Hemicelluloses: Science and Technology - American Chemical Society

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Chapter 6

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Characterization of Hemicelluloses from Wood Employing Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Olof B. Dahlman, Anna Jacobs, and Maria Nordstrom Swedish Pulp and Paper Research Institute, STFI, Box 5604, S-114 86, Stockholm, Sweden

This chapter describes some analytical procedures employing Matrix-Assisted-Laser-Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry for the characterization of hemicelluloses. The molar mass parameters for glucuronoxylans, hexenuronoxylans and glucomannans were determined by using an analytical procedure involving size exclusion chromatography followed by off-line MALDI-MS analysis. In the case of O-acetylated glucuronoxylans and glucomannans, the degree of substitution with acetyl moieties was determined on the basis of the MALDI spectra obtained prior to and following deacetylation. By using this MALDI­ -MS procedure O-acetyl residues were found to be present in hardwood glucomannans. The distribution of 4-O-methyl­ -glucuronic acid residues along the polysaccharide chains of hardwood and softwood xylans was studied employing MALDI-MS analysis. In the case of softwood xylans, the 4-O­ -methylglucuronic acid residues were distributed regularly within the polysaccharide. In contrast, the corresponding uronic acid residues in hardwood xylans were found to be distributed irregularly along the polysaccharide chains.

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© 2004 American Chemical Society In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

81 In 1996 we described (1) the successful application of Matrix-Assisted-LaserDesorption/Ionization Time-Of-Flight Mass Spectrometry ( M A L D I - T O F - M S ) for characterizing hemicelluloses isolated from wood and pulps. Since then this approach has been improved considerably (2,3) and several different procedures for wood and pulp constituents have been reported (3-8). In the present article, a selection of our previously reported analytical procedures are discussed together with some hitherto unpublished results from our laboratory.

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M A L D I is a soft ionization and desorption technique that was first described in 1988 by Karas and Hillenkamp (9,10). Among other applications, they employed this new technique for characterizing biomacromolecules (e.g., proteins and polysaccharides) and synthetic polymers (10,11). The major advantages associated with the use of M A L D I - M S include a high level of sensitivity; applicability to molecules exhibiting a wide range of masses; little or no fragmentation of the molecules being analyzed and the rapidity with which results can be obtained. Interpretation of data is facilitated by the fact that almost only single-charged ions are generated. This fact also implies that the mass to charge ratios of the ions formed in connection with M A L D I analysis are directly related to the absolute molar masses of the molecules present. The present article discusses the analytical information that can be obtained by M A L D I - M S analysis of different types of non-acetylated and O-acetylated hemicelluloses derived from wood and pulp. Furthermore, a procedure for analyzing the molar mass parameters for hemicelluloses, utilizing size exclusion chromatography (SEC) followed by M A L D I - M S analysis, is discussed.

MALDI-MS analysis of glucomannans The glucomannans are linear polysaccharides composed of P-(l-4)-linked glucopyranosyl and mannopyranosyl residues. Several kinds of glucomannans, which might contain P-(l-6)-linked galactopyranosyl residues, have been isolated from both soft- and hardwoods. Glucomannans from softwood have galactose and O-acetyl groups linked to the mannose residues and a glucose-tomannose ratio of 1:3 to 1:4. The M A L D I - M S spectrum of a low molar mass fraction of an alkali-treated glucomannan from spruce wood is depicted in Figure 1. This spectrum contains a distribution of well-resolved signals separated by 162 mass units (i.e., the mass of a hexose residue). These peaks originate from different oligomers with varying chain length. From the mass (i.e., the m/z ratio) associated with its M A L D I - M S signal, the number of hexose residues present in each oligomer can

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Figure 1. MALDI-MS spectrum (positive-ion mode) of a partly depolymerized alkali-treated spruce glucomannan demonstrating a distribution of wellresolved signals corresponding to oligo- and polysaccharide chains of increasing lengths. be calculated. In Figure 1, the mass peaks corresponding to hexoseg and hexose are indicated. The precise sugar composition of each oligomer cannot be derived since the constituent sugars, i.e., glucose, mannose and galactose residues, all have same molar mass (i.e., 162). However, the absolute molar mass, the molar mass distribution and the degree of polymerization (DP) for this glucomannan sample are obtained with a high accuracy from the M A L D I - M S spectrum.

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Glucomannan present in softwood contains O-acetyl substituents at certain of the mannose residues. In Figure 2 (upper spectrum), the M A L D I - M S spectrum of a partly depolymerized O-acetyl-glucomannan, obtained by hydrothermal treatment of spruce wood, is depicted (7). This spectrum demonstrates a number of partially overlapping series of signals separated by 42 mass units (i.e., the mass of an acetyl group). These signals originate from Oacetyl-glucomannans with different numbers of hexose residues and 0-acetyl groups. The number of hexose residues and 0-acetyl groups in each oligomer can be calculated from the m/z ratio of its M A L D I - M S peak. The mass peaks corresponding to hexose -Ac and hexosei -Ac are indicated in the spectrum. As expected for softwood glucomannan, the signal pattern indicates an irregular distribution of the acetyl groups along the polysaccharide chains. After alkaline hydrolysis, the M A L D I - M S spectrum (lower spectrum, Figure 2), contained only a single series of sharp peaks originating from the sodiated molecular ions, [MNa] , of glucomannan polysaccharide chains. The average degree of substitution (DS-0.3) with acetyl substituents of the O-acetyl-glucomannan was determined from the difference in the peak-average mass, M p , values obtained by M A L D I M S prior to and following removal of the O-acetyl substituents. 14

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83 Hardwoods contain small amounts of glucomannans with a glucose-tomannose ratio 1:1 to 1:2. Galactose substituents can also be present in hardwood glucomannans. We recently reported (7) the M A L D I - M S identification of 0acetyl moieties present in glucomannan isolated from aspen wood. The M A L D I M S spectra of O-acetyl-glucomannans from aspen and birch demonstrate similar pattern of signals as that of the O-acetyl-glucomannan from spruce. Figure 3 (upper spectrum), depicts the M A L D I - M S spectrum of the 0-acetylglucomannan isolated from birch by extraction with water. The signals in this spectrum, assigned to O-acetylated oligosaccharides with increasing numbers of hexose residues, indicate an irregular distribution of the acetyl groups along the polysaccharide. The assignment was supported by the M A L D I - M S spectrum of the glucomannan following alkaline deacetylation (lower spectrum, Figure 3). This spectrum demonstrates signals corresponding to the molecular ions, [M+Na] , of glucomannans with chain lengths ranging up to 18 hexose residues. Also in this case, the degree of substitution with acetyl moieties (DS-0.3) was determined from the M A L D I - M S analyses prior to and following deacetylation. +

MALDI-MS analysis of glucuronoxylans The glucuronoxylans are composed of linear chains of p-(l->4) xylopyranosyl residues branched with o c - ( l - » 2 ) - l i n k e d 4-0-methylglucuronopyranosyl residues (4-0-MeGlcA). The 4-0-MeGlcA-to-xylose ratio is -1:6 for softwood and -1:10 for hardwood xylans. 0-acetyl-(4-0-methylglucurono)-xylan is the major hemicellulose present in hardwood, whereas arabino-(4-0-methylglucurono)xylan is present in softwood. During acidic sulfite cooking of softwood, the (3-(l->3) arabinofuranosyl side-groups are cleaved off from the backbone while some of the 4-O-methylglucuronic acid residues remain linked to the xylan chains. The M A L D I - M S spectrum of a 4-0-methyl-glucuronoxylan, isolated from a softwood sulfite pulp, is depicted in Figure 4 (3,12). The spectrum contains a broad range of wellresolved peaks, which originate from different oligomers with varying sugar compositions. The composition of each oligomer can be derived from the mass associated with its M A L D I - M S signal (i.e., the m/z ratio), since xylose and 4-0M e G l c A residues have different masses (i.e., 132 and 190, respectively). This M A L D I - M S spectrum is evidently composed of two major, partially overlapping series of mass peaks, in which the distance between adjacent peaks in each series corresponds to 132 mass units (the mass of a xylose residue). The first series contains oligosaccharides with an odd number of 4-0-MeGlcA residues linked to the xylan chains (denoted o in Figure 4). In contrast, the second series contains oligosaccharides containing an even number of 4-0M e G l c A moieties as substituents on the backbone (denoted x in Figure 4).

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Figure 2. MALDI-MS spectrum (positive-ion mode) of an O-acetylglucomannan (upper spectrum) isolated from hydrothermally treated spruce wood. Spectral analysis of the same spruce glucomannan following alkaline hydrolysis (lower spectrum).

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In the case of 4-0-methyl-glucuronoxylan from hardwood, which, in addition to 4-0-MeGlcA residues, has different numbers of 0-acetyl groups linked to the polysaccharide backbone, the differences in molar mass between different oligosaccharides chains were often too small to be resolved by the M A L D I instrument at our laboratory. The M A L D I - M S spectrum of a partly depolymerized xylan isolated from hydrothermally treated aspen wood, is depicted in Figure 5 (upper spectrum) (7). This spectrum contains a distribution of more-or-less poorly resolved mass peaks, but it is possible to observe several signals separated by 42 mass units (i.e., the mass of an acetyl moiety). Hence, these mass peaks originate from 0-acetyl-(4-0-methylglucurono)xylan oligosaccharides with different D P values and numbers of 0-acetyl substituents. After deacetylation by alkaline hydrolysis, the M A L D I - M S spectrum of the aspen xylan (Figure 5, lower spectrum) revealed that this xylan contained acidic xylooligosaccharides with one or two 4-0-MeGlcA substituents (DP 10-28). Thus, the molar mass parameters (i.e., average molar masses) and the degree of polymerization and the degree of acetyl substitution of this xylan could be conveniently determined by this M A L D I - M S analysis procedure. In a recent study (6), the distributions of 4-0-MeGlcA residues along the xylan chains of softwood and hardwood xylans were characterized by employing partial acid hydrolysis and subsequent M A L D I - M S analysis of the oligo­ saccharides obtained. The negative-ion M A L D I - M S spectrum of the oligosaccharides obtained from a softwood xylan (spruce) after such hydrolysis is presented in Figure 6. The contents of xylose and 4-0-MeGlcA in the acidic oligosaccharides present could be calculated without ambiguity on the basis of their molar masses determined by M A L D I - M S . Thus, the signals in the spectrum originated from oligo- and polysaccharides containing increasing numbers of xylose residues and from one up to five 4-0-MeGlcA substituents. It is evident from this spectrum (Figure 6) that each series of uronic acidcontaining xylosaccharides exhibits a quite limited range of sizes. Thus, the peak patterns revealed by M A L D I - M S analysis indicate that the 4-0-MeGlcA substituents are distributed periodically along the backbones of the original softwood xylan. If the 4-0-MeGlcA residues were randomly distributed instead, the distributions of xylosaccharides containing 4-0-MeGlcA substituents would have been considerably broader (as in the case for hardwood xylan in Figure 7). The positive-ion M A L D I mass spectrum obtained for the oligo- and polysaccharides obtained from a hardwood (birch) xylan after mild acid hydrolysis is shown in Figure 7 (upper spectrum). This spectrum consists of several partially over-lapping series of peaks, which were identified on the basis of their exact molar masses as corresponding to neutral xylosaccharides with DP up to 30 and acidic xylosaccharides with one or two 4-0-MeGlcA substituents.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Figure 4. MALDI-MS spectrum (negative-ion mode) of a 4-O-methylglucuronoxylan from a sulfite pulp (spruce). The mass peaks correspond to xylooligosaccharides of increasing lengths containing an odd (o) or even (x) number of 4-O-methylglucuronic acid residues. Reproduced from Reference 3. Copyright 2001 American Chemical Society.

A = 42 (O-Acetyl substituent) O-Acetyl-4-O-methylglucuronoxylan

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Figure 5. MALDI-MS analysis of O-acetyl-4-O-methyglucuronoxylan obtained from hydrothermally treated aspen (upper spectrum) showing series of weak signals (A) separated by 42 mass units (i.e., the mass of an acetyl substituent). Following deacetylation (lower spectrum) acidic xylooligosaccharides with one or two 4-O-metylglucuronic acid residues are detected.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Figure 6. MALDI mass spectrum (negative-ion mode) of the oligosaccharides obtained from a spruce xylan after mild acid hydrolysis. Reproduced from Reference 6. Copyright 2001 American Chemical Society.

Positive-ion MALDI (Neutral and acidic polysaccharides)

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Figure 7. Positive- (upper) and negative- (lower) ion MALDI mass spectra of the oligosaccharides obtained from birch wood xylan by mild acid hydrolysis. In positive ion mode, both neutral and acidic oligosaccharides are detected, whereas acidic oligosaccharides only are detected by negative-ion MALDI-MS. Reproduced from Reference 6. Copyright 2001 American Chemical Society.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

88 The lower spectrum in Figure 7 depicts the corresponding negative-ion M A L D I - M S for the oligosaccharides in the hydrolysate obtained from the birch wood xylan. In M A L D I - M S , only the oligo- and polysaccharides containing acidic groups generate negative ions. Consequently, this spectrum contains two broad series of peaks corresponding to acidic xylosaccharides of increasing chain-length and with one or two 4-0-MeGlcA residues. Altogether, these M A L D I spectra indicate that the 4-O-methylglucuronic acid residues are distributed irregularly along the backbones of hardwood xylans.

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MALDI-MS analysis of hexenuronoxylans Under alkaline cooking conditions some of the 4-O-MeGlcA residues present in hardwood and softwood xylans are converted into unsaturated hexenuronic acid residues (HexA) by P-elimination of methanol. In an early study (4) employing M A L D I - M S , we were able to identify a number of different HexA and 4-0M e G l c A containing xylooligosaccharides in the hydrolysate obtained after endoxylanase treatment of an unbleached hardwood kraft pulp.

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Figure 8. MALDI mass spectrum (negative-ion mode) of the acidic xylooligosaccharides obtained by endoxylanase treatment of a hexenuronoxylan isolated from alkaline peroxide bleached hardwood kraft pulp. The spectrum consists of two series ofpeaks assigned to the molecular ions [M-H]' of xylooligosaccharides with varying chain lengths and containing one hexenuronic acid (HexA) residue or one 4-0-methylglucuronic acid (4-0MeGlcA) residue.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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89 Figure 8 depicts the negative-ion M A L D I - M S spectrum of a partly depolymerized enzymatically (endoxylanase) treated hexenuronoxylan, originating from an oxygen delignified and alkaline peroxide bleached hardwood kraft pulp. This M A L D I - M S spectrum demonstrates a broad distribution of mass peaks, which originates from HexA and 4-O-MeGlcA containing xylo­ oligosaccharides. The composition of each oligosaccharide can be derived from its M A L D I - M S peak, since xylose, HexA and 4-O-MeGlcA residues all have different molar masses (i.e., 132, 158 and 190, respectively). The M A L D I - M S spectrum is evidently composed of two partially overlapping series of signals, in which the distance between adjacent signals in each series corresponds to one xylose residue. The first series originates from oligosaccharides with one HexA residue linked to the xylan backbone. The second series corresponds to oligosaccharides containing one 4-0-MeGlcA residue on the backbone. Furthermore, in the high mass part of the spectrum several oligosaccharides containing both HexA and 4-O-MeGlcA residues, can be detected.

SEC/MALDI-MS analysis of hemicelluloses It has been reported that in the case of mixtures of polymers exhibiting a wide range of molar masses, M A L D I - M S may yield somewhat inaccurate values for the average molar mass, due to discrimination of signal from the larger polymers (8). However, this problem can be minimized or eliminated simply by separating the polymer mixture into fractions each containing components within a relatively narrow size range by S E C prior to M A L D I - M S analysis (so-called S E C / M A L D I - M S ) (3,7,8). Figure 9 depicts a schematic representation of the analytical procedure developed by us to characterize the absolute molar mass parameters of hemi­ celluloses derived from wood and pulp. In this procedure, S E C separates the hemicellulose into fractions each containing components with a narrow range of molar masses and the accurate peak-average molar mass (Mp) of each fraction is subsequently determined by M A L D I - M S . The molar mass parameters for the hemicelluloses are then calculated on basis of the S E C distribution curve and utilizing the M A L D I - M S calibrated mass scale. In the case of water-soluble 0-acetylated xylans and glucomannans, an ammonium acetate solution with a p H of 7 was employed as eluent in connection with the S E C separation step (7). However, for alkali extracted xylans and glucomannans, a more alkaline (pH 13) sodium hydroxide/acetate eluent was required (3). In all cases pretreatment of the S E C fractions by passage through a cation-exchange resin prior to M A L D I - M S analysis and/or the use of M A L D I probes coated with a Nafion film (2), were necessary in order to minimize the disturbance by buffer ions during the M A L D I analysis step.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Retention time Calibration of SEC using MALDI-MS Mp values 1*234567

Fractionation of the sample by S E C

MALDI-MS analysis of SEC fractions

Figure 9. Schematic representation of the analytical procedure employed to characterize the molar mass parameters for hemicelluloses. SEC separates the hemicellulose into fractions with a narrow range of molar masses, the absolute molar mass (Mp) of each fraction is determined by MALDI-MS and, finally, the molar mass parameters for the entire hemicellulose are calculated from the SEC distribution curve using the MALDI-MS calibrated mass scale.

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Figure 10. SEC chromatogram and MALDI-MS spectra of three sequential fractions (a, b and c; shaded) obtained with an arabino-4-O-methylglucuronoxylanfrom spruce wood. Reproduced from Reference 3. Copyright 2001 American Chemical Society.

In Hemicelluloses: Science and Technology; Gatenholm, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2003.

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Dissolving pulp xylan DP = 24 '

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Absolute Molar Mass Figure 11. The SEC molar mass distribution curves for arabino-4-O-methylglucuronoxylan (from spruce holocellulose), arabinohexenuronoxylan (kraft pulp) and two different 4-O-methylglucuronoxylans (sulfite and dissolving pulp) obtained by the SEC/MALDI-MS procedure. Each xylan was analyzed separately employing the alkaline (pH 13) buffer SEC system and subsequently graphed on the same MALDI-MS calibrated absolute molar mass scale. Figure 10 illustrates fractionation of an arabino-4-O-methylglucuronoxylan by S E C and the subsequent M A L D I - M S analysis of three of the fractions obtained. These fractions gave rise to relatively symmetric distributions of M A L D I signals up to a m/z value of at least 35 000. However, no signals originating from individual polysaccharide constituents can be resolved in this case. These M A L D I - M S spectra are nonetheless of sufficiently high quality to provide reliable values for the peak-average molar mass (Mp). Subsequently, the logarithms of the M p values determined by M A L D I - M S were used together with the S E C distribution curve to obtain the average molar mass values and the polydispersity index for this xylan from spruce (3). The S E C distribution curves and the degree of polymerization (DP) values for four different softwood xylans, determined by this S E C / M A L D I - M S procedure with the sodium hydroxide/acetate (pH 13) eluent system are documented in Figure 11. As can be seen from this figure, the arabino-4-0methylglucuronoxylan extracted from the spruce holocellulose demonstrates a higher DP than does the arabinohexenuronoxylan extracted from the softwood kraft pulp. The 4-