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).
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.
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 angiosperms3-6. 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 laboratory10,12-13 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).
1. Labelling of the Reducing End (RE) Sugar Residue of W-sol AXs with 2-aminobenzamide (2AB)
2. Generation of Xylo-oligosaccharides from 2 AB Labelled W-sol AXs
3. Generation of Xylo-oligosaccharides from KOH-sol AXs and 2AB Labelling
4. MALDI-TOF-MS
5. ESI-QTOF-MS
6. ESI-MSn
7. H1 NMR Spectroscopy
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 by MSn techniques. Here we present a representative, rather than comprehensive, example of the approach.
The signals in the MALDI-TOF-MS spectrum of oligosaccharides derived from 2AB-labelled native W-sol AXs (Figure 2A) include a highly abundant pseudo-molecular ion series at m/z 701, 833, and 965 representing a series of unlabeled neutral internal region oligosaccharides with 5-7 pentosyl residues (P5-7), respectively. A series of signals at m/z 745, 877, and 1009, designate that an unlabeled acidic oligosaccharide series, with P4-6+HexA1 (Hexuronic acid), is also present in this fraction (Figure 2A). Pseudo-molecular ions at m/z 821 and 953 indicate the presence of the 2AB-labelled original RE oligosaccharides P5-6+2AB, respectively.
The ESI-QTOF-MS analysis for the native oligosaccharides with an on-line chromatographic fractionation of oligosaccharides by RP C-18 HPLC is then performed. Figure 2B and 2C, shows the selected ion scans, extracted from ESI-QTOF-MS total ion chromatogram (TIC), of oligosaccharides released by endoxylanase from W-sol Fr AXs. The signals include a pseudo-molecular ion series assigned as [M + NH4]+ at m/z 696, 828, 960, 1092, 1224, and 1356 representing a series of internal region neutral oligosaccharides with 5-10 pentosyl residues (P5-10), respectively (Figure 2B). Several isomeric structures are possible for an oligosaccharide of a defined mass (see below ESI-MSn analysis). Hence the multiple peaks for each of the molecular ion scans are possible as observed in Figure 2B. Pseudo-molecular ions assigned as [M + H]+ at m/z 271, 403, 535, 667, 799, and 931 indicate the presence of a 2AB labelled RE oligosaccharide series P1-6+2AB, respectively (Figure 2C). The signals detected as [M + H]+ ions at m/z 613, 745, 877, 1009, 1141, and 1273 indicate the presence of an acidic oligosaccharides with P3-8+HexA1, respectively (Figure 2C). In the commelenid monocots, the xylan backbone can also be substituted with phenolic acids, primarily ferulic acid (and also p-coumaric acid), which has the same molecular mass as glucuronic acid and can be detected in W-sol AXs of wheat endosperm cell walls. However, further analysis of W- and KOH-sol AXs using ESI-MSn (and compositional analyses by GC-MS of TMS derivatives following methanolysis; not shown here) confirm the acidic oligosaccharides with P3-8+HexA1 in wheat endosperm AXs.
The signals assigned as [M + H]+ ions of ESI-Q-TOF full scan spectrum of the region between 3.10-3.48 min (Figure 2D) includes the series of 2AB labelled RE oligosaccharides: m/z 271, 403, 535, 667, 799, 931, 1063, and 1195 (P1-8+2AB, respectively). A series of pseudo-molecular ions in the ESI-Q-TOF full scan spectrum of the region between 3.59-4.05 min (Figure 2E) represent the internal region acidic oligosaccharides observed as both [M+Na]+ ions: m/z 613, 745, 877, 1009, and 1141 (P3-7+HexA1, respectively) and [M + NH4]+ ions: m/z 740, 872, 1004, 1136, and 1268 (P4-8+HexA1, respectively).
In order to sequence the individual oligosaccharides, we performed ESI-MSn on the per-O-methylated oligosaccharides rather than on the native oligosaccharides obtained from W-sol and KOH-sol AXs since it is challenging to unequivocally assign structures by sequencing native oligosaccharides. In addition, it also requires greater quantities of material. Methylation of the oligosaccharides was carried out as described by Pettolino et al.1. The ESI-MSn investigations performed on the RE neutral oligosaccharide alditols derived from KOH-sol AXs, and 2AB labelled neutral RE oligosaccharide derived from W-sol AXs are described below as an example to assist in the interpretation of the spectra and deduced structures. The same approach can be applied to all the oligosaccharides generated from enzymic hydrolysis. The fragment ions in the ESI-MSn spectra were identified as Y and B ions according to Domon & Costello.16 Un-methylated hydroxyl group(s) generated during gas-phase fragmentation of per-O-methylated oligosaccharides in MSn provides a 14Da mass difference "scar" that can be used to identify the branching pattern and the glycosyl sequences.12-13 Each scar generated by the fragmentation event is marked as a solid line (Figures 3 and 4). As several isomeric structures are possible for a defined mass, then in these isomeric structures, Y and B ions are labelled in red and black, respectively.
The ESI-MS2, ESI-MS3 and ESI-MS4 spectra of per-O-methylated RE neutral oligo-glycosyl alditol generated from the fragmentation of the pseudo-molecular ion m/z 885 (P4+Xylol) is shown in Figure 3. The ESI-MS2 spectrum includes the abundant Y ions at m/z 711, 551, and 391 generated by the loss of one, two and three pentosyl residues, respectively, from the parent ion. The abundant m/z 711 ion can be generated by either the loss of a non-reducing terminal end Xyl residue or by the loss of a terminal side chain Ara residue. The diagnostic Y m/z 391 ion in the resulting spectrum can be generated from the loss of three non-reducing end Xyl residues from the RE oligosaccharide which has a side chain Ara residue on the RE Xylol residue or the RE oligosaccharide which has a side chain Ara residue on the 2nd Xyl residue from the RE Xylol residue. Although there is a formal possibility that other structures, such as Xyl4-Xylol, and (Ara)Xyl-Xyl-Xyl-Xylol would give rise to this fragment ion these structures are excluded from consideration as the specificity of the endo-xylanase used to cleave the polysaccharide would either degrade or not cleave at the glycosidic linkage adjacent to a branch point, respectively. Correspondingly, the diagnostic Y m/z 551 ion can be generated from the loss of two non-reducing end Xyl residues from the RE oligosaccharide which has the side chain Ara residue on either the RE Xylol residue or the RE oligosaccharide which has the side chain Ara residue on the penultimate Xyl residue. Thus, four possible isomeric structures can be proposed (Figure 3: I, II, III and IV). The abundant Y ion m/z 377 (see Figure 3: Ia and IIa) and B ion m/z 503 (see Figure 3: Ia, Ib, IVa and IVb) generated from further fragmentation of isomeric precursor m/z 711 ion are also observed in this spectrum. The ESI-MS3 spectrum (Figure 3) recorded by the fragmentation of isomeric precursor m/z 711 ions included a major peak at m/z 537 (Y ion of P2+Xylol with two scars; generated from the precursor ion Ia, Ib, IIa, IIb, IIIa, IIIb, IVa and IVb) and m/z 391 (Y ion of P1+ Xylol with one scar; generated from the precursor ion IIb, IIIa, IIIb, IVa and IVb). These two major peaks (m/z 537and m/z 391) can be generated by the loss of one and two non-reducing terminal Xyl residues, respectively, from the isomeric precursor m/z 711 ion generated from the fragmentation of the pseudo-molecular parent ion m/z 885 (P4+ Xylol) during ESI-MS2. Relatively lower abundance peaks at m/z 377 (Y ion of P1+ Xylol with two scars; generated from the precursor ion Ia and IIa) and 551 (Y ion of P2+ Xylol with one scar; generated from the precursor ion Ib and IIb) were also observed in this spectrum.
The ESI-MS4 of the fragmentation of the isomeric precursor m/z 551 ions included a major peak at m/z 377 (Y ion of P1+Xylol with two scars) and m/z 391 (Y ion of P1+ Xylol with one scar) generated from the precursor ion of structures I and II. Therefore the collective evidence suggested that the RE glycosyl sequence consists of the Ara side chain attached to the RE Xylol residue (diagnostic fragmentation pathway m/z 885→711→551→391; Figure 3II), Ara side chain attached to both RE Xylol and the penultimate (1st Xyl residue from RE Xylol) Xyl residue (diagnostic fragmentation pathway m/z 885→711→537→391; Figure 3III), Ara side chain attached to the penultimate (1st Xyl from RE Xylol) Xyl residue (diagnostic fragmentation pathway m/z 885→711→537→377; Figure 3I) and the Ara side chain attached on the 2nd Xyl residue from the RE Xylol (Figure 3IV).
The presence of these ions confirms the proposed isomeric structures I, II, III and IV and the neutral RE oligosaccharide structure of: -[Araf-(1→3)](+/-)-Xylp-(1→4)-[Araf-(1→3)](+/-)-Xylp-(1→4)-[Araf-(1→3)](+/-)-Xylp.
The ESI-MS2 spectrum of per-O-methylated 2AB labelled RE oligosaccharide generated from the fragmentation of the quasi-molecular [M+Na]+ ion at m/z 871 (P4+2AB) is shown in Figure 4. This spectrum includes the most abundant Y ion at m/z 697 (P3+2AB with one scar), m/z 537 (P2+2AB with one scar) and m/z 377 (P1+2AB with one scar), generated by the loss of either one, two or three non-reducing pentosyl residues, respectively. The m/z 697 ion can be generated by either the loss of a non-reducing terminal end Xyl residue or by the loss of a terminal Ara residue whereas the m/z 537 ion can only be generated by the loss of two terminal Xyl residues. The diagnostic fragmentation pathway (m/z 871→697→537→377) suggested that the existence of linear un-branched xylan backbone oligosaccharide(s) at the RE corresponding to the quasi-molecular ion m/z 871 (P4+2AB). However the linear un-branched xylosyl backbone (P4+2AB) is susceptible to site specific endoxylanase for further digestion. Therefore two isomeric m/z 697 ions can exist. Accordingly two possible isomeric structures are proposed (Figure 4: I and II). In these isomeric structures Y and B ions are labelled in red and black, respectively. The ESI-MS3 spectrum recorded from the fragmentation of isomeric precursor m/z 697 ion generates m/z 523 ion (Y ion of P2+2AB with two scars; Figure 4, structures Ia, Ib, IIa and IIb), m/z 363 ion (Y ion of P1+2AB with two scars; Figure 4, structure IIa) and Y ion at m/z 377 (P1+2AB with one scar; Figure 4, structures Ia and Ib). The fragment ion at m/z 377 can only arise from the proposed structure I and the fragment ion at m/z 363 can only arise from the proposed structure II. The simultaneous presence of these two ions confirms the proposed structures I and II. Thus the RE glycosyl sequence of the xylan chain of wheat endosperm AXs consist of an Ara branch attached to the RE Xyl residue (fragmentation pathway m/z 871→697→523→363) and/or penultimate Xyl residue (fragmentation pathway m/z 871→697→523→377).
NMR analysis of the AXs
MS-based analyses do not provide information on either the anomeric configuration (α/β) or the D/L configuration of the sugars that must be obtained by other approaches, including enzymic and physical (e.g., NMR). For heteroxylans with the characteristic RE reduced tetrasaccharide (Xyl-Rha-GalA-Xylol), the NMR spectrum contains anomeric signals leading to the identification and sequencing of this RE oligosaccharide. We describe the use of 600 MHz 1D 1H-NMR spectroscopy as a single step method to determine the complete glycosyl sequence of the wheat endosperm AX RE oligosaccharide on the KOH-sol Fr, including the anomeric configuration (α/β) and the D/L configuration of the sugars. Resonances were assigned on the basis of published assignments of wheat AX oligosaccharides 17-18 (Figure 5, Table 1). The 1H-NMR spectrum of the AX extracted from wheat endosperm is dominated by the anomeric chemical shifts at 5.39, 5.27 and 5.22 ppm that are assigned to the proton of a terminal α-L-Araf residue, attached to O-3 position (T-α-L-Araf →3S) of the singly branched (1,4)-β-Xylp backbone residues and both O-3 and O-2 positions (T-α-L-Araf →3D and T-α-L-Araf →2D) of doubly branched (1,4)-β-Xylp backbone residues, respectively (Figure 5 and Table 1).
The signals at 5.41 are assigned to the (T-α-L-Araf→3S+D) H1 signal of α-L-Araf side chain attached to the O-3 position of the singly branched β-D-Xylp residue with adjoining doubly branched β-D-Xylp. The signal at 5.29 is assigned to the (T-α-L-Araf→3D+D) H1 signal of α-L-Araf side chain attached to the O-3 position of the doubly branched β-D-Xylp residue with adjoining doubly branched β-D-Xylp. The signal at 5.24 is assigned to the (T-α-L-Araf→2D+D) H1 signal of α-L-Araf side chain attached to the O-2 position of the doubly branched β-D-Xylp residue with adjoining doubly branched β-D-Xylp.
Figure 1. Summary of Experimental Approach. A summary of the strategy employed in generating, purifying and sequencing the reducing end (RE) and internal region oligosaccharides of wheat endosperm arabinoxylans (AXs)is shown. This figure has been reproduced with permission from Ratnayake et al. (2014)2. Please click here to view a larger version of this figure.
Figure 2. MALDI-TOF MS (A) and ESI-QTOF MS (B–E) analysis of native oligosaccharides released by endoxylanase from 2AB labelled W-sol AXs as outlined in Figure 1. MALDI-TOF MS spectrum: (A) (The signals are identified as [M+Na]+ adduct ions); Selected ion scans of the ESI-QTOF MS chromatograms: B = P5-10 derived from internal region oligosaccharides (The signals are identified as [M + NH4]+ adduct ions); C = P1-6+2AB derived from RE oligosaccharides (The signals are identified as [M+H]+adduct ions) & P3-8G derived from acidic oligosaccharides (The signals are identified as [M + Na]+ adduct ions); D = ESI-Q-TOF full scan spectrum: region between 3.10-3.48 min, E = ESI-Q-TOF full scan spectrum: region between 3.59-4.05 min (The signals are identified as both [M + Na]+ and [M + NH4]+ adduct ions); P = pentosyl unit (either Ara or Xyl); G = uronosyl residue (GlcA); RE = reducing end oligosaccharides; 2AB = 2 aminobenzamide; EIC: extracted ion chromatogram. Please click here to view a larger version of this figure.
Figure 3. The ESI-MS2, ESI-MS3 and ESI-MS4 spectra of per-O-methylated RE neutral glycosyl alditol (P4+Xylol) – m/z 885. The signals are assigned as the [M + Na]+ pseudo-molecular ion adducts. As several isomeric structures for a defined mass are possible then in isomeric structures Y and B ions are labelled in red and black, respectively. Each "scar" generated by the fragmentation event is marked as a solid line. X = Xylosyl residue; A = Arabinosyl residue. The ESI-MS3 spectra has been reproduced with permission from Ratnayake et al. (2014)2. Please click here to view a larger version of this figure.
Figure 4. The ESI-MS2 and ESI-MS3 spectra of per-O-methylated 2AB labelled neutral RE oligosaccharide (P4+2AB) – m/z 871. The signals are assigned as the [M + Na]+ pseudo-molecular ion adducts. As several isomeric structures for a defined mass are possible in isomeric structures, Y and B ions are labelled in red and black respectively. Each "scar" generated by the fragmentation event is marked as a solid line. X = Xylosyl residue; A = Arabinosyl residue. This figure has been reproduced with permission from Ratnayake et al. (2014)2. Please click here to view a larger version of this figure.
Figure 5. Anomeric region of the 600 MHz 1D 1H-NMR spectrum of the AX oligosaccharides generated by endoxylanase treatment of the KOH- Sol Fr. 1H chemical shift referenced to an internal standard of acetone at 2.225 ppm. T-α-L-Araf→3S: H1 signal of α-L-Araf side chain attached to the O-3 position of the singly branched β-D-Xylp residue; T-α-L-Araf→2D: H1 signal of α-L-Araf side chain attached to the O-2 position of the doubly branched β-D-Xylp residue; T-α-L-Araf→3D: H1 signal of α-L-Araf side chain attached to the O-3 position of the doubly branched β-D-Xylp residue; T-α-L-Araf→3S+D: H1 signal of α-L-Araf side chain attached to the O-3 position of the singly branched β-D-Xylp residue with adjoining doubly branched β-D-Xylp; T-α-L-Araf→2D+D: H1 signal of α-L-Araf side chain attached to the O-2 position of the doubly branched β-D-Xylp residue with adjoining doubly branched β-D-Xylp; T-α-L-Araf→3D+D: H1 signal of α-L-Araf side chain attached to the O-3 position of the doubly branched β-D-Xylp residue with adjoining doubly branched β-D-Xylp; 2-α-L-Araf→3S: H1 signal of 2-α-L-Araf side chain residue attached to the O-3 position of the singly branched β-D-Xylp residue; Please click here to view a larger version of this figure.
Sugar residues | H-1/C-1 | H-2/C-2 | H-3/C-3 | H-4/C-4 | H-5eq/C-5 | H-5ax/C-5 |
T-α-L-Araf →3S | 5.396/107.6 | 4.16 | 3.95 | 4.3 | 3.82 | 3.72 |
T-α-L-Araf | 5.415/ | 4.18 | 3.95 | |||
→3S +D | ||||||
T-α-L-Araf →2D | 5.223/ | 4.16 | 3.97 | 3.82 | 3.74 | |
T-α-L-Araf →2D+D | 5.243/ | 4.16 | 3.98 | |||
T-α-L-Araf →3D | 5.272/108.9 | 4.18 | 3.96 | 4.26 | 3.79 | 3.74 |
T-α-L-Araf →3D+D | 5.298/ | 4.18 | 3.96 | |||
2-α-L-Araf →3S | 5.548/106.4 | 4.27 | 4.06 | 4.3 | 3.82 | |
α-Xylp (Reducing) | 5.185/91.9 | 3.55 | 3.72 | |||
β-Xylp (Reducing) | 4.580/96.6 | 3.29 | 3.48 | 3.64 | 4.06 | 3.38 |
β-4-Xylp | 4.470/ | 3.32 | 3.58 | |||
β -4-Xylp+S | 4.461 | 3.31 | 3.56 | 3.75 | 4.08 | 3.36 |
β-4-Xylp+D | 4.448 | 3.31 | 3.56 | 3.75 | 4.08 | 3.36 |
β-3,4-Xylp | 4.518/ | 3.44 | 3.85 | |||
S+β-3,4-Xylp | 4.514/ | 3.47 | 3.75 | 3.86 | 4.14 | 3.43 |
D+β-3,4-Xylp | 4.505 | |||||
β-3,4-Xylp+S | 4.492 | 3.45 | 3.74 | |||
β-3,4-Xylp+D | 4.482 | 3.45 | 3.74 | |||
β-2,3,4-Xylp | 4.638 | |||||
S+β-2,3,4-Xylp | 4.627 | 3.59 | 3.87 | 3.88 | ||
D+β-2,3,4-Xylp | 4.616 | |||||
β-2,3,4-Xylp+S | 4.593 | |||||
β-2,3,4-Xylp+D | 4.593 | |||||
Chemical shifts are reported relative to internal acetone, δ 2.225. | ||||||
S = Singly branched β-Xylp | ||||||
S+D = Singly branched β-Xylp + Doubly branched β-Xylp | ||||||
D = Doubly branched β-Xylp | ||||||
D+D = Doubly branched β-Xylp + Doubly branched β-Xylp |
Table 1. 1H-NMR signals of the xylo-oligosaccharides generated by endoxylanase treatment of the wheat endosperm KOH-sol Fr.
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 provided a powerful tool to degrade polysaccharides to oligosaccharides that can then be chromatographically fractionated, and when used in combination with chemical and physical techniques completely sequenced. The challenge is to then re-assemble these complex mixtures into the original polysaccharide sequence- one that is still to be successfully addressed.
Here we describe an approach (whose order of application can be varied) that relies on the integration of established enzymic, chemical and physical techniques for the structural characterization of both the reducing end (RE) and internal region glycosyl sequence(s) of heteroxylans. An additional complementary technique not described here that has proved very useful for characterizing oligosaccharides is PACE (polysaccharide analysis by carbohydrate gel electrophoresis) developed by the Dupree19 group and it could easily be integrated into this protocol if the equipment is available. Furthermore, variations on the LC chromatography can also be useful, such as tandem inline hydrophilic interaction chromatography (HILIC) followed by RP chromatography offering the possibility of separating both untagged/tagged oligosaccharides in a single step. The techniques rely on tagging (with 2 aminobenzamide (2AB)) the reducing end (RE) of the heteroxylan chain prior to enzymatic (endoxylanase) hydrolysis. 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. In this latter scenario the original RE of the KOH-sol AX would not be labelled with 2AB since it had been reduced to the glycosyl-alditol during the alkali extraction that contained the reductant, sodium borohydride (NaBH4). Therefore, the 2AB-labelled oligosaccharides generated post-xylanase digestion, will originate from "internal" oligosaccharides and the original RE oligosaccharide will contain a RE alditol without a 2AB tag (see Figure 1). This approach can also be applied to other classes of polysaccharides using (where available) the appropriate endo-hydrolases.
The MS-based approach is significantly enhanced by methylation of the oligosaccharides generated after endoxylanase treatment since the un-methylated hydroxyl group(s) generated during gas-phase fragmentation of per-O-methylated oligosaccharides in MSn provides a 14Da mass difference "scar" that can be used to assist in the identification of the branching pattern and the glycosyl sequence.5-6 The identity of the pentosyl residues (and any sugar residue) cannot be made from the MS data alone but comes from having a knowledge of the composition of the molecule; where this is not available then the relevant monosaccharide and linkage analyses must be performed prior to making these assignments in MSn. Furthermore the signals corresponding to the RE acidic oligosaccharide alditol, generated from KOH-sol Fr (Xyl3-MeGlcA-Xylitol: m/z 761) and the characteristic dicot xylan RE glycosyl sequence (Xyl2-Rha-GalA-Xylitol: m/z 761), if present, are not able to be distinguished as both have the same molecular mass in native form but can be distinguished from their MS fragmentation (MSn) spectra which is best performed on the methylated oligosaccharides. Finally, MS-based techniques are unable to provide information on either the anomeric configuration (α/β) of the glycosidic linkage or the D/L configuration of the sugars- this must be determined by alternate methods, including enzymic and physical (e.g., NMR).
The authors have nothing to disclose.
This project was supported by funds from Commonwealth Scientific and Research Organisation Flagship Collaborative Research Program, provided to the High Fibre Grains Cluster via the Food Futures Flagship. AB also acknowledges the support of an Australia Research Council (ARC) grant to the ARC Centre of Excellence in Plant Cell Walls (CE110001007).
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 |