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Dec 2019

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Single-run HPLC Quantification of Plant Cell Wall Monosaccharides
Single-run HPLC定量分析植物细胞壁单糖   

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Abstract

The plant cell wall is a complex network of polysaccharides and proteins that provides strength and structural integrity to plant cells, as well as playing a vital role in growth, development, and defense response. Cell wall polysaccharides can be broadly grouped into three categories: cellulose, pectins, and hemicelluloses. Dynamic interactions between polysaccharides and cell wall-associated proteins contribute to regions of flexibility and rigidity within the cell wall, allowing for remodeling when necessary during growth, environmental adaptation, or stress response activation. These polysaccharide interactions are vital to plant growth, however they also contribute to the level of difficulty encountered when attempting to analyze cell wall structure and composition. In the past, lengthy protocols to quantify cell wall monosaccharides contributing to cellulose as well as neutral and acidic cell wall polysaccharides have been used. Recently, a streamlined approach for monosaccharide quantification was described. This protocol combines a simplified hydrolysis method followed by several runs of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Here, we present an updated version of this protocol in which we can analyze all nine cell wall monosaccharides in a single high-performance liquid chromatography HPAEC-PAD gradient profile. The inclusion of an enzymatic starch degradation, as well as alternate internal standards for added quantification accuracy, and a ready-to-use Python script facilitating data analysis adds a broadened scope of utility to this protocol. This protocol was used to analyze Arabidopsis light-grown seedlings and dark-grown hypocotyls, but is suitable for any plant tissues.

Keywords: Cell wall (细胞壁), Cellulose (纤维素), HPLC (高效液相色谱法), Monosaccharides (单糖), Saeman hydrolysis (Saeman水解), Arabidopsis (拟南芥)

Background

Understanding plant cell wall structure and composition have been at the forefront of both academic and industrial plant research for many years. Polysaccharide and cell wall protein interactions provide structure to plant cells, while also actively playing important biological roles during growth and adaptation to diverse external conditions (Cosgrove, 2005 and 2016; Kesten et al., 2017). The plant cell wall acts as an important physical barrier during biotic or abiotic interactions, but can also provide an endogenous source of signaling molecules released during stress response (Cosgrove, 2005 and 2016; Kesten et al., 2017 and 2019). These cell wall-derived molecules can activate signaling cascades to alert the plant host of an invading pathogen or a need to redirect growth resources. Cell wall polysaccharides are also important for industrial uses–pectins are widely used in the food and cosmetics industries, while cellulose is important for the food, paper, and textile industries (Pettolino et al., 2012). As such, efficient and informative methods for characterizing plant cell wall polysaccharides are vital in both academia and industry to further understand the biological role of the plant cell wall and improve ease of polymer extraction while reducing waste during industrial applications.

Experiments from the 1940s conducted in wood established the use of dilute acid hydrolysis at high temperature to study sugar decomposition into monomers (Saeman, 1945). Such studies have provided a foundation for many current protocols, including the one presented here. Streamlining this analysis has allowed us to improve both the accuracy and efficiency of our measurements. Other recently described protocols have established the combination of the widely-used trifluoroacetic acid (TFA) hydrolysis approach with a single HPLC run, or a dual sulfuric acid-based h ydrolysis method, referred to as a “one-step two-step sulfuric acid hydrolysis” approach, with multiple HPAEC-PAD runs (Zhang et al., 2012; Voiniciuc et al., 2016; Yeats et al., 2016a and 2016b). This “one-step two-step” approach makes use of a Saeman hydrolysis procedure followed by what has been previously referred to as a “matrix hydrolysis” (Yeats et al., 2016a). Saeman hydrolysis, the “two-step” portion of this approach, is essentially the addition of concentrated sulfuric acid hydrolysis (72% sulfuric acid) directly to plant material in order to swell the sample. After one hour at room temperature, water is added to the sample to dilute the sulfuric acid concentration to 4%. This portion of the hydrolysis is then performed at 121 °C for one hour. The use of a dilute acid hydrolysis immediately following a strong acid swelling hydrolysis allows the release of glucose from all possible sources, including heavily cross-linked and rigidly packed cell wall components, such as crystalline cellulose. The “one-step” portion of the hydrolysis, also called the “matrix hydrolysis”, refers to the hydrolysis of samples only at 121 °C with 4% sulfuric acid. By simply treating samples with a “matrix hydrolysis”, only monosaccharides from easily hydrolysable sources will be released. Therefore, the difference between these two hydrolyses allows the quantification of crystalline cellulose in what is referred to as a “one-step two-step” hydrolysis approach. In our protocol, we combine the efficient “one-step two-step” sulfuric acid hydrolysis described by Yeats et al. (2016a) with the inclusion of an optional enzymatic starch degradation and the use of alternate internal standards to elute all cell wall sugars and quantify glucose derived from crystalline cellulose in a single HPAEC-PAD run. Our additions to the previously mentioned protocols contribute significant improvements in both ease and accuracy of monosaccharide measurement that will greatly benefit the field of plant cell wall research.

Materials and Reagents

  1. 2 ml screw-cap tubes (Sarstedt, catalog number: 72.694.006 )
  2. 50 ml conical tubes (Greiner Bio-One, catalog number: 7.210 261 )
  3. 500 μl autosampler Snap Ring vials (Sigma, catalog number: 27422 )
  4. Autosampler vial lids (Sigma, catalog number: 24757 )
  5. Stainless steel 25 ml grinding jars (Retsch, catalog number: 02.462.0119 )
  6. Stainless steel 12 mm grinding balls (Retsch, catalog number: 05.368.0032 )
  7. Tin weigh boats, 5 x 9 mm (Santis Analytical AG, catalog number: SA76981103 )
  8. 0.22 μm sterile PES-membrane filter (Life Systems Design AG, catalog number: 99255 )
  9. Metal spatula
  10. Plastic weighing papers (HuberLab, catalog number: 12.9702.080 )
  11. Conical tubes (Grenier Bio, catalog number: 7.210 261 )
  12. Nylon mesh, 60 μm pore size (Sefar Nitex, catalog number: 3A03-0060-110-00 )
  13. Square Petri plates (Greiner Bio-One, catalog number: 7.688.102 )
  14. Homogenizing beads (depending on amount of material):
    1. Small glass beads (2.85-3.45 mm beads, Roth, catalog number: A557.1 )
    2. Large metal balls (12 mm beads, Retsch, catalog number: 05.368.0032 )
  15. CarboPac PA20 column (3 x 150 mm, Thermo Fisher Scientific, catalog number: 0 60142 )
  16. CarboPac PA20 guard column (3 x 30 mm, Thermo Fisher Scientific, catalog number: 0 60144 )
  17. Arabidopsis thaliana
  18. Ethanol (HCI Shop, ETH Zurich, catalog number: 0 2000107 )
  19. Chloroform (Sigma, catalog number: 25693 )
  20. Methanol (HCI Shop, ETH Zurich, catalog number: 0 2000342 )
  21. Acetone (Sigma, catalog number: 24201-4X2.5L-GL-R )
  22. (Optional) Enzymatic starch degradation
    1. Amyloglucosidase (Sigma, catalog number: 10102857001 )
    2. α-Amylase (Sigma, catalog number: 10102814001 )
  23. Sulfuric acid (Sigma, catalog number: 258105 )
  24. Ultrapure water (Milli-Q or equivalent)
  25. Monosaccharide analysis standards:
    1. L-fucose (Sigma, catalog number: F2252-5G )
    2. D-glucose (Sigma, catalog number: G7528-1KG )
    3. D-galactose (Sigma, catalog number: 48260 )
    4. D-xylose (Sigma, catalog number: W360600-SAMPLE )
    5. D-mannose (Sigma, catalog number: 63579 )
    6. L-arabinose (Roth, catalog number: 5118.2 )
    7. L-rhamnose (Sigma, catalog number: W373011-SAMPLE-K )
    8. D-galacturonic acid monohydrate (Sigma, catalog number: 48280 )
    9. D-glucuronic acid (Sigma, catalog number: G5269-10G )
  26. (Optional) Alternate internal standards
    1. D-sedoheptulose (CarboSynth, catalog number: MS139006 )
    2. D-ribose (Sigma, catalog number: R7500-5G )
  27. Sodium hydroxide, 50% solution in water (Sigma, catalog number: 415413 )
  28. Sodium acetate, anhydrous (Sigma, catalog number: 32319-1KG-R )
  29. Lugol solution (Sigma, catalog number: 32922-6X1L )
  30. Liquid nitrogen

Equipment

  1. Freeze-dryer (Christ, Alpha 2-4)
  2. Microcentrifuge (Eppendorf, model: 5424 R )
  3. Sample concentrator (Stuart, model: SBHCONC/1 )
  4. Autoclave-compatible rack (Karter Scientific, catalog number: 125A7 )
  5. Microbalance (Mettler Toledo MX5)
  6. Tissue homogenizer (Retsch MM200)
  7. Micro-centrifuge tube shaker (Eppendorf ThermoMixer F1.5)
  8. Tube rotator (Labinco LD79, catalog number: 79000 )
  9. Speed-vacuum centrifuge (Eppendorf Concentrator Plus)
  10. Autoclave (Thermo Fisher Scientific, Sterico, Varioklav)
  11. Heating block (Stuart, model: SBH130D )
  12. Autosampler (Dionex, model: AS-1 )
  13. Dionex ICS-5000 (Dionex, model: DC-5 )
    1. ED Electrochemical Detector (without cell, product number: 072042)
    2. ED Cell (no reference or working electrode, product number: 072044)
    3. Gold (Au) on Polytetrafluororethylene (PTFE) Disposable Electrode (product number: 066480)

Software

  1. Chromeleon 8 (Thermo Fisher Scientific)
    Available for a fee at https://www.thermofisher.com/order/catalog/product/CHROMELEON7
  2. Microsoft Excel
    Available for a fee at https://www.office.com
  3. GraphPad Prism
    Available for a fee at https://www.graphpad.com
  4. Spyder5 (Anaconda3)
    Freely available at https://www.anaconda.com
  5. Python 3.6
    Freely available at https://www.python.org

Procedure

  1. Generating and preparing plant material
    1. Grow plants in desired conditions
      Note: Plant samples can be collected from any desired growth method; we suggest the following in vitro method for Arabidopsis material generated from either light-grown (any day cycle) seedlings or dark-grown hypocotyls.
      1. Gas or liquid sterilize seeds and stratify at 4 °C for 2-3 days.
      2. Plates: prepare ½ MS + 1% sucrose + 0.9% agar square Petri plates; sterilize nylon mesh and lay on top of agar plates; sow approximately 100-120 seeds at top of plates; collect material at desired time point in 50 ml conical tubes; remove seeds before processing.
      3. For light-grown plants, grow plants in light for 14 days under long-day conditions (16 h light, 8 h dark) with light optimized for Arabidopsis (130-150 μE m-2 s-1) at 20-22 °C.
      4. For dark-grown hypocotyls, sow multiple lines of seed per plate (without nylon); leave seeds sown on plates in light for 2-4 h; cover in several layers of aluminum foil and allow to grow in climate-controlled chamber for 5 days at 20-22 °C.
    2. Leave plants in the dark for 24-48 h prior to harvesting to deplete starch reserves; if not possible, an enzymatic starch degradation can be performed after homogenization of plant material (proceed to Step A5).
    3. If analyzing aerial plant tissues (containing chlorophyll), harvest material in enough 70% ethanol to fully submerge plant material, and continue exchanging ethanol until chlorophyll is depleted and the liquid no longer has any trace of green colour.
      If non-chlorophyll-containing plant portions are to be analyzed, harvest and flash freeze samples with liquid nitrogen and proceed to grinding in Step A5.
    4. Remove ethanol and use freeze-dryer to dry material over 2 days (or longer if necessary).
    5. Grind all plant material using tissue homogenizer with 12 mm diameter metal balls and metal containers.
      Notes:
      1. If generating only a small amount of material, it is possible to harvest roots into microcentrifuge tubes and use a tissue homogenizer with racks for microcentrifuge tubes and glass beads for grinding. In this case, aliquot ≤ 200 mg plant material to ensure thorough homogenization.
      2. When collecting plant material, it is imperative to avoid collection of agar, soil, or any growth media containing sugars with samples (N.B. agar will contribute to galactose quantification). Nylon mesh can be used as described above to prevent agar adhesion to roots.

  2. Starch degradation conducted as previously described (Hostettler et al., 2011) with modifications as follows:
    1. Aliquot ground plant material up to ~0.5 ml in a 2 ml micro-centrifuge tube.
    2. Add 1 ml 80% ethanol (v/v) and heat samples at 95 °C for 10 min. Mix well by vortexing each sample for 10-15 s.
    3. Centrifuge at room temperature (RT) at 3,000 x g for 5 min; discard supernatant.
      Note: All centrifugation steps should be performed at RT unless otherwise indicated.
    4. Continue with the following washing steps: shaking in a ThermoMixer for 10 min during each wash, centrifuging at 3,000 x g for 5 min, vortexing to re-suspend pellet between washes, discard supernatant, and add the next washing solution: 1 ml 50% (v/v) ethanol, 1 ml 20% (v/v) ethanol, 1 ml water, and finally 1 ml 80% (v/v) ethanol.
      Note: After Step B4, the final wash should be mostly clear, but pellet may still be green.
    5. Dry the pellet at room temperature, using a speed-vacuum centrifuge, or in an oven at 60 °C for at least 30 min or over-night until completely dry. Re-suspend in 400 μl water and vortex to mix.
    6. Boil at 95 °C for 10-15 min–do not cool on ice.
    7. Prepare a digestion mixture of 9 parts amyloglucosidase and 1 part α-amylase.
      Note: Calculate volume of digestion mixture needed based on total number of samples, keeping in mind that 20 μl of the mixture is required per sample. One sample requires 18 μl of amyloglucosiades and 2 μl α-amylase.
    8. Add 380 μl 0.22 M sodium acetate to 20 μl of the prepared 9:1 amyloglucosidase: α-amylase mixture; combine with 400 μl sample.
      Note: If processing larger sample volumes, sample can be resuspended in larger volume of water. For digestion, simply combine equal parts sample with digestion mixture and proceed as indicated.
    9. Digest at 37 °C for a minimum of 2 h.
    10. Check if there is remaining starch by staining a small portion of the plant material with Lugol solution.
      Note: Lugol staining suggested procedure:
      1. Mix 20 μl sample with 80 μl 100% ethanol.
      2. Boil for 5 min at 90 °C.
      3. Centrifuge for 5 min at 5,000 x g. Discard supernatant.
      4. Add 25 μl Lugol solution; check for colour change after 5 min.
        1. Lugol solution can be used at full concentration without dilution of purchased solution.
        2. Colour change to a deep blue/black colour indicates presence of starch.
        3. If colour change is observed, continue digestion overnight and check with Lugol again. Continue digestion until colour change is no longer observed (Figure 1).
        4. Once no colour change is observed, proceed to Step A11.
    11. Centrifuge samples at 5,000 x g for 5 min and proceed with the insoluble fraction remaining.


      Figure 1. Lugol stain reveals presence or absence of starch. A. Full concentration Lugol alone has a faint yellow colour (left tube), while Lugol added directly to starch extracted from corn shows a strong dark brown/purple colour change (right tube). B. Both tubes contain cell wall AIR material; the tube on the left has been subjected to starch degradation, while the tube on the right has not. Following the procedure described in Step A10, addition of 25 μl full concentration Lugol will stain AIR samples containing starch a dark brown/purple colour, as observed in the right tube. If the Lugol remains yellow when added to the sample, starch is not present, and one may proceed with Step A11.

  3. Cleaning and production of final cell wall-derived alcohol insoluble residue (AIR) preparation
    1. Aliquot de-starched insoluble fraction up to 0.5 ml in a 2 ml micro-centrifuge tube.
    2. Add 1.5 ml of a 1:1 methanol:chloroform (v/v) mixture to sample and vortex to mix thoroughly. Mix samples for 2 h using the tube rotator set to 15 rpm, or any standard tube mixer.
    3. Centrifuge at 10,000 x g for 5-10 min at room temperature and remove supernatant.
    4. Add 1.5 ml RT acetone and vortex to mix thoroughly. Mix for 30 min using a tube rotator set to 15 rpm or any standard tube mixer.
    5. Centrifuge at 10,000 x g for 5-10 min at room temperature and remove supernatant.
    6. Dry the final pellet at room temperature, using a speed-vacuum centrifuge, or in an oven at 60 °C for at least 30 min or overnight until completely dry. The final product is the cell wall-derived alcohol insoluble residue (AIR).

  4. Sample and standard hydrolysis conducted as described in Yeats et al. (2016a) (see Figure 2 in this manuscript). Briefly, the method is summarized as follows:


    Figure 2. Gradient profile of single HPLC run to elute all cell wall monosaccharides. Each block of colour corresponds to a particular eluent, indicated in the legend on the right. The primary y-axis (left) features percentage, referring to what percentage each eluent comprises the final mixed eluent that flows through the column, with the total always equaling to 100%. The secondary y-axis (right) indicates the volume (ml/minute) used up per eluent, relating to the percentage of the total mixed eluent to which each individual eluent contributes. The x-axis indicates the amount of time (minutes) for which the indicated eluent composition should proceed.

    1. Weigh 1 ± 0.1 mg AIR per technical replicate into 2 ml screw-cap tubes using a microbalance and tin weigh boats; record final weight (required for analysis steps).
      Notes:
      1. If spatula used for weighing AIR needs to be cleaned in between samples, do not use any kind of tissue/paper towel. Use the plastic weighing papers to wipe and rinse thoroughly with 100% ethanol in between samples.
      2. We recommend a minimum of two technical replicates per sample per hydrolysis (meaning, 2 technical replicates for samples subjected only to matrix hydrolysis, and 2 technical replicates subjected to Saeman hydrolysis + matrix hydrolysis) and three biological replicates for each experimental analysis.
      3. Prior to sample hydrolysis, internal standards (sedoheptulose or ribose) can be added directly to weighed AIR aliquots. Allow internal standards to completely dry for 30 min or longer as necessary, either at room temperature or using a sample concentrator.
        1. For our assays, we added 150 μg of sedoheptulose to AIR material; however, this amount must be optimized based on the sample dilution that will be measured considering the working range of the instrument of choice.
        2. Addition of the standards (ribose or sedoheptulose) is not mandatory. However, they provide an added measure of certainty to ensure consistency of hydrolysis and quantification.
        3. Sedoheptulose worked better for this analysis as we observed a “background” peak with the same retention time as ribose. However, with a slightly different instrument or sample, ribose may be used. This should be tested prior to hydrolysis of all samples and standards.
    2. One sample set will be subjected to Saeman as well as matrix hydrolysis, while a second sample set will be subjected only to matrix hydrolysis.
      Note: Previously, this method was described using autoclaving as the hydrolysis method (Yeats et al., 2016a). We confirm this as an efficient method of hydrolyzing upwards of 50 samples at once. However, we also confirmed the validity of the HPLC analysis and quantification if hydrolysis is accomplished using a heating block at 121 °C for 1 h. However, data is more consistent when using autoclaving to accomplish hydrolysis (Figure 3), as is demonstrated by reduced error. Further, it appears hydrolysis of galacturonic acid may be incomplete using heating block hydrolysis, although all other monosaccharide values seem to be consistent across either method. It may also be possible to use a heating block at a lower temperature (80-100 °C) for a longer hydrolysis time, however this should be tested and optimized.


      Figure 3. Sample hydrolysis may be accomplished using either a heating block or autoclave, with minor differences. Monosaccharide elution profile of cell wall AIR derived from light-grown wild-type Col-0 seedlings. A minimum of two technical replicates per sample per hydrolysis method were used. Bars represent average of 2 biological replicates ± standard error.

    3. For standard curve analysis, make a 100 μg stock solution containing all monosaccharides for quantification as well as the appropriate internal standard. Dilute into appropriate standard concentrations based on assaying range.
      Note: Recommended concentrations to generate the standard curve are as follows: 0.05 μg, 0.1 μg, 0.5 μg, 1 μg, 2 μg, 5 μg.
    4. To consider sugar-specific losses during hydrolysis and calculate monosaccharide-specific correction factors, prepare two recovery standards by combining 500 μl of the 100 μg standard mixture with 900 μl water.
      1. One recovery standard is subjected to the same conditions as the matrix hydrolysis samples (water + acid, hydrolysis for 1 h at 121 °C).
      2. The second recovery standard is treated with acid, but is not subjected to heat hydrolysis.
    5. After hydrolyses are complete, allow samples to cool at room temperature and centrifuge for 1 min at 20,000 x g to pellet any insoluble material. The supernatant is used in the next setp.

  5. HPLC analysis
    1. Dilute sample supernatants as required before injection (1:10, 1:20, 1:50, or 1:100 dilutions may be used depending on the starting material and detector sensitivity) and pipet into autosampler vials.
      Notes:
      1. Appropriate standards and dilutions may vary based on samples or detector sensitivity; it is recommended to test standards as well as sample dilutions thoroughly to optimize conditions before completing processing of all material.
      2. It is strongly recommended to randomize order of sample analysis and run a standard after every 10-15 samples to ensure sensitivity and accuracy of measurement is consistent.
    2. Make eluents; purge with and maintain under helium gas, or as directed by manufacturer (Rohrer, 2017). Any eluents containing sodium acetate must be filtered using a 0.22 μm PES filter. All eluents must be purged for a minimum of 10 min before addition of 50% sodium hydroxide solution, followed by additional purging of a minimum of 10 min after addition.
      Eluent A = water
      Eluent B = 50 mM sodium hydroxide
      Eluent C = 100 mM sodium hydroxide, 100 mM sodium acetate
      Eluent D = 200 mM sodium hydroxide
    3. Inject 10 μl of each standard, recovery standard, and sample onto a 3 x 150 mm CarboPac PA20 column equipped with a 3 x 50 mm CarboPac PA20 guard column.
    4. Maintain column temperature at 36 °C with a flow rate of 0.4 ml/min.
    5. Use the following elution profile to elute all monosaccharides and standards (Figure 4, Table 1): 0-18 min 4.8% B, 95.2% A; 18-20 min linear gradient to next condition; 20-30 min 50% D, 50% A; 30-40 min linear gradient next condition; 40-56 min 100% C; 56-56.1 min linear gradient to 50% D; 56.1-60 min 50% D; 60-60.1 min change to next condition; 60.1-80 min 4.8% B, 95.2% A to equilibrate column back to starting conditions:


      Figure 4. Elution profile of cell wall monosaccharides in a single HPAEC-PAD run. Four separate injections are presented: 1 μg/ml standards mixture using ribose as an internal standard (blue), 1 μg/ml standards mixture using sedoheptulose as an internal standard (black), and an example of a wild type Col-0 matrix hydrolysis profile (hydrolyzed using a heating block) (pink), and an example of a wild type Col-0 Saeman hydrolysis + matrix hydrolysis profile with sedoheptulose added as an internal standard (hydrolyzed using a heating block) (brown). After sample hydrolysis (pink), a background peak (*) appeared that clearly overlaps with the ribose peak (blue).

      Notes:
      1. Retention times of peaks may shift as more samples run, therefore regular “column flushing” and monitoring of column performance may be necessary.
      2. A short “column flushing” period is incorporated into the gradient profile (~5 min of 100 mM NaOH at 56 min); however, it is also possible to run a flushing profile periodically as follows: 30 min 100% Eluent C followed by 30 min 100% Eluent D with a short equilibration step (approximately 10-15 min) back to starting conditions (95.2% Eluent A, 4.8% Eluent B).
      3. If significant changes in column performance are observed, immediate action to fully flush the column as per the manufacturer’s instructions must be taken. In short, we accomplished column flushing by conducting the following:
        1. Disconnect column from detector and general machine flow by turning the electrode/detector off and unscrewing the column outlet.
        2. Wash column with 2 M NaOH (allowing flow-through to drip into a waste receptacle) for 1 h; adjust flow rate until pressure reaches similar level to running pressure during sample analysis (in this case, ~2,200 psi).
        3. Re-equilibrate column with starting conditions (in the case of this protocol, 4.8% Eluent B, 95.2% Eluent A) for 30 min; again, adjust flow rate until pressure reaches similar level to running pressure during sample analysis, and collect flow-through in a waste receptacle.
        4. Re-attach column to the system, and run a water sample followed by a standard before continuing with sample analysis using the normal gradient profile.
      4. Technical notes from the manufacturer were consulted thoroughly for this analysis (Basumallick and Rohrer, 2017).

      Table 1. Plant cell wall monosaccharide elution gradient steps

Data analysis

  1. All standard curve and sample peaks were integrated using Chromeleon 8.0 software and analyzed using Microsoft Excel as described in Yeats et al. (2016a). GraphPad Prism was used for statistical analyses and generating graphs.
  2. In order to facilitate data entry into Microsoft Excel, a customized peak-calling analysis method in Chromeleon and a Python script can be used to copy integrated curve values into a sorted, transposed Excel spreadsheet. Values can subsequently be copied into a similar analysis sheet as the one described in Yeats et al. (2016a).
    1. Raw data from Chromeleon runs must be saved as Excel spreadsheets within the same folder as the Python script.
    2. Peak calling can be adjusted directly in Chromeleon to limit output to only sugars desired for analysis.
    3. The Python script can be modified to analyze all cell wall sugars, as well as glucosamine if desired, or other additional sugars.
      Note: If using the Python script for analysis, adjust the number of inputs in Line 9 of the script to reflect the number of peaks that will be called by the Chromeleon software based on the test parameters chosen in the software.

Conclusions:
This protocol can be used to efficiently quantify all cell wall monosaccharides using a single HPAEC-PAD gradient profile. This quantification will allow the reliable characterization of monosaccharide composition, and allows for the determination of proportions of glucose that come from both the crystalline and non-crystalline portions of the plant cell wall. For example, using wildtype (Col-0), and the well-characterized cellulose-deficient mutant and prc1-1, we demonstrate that this method is sufficient for resolving differences between cell wall mutants (Figure 5). Additionally, this analysis has already been used in a recent study to quantify differences in glucose derived from crystalline cellulose (Kesten et al., 2019). This method is simple, reliable, and consistent, and can be used to better understand cell wall monosaccharide compositional changes in a biological context. Past methods rely on multiple hydrolyses approaches or HPLC gradients to quantify neutral cell wall monosaccharaides, uronic acids, and cellulose separately. Therefore, this analysis represents a streamlined alternative to total cell wall monosaccharide analysis.


Figure 5. Cell wall mutants can be clearly distinguished from wild-type using this hydrolysis and analysis method. Monosaccharide elution profile of cell wall AIR derived from dark-grown Col-0 or cellulose-deficient prc1-1 hypocotyls. A minimum of two technical replicates per sample per hydrolysis method were used. Bars represent average of 2 or 3 biological replicates (for Col-0 or prc1-1, respectively) ± standard error.

Acknowledgments

We gratefully acknowledge S. Zeeman (ETH Zürich) and T. Yeats (Cornell University) for technical advice, C. Kesten (ETH Zürich) for scientific discussion, and S. Dora (ETH Zürich) and A. García Moreno (University of Málaga) for technical support during development and optimization of this protocol. As previously mentioned, this protocol was adapted and modified from Yeats, T., Vellosillo, T., Sorek, N., Ibáñez, A.B., and Bauser, S. (2016). Bio-protocol 6(20): e1978. This work has been financially supported by the Swiss National Foundation to C.S.-R. (SNF 31003A_163065/1; A.M.).

Competing interests

There are no conflicts of interest or competing interest.

References

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  13. 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

[摘要] 植物细胞壁是由多糖和蛋白质组成的复杂网络,可为植物细胞提供强度和结构完整性,并且在生长,发育和防御反应中起着至关重要的作用。细胞壁多糖可大致分为三类:纤维素,果胶和半纤维素。多糖和细胞壁相关蛋白之间的动态相互作用有助于细胞壁内的柔韧性和刚性区域,从而在生长,环境适应或应激反应激活过程中在必要时进行重塑。这些多糖相互作用对于植物生长至关重要,但是它们也增加了植物的难度。 在尝试分析细胞壁结构和组成时进行了反驳。过去,已经使用冗长的方案来量化对纤维素有贡献的细胞壁单糖以及中性和酸性细胞壁多糖。近来,STREA 毫升为单糖定量描述INED方法。该方案结合了简化的水解方法,随后进行了多次运行的高性能阴离子交换色谱和脉冲安培检测(HPAEC-PAD)。在这里,我们介绍了该协议的更新版本,其中我们可以在单个高效液相色谱HPAEC-PAD梯度曲线中分析所有九种细胞壁单糖。包括酶促淀粉降解以及替代的内部标准以提高定量准确性,以及便于数据分析的即用型Python脚本为该协议增加了广泛的应用范围。该方案用于分析拟南芥的浅色幼苗和深色的胚轴,但适用于任何植物组织。

[背景] 了解植物细胞壁的结构和组成多年来一直是学术和工业植物研究的最前沿。多糖和细胞壁蛋白的相互作用为植物细胞提供了结构,同时在生长和适应各种外部条件的过程中也起着重要的生物学作用(Cosgrove,2005和2016;Kesten 等,2017)。所述植物细胞壁充当期间生物或非生物相互作用的一个重要物理屏障,而且还可以提供信令应激反应期间释放的分子的内源性来源(科斯格罗夫,2005年和2016; Kesten 。等人,2017 和2019 )。这些细胞壁衍生的分子可以激活信号传导级联,以提醒植物宿主入侵的病原体或需要重定向生长资源。细胞壁多糖对工业用途也很重要- 果胶广泛用于食品和化妆品工业,而纤维素对食品,造纸和纺织工业也很重要(Pettolino 等人,2012)。因此,表征植物细胞壁多糖的有效且有用的方法在学术界和工业界都至关重要,以进一步了解植物细胞壁的生物学作用并提高聚合物的提取难度,同时减少工业应用中的浪费。

1940年代在木材中进行的实验建立了在高温下使用稀酸水解来研究糖分解为单体的方法(Saeman,1945年)。此类研究为许多当前协议(包括此处介绍的协议)提供了基础。简化此分析使我们能够提高测量的准确性和效率。最近其他描述的方案已建立了广泛使用的三氟乙酸(TFA)水解方法与单次HPLC运行或基于双硫酸的水解方法(称为“一步两步硫酸”)的组合。水解”方法,并运行多个HPAEC-PAD(Zhang 等人,2012;Voiniciuc 等人,2016; Yeats 等人,2016a 和2016b)。这种“一步到两步”方法利用了Saeman 水解程序,随后使用了以前称为“矩阵水解”的方法(Yeats 等,2016a)。Saeman 水解是该方法的“两步”部分,本质上是将浓硫酸水解(72%硫酸)直接添加到植物原料中以使样品溶胀。在室温下一小时后,将水添加到样品中以将硫酸浓度稀释至4%。然后将这部分水解在121 ℃下进行1小时。在强酸溶胀水解后立即使用稀酸水解,可以使葡萄糖从所有可能的来源中释放出来,包括严重交联且刚性堆积的细胞壁成分,例如结晶纤维素。水解的“一步”部分,也称为“基质水解”,是指仅在121 °C下用4%的硫酸水解样品。通过简单地用“基质水解”处理样品,仅会释放来自易于水解来源的单糖。因此,这两种水解之间的差异允许以所谓的“一步两步”水解方法定量结晶纤维素。在我们的方案中,我们结合了Yeats 等人描述的有效的“一步两步”硫酸水解。(2016a )包括可选的酶促淀粉降解,并使用替代内部标准品洗脱所有细胞壁糖,并在一次HPAEC-PAD运行中量化源自结晶纤维素的葡萄糖。我们对先前提到的方案的补充为单糖测量的简便性和准确性做出了重大改进,这将大大有益于植物细胞壁研究领域。

关键字:细胞壁, 纤维素, 高效液相色谱法, 单糖, Saeman水解, 拟南芥

材料和试剂


 


2 ml螺帽管(Sarstedt ,目录号:72.694.006)
50 ml锥形管(Greiner Bio-One,目录号:7.210 261)
500 微升自动进样器卡环小瓶(Sigma,目录号:27422)
自动进样器样品瓶盖(Sigma,目录号:24757)
25毫升不锈钢研磨罐(Retsch ,目录号:02.462.0119)
12毫米不锈钢磨球(Retsch ,目录号:05.368.0032)
锡称量舟,5 x 9 毫米(Santis Analytical AG,货号:SA76981103)
0.22 μ 米无菌PES膜过滤器(生命系统设计AG,目录号:99255)
金属锅铲
塑料称量纸(HuberLab ,目录号:12.9702.080 )
锥形管(Grenier Bio,目录号:7.210 261)
尼龙网,60 微米孔径(Sefar公司Nitex ,目录号:3A03-0060-110-00)
正方形培养皿板(Greiner Bio-One,目录号:7.688.102)
均质珠(取决于材料的数量):
小玻璃珠(2.85-3.45 mm珠,Roth,目录号:A557.1)
大型金属球(12 毫米珠,Retsch ,目录号:05.368.0032)
CarboPac PA20色谱柱(3 x 150 mm, Thermo Fis her Scientific,目录号:060142)
CarboPac PA20保护柱(3 x 30毫米,Thermo Fis her Scientific,目录号:060144)
拟南芥拟南芥
乙醇(苏黎世联邦理工学院HCI商店,目录号:02000107)
氯仿(Sigma,目录号:25693)
甲醇(苏黎世联邦理工学院HCI商店,目录号:02000342)
丙酮(Sigma,目录号:24201-4X2.5L-GL-R)
(可选)酶促淀粉降解
淀粉葡糖苷酶(Sigma,目录号:10102857001)
α- 淀粉酶(Sigma,目录号:10102814001)
硫酸(Sigma,目录号:258105)
超纯水(Milli-Q或同等水平)
单糖分析标准:
L-岩藻糖(Sigma,目录号:F2252-5G)
D-葡萄糖(Sigma,目录号:G7528-1KG)
D-半乳糖(Sigma,目录号:48260)
D-木糖(Sigma,货号:W360600-SAMPLE)
D-甘露糖(Sigma,目录号:63579)
L-阿拉伯糖(Roth,目录号:51 18.2)
L-鼠李糖(Sigma,目录号:W373011-SAMPLE-K)
D-半乳糖醛酸一水合物(Sigma,目录号:48280)
D-葡萄糖醛酸(Sigma,目录号:G5269-10G)
(可选)替代内部标准
D- 七庚糖(CarboSynth ,目录号:MS139006)
D-核糖(Sigma,目录号:R7500-5G)
氢氧化钠,在水中的50%溶液(Sigma,目录号:415413)
无水乙酸钠(Sigma,目录号:32319-1KG-R)
Lugol 解决方案(Sigma,目录号:32922-6X1L)
液氮
 


设备


 


冷冻干燥机(基督,阿尔法2-4)
微量离心机(Eppendorf,型号:5424 R)
样品浓缩器(Stuart,型号:SBHCONC / 1)
高压灭菌器兼容机架(Karter Scientific,目录号:125A7 )
微量天平(Mettler Toledo MX5)
组织匀浆器(Retsch MM200)
微量离心管摇床(Eppendorf ThermoMixer F1.5)
旋转管(Labinco LD79,目录号:79000)
高速真空离心机(Eppendorf Concentrator Plus)
高压灭菌器(Thermo Fis 她的科学,Sterico ,Varioklav )
加热块(Stuart,型号:SBH130D)
自动进样器(Dionex ,型号:AS-1)
Dionex ICS-5000(Dionex,型号:DC-5)
ED电化学检测器(无电池,产品编号:072042)
ED Cell(无参比电极或工作电极,产品编号:072044)
聚四氟乙烯(PTFE)一次性电极上的金(Au)(产品编号:066480)
 


软件


 


Chromeleon 8(Thermo Fisher Scientific)
可通过https://www.thermofisher.com/order/catalog/product/CHROMELEON7付费获得


微软Excel
可通过https://www.office.com付费使用


GraphPad棱镜
可通过https://www.graphpad.com付费使用


Spyder5(Anaconda3)
可从https://www.anaconda.com免费获得


Python 3.6
可在https://www.python.org免费获得


 


程序


 


A. 生成和准备植物材料      


的Gr 流在所希望的条件下的植物
注意:可以从任何所需的生长方法中收集植物样品。我们建议采用以下体外方法从光生(每天循环)幼苗或暗生下胚轴产生的拟南芥材料中分离。


气体或液体将种子灭菌并在4°C下分层2-3天。
板:准备?MS + 1%蔗糖+ 0.9%琼脂方形培养皿;消毒尼龙网并放在琼脂板上; 在平板顶部播种约100-120粒种子;在所需的时间点将材料收集在50 ml锥形管中;加工前去除种子。
对于光照生长的植物,植物生长在升飞行持续14天,长日照条件下(16岁以下小时光照,8 小时黑暗)的光为拟南芥(130-150优化μE 米-2 小号-1 在20-22)° C。
对于深色的胚轴,每盘播种多行种子(不播种尼龙)。将种子在光照下播种2-4小时;覆盖几层铝箔,并在20-22°C的恒温箱中生长5天。
离开工厂的黑暗HARVES 24-48小时之前婷耗尽淀粉储备; 如果不可能的话,可以在植物材料均质化之后进行酶促淀粉降解(进行至S tep A5 )。
如果分析气生植物组织(含叶绿素),收获材料在足够的70%的乙醇,以完全浸没的植物材料,并且直到叶绿素耗尽继续交换乙醇和液体不再具有绿色的任何痕迹颜色。
如果包含非叶绿素植物部分是要被分析,收获及闪烁冷冻样品用液氮,然后继续在磨削小号TEP A5。


除去乙醇,并使用冷冻干燥机将物料干燥2天(如果需要,可以更长)。
使用组织均质器,直径为12 mm的金属球和金属容器研磨所有植物材料。
笔记:


              如果仅产生少量物质,则可以将根部收获到微量离心管中,并使用带有机架的组织均质器用于微量离心管,并使用玻璃珠进行研磨。在这种情况下,分装≤ 200 毫克的植物材料,以确保彻底均化。
收集植物材料时,必须避免收集琼脂,土壤或样品中含有糖的任何生长培养基(NB琼脂将有助于半乳糖定量)。尼龙网可如上所述使用,以防止琼脂粘附到根部。
 


B. 淀粉降解,如先前所述(Hostettler 等,2011),进行了如下修改:      


等分试样地面的植物材料高达?0.5毫升在2 ml微离心管中。
加入1 ml 80%乙醇(v / v),并在95°C加热样品10分钟。将每个样品涡旋10-15 s,使其充分混合。
室温(RT)于3,000 xg离心5分钟; 丢弃上清液。
注意:除非另有说明,所有离心步骤均应在室温下进行。


继续以下洗涤步骤:每次洗涤在ThermoMixer中摇动10分钟,以3,000 xg离心5分钟,涡旋以在洗涤之间重新悬浮沉淀,弃去上清液,然后添加下一种洗涤溶液:1 ml 50%( v / v)乙醇,1 ml 20%(v / v)乙醇,1 ml水,最后是1 ml 80%(v / v)乙醇。
注意:在步骤B4之后,最后的洗涤应该基本清除,但沉淀可能仍是绿色的。


在室温下使用高速真空离心机或在60°C的烤箱中干燥沉淀至少30分钟或整夜,直至完全干燥。重悬于400μl 水中并涡旋混合。
在95 °C下煮沸10-15分钟- 请勿在冰上冷却。
准备9份淀粉葡糖苷酶和1份α-淀粉酶的消化混合物。
注意:消化混合物的体积计算需要根据样品的总数,牢记20 微升所述混合物的每样品所需。一个样品需要18 微升的amyloglucosiades 和2 微升α淀粉酶。


添加380 微升0.22M的醋酸钠至20 微升的制备9:1 淀粉葡糖苷酶:α淀粉酶混合物; 与400μl 样品合并。
注意:如果要处理更大的样品量,可以将样品重悬在更大体积的水中。对于消化,只需将等份样品与消化混合物合并,然后按照指示进行即可。


在37°C下消化至少2小时。
用Lugol 溶液将一小部分植物材料染色,检查是否残留淀粉。
注意:Lugol 染色建议的步骤:


混合20 微升80样品微升的100%乙醇。
在90°C下煮沸5分钟。
以5,000 x g离心5分钟。丢弃上清液。
添加25 微升的Lugol 溶液; 检查的colo UR 5分钟后的变化。
Lugol 溶液可以全浓度使用,而无需稀释所购溶液。
颜色更改为深蓝色/黑色颜色表示淀粉存在。
如果观察到颜色变化,请继续消化过夜,然后再次使用Lugol 检查。继续消化直至不再观察到颜色变化(图1)。
一旦未观察到颜色变化,请继续执行步骤A11。
将样品以5,000 xg离心5分钟,然后保留不溶部分。
 


 


D:\ Reformatting \ 2020-1-6 \ 1902838--1288克拉拉·桑切斯-罗德里格斯819822 \ Figs jpg \ fig1.jpg


图1. Lugol 染色表明存在或不存在淀粉。一。充分浓度的Lugol 单独具有淡黄色颜色(左管),而复方碘直接加入到淀粉从玉米中提取示出强烈的暗棕色/紫色的颜色变化(右管)。B.两个管都包含细胞壁AIR材料;左侧的试管没有经过淀粉降解,而右侧的试管则没有。下列中描述的方法小号TEP A10,加入25 微升全浓度的Lugol 会弄脏含淀粉暗棕色/紫色空气样品颜色,如在右管观察到。如果将Lugol 添加到样品中后仍保持黄色,则说明不存在淀粉,可以继续进行S tep A11。


 


C. 清洁和生产最终细胞壁衍生的不溶性酒精(AIR)制剂      


将等分试样在2 ml微量离心管中解淀粉,直至0.5 ml。
添加1.5毫升的1:1 甲基的ANO1 :氯仿(体积/体积)混合物中以样品并涡旋以彻底混合。使用设置为15 rpm的试管旋转器或任何标准试管混合器将样品混合2 h。
在室温下以10,000 xg离心5-10分钟,然后除去上清液。
加入1.5 ml RT丙酮并涡旋充分混合。使用设置为15 rpm的试管旋转器或任何标准试管混合器混合30分钟。
在室温下以10,000 xg离心5-10分钟,然后除去上清液。
使用高速真空离心机或在60°C的烤箱中于室温下干燥最终沉淀至少30分钟或过夜,直至完全干燥。最终产物是细胞壁来源的不溶性酒精残留物(AIR)。
 


D. 如Yeats 等人所述进行样品和标准水解。(2016a )(参见图2 中这个手稿)。简要地,该方法总结如下:      


 


 


 


D:\ Reformatting \ 2020-1-6 \ 1902838--1288克拉拉·桑切斯-罗德里格斯819822 \ Figs jpg \ fig2.jpg


图2 。单个HP LC的梯度曲线可洗脱所有细胞壁单糖。每个颜色块对应一个特定的洗脱液,如右图例所示。y轴的主要特征(左侧)为百分比,是指每种洗脱液占流经色谱柱的最终混合洗脱液所占的百分比,总含量始终等于100%。二次y轴(右)表示体积(米升每洗脱液用完/分钟),与全混合洗脱,其中每个单独的洗脱剂有助于的百分比。x轴指示所指示的洗脱液组合物应进行的时间量(分钟)。


 


          使用微量天平和锡称量舟,将每个技术复制品称量1±0.1 mg AIR到2 ml螺帽管中;记录最终重量(分析步骤所需)。
笔记:


如果需要在两次采样之间清洁用于称量AIR的刮刀,请勿使用任何种类的纸巾/纸巾。使用塑料称量纸在两次样品之间用100%乙醇彻底擦洗。
我们建议每次水解至少每个样品进行两次技术重复(即,仅对基质进行水解的样品进行2次技术重复,对进行Saeman 水解+基质水解的样品进行2次技术重复),每次实验分析均进行3次生物学重复。
在样品水解之前,可以将内标(七庚糖或核糖)直接添加到称量的AIR等分试样中。允许内部标准,以完全干燥30分钟或更长的时间在必要时,无论是在室温下或用样品浓缩。
对于我们的测定,我们向AIR材料中添加了150μg 的七庚糖;但是,必须根据所选仪器的工作范围进行测量的样品稀释度来优化此量。
标准(核糖或七庚糖)的添加不是强制性的。但是,它们提供了确定性的附加度量,以确保水解和定量的一致性。
七羟庚糖在该分析中效果更好,因为我们观察到了一个与核糖具有相同保留时间的“背景”峰。但是,如果仪器或样品稍有不同,则可以使用核糖。应在水解所有样品和标准品之前进行测试。
          一个样品组将经受Saeman 以及基质水解作用,而第二个样品组将仅经受基质水解作用。
注意:以前,此方法是使用高压灭菌法作为水解方法来描述的(Yeats 等,2016a )。我们确认这是一次可水解多达50个样品的有效方法。但是,如果在121°C下加热1小时完成水解,则我们也确认了HPLC分析和定量的有效性。但是,使用高压灭菌法完成水解时,数据更加一致(图3 ),这可以减少误差。此外,使用加热块水解似乎半乳糖醛酸的水解可能是不完全的,尽管所有其他单糖值似乎在两种方法中都保持一致。也可以在较低温度(80-100 °C)下使用加热块以延长水解时间,但是应对此进行测试和优化。


 


D:\ Reformatting \ 2020-1-6 \ 1902838--1288克拉拉·桑切斯-罗德里格斯819822 \ Figs jpg \ fig3.jpg


图3. 样品水解可以使用加热块或高压釜完成,但有微小差异。来源于光生长的野生型Col-0幼苗的细胞壁AIR的单糖洗脱曲线。每种水解方法每个样品至少要进行两次技术重复。条形表示2个生物学重复的平均值±标准误差。


 


          为了进行标准曲线分析,请制成100μg 储液,其中包含用于定量的所有单糖以及适当的内标。根据测定范围稀释成适当的标准浓度。
是推荐的浓度,以产生标准曲线,如下所示:注0.05 微克,0.1 微克,0.5 微克,1 微克,2 微克,5 微克。


          为了在水解和计算特定的单糖-校正因子考虑糖特异性的损失,通过结合500准备两个恢复标准微升的100 微克具有900标准混合物微升水。
一种回收标准品与基质水解样品(水+酸,在121°C下水解1小时)处于相同条件下。
第二回收标准用酸处理,但不进行热水解。
          水解完成后,让样品在室温下冷却,并以20,000 xg 离心1分钟以沉淀任何不溶物。上清液用于下一步骤。 
 


E. HPLC分析      


                  进样前根据需要稀释样品上清液(根据起始材料和检测器的灵敏度,可以使用1:10、1:20、1:50或1:100稀释液),然后移入自动进样瓶中。
笔记:


适当的标准液和稀释液可能会根据样品或检测器的灵敏度而有所不同;建议在完成所有材料处理之前彻底测试标准液和样品稀释液以优化条件。
强烈建议将样品分析的顺序随机化,并每隔10-15个样品运行一次标准液,以确保测量的灵敏度和准确性始终如一。
                  制备洗脱液;用氦气吹扫并保持在氦气下,或按照制造商的指示进行吹扫(Rohrer,2017)。含有乙酸钠任何洗脱液必须使用0.22被过滤微米PES过滤器。在添加50%氢氧化钠溶液之前,必须将所有洗脱液吹扫至少10分钟,然后在添加后至少吹扫至少10分钟。
洗脱液A =水


洗脱液B = 50 mM 氢氧化钠


洗脱液C = 100 mM氢氧化钠,100 mM乙酸钠


洗脱液D = 200 mM氢氧化钠


                  将每种标准品,回收标准品和标准品的10μl 注入配有3 x 50 mm CarboPac PA20保护柱的3 x 150 mm CarboPac PA20 色谱柱。
                  将色谱柱温度保持在36 °C,流速为0.4 ml / min。
                  使用以下洗脱曲线洗脱所有单糖和标准品(图4 ,表1 ):0-18 分钟4.8%B,95.2%A;18-20 分钟线性梯度到下一个条件;20-30 分钟50%D,50%A; 30-40 分钟线性梯度下一个条件;40-56 分钟100%C; 56-56.1 min 线性梯度到50%D; 56.1-60 分钟50%D; 60-60.1 分钟更改为下一个条件;60.1-80 分钟,4.8%B,95.2%A使柱回到起始条件:
 


 


D:\ Reformatting \ 2020-1-6 \ 1902838--1288克拉拉·桑切斯-罗德里格斯819822 \ Figs jpg \ fig4.jpg


图4.单个HPAEC-PAD运行中细胞壁单糖的洗脱曲线。四个独立的注射被呈现:1 微克/ ml的标准混合物使用核糖作为内标(蓝色),1 微克/毫升使用标准混合物景天庚酮糖作为内标(黑色),和野生型Col-0中基质水解的一例(使用加热块水解)(粉红色),以及野生型Col-0 Saeman 水解+基质水解曲线的实例,其中添加了七庚糖作为内标(使用加热块水解)(棕色)。样品水解后(粉红色),出现一个背景峰(*),该峰明显与核糖峰(蓝色)重叠。


 


笔记:


随着更多样品的运行,峰的保留时间可能会发生变化,因此可能需要定期进行“色谱柱冲洗”并监测色谱柱性能。
短“列冲洗”期间被结合到梯度分布(?5分钟的100mM的NaOH在56分钟的); 但是,也可以定期运行冲洗程序,如下所示:30分钟100%洗脱液C,然后30分钟100%洗脱液D,经过短暂的平衡步骤(约10-15分钟)回到起始条件(95.2%洗脱液A ,4.8%洗脱液B)。
如果观察到色谱柱性能发生重大变化,则必须立即采取措施,按照制造商的说明完全冲洗色谱柱。简而言之,我们通过执行以下操作完成了列冲洗:
通过关闭电极/检测器并旋松色谱柱出口来断开色谱柱与检测器和常规机器的连接。
用2 M NaOH (允许流通液滴入废液容器)洗涤柱1小时;直到压力达到调节流速相似的水平在样品分析期间运行压力(在这种情况下,?2 ,200磅)。
用起始条件(在本方案中为4.8%洗脱液B,95.2%洗脱液A)重新平衡色谱柱30分钟;再次,调整流速直到样品分析过程中压力达到与运行压力相似的水平,然后将流过的液体收集在废物容器中。
重新连接? olumn到该系统,并且运行一个水样品,随后使用普通的梯度分布与样品分析继续之前的标准。
对于此分析,已充分参考制造商的技术说明(Basumallick 和Rohrer,2017年)。
 


表1.植物细胞壁单糖洗脱梯度步骤


时间(分钟)


洗脱液A(%)


洗脱液B(%)


洗脱液C(%)


洗脱液D(%)


0-18


95.2


4.8


0


0


20


50


0


0


50


30


50


0


0


50


40


0


0


100


0


56


0


0


100


0


56.1


0


0


0


100


60


0


0


0


100


60.1


95.2


4.8


0


0


80


95.2


4.8


0


0


 


数据分析


 


          所有标准曲线和样品峰均使用Chromeleon 8.0软件进行积分,并使用Microsoft Excel进行分析(如Yeats 等人所述)。(2016年a )。Grap hPad Prism用于统计分析和生成图形。
          为了便于将数据输入Microsoft Excel,可使用Chromeleon中的自定义峰调用分析方法和Python脚本将积分曲线值复制到经过排序的转置Excel电子表格中。随后可以将值复制到与Yeats 等人中描述的方法类似的分析表中。(2016年a )。
来自Chromeleon 运行的原始数据必须另存为Excel电子表格,与Python脚本位于同一文件夹中。
可以在Chromeleon中直接调整峰调用,以将输出限制为仅分析所需的糖。
的Python脚本可以被修改以分析所有细胞壁菅RS,因为如果以及葡糖胺需要,或其它附加的糖。
注意:如果使用Python脚本进行分析,请根据脚本中第9行的输入参数调整Chromeleon 软件将调用的峰数,以反映Chromeleon 软件将调用的峰数。


 


结论:


该协议可用于高效定量所有细胞壁单糖,例如使用HPAEC-PAD梯度图。该定量将允许可靠地表征单糖组成,并允许确定来自植物细胞壁的结晶和非结晶部分的葡萄糖的比例。例如,使用野生型(Col-0),以及表征充分的纤维素缺乏突变体和prc1-1,我们证明了该方法足以解决细胞壁突变体之间的差异(图5)。另外,该分析已经在最近的研究中用于量化源自结晶纤维素的葡萄糖的差异(Kesten 等,2019)。该方法简单,可靠和一致,可用于更好地了解生物学背景下细胞壁单糖的组成变化。过去的方法依赖于多种水解方法或HPLC梯度来分别定量中性细胞壁单糖,糖醛酸和纤维素。因此,该分析代表了总细胞壁单糖分析的简化替代方案。


 


D:\ Reformatting \ 2020-1-6 \ 1902838--1288克拉拉·桑切斯-罗德里格斯819822 \ Figs jpg \ fig5.jpg


图5 。使用这种水解和分析方法,可以将细胞壁突变体与野生型区分开来。来自暗生长的Col-0或缺乏纤维素的prc1-1 下胚轴的细胞壁AIR的单糖洗脱曲线。每种水解方法每个样品至少要进行两次技术重复。条形代表2或3个生物学重复的平均值(分别对于Col-0或prc1-1 )±标准误差。


 


致谢


 


非常感谢S. Zeeman(ETHZürich)和T. Yeats(康奈尔大学)的技术建议,C. Kesten (ETHZürich)的科学讨论,以及S. Dora(ETHZürich)和A.GarcíaMoreno(马拉加大学))在此协议的开发和优化过程中提供技术支持。如前所述,该协议是从Yeats,T.,Vellosillo ,T.,Sorek ,N.,Ibá?ez,AB和Bauser ,S. (2016 )改编和修改的。生物协议6(20):e1 978 。这项工作得到了瑞士国家基金会对CS-R的资助。(SNF 31003A_163065 / 1; AM)。


 


 


 


利益争夺


 


没有利益冲突或利益冲突。


 


参考文献


 


Basumallick ,L.,Rohrer,J. (2017年)。木质水解产物中糖醛酸和木糖的测定。赛鱼呃科学entific。
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引用:Menna, A., Fischer-Stettler, M., Pfister, B., Sancho Andrés, G., Holbrook-Smith, D. and Sánchez-Rodríguez, C. (2020). Single-run HPLC Quantification of Plant Cell Wall Monosaccharides. Bio-protocol 10(5): e3546. DOI: 10.21769/BioProtoc.3546.
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