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Structural analysis of hemicelluloses by enzymatic digestion and MALDI-TOF MS
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Structural analysis of hemicelluloses by enzymatic digestion and MALDI-TOF MS

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Category
Plant cell wall analysis
Protocol Name

Structural analysis of hemicelluloses by enzymatic digestion and MALDI-TOF MS

Authors
Tuomivaara T., Sami
Complex Carbohydrate Research Center, University of Georgia

Kulkarni, Ameya
Complex Carbohydrate Research Center, University of Georgia

York S., William *
Complex Carbohydrate Research Center, University of Georgia
*To whom correspondence should be addressed.
KeyWords
Reagents

Xyloglucan polymer preparation from medium of suspension cultured sycamore maple cells

Aspergillus aculeatus xyloglucan-specific endoglucanase, XEG (Novozymes, Bagsvaerd, Denmark)

25 mM ammonium acetate, pH 5.0

2,5-dihydroxybenzoic acid, DHB (Sigma-Aldrich, St. Louis, MO)

Methanol, analytical grade (Sigma-Aldrich)

Ethanol, technical grade (Sigma-Aldrich)

Ultrapure water

Instruments

MALDI-TOF MS instrument (LT MicroFlex, Bruker Daltonics Inc. Billerica, MA)

MALDI-TOF MS sample plate with ground stainless steel surface (Bruker Daltonics Inc.)

Methods
1.

Generation of hemicellulosic oligosaccharides by enzymatic hydrolysis

1) 

 Dissolve the XG (Note 1) preparation, from which small molecular weight material has been removed by dialysis or some other method (Note 2), in 25 mM ammonium acetate, pH 5.0 buffer (Note 3) to a final concentration of 10 mg/mL.

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2) 

 Add XEG to a final amount of 1 U/mL and incubate at room temperature for 1 h (see Note 4).

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3) 

 Add three volumes of absolute ethanol to precipitate XEG and the remaining unhydrolyzed polysaccharide and incubate at +4°C for at least 1 h (see Note 5).

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4) 

 Centrifuge the mixture at 1,000 × g for at least 10 min to remove the precipitated material.

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5) 

 Transfer the supernatant to a fresh tube and dry in air or on a heating block until ethanol is evaporated.

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6) 

 Dissolve the oligosaccharide preparation in ultrapure water, freeze and and lyophilize the solution to give fluffy oligosaccharide preparation that can be weighed or otherwise quantified (Note 6).

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7) 

 Dissolve the oligosaccharides in ultrapure water and store the solution at −20°C.

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2.

Preparation of MALDI-TOF Matrix

1) 

 Dissolve 2,5-dihydroxybenzoic acid (DHB) in methanol to a final concentration of 40 mg/mL by vigorous vortexing.

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2) 

 Add equal volume of ultrapure water and vortex thoroughly.

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3) 

 Centrifuge at 1,000 × g for at least 10 min to remove undissolved material.

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4) 

 Store the ready-to-use supernatant at room temperature protected from light (see Note 7).

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3.

MALDI-TOF MS Analysis of Oligosaccharides

1) 

 Mix 1 μL of matrix and 1 mg/mL oligosaccharide solutions and transfer to a MALDI sample plate (Note 8). Further information on XG analysis by MALDI-TOF MS can be found in Peña et al. (2012).

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2) 

 Air-dry the sample matrix drop. This usually takes 5 to 10 min.

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3) 

 Place the sample plate in the sample plate holder, transfer it into the instrument and wait for the vacuum to form.

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4) 

 Select appropriate instrument settings. These settings include the polarity (positive), detected m/z range (1000 to 1700), laser power (30%), number of summed laser shots (50). All parameters can be adjusted to maximize the MS data quality (Note 9).

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5) 

 Initiate laser firing and data acquisition. The focal point of the laser on the sample spot can be adjusted to find a “sweet spot” to obtain maximum quality MS data.

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Notes
  1. This protocol is applicable to a wide variety of polysaccharides (XG, xylans, mannans, pectins, and many others). MGX and wheat AX are available from Sigma-Aldrich and other sources. Further information on xylan analysis can be found in Kulkarni et al. (2012).
  2. It is imperative that the contaminants with molecular masses at the intended range of MALDI-TOF MS detection have been removed prior to enzymatic hydrolysis. Suitable methods include size-exclusion chromatography, dialysis, or ethanol precipitation (see Methods, Section 1.3).
  3. Ammonium acetate is a preferred buffer since it buffers well at pH 5.0, which coincides with the optimum pH of majority of hemicellulose hydrolyzing glycoside hydrolases. Also, ammonium acetate is a volatile buffer that generally doesn’t interfere with MALDI-TOF analysis at millimolar concentrations, and can be removed by freeze-drying, if necessary.
  4. Various xylan polymers can be hydrolyzed by Trichoderma viride M1 endoxylanase (Megazyme International Ireland, Wicklow, Ireland). The amount of enzyme (in terms of the enzyme units), incubation time and temperature can be adjusted for optimum hydrolysis.
  5. The suitable final ethanol concentration depends on the polymer. Typically, final concentrations of 75% should be used for XG, and 60% for xylans, such as MGX and AX. Ethanol can be precooled to 4°C or even lower temperature, to facilitate the precipitation. This step along with step 4 can be omitted if the polysaccharide preparation is very pure and no contaminating polymers are present. If the sample contains too much material, even outside the detected m/z range, MALDI-TOF MS signal quality drops due to suboptimal crystal formation.
  6. Even though ammonium acetate is volatile, several rounds of freeze-drying might be needed to remove it completely. A glassy pellet after freeze-drying indicates the presence of residual salt in the sample, whereas a fluffy sample is consistent with a pure oligosaccharide sample.
  7. The matrix solution is stable for months at room temperature if protected from light. Strong coloration appears with prolonged exposure to light.
  8. As little as few ng of single oligosaccharide can be detected, so sample concentrations lower than 1 mg/mL are suitable for analysis. However, this requires very pure sample without salt or polymer contaminants.
  9. The detected m/z range can be adjusted to coincide with the expected m/z range of the produced oligosaccharides. m/z range was set to 700–1600 for MGX and 650–4000 for AX. The laser power and number of summed laser shots should be adjusted separately for each sample spot. Matrix signals dominate the spectrum usually below m/z 500, so it is difficult to identify mono-, di- and trisaccharides using MALDI. Larger oligosaccharides typically ionize less efficiently than smaller ones, resulting in low signal intensity that sometimes prevents their detection. It is possible to compensate for this by increasing the laser power, but this can lead to signal saturation and increased peak widths, making accurate mass determination and quantification more difficult.
Initial amount

In favorable cases, MALDI-TOF MS allows the detection of as little as few ng of single oligosaccharide per sample spot.

Produced amount

Not applicable.

Discussion

In a full MS spectrum, each signal corresponds to ions with a particular m/z value, within limits of the mass resolution of the instrument. Composition of an oligosaccharide corresponding to a given signal can be deduced by adding the incremental masses of suspected residues, the mass of a water molecule (to account for atoms at the ends of the oligosaccharide) and the ionizing species (e.g., Na+) until their sum equals that of the signal m/z value. Incremental masses of some common residues in oligosaccharides are: 176 for hexuronic acids (for example glucuronic acid, GlcA), 162 for hexoses (glucose, Glc; and galactose, Gal), 146 for deoxyhexoses (fucose, Fuc), 132 for pentoses (xylose, Xyl; and arabinose, Ara), 42 for acetyl (Ac) group, and 14 for methyl (Me) group. Combinations of these structures can form larger residues, such as 4-O-methylglucuronic acid (MeGlcA) with a residue mass of 190. Further, the MeGlcA and other hexuronic acids can form a neutral salt by replacement of the carboxylic proton with a monovalent cation such as sodium (Na, atomic mass of 22) or potassium (K, atomic mass of 38) with a concomitant increase in the residue mass to 211 or 227, respectively. Detected ions in positive mode MALDI-TOF MS are mostly singly charged adducts, typically with either a Na+ or K+ cation. Mass accuracy of MALDI-TOF MS is usually sufficient to correlate the nominal measured and theoretical m/z values of an oligosaccharide, making assignment of the oligosaccharide’s residue composition straightforward. Mass calibration of MALDI-TOF MS instrument (performed with sample of known compositions, such as a series of malto-oligomers) is crucial to the data analysis since m/z values are the primary information from MS spectrum.

 

In Figure 1, some structural features of XG secreted by suspension cultured sycamore maple cells are probed. The general structure of XG consist of β(1,4)-D-Glcp residues forming the backbone, with nearly all backbone residues carrying a side chain. In the XG nomenclature (Fry et al., 1993), unbranched backbone residue is denoted by G. Side chain structures vary from the simplest α(1,6)-d-Xylp residue (denoted by X), to more complex ones, where the Xyl residue can be extended by β(1,2)-d-Galp residue (L), which can further be extended by α(1,2)-l-Fucp (F) (see Fig. 1 for the structures). The structures of these oligosaccharides have been unambiguously elucidated by NMR spectroscopy (Kiefer et al., 1989). Some of the Gal residues, especially in the F side chain, can be further acetylated at the 3, 4, and/or 6 positions. A small portion of Gal in the XXLG oligosaccharide (KEGG Glycan (Hashimoto et al., 2006) ID G00777) can be acetylated as well. There is little redundancy in the molecular masses of XG oligosaccharides, which usually makes structural assignments very straightforward, and unambiguous structures can be assigned to each signal in MALDI-TOF MS spectrum. The signal at m/z 1393 almost always corresponds to the pseudomolecular ion [XXFG + Na]+. The peaks in the triplet of signals at m/z 1393, 1435 and 1477 are separated by 42 mass units and correspond to various acetylated forms of XXFG (KEGG Glycan ID G00433). Inspection of the signal intensities reveals that XXFG (including all acetylation forms) is the most abundant oligosaccharide, and single acetylation is the most prevalent modification of XXFG.

 

In Figure 2, the pseudomolecular ion at m/z 891 from endoxylanase treatment of MGX corresponds to oligosaccharide with five pentosyl (P) and one methylhexuronosyl (M) residue ([P5M + Na]+). Contributions from the residues and adducts add up as follows: P residues contribute 5 × 132 = 660, M residue contributes 190, the reducing end contributes 18, and Na+ adduct contributes 23, totaling 891. The mass difference of 22 between the doublet of signals at m/z 891 and 913 indicate that the latter is a Na salt. The structure of MGX has been previously deduced to consist of a linear backbone of β(1,4)-D-Xylp residues, with α(1,2)-d-MeGlcpA side chains randomly distributed on the backbone. The full mass spectrum does not allow unambiguous sequencing of the isobaric oligosaccharides that may be present, but reveals that these oligosaccharides contain exactly six Xyl residues and one MeGlcA side chain.

 

In Figure 3, the pseudomolecular ion at m/z 701, in the spectrum of oligosaccharides prepared by endoxylanase treatment of wheat AX, corresponds to the Na+ adduct of oligosaccharide with five pentosyl (P) residues, [P5 + Na]+. The residue masses add up as follows: P residues contribute 5 × 132 = 660, reducing end contributes 18, and Na+ adduct contributes 23, totaling 701. Despite the multitude of signals, the spectrum is simple to interpret owing to the homogenous laddering of the signals, with equal spacing of 132 mass units. Due to the redundancy of the molecular masses of the pentoses (Xyl and Ara), full mass spectrum cannot give any sequence information without auxiliary information, such as the enzyme specificity, or sugar composition analysis. NMR spectroscopy has shown that the backbone consists of β(1,4)-d-Xylp residues, whereas the side-chains consist of α(1,2) and α(1,3)-L-Araf residues randomly distributed on the backbone.

Figure & Legends

Figure & Legends

 

 

Fig. 1. Positive mode MALDI-TOF spectrum of the oligosaccaharides generated by Aspergillus aculeatus XEG treatment of xyloglucan from the medium of suspension cultured sycamore maple cells.

 The major signals correspond to sodium adducts of oligosaccharides ([M + Na]+) and are labeled with their verified chemical structures (rendered using the Consortium for Functional Glycomics nomenclature), along with shorthand nomenclature (see Data Analysis), and measured m/z values. Each of these signals is accompanied by a significantly less intense signal at 16 mass units higher m/z value, corresponding to the potassium adduct ([M + K]+) of the oligosaccharide.

 

 

 

Fig. 2. Positive mode MALDI-TOF spectrum of the oligosaccharides generated by Trichoderma viride M1 endoxylanase treatment of 4-O-methylglucuronoxylan.

 The major signals labeled with most likely sugar composition and m/z value are from sodium adducts of oligosaccharides ([M + Na]+). Here, P and M denote pentose and MeGlcA residues, respectively, and the subscript indicates the number of residues. For example, signal labeled as P5M at m/z 891 corresponds to a sodium adduct of an oligosaccharide with five Xyl and two MeGlcA residues. The cartoon structure (rendered using the Consortium for Functional Glycomics nomenclature) depicts one possible structure corresponding to the P5M signal. The other major signals labeled only by their m/z values are from sodium salts of the corresponding oligosaccharides.

 

 

Fig. 3. Positive mode MALDI-TOF MS spectrum of the oligosaccharides generated by treating wheat arabinoxylan with Trichoderma viride M1 endoxylanase.

 All peaks correspond to sodium adducts of oligosaccharides ([M + Na]+) with various numbers of pentosyl (P) residues, as indicated by the subscript. The cartoon structure depicts one possible structure corresponding to the P5 signal.

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