 0.2 ml min-1. If the CarboPac PA20 column is being used with an HPAEC-PAD system that is equipped with an eluent generator, it will need to be temporarily moved to a different system or port in order to complete this procedure.

Data analysis

  1. Integrate all standard curve and hydrolysis samples using Chromeleon 7 software. For the calibration curves, a quadratic curve that is forced through the origin is used. For some samples, peak identity may need to be manually assigned as retention times tend to shift earlier with multiple runs. Figures 3-5 show representative chromatograms from the three HPAEC-PAD methods.


    Figure 3. HPAEC-PAD analysis #1 representative chromatograms. A. Standard mixture. B. Saeman hydrolysis sample. C. Matrix hydrolysis sample. The peak at ~8.2 min contains both Rha and Ara and is not quantified in this method.


    Figure 4. HPAEC-PAD analysis #2 representative chromatograms. A. Standard mixture. B. Matrix hydrolysis sample. Only Rha and Ara are quantified in these runs. Fuc elutes at ~3.4 min, Gal elutes at ~7.6 min, Glc elutes at ~8.2 min. The peak at ~9.0 min is a mixture of Xyl and Man.


    Figure 5. HPAEC-PAD analysis #3 representative chromatograms. A. Standard mixture. B. Matrix hydrolysis sample.

  2. From each run, the results (assayed monosaccharide concentration, µg ml-1) can be copied from the results table into a Microsoft Excel spreadsheet. Set up a table with results for each sugar in columns and a column with the AIR mass for each sample (Figure 6).
  3. Calculate correction factors for each sugar by dividing the concentration of the recovery standard by the concentration of the respective sugar in the recovery standard control. These are typically > 0.85 for neutral sugars, but are lower for uronic acids (~0.6-0.8). The same Glc recovery factor is used for both Saeman and Matrix hydrolysis samples. Lower than expected correction factors indicate excessive sample loss that can occur due to prolonged high temperature treatment. Repeat the hydrolysis and ensure that samples are cooled and diluted as soon as the autoclave run is complete.
  4. In a new column, calculate the recovery-standard corrected monosaccharide amounts as µg per mg of AIR using the following formula:

    Where,
    c = Assayed concentration (µg ml-1), 
    d = 100 (dilution factor),
    v = 1.450 (volume, ml),
    r = Recovery correction factor (From step C3),
    m = AIR mass (mg).
  5. At this point, technical replicates can be validated to check if there is any systematic difference in monosaccharide quantification that would justify discarding a technical replicate. (This would be indicative of sample loss or a weighing error.). Fuc, Gal, and Xyl should have consistent values in both the Saeman and Matrix hydrolysis samples. Glc will be much higher in the Saeman hydrolysis samples, and Man is expected to be more abundant in these samples as well since Glc isomerization to Man is a known side reaction during Saeman hydrolysis (Carpita and Shea, 1989). The extent of this side reaction is only ~2%, so it is ignored for the purpose of cellulose quantification.
  6. Average technical replicates into a new column. Only the Matrix hydrolysis samples, which were subjected to all three HPAEC-PAD methods, are used for reporting neutral sugars and uronic acids.
  7. To calculate cellulose amount, subtract Glcmatrix (from Matrix hydrolysis samples) from Glctotal (from Saeman hydrolysis samples) and multiply by 0.9. The 0.9 correction factor reflects the difference in molar mass between glucose (180.16 g mol-1) and anhydroglucose (162.14 g mol-1), as it occurs in the cellulose polymer prior to hydrolysis.
    Cellulose (µg mg-1 AIR) = 0.9 × (Glctotal (µg mg-1) - Glcmatrix (µg mg-1))
  8. For statistical analysis, we typically use at least three biological replicates of AIR prepared from independent plants or pools of seedlings. Results are reported as means and standard deviation and statistical significance is assessed by Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test.


    Figure 6. Screenshot of Excel spreadsheet illustrating the calculation of monosaccharide and cellulose from HPAEC-PAD integration data. The sample analyzed was AIR prepared from 5d old dark-grown Arabidopsis seedlings.

Notes

  1. Any starch present in the sample will contribute to glucose quantified in both hydrolysis regimes. If quantification of glucose derived from matrix polysaccharides is desired, starch can be reduced by moving light-grown plants to the dark for 24 h before tissue is collected (Sorek et al., 2015). Alternatively, starch can be enzymatically removed during preparation of the AIR (Pettolino et al., 2012) or can be independently assayed from the same AIR material (Yeats et al., 2016). Starch will not impact the determination of cellulose since it will contribute equally to the glucose quantified in the Saeman and matrix hydrolysis samples.
  2. If a freeze-dryer is not available, it is possible to prepare AIR directly from frozen tissue that is homogenized with a mortar and pestle or a ball mill. In both cases, the sample must remain frozen during homogenization: the mortar and pestle or ball mill tube rack should be immersed in liquid nitrogen before use. Tubes are more brittle at low temperatures and we have noted an increased rate of tubes breaking during homogenization in the ball mill, so we prefer to work with dried tissue at room temperature.
  3. The terms AIR and cell wall material are often used interchangeably, however, it is clear from the mass balance of quantified monosaccharides that AIR prepared from seedlings is only ~50% cell wall material. More extensive washing of AIR with an aqueous solution of SDS and sodium metabisulfite removes additional non-cell wall material (Dick-Pérez et al., 2011). However, this protocol is lengthy and inconvenient if multiple samples are to be analyzed. Since there can be variation in the efficiency of extraction and washing during AIR preparation, it is best practice to prepare all AIR samples to be analyzed at the same time. We find it is convenient to prepare up to ~60 AIR samples simultaneously, with centrifuge capacity being a limiting factor. For hydrolysis steps, the number of tubes multiplies by 4, so once AIR is prepared, we proceed in batches of 15-20 samples (a total of 60-80 hydrolysis samples). For a large project, we recommend including a control sample with each batch.
  4. Handling the dry AIR material can be difficult due to the buildup of static charge. For moving the AIR to Sarstedt tubes, we use disposable anti-static polypropylene powder scoops (Cole-Parmer). Laboratory humidity > 40% will help to reduce static.
  5. Sugar losses occurring during hydrolysis are somewhat concentration dependent. For convenience, a standard mixture with equal amounts of each sugar is prepared. For more accurate absolute quantification, recovery standards can be prepared based on the expected composition of the samples being analyzed.
  6. LC elution with the same concentration of NaOH instead of KOH gives comparable results. In our work, we have used an HPAEC-PAD equipped with a KOH eluent generator module for the CarboPac PA-20 analyses. User-prepared eluents are typically prepared with NaOH. The procedure for eluent preparation is detailed in the CarboPac PA-20 manual. Following the outlined procedure is particularly important for avoiding carbonate entering the system, which drastically affects the chromatography of sugars.

Acknowledgments

We thank Will Barnes (Penn State University) and Chris Somerville (University of California, Berkeley) for helpful comments on this protocol. Funding for this work is from the Energy Biosciences Institute and the Philomathia Foundation. This protocol is based on the following previously published reports: Bauer and Ibáñez, 2014; Chen et al., 2016; Sorek et al., 2015; and Yeats et al., 2016.

References

  1. Bauer, S. and Ibanez, A. B. (2014). Rapid determination of cellulose. Biotechnol Bioeng 111(11): 2355-2357.
  2. Carpita, N. C. and Shea, E. M. (1988). Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of parially methylated alditol acetates. In Biermann, C. J. and McGinnis, G. D. (Eds). Analysis of carbohydrates by GLC and MS. CRC Press, 157-216.
  3. Chen, S., Jia, H., Zhao, H., Liu, D., Liu, Y., Liu, B., Bauer, S. and Somerville, C. R. (2016). Anisotropic cell expansion is affected through the bidirectional mobility of cellulose synthase complexes and phosphorylation at two critical residues on CESA3. Plant Physiol 171(1): 242-250.
  4. Dick-Perez, M., Zhang, Y., Hayes, J., Salazar, A., Zabotina, O. A. and Hong, M. (2011). Structure and interactions of plant cell-wall polysaccharides by two- and three-dimensional magic-angle-spinning solid-state NMR. Biochemistry 50(6): 989-1000.
  5. Gao, X., Kumar, R. and Wyman, C. E. (2014). Fast hemicellulose quantification via a simple one-step acid hydrolysis. Biotechnol Bioeng 111(6): 1088-1096.
  6. Pettolino, F. A., Walsh, C., Fincher, G. B. and Bacic, A. (2012). Determining the polysaccharide composition of plant cell walls. Nat Protoc 7(9): 1590-1607.
  7. Saeman, J. F., Bubl, J. L. and Harris, E. E. (1945). Quantitative saccharification of wood and cellulose. Ind Eng Chem Anal Ed 17: 35-37.
  8. Sorek, N., Szemenyei, H., Sorek, H., Landers, A., Knight, H., Bauer, S., Wemmer, D. E. and Somerville, C. R. (2015). Identification of MEDIATOR16 as the Arabidopsis COBRA suppressor MONGOOSE1. Proc Natl Acad Sci U S A 112(52): 16048-16053.
  9. Updegraff, D. M. (1969). Semimicro determination of cellulose in biological materials. Anal Biochem 32(3): 420-424.
  10. Voiniciuc, C. and Günl, M. (2016). Analysis of monosaccharides in total mucilage extractable from Arabidopsis seeds. Bio-protocol 6(9): e1801.
  11. Yeats, T. H., Sorek, H., Wemmer, D. E. and Somerville, C. R. (2016). Cellulose deficiency is enhanced on hyper accumulation of sucrose by a H+-coupled sucrose symporter. Plant Physiol 171(1): 110-124.
  12. Zhang, Z., Khan, N. M., Nunez, K. M., Chess, E. K. and Szabo, C. M. (2012). Complete monosaccharide analysis by high-performance anion-exchange chromatography with pulsed amperometric detection. Anal Chem 84(9): 4104-4110.

简介

植物细胞壁主要由多糖纤维素,半纤维素和果胶组成。这些组分的结构和组成复杂性对于确定植物生长期间的细胞壁功能是重要的。此外,细胞壁结构限定了植物来源的生物质的多种功能性质,例如食品的流变性质和用于生产纤维素生物燃料的原料适用性。分子生物学实验室中细胞壁化学的典型表征包括用于定量半纤维素和果胶衍生单体的温和酸水解和通过Updegraff方法对纤维素的单独分析。我们采用了一个简化的"一步两步"水解方案,允许通过配对的脉冲安培检测(HPAEC-PAD)的高效阴离子交换层析同时测定纤维素含量,中性糖和糖醛酸样品。在我们的工作中,该方案已经在很大程度上替代了Updegraff纤维素定量和用2μMTFA水解以在微量级上测定基质多糖组成。

[背景] 是基于在121℃下在4%(w/v)硫酸中水解的样品的配对分析。一组样品首先用72%(w/w)硫酸预处理以使纤维素膨胀并使其易于稀释酸水解(图1中的Saeman水解; Saeman等人,1945)。另一组样品不进行这种预处理,导致非结晶基质多糖的水解(图1中的基质水解)。从每个水解制度中回收的葡萄糖的比较允许纤维素量的计算,其与更劳动密集的Updegraff(1969)方案(Bauer和Ibáñez,2014)很好地一致。除了葡萄糖(Glc),源自基质多糖的其它糖可以从基质水解样品中量化(Gao等人,2014)。因此,使用相对少的手动操作,可以从两个水解样品和总共四个HPAEC-PAD实验定量基质单糖和纤维素。尽管该方案需要进行色谱分离,但准备样品所需的"实际操作"时间大大减少使得该技术非常适合于高通量分析。虽然我们已经发现,对于鼠李糖(Rha),阿拉伯糖(Ara),甘露糖(Man)和木糖(Xyl)的稳定和可重现的分析需要多个HPAEC-PAD运行,报道描述同时定量单一的中性和酸性糖运行可以允许进一步改进该协议的吞吐量(Zhang等人,2012; Voiniciuc和Grünl,2016)。此处描述的协议步骤如图1所示。



图1.协议概述。 AIR =酒精不溶残渣。

关键字:纤维素, 细胞壁, 半纤维素, 离子色谱法

材料和试剂

  1. 2ml安全锁微量离心管(Eppendorf,目录号:022363352)
  2. 铝箔
  3. 2ml具有螺帽的Sarstedt管(SARSTEDT,目录号:72.694.007)
  4. 2ml螺旋盖自动进样器小瓶(Thermo Fisher Scientific,Thermo Scientific ,目录号:C4000-1W)*
  5. 带预切口隔片的自动进样器样品瓶盖(Phenomenex,目录号:AR0-8977-13-B)*
  6. 5/32"研磨球,440C不锈钢处理(OPS诊断,目录号:GBSS 156-5000-01)*
  7. 一次性抗静电聚丙烯粉末勺(Cole-Parmer仪器,目录号:06277-60)
    注意:此产品已停产。
  8. 乙醇(Decon Labs,目录号:V1016)*
  9. 氯仿(Thermo Fisher Scientific,Fisher Scientific,目录号:C607-4)*
  10. 甲醇(Thermo Fisher Scientific,Fisher Scientific,目录号:A412P4)*
  11. 丙酮(Thermo Fisher Scientific,Fisher Scientific,目录号:A184)*
  12. 72%(w/w)硫酸溶液(RICCA Chemical,目录号:R81916001A)*
  13. 超纯水(Milli-Q或等效物)*
  14. 9糖:
    L-岩藻糖(Fuc)(Sigma-Aldrich,目录号:F2252)*
    D-葡萄糖(Glc)(Sigma-Aldrich,目录号:G8270)*
    D-半乳糖(Gal)(Sigma-Aldrich,目录号:G0750)* D-木糖(Xyl)(Sigma-Aldrich,目录号:X1500)*
    D-甘露糖(Man)(Sigma-Aldrich,目录号:M8574)*
    L-阿拉伯糖(Ara)(Sigma-Aldrich,目录号:A3256)*
    L-鼠李糖(Rha)(Sigma-Aldrich,目录号:W373011)*
    D-半乳糖醛酸一水合物(GalA)(Sigma-Aldrich,目录号:48280)*
    D-葡萄糖醛酸(GluA)(Sigma-Aldrich,目录号:G5269)*
  15. 氢氧化钠溶液(50%,w/w)(Thermo Fisher Scientific,Fisher Scientific,目录号:SS254)
  16. 无水乙酸钠(Sigma-Aldrich,目录号:71183)
  17. 液氮

*注意:这些项目可以可靠地替换为来自不同供应商的实验室级别的等同项目。

设备

  1. 冷冻干燥机(Labconco,型号:FreeZone 12)*(可选)
  2. 磁铁
  3. 吸气器
  4. 金属刮刀
  5. 微量离心机(Eppendorf,型号:5417R)*
  6. 与高压灭菌器兼容的机架
  7. 自动取样器
  8. 微量天平(Mettler Toledo,型号:XS105)*
  9. 球磨机(Retsch,型号:MM 400)*
  10. 任选配备有洗脱液发生器(Thermo Fisher Scientific,Fisher Scientific,型号:ICS-5000 + SYSTEM)的Dionex ICS-5000 HPAEC-PAD系统
  11. CarboPac PA-20分析柱,3×150mm(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:060142)
  12. CarboPac PA-20保护柱,3×30mm(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:060144)
  13. CarboPac PA-200分析柱,3×250mm(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:062896)
  14. CarboPac PA-200 Guard柱,3×50mm(Thermo Fisher Scientific,Thermo Scientific TM,目录号:062895)

*注意:这些项目可以可靠地替换为来自不同供应商的实验室级别的等同项目。

软件

  1. Chromeleon 7(Thermo Fisher Scientific)
  2. Microsoft Excel

程序

  1. 醇不溶残渣(AIR)的制备
    1. 快速冷冻300 mg新鲜组织在2 ml Eppendorf管(注1)。如果使用更多的组织,均化和溶剂萃取将是低效的。如果植物来自琼脂固化的平板,必须小心避免与幼苗接触任何琼脂,因为它将在后面的步骤中水解和定量为Gal。如果冷冻干燥机不可用,请参见注释2并跳到步骤A6
    2. 留下管的盖子打开,但用一小块铝箔覆盖开口。通过用针或移液管尖端戳一个小孔来确保箔片通风
    3. 冻干样品2-3天。
    4. 每管加3个钢球,关闭盖子。
    5. 将干燥的样品均质化2分钟,在室温下在25Hz下摇动。反转管夹的方向,并在25 Hz下再摇动2分钟。
    6. 确保在进行之前获得没有团块的细粉末。
    7. 向管中加入1.5ml 70%(v/v)乙醇并涡旋。使用磁铁从浆料中取出钢球,并将其丢弃
    8. 以20,000×g离心10分钟。
    9. 使用与有机溶剂或移液管相容的吸气器弃去上清液。
    10. 加入1.5ml 1:1(v:v)氯仿:甲醇。用小金属刮刀破碎沉淀,然后彻底涡旋样品
    11. 重复步骤A8-10两次,用氯仿:甲醇洗涤总共3次。
    12. 如前所述离心并除去溶剂(步骤A8-9)
    13. 加入1.5ml丙酮,破碎沉淀,涡旋,离心,弃去上清液,如前
    14. 通过使用真空离心机干燥最终的沉淀至少30分钟或者空气干燥过夜。所得材料是AIR(注3,图2)。


      图2.步骤A14后的AIR材料

  2. 一步两步水解
    1. 对于每个AIR样品,在分析天平上(注4),精确称量?1mg材料至四个2ml Sarstedt管中的每一个中。避免材料块。记录每个管中AIR的精确重量。这四个管是技术重复,将经历两个不同的水解方案作为重复
    2. Saeman水解样品
      1. 向含有AIR的两个管中,加入50μl的72%(w/w)硫酸。立即盖上试管,剧烈涡旋,并短暂离心以收集试管底部的样品。延迟或不充分的初始混合可导致样品的再水合不良和水解不完全
      2. 在室温下孵育管1小时。每10分钟或使用Eppendorf恒温混合器连续涡旋
      3. 向每个管中加入1,400μl水以将硫酸浓度调节至4%(w/v)。涡旋混合。
    3. 基质水解样品
      1. 向第二组两个管中,首先加入1400μl水,然后加入50μl72%(w/w)硫酸以得到4%(w/v)浓度的硫酸。盖上管和涡流。
    4. 恢复标准
      1. 制备由9种糖(Fuc,Rha,Ara,Gal,Glc,Xyl,Man,GalA和GluA)组成的标准混合物,每种浓度为100μg/ml。建议单独制备每种糖的浓缩原液,然后混合。如果使用水合糖,则计算额外的水量。准备至少足够用于本部分的协议(1毫升)以及准备标准曲线在下一部分的协议(138微升)。糖标准可以冷冻储存一次。
      2. 为了解释水解期间的糖特异性损失,使回收标准经受与基质水解样品相同的条件(注5)。移取500微升的标准混合物和900微升的水到2毫升的Sarstedt管。加入50μl的72%(w/w)硫酸。用第二个管重复。盖上管和涡流。
    5. 除了一个回收标准之外,在高压灭菌器兼容的架子和高压灭菌器中,将所有试管在121℃下放置60分钟。第二回收标准管不经高压灭菌,并且用作计算单糖特异性校正因子的对照
    6. 将样品冷却至室温,并在20,000×g离心1分钟,以沉淀任何不溶性物质。
    7. 稀释样品,回收标准品和回收标准品对照100倍,将10μl转移到含有990μlMilliQ水的自动进样器样品瓶中。将样品在4°C储存长达两个星期。如果样品在-20°C下冷冻,可以保存一个月或更长时间。对于从茎,木材或其它次级组织制备的更多富含纤维素的AIR,对于从拟南芥幼苗分离的AIR,该稀释因子是方便的,应当使用更高的稀释因子(200-500倍)。
    8. 标准曲线样本
      1. 稀释来自步骤B4的标准混合物(9种糖,各100μg/ml),得到0.05μg/ml和5μg/ml之间的标准曲线。 1 。将其直接制备到具有预切口隔片的2ml自动进样器小瓶中(表1)。该浓度范围通常覆盖仪器的完整线性范围  
        表1.准备标准曲线样本

        *注意:标准8进一步稀释,以准备标准1-4。

  3. HPAEC-PAD分析
    1. HPAEC-PAD分析#1(所有样品)
      1. 将25μl每种标准品,回收标准品和样品注入3 x 150 mm CarboPac PA-20分析柱,配备相同材料的3 x 50 mm保护柱。用0.4mM min -1的流速用2mM KOH洗脱化合物30分钟(注释6)。这个运行将解决Fuc,Gal,Glc,Xyl和Man。 Rha和Ara不能用这些条件一致地分开,因此它们通过只对基质水解样品必需的第二次分析来量化。
      2. 根据我们的经验,峰值的保留时间将随着更多样品的运行而更早移动,在?50-100个样品后,需要用乙酸钠进行柱子冲洗(参见下面的步骤C4)。
    2. HPAEC-PAD分析#2(仅基质水解样品)
      1. 将25μl每种标准品,回收标准品和样品注入3 x 150 mm CarboPac PA-20分析柱,配备相同材料的3 x 50 mm保护柱。用18mM KOH 0.4ml min -1 -1洗脱化合物30分钟。此运行将解决Rha和Ara。
      2. 在运行?50-100个样品后可能需要进行柱冲洗(见下面的步骤C4)。
    3. HPAEC-PAD分析#3(仅基质水解样品)
      1. 将25μl每种标准品,回收标准品和样品注入3 x 150 mm CarboPac PA-200分析柱,配备相同材料的3 x 50 mm保护柱。用100mM NaOH中50mM至200mM乙酸钠的10分钟梯度洗脱化合物,流速为0.4ml min -1 -1(这是用5%-20%梯度的5%-20%洗脱液B [100mM NaOH,1M乙酸钠],洗脱液A由100mM NaOH组成,剩余体积)。此运行将分解GalA和GluA
    4. 柱冲洗程序
      1. CarboPac PA20柱的性能在运行?50-100个样品后降解,导致越来越早的保留时间,直到峰不能充分分辨。这通过用100mM NaOH/1M乙酸钠冲洗柱30分钟,然后用0.2ml min -1 -1柱冲洗30分钟来补救。如果CarboPac PA20色谱柱与配有洗脱液发生器的HPAEC-PAD系统一起使用,则需要将其临时移至不同的系统或端口,以完成此过程。

数据分析

  1. 使用Chromeleon 7软件集成所有标准曲线和水解样品。对于校准曲线,使用强制通过原点的二次曲线。对于一些样品,峰标识可能需要手动分配,因为保留时间倾向于在多次运行时更早移动。图3-5显示了三种HPAEC-PAD方法的代表性色谱图

    图3. HPAEC-PAD分析#1代表性色谱图。 A.标准混合物。 B.Saeman水解样品。 C.基质水解样品。在?8.2min的峰包含Rha和Ara,并且在该方法中没有定量

    图4. HPAEC-PAD分析#2代表性色谱图。 A.标准混合物。 B.基质水解样品。只有Rha和Ara在这些运行中被定量。 Fuc在?3.4分钟洗脱,Gal在?7.6分钟洗脱,Glc在?8.2分钟洗脱。 ?9.0分钟的峰是Xyl和Man的混合物

    图5. HPAEC-PAD分析#3代表性色谱图。 A.标准混合物。 B.基质水解样品
  2. 从每次运行,可以将结果(测定的单糖浓度,μgml -1 )从结果表复制到Microsoft Excel电子表格中。设置一个表格,其中列出每个糖的结果,以及每个样品的AIR质量的列(图6)。
  3. 通过将回收标准品的浓度除以回收标准品对照品中各糖的浓度,计算每种糖的校正因子。这些通常是> 0.85对中性糖,但对于糖醛酸较低(?0.6-0.8)。相同的Glc回收系数用于Saeman和Matrix水解样品。低于预期的校正因子表明由于长时间的高温处理可能发生的样品损失过多。重复水解,确保样品冷却,并在高压釜运行完成后立即稀释。
  4. 在新栏中,使用下式计算恢复标准校正的单糖量,以mg/mg AIR计:

    在哪里,
    c =测定浓度(μg -1 ),
    d = 100(稀释因子),
    v = 1.450(体积,ml),
    r =恢复修正系数(从步骤C3开始),
    m = AIR质量(mg)。
  5. 在这一点上,可以验证技术重复,以检查在单糖定量中是否存在任何系统差异,这将证明丢弃技术重复是合理的。 (这将指示样品损失或称重误差。 Fuc,Gal和Xyl应在Saeman和Matrix水解样品中具有一致的值。 Glc在Saeman水解样品中将高得多,并且Man在这些样品中预期更丰富,因为Glc异构化为Man是Saeman水解期间的已知副反应(Carpita和Shea,1989)。这种副反应的程度仅为?2%,因此为了纤维素定量的目的而被忽略
  6. 平均技术复制到新列中。只有使用所有三种HPAEC-PAD方法的Matrix水解样品用于报告中性糖和糖醛酸。
  7. 为了计算纤维素量,从Glc总(来自Saeman水解样品)中减去Glc基质(来自基质水解样品)并乘以0.9。 0.9校正因子反映了葡萄糖(180.16g mol -1 -1)和葡糖酐(162.14g mol -1 -1)之间的摩尔质量差异,因为其发生在纤维素聚合物中水解前。
    纤维素(μgmg -1 AIR)= 0.9×(Glc 总的<μg> )< > (μgmg -1 ))
  8. 对于统计分析,我们通常使用从独立植物或幼苗池制备的AIR的至少三个生物重复。结果报告为平均值,标准偏差和统计学显着性通过Student's t检验或单因素方差分析,接着通过Tukey多重比较检验来评估。


    图6.示出从HPAEC-PAD整合数据计算单糖和纤维素的Excel电子数据表的屏幕截图。分析的样品是从5d老黑暗生长的拟南芥幼苗制备的。

笔记

  1. 样品中存在的任何淀粉将有助于在两种水解方案中量化的葡萄糖。如果需要定量来自基质多糖的葡萄糖,则可以在收集组织之前通过将光生长的植物移动到暗处24小时来减少淀粉(Sorek等人,2015)。或者,淀粉可以在AIR的制备期间被酶去除(Pettolino等人,2012),或者可以从相同的AIR材料独立地测定淀粉(Yeats等人, 2016)。淀粉不会影响纤维素的测定,因为它将同样对在Saeman和基质水解样品中定量的葡萄糖有贡献。
  2. 如果没有冷冻干燥器,可以直接从用研钵和杵或球磨机均质化的冷冻组织制备AIR。在这两种情况下,样品在均质化期间必须保持冷冻:在使用之前,砂浆和杵或球磨管架应浸入液氮中。管在低温下更脆,并且我们注意到在球磨机中均质化期间管的破裂速率增加,因此我们优选在室温下用干燥的组织工作。
  3. 术语AIR和细胞壁材料通常可互换使用,然而,从定量单糖的质量平衡显而易见,从幼苗制备的AIR仅为?50%细胞壁材料。用SDS和偏亚硫酸氢钠的水溶液更广泛地洗涤AIR除去额外的非细胞壁物质(Dick-Pérez等人,2011)。然而,如果要分析多个样品,则该方案是冗长的和不方便的。由于在AIR制备过程中提取和洗涤的效率可能存在差异,因此最佳做法是同时制备所有待分析的空气样品。我们发现,同时准备高达?60个AIR样品是方便的,离心机容量是一个限制因素。对于水解步骤,管的数目乘以4,因此一旦制备AIR,我们以15-20个样品(总共60-80个水解样品)的批次进行。对于大型项目,我们建议在每个批次中包括一个对照样品
  4. 由于静电荷的累积,处理干燥的AIR材料可能是困难的。为了将AIR移动到Sarstedt管,我们使用一次性防静电聚丙烯粉末勺(Cole-Parmer)。实验室湿度> 40%有助于减少静电
  5. 在水解期间发生的糖损失有些浓度依赖性。为了方便,制备具有等量的每种糖的标准混合物。为了更准确的绝对定量,可以根据正在分析的样品的预期组成来制备回收标准
  6. 用相同浓度的NaOH代替KOH的LC洗脱得到可比较的结果。在我们的工作中,我们使用配有KOH洗脱液发生器模块的HPAEC-PAD进行CarboPac PA-20分析。用户制备的洗脱剂通常用NaOH制备。在CarboPac PA-20手册中详细描述了洗脱液制备程序。遵循概述的程序对于避免碳酸盐进入系统是特别重要的,这极大地影响糖的色谱法

致谢

我们感谢Will Barnes(Penn州立大学)和Chris Somerville(加利福尼亚大学伯克利分校)对本协议提出有益的意见。这项工作的资金来自能源生物科学研究所和Philomathia基金会。该协议基于以下先前发布的报告:Bauer和Ibá?ez,2014; Chen 。,2016; Sorek 。,2015;和Yeats 。,2016。

参考文献

  1. Bauer,S。和Ibanez,AB(2014)。  快速测定纤维素。 111(11):2355-2357。
  2. Carpita,NC和Shea,EM(1988)。  各向异性细胞扩增受纤维素合酶复合物的双向迁移和两个关键残基的磷酸化影响CESA3。 植物生理学 171(1):242-250
  3. Dick-Perez,M.,Zhang,Y.,Hayes,J.,Salazar,A.,Zabotina,OA和Hong,M.(2011)。  通过二维和三维魔角旋转固态NMR的植物细胞壁多糖的结构和相互作用。 a> Biochemistry 50(6):989-1000。
  4. Gao,X.,Kumar,R.和Wyman,CE(2014)。  通过简单的一步酸水解快速半纤维素定量。 111(6):1088-1096。
  5. Pettolino,FA,Walsh,C.,Fincher,GB和Bacic,A。(2012)。  确定植物细胞壁的多糖组成 Nat Protoc 7(9):1590-1607。
  6. Saeman,JF,Bubl,JL和Harris,EE(1945)。  Quantitative糖化木材和纤维素。 Ind Eng Chem Anal Ed 17:35-37。
  7. Sorek,N.,Szemenyei,H.,Sorek,H.,Landers,A.,Knight,H.,Bauer,S.,Wemmer,DE和Somerville,CR(2015)。< a class ="ke- insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/5361396"target ="_ blank">将MEDIATOR16鉴定为拟南芥 COBRA抑制剂MONGOOSE1。 Proc Natl Acad Sci USA 112(52):16048-16053。
  8. Updegraff,DM(1969)。  半微确定纤维素生物材料。 Anal Biochem 32(3):420-424
  9. Voiniciuc,C.和Günl,M.(2016)。  单糖分析可以从拟南芥种子中提取的总粘液。 生物协议 6(9):e1801。
  10. Yeats,TH,Sorek,H.,Wemmer,DE和Somerville,CR(2016)。  纤维素缺乏通过H sup + +/- 偶联的蔗糖同向转运体在蔗糖的超累积上增强。植物生理学171(1) :110-124。
  11. Zhang,Z.,Khan,NM,Nunez,KM,Chess,EK和Szabo,CM(2012)。 
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
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. Yeats, T., Vellosillo, T., Sorek, N., Ibáñez, A. B. and Bauer, S. (2016). Rapid Determination of Cellulose, Neutral Sugars, and Uronic Acids from Plant Cell Walls by One-step Two-step Hydrolysis and HPAEC-PAD. Bio-protocol 6(20): e1978. DOI: 10.21769/BioProtoc.1978.
  2. Yeats, T. H., Sorek, H., Wemmer, D. E. and Somerville, C. R. (2016). Cellulose deficiency is enhanced on hyper accumulation of sucrose by a H+-coupled sucrose symporter. Plant Physiol 171(1): 110-124.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。

Trevor Yeats
Energy Biosciences Institute, University of California, USA
I am the primary author of this protocol and I have two suggestions regarding this protocol based on my experience implementing it in a different lab:

1. Hydrolysis time in the autoclave may need to be optimized, likely because of differences in the speed of cooling and exhausting. The polymer that is most recalcitrant to sulfuric acid hydrolysis is polygalacturonic acid (PG), and the released galacturonic acid is degraded during hydrolysis conditions as well. My initial experience was that a 1 hour hydrolysis was insufficient when working with Arabidopsis cell wall material and an autoclave that exhausted relatively quickly. Incomplete hydrolysis of PG yields oligomers that can be detected by HPAEC-PAD with a sodium acetate gradient, which can be helpful in optimizing hydrolysis conditions. The ratio of the peak area between the monomer and the dimer is a particularly good metric of the extent of hydrolysis: Initial experiments with 60 minute hydrolysis gave a ratio of 6, while 120 minutes of hydrolysis gave a ratio of 18. In these conditions, the correction factor for GalUA (0.54) has a relatively large impact on the quantification, underscoring that product degradation and extent of hydrolysis must be optimized in order to obtain good results. Commercial pectin and PG of know GalA content are useful for such optimization experiments.

2. Analysis of uronic acids can be performed on a CarboPac PA20 column using the same conditions indicated in this protocol or by isocratic elution with 170 mM sodium acetate, 100 mM NaOH. I have found the latter to be more convenient as the baseline does not increase during the run.
4/19/2017 10:15:04 AM Reply