Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Summary

This protocol describes the specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans by tagging the RE with 2 aminobenzamide prior to enzymatic (endoxylanase) hydrolysis and then analysis of the resultant oligosaccharides using mass spectrometry (MS) and nuclear magnetic resonance (NMR).

Abstract

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. De-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR)¹ and sequentially extracted with water (W-sol Fr) and 1 M KOH containing 1% NaBH₄ (KOH-sol Fr) as described by Ratnayake et al. (2014)². Two different approaches (see summary in Figure 1) are adopted. In the first, intact W-sol AXs are treated with 2AB to tag the original RE backbone chain sugar residue and then treated with an endoxylanase to generate a mixture of 2AB-labelled RE and internal region reducing oligosaccharides, respectively. In a second approach, the KOH-sol Fr is hydrolyzed with endoxylanase to first generate a mixture of oligosaccharides which are subsequently labelled with 2AB. The enzymically released ((un)tagged) oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, RP-HPLC-ESI-QTOF-MS and ESI-MSⁿ. Endoxylanase digested KOH-sol AXs are also characterized by nuclear magnetic resonance (NMR) that also provides information on the anomeric configuration. These techniques can be applied to other classes of polysaccharides using the appropriate endo-hydrolases.

Introduction

Heteroxylans are a family of polysaccharides that are the predominant non-cellulosic polysaccharides of the primary walls of grasses and the secondary walls of all angiosperms^3-6. The xylan backbones differ in their types and patterns of substitution with glycosyl (glucuronic acid (GlcA), arabinose (Araf)) and non-glycosyl (O-acetyl, ferulic acid) residues depending upon tissue type, developmental stage and species⁷.

Walls from wheat (Triticum aestivum L.) endosperm are composed primarily of arabinoxylans (AXs) (70%) and (1→3)(1→4)-β-D-glucans (20%) with minor amounts of cellulose and heteromannans (2% each)⁸. The xylan backbone may be variously un-substituted and predominantly mono-substituted (primarily O-2 position and to a lesser extent O-3 position) and di-substituted (O-2 and O-3 positions) with α-L-Araf residues⁹. The reducing end (RE) of heteroxylans from dicots (for example, Arabidopsis thaliana)¹⁰ and gymnosperms (for example, spruce (Picea abies))¹¹ contains a characteristic tetrasaccharide glycosyl sequence; -β-D-Xylp-(1→3)-α-L-Rhap-(1→2)-α-D-GalpA-(1→4)-D-Xylp. To understand heteroxylan biosynthesis and function (biological and industrial), it is important to fully sequence the xylan backbone to understand the types and the patterns of substitutions as well as the sequence of the reducing end (RE).

Specific techniques used for the structural characterization of reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans are described in this manuscript. The techniques rely on fluorophore tagging (with 2 aminobenzamide (2AB)) the reducing end (RE) of the heteroxylan chain prior to enzymatic (endoxylanase) hydrolysis. This approach, particularly for the RE sequencing, was first reported by the York laboratory^10,12-13 but is now extended to include the internal region sequencing and is a combination of established techniques that is equally adaptable to all heteroxylans independent of their source of isolation. This approach can also be applied to other classes of polysaccharides using (where available) the appropriate endo-hydrolases.

In the present study, de-starched wheat endosperm cell walls were isolated as an alcohol-insoluble residue (AIR) and sequentially extracted with water (W-sol Fr) and 1M KOH containing 1% NaBH₄ (KOH-sol Fr) as described in Ratnayake et al. (2014)². The released oligosaccharides from both W- and KOH-sol Frs are then methylated and the detailed structural analysis of both the native and methylated oligosaccharides is performed using a combination of MALDI-TOF-MS, ESI-QTOF-MS-coupled with HPLC with the online chromatographic separation using a RP C-18 column and ESI-MSⁿ. Endoxylanase digested KOH-sol AXs was also characterized by nuclear magnetic resonance (NMR).

Protocol

1. Labelling of the Reducing End (RE) Sugar Residue of W-sol AXs with 2-aminobenzamide (2AB)

1. Incubate W-sol AXs with 2AB (0.2 M) in the presence of 1 M NaBH₃CN (sodium cyanoborohydride) (pH 5.5) for 2 hr at 65 °C to convert the reducing ends of the polysaccharide backbone chains to their fluorescent derivatives.
   CAUTION: The following step should be performed in the fume hood as NaBH₃CN releases poisonous cyanide gas when it is in contact with water.
   1. Weigh out NaBH₃CN (62.8 mg) and dissolve in water (1 ml) in a microcentrifuge tube (1.5 ml) to prepare a 1 M NaBH₃CN solution. Dissolve 2AB reagent (27.2 mg) in 1 M NaBH₃CN solution (1 ml) by heating at 65 °C and adjust the pH of the reaction mixture (0.2 M 2AB, 1 M NaBH₃CN) to pH 5.5 with 10% acetic acid.
   2. Add 200 µl of reaction mixture (0.2 M 2AB, 1 M NaBH₃CN) to W-sol AXs (1 mg) in a glass tube with a cap and mix using a vortex mixer. Incubate for 2 hr at 65 °C in a fume hood. Cool the suspension to RT and add 4 vols. of absolute ethanol.
   3. Place the suspension in a cold storage (4 °C) O/N to precipitate polysaccharides.
   4. Centrifuge (1,500 x g, 10 min, RT) to remove supernatant. Wash the pellet extensively with absolute ethanol (4x), acetone (1x) and methanol (1x), centrifuging between each wash. Vacuum dry at 40 °C O/N.
      Note: Extensive washing also removes residual 2AB.

2. Generation of Xylo-oligosaccharides from 2 AB Labelled W-sol AXs

1. Dissolve 2AB labelled W-sol AXs (1 mg) in 500 µl of sodium acetate buffer (100 mM, pH 5) in a microcentrifuge tube (1.5 ml). Add 4 units of endoxylanase (GH 11, [M1]) and incubate at 37 °C for 16 hr.
2. Destroy enzyme activity by heating the reaction mixture for 10 min in a boiling water bath. Cool the suspension to RT and transfer to glass tube with a cap. Add 4 vols. of absolute ethanol and place the suspension in a cold storage (4 °C) O/N to precipitate any undigested polysaccharides.
3. Centrifuge (1,500 x g, 10 min, RT) to separate undigested polysaccharides (pellet) and endoxylanase generated xylo-oligosaccharides (supernatant). Decant supernatant into a clean glass tube and place it into a warm water bath (40 °C).
4. Evaporate the ethanol under a stream of nitrogen gas to an end point volume (~500 µl). Freeze the supernatant at -80 °C for 4 hr and dry the frozen supernatant in a freeze dryer to recover xylo-oligosaccharides.

3. Generation of Xylo-oligosaccharides from KOH-sol AXs and 2AB Labelling

1. Treat KOH-sol AXs with endoxylanase (GH 11, [M1]) to generate xylo-oligosaccharides as described above (sections 2.1-2.4).
2. Treat endoxylanase generated xylo-oligosaccharides from KOH-sol AXs with 2AB reaction mixture (0.2 M 2AB, 1 M NaBH₃CN) as described above (sections 1.1.1-1.1.2).
3. Decant supernatant into a clean glass tube and place it into a warm water bath (40 °C). Evaporate the ethanol under a stream of nitrogen gas to an end point volume (~500 µl). Freeze the supernatant at -80 °C for 4 hr and dry the frozen supernatant in a freeze dryer to recover xylo-oligosaccharides.

4. MALDI-TOF-MS

1. Preparation of MALDI-matrix Solution
   1. Add a small scoop of 2, 5-dihydroxbenzoic acid (DHB) to 50% acetonitrile (500 ml) containing 0.1% formic acid in a tube (MALDI-matrix solution). Mix using a vortex, if it dissolves quickly, add another small scoop of DHB. Note: Ideal concentration of MALDI-matrix solution is 10 µg/µl^-1.
2. Preparation of MALDI-target Plate
   1. Deposit the aqueous oligosaccharide (native) samples (5-10 µg) (W-sol and/or KOH-sol) onto a MALDI-target plate. Add 0.3 µl MALDI-matrix solution using a separate tip and mix by pipetting up and down. Allow the mixture to dry at RT.
      Note: Properly dried samples should consist of long needle-shaped crystals pointing toward the center of the spot. If the deposit is sticky and/or smeary the sample may either be too concentrated or consist of salts, such deposits are unlikely to produce good spectra and the sample should be purified further.
   2. Introduce the target plate into the MS source and operate in positive (+ive) ion mode. Adjust the accelerator voltage to 19.0 kV at ion source 1 and 16.3 kV at ion source 2. Adjust the laser power to greater than 70%. Select the sample spot on the MALDI-target plate and click start to begin laser shots.
      Note: All areas of the target will not yield signals. A particular spot will only give a signal for a few laser shots due either to depletion of the sample/matrix mixture or characteristics of the crystal.
      1. Move the laser to different areas of the target during acquisition and average approximately 200 random spectra to obtain satisfactory signal-to-noise.

5. ESI-QTOF-MS

1. Analyze endoxylanase-generated oligosaccharides (native) using nano-HPLC coupled with an electrospray ionization (ESI) quadrupole time of flight (QTOF) MS instrument with online chromatographic separation using a RP C-18 column (75 µm x 150 mm; 3.5 µm bead size).
   1. Transfer the endoxylanase-generated aqueous oligosaccharides mixture into a vial and place into the HPLC auto sampler. Program the elution gradient of 5-80% with the mobile phases 0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile, respectively, over 60 min.
   2. Adjust the flow rate to 0.2 µl/min. Adjust the positive-ion mode in the scan range of 300-1,600 m/z and a scan-rate of 0.5 scans/second using the ESI source conditions as follows: curtain gas 10, GS1 4, source temperature 100 °C, ion spray voltage 2,300 V, and de-clustering potential 50 V. Run the LC chromatographic program and elute oligosaccharides. The resultant total ion chromatogram (TIC) is saved automatically by the software.
   3. Open the saved TIC and select extract chromatogram. Type the expected masses (e.g., 271, 403, 535, 667, 799 and 931 m/z) at the command line. Scan chromatogram by clicking Enter. Process the selected ion scans of the ESI-QTOF MS chromatogram data using software according to the manufacturer's instructions¹⁴.

6. ESI-MSⁿ

1. Insert 1 to 2 µl of per-O-methylated oligosaccharide (methylated as described by Pettolino et al.¹) sample in 50% acetonitrile into a nanospray tip using a syringe. Trim the nano-spray tip using a glass cutter to fit into the discrete nano-spray holder attached to the MS.
2. Set the mass according to the expected mass range (200-1,500 m/z) and curtain gas to 10, ionspray voltage at 1,900 V and polarity to positive.
3. Press the acquire button to open relevant window, enter data file name to obtain a total ion scan (ESI-MS¹). Then press the STOP button.
4. Change scan type from product ion to fragment a peak of interest. Enter the mass of interest (e.g., 885 m/z) to fragment and adjust the mass range (200-900 m/z). Press the acquire button and adjust the collision energy (up and/or down in the compound tab) to achieve entire fragmentation of parent ion (885 m/z) and acquire a fragment ion scan (ESI-MS²).
5. Enter the mass of fragment ion of interest (e.g., 711 m/z) and adjust the mass range (200-720 m/z). Press the acquire button and adjust the collision energy to achieve entire fragmentation of precursor ion (711 m/z) and acquire a fragment ion scan (ESI-MS³).

7. H¹ NMR Spectroscopy

1. Dissolve endoxylanase generated mixture of oligosaccharides (KOH-sol, native form) (~500 µg) in D₂O (1.0 ml, 99.9%) in a plastic test tube (15 ml). Freeze the suspension at -80 °C for 4 hr and dry the frozen suspension in a freeze dryer to recover xylo-oligosaccharides.
2. Repeat 7.1 twice in order to fully exchange the H₂O with D₂O.
3. Dissolve dried oligosaccharides in D₂O (0.6 ml, 99.9%) and add 0.5 µl of acetone (5% in D₂O) as an internal standard. Transfer the deuterated oligosaccharides into the NMR sample tube.
4. Hold the NMR tube containing sample by the top and insert the sample tube in a plastic spinner. Place the spinner in the sample depth gauge. Push or pull the sample tube to adjust the depth of the sample to ensure that the center line of the sample top and bottom depth gauges are equal.
5. Remove the depth gauge and insert the sample into the auto sampler attached to a 600 MHz NMR spectrometer equipped with a cryo-probe.
6. Login and open spectrometer control software. Enter the sample file name. Open an existing dataset and then use the "edc" command to save it to under a new name. Type the position number of the sample in the auto sampler and press "ENTER".
   Note: Pressing the "ENTER" button will drop the sample tube gently to the magnet bore where it will be positioned at the top of the probe.
7. Set the desired sample temperature by typing "edte" at the command line. Wait for the sample temperature to reach the desired value before proceeding to the next step. Enter " lock" at the command line and select appropriate solvent (D₂O). Wait for the " lock finished" message to appear at the bottom of the window.
8. Type "atmm" at the command line and click "optimize" at the top of the atmm menu bar. Choose start for tuning and matching of the probe for the selected channel (¹H in this case).
9. Type "topshim" at the command line for the shimming process in which minor adjustments are made to the magnetic field until uniform magnetic field is achieved around the sample.Acquire the signal by typing "zg" at the command line and enter.
10. Analyze spectra using software¹⁵ according to the manufacturer's instructions with ¹H chemical shift referenced to an internal standard of acetone at 2.225 ppm.

Results

Endoxylanase digestion of 2AB-labelled W-sol AXs generates a mixture of 2AB-labelled RE oligosaccharides and a series of un-labeled (without 2AB label) oligosaccharides derived from the internal regions of the xylan chain (Figure 1; from Ratnayake et al.²). A series of chromatographic approaches is then employed to fractionate the complex mixture of isomers. Finally, MS techniques are utilized to identify the isomeric structures that are then sequenced...

Discussion

Most matrix phase cell wall polysaccharides have seemingly randomly substituted backbones (with both glycosyl and non-glycosyl residues) that are highly variable depending upon the plant species, developmental stage and tissue type³. Since polysaccharides are secondary gene products their sequence is not template derived and there is therefore no single analytical approach, such as exists for nucleic acids and proteins, for their sequencing. The availability of purified linkage-specific hydrolytic enzymes has ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2 aminobenzamide (2AB)	Sigma-Aldrich (www.sigmaaldrich.com)	A89804
sodium borohydride (NaBH4)	Sigma-Aldrich (www.sigmaaldrich.com)	247677	Hazardous, handle with care
sodium cyanoborohydride (NaBH3CN)	Sigma-Aldrich (www.sigmaaldrich.com)	156159	Hazardous, handle with care
endo-1,4-β-Xylanase M1 (from Trichoderma viride) (120101a)	Megazyme (www.megazyme.com)	E-XYTR1
Deuterium Oxide (D2O)	Sigma-Aldrich (www.sigmaaldrich.com)	151882
Freeze dryer (CHRIST-ALPHA 1-4 LD plus)
RP C18 Zorbax eclipse plus column	Agilent		(2.1×100 mm; 1.8 µm bead size)
MicroFlex MALDI-TOF MS   (Model - MicroFlex LR)	(Bruker Daltonics, Germany)
(ESI) -(QTOF) MS   (Model # 6520)	(Agilent, Palo Alto, CA )
ESI-MSn  - ion-trap  (Model # 1100 HCT)	(Agilent, Palo Alto, CA).
Bruker Avance III 600 MHz -NMR	Bruker Daltonics, Germany
Topspin (version 3.0)-Biospin- software	Bruker
GC-MS (Model # 7890B)	Agilent