A Comparative Study of Matrix- and Nano-assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry of Isolated and Synthetic Lignin

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1 Research Article Received: 8 April 2011; Revised: 29 June 2011; Accepted: 29 June 2011 Published online in Wiley Online Library: 7 September 2011 (wileyonlinelibrary.com) DOI /pca.1350 A Comparative Study of Matrix- and Nano-assisted Laser Desorption/Ionisation Time-of-Flight Mass Spectrometry of Isolated and Synthetic Lignin Koichi Yoshioka, Daisuke Ando and Takashi Watanabe* ABSTRACT: Introduction Lignin is the second most abundant biopolymer next to cellulose. However, because of the complexity of the heterogeneous macromolecules, it is difficult to elucidate the polymeric structures of lignin by conventional analytical methods. Objective To obtain the detailed structures of lignin, we comparatively applied nano-assisted laser desorption/ionisation time-of-flight mass spectrometry (NALDI-TOF MS) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Methodology Synthetic lignin from coniferyl alcohol and an isolated lignin from Pinus densiflora were subjected to NALDIand MALDI-TOF MS. Results We first obtained NALDI-TOF MS of synthetic and isolated lignin. Mass increments of 178 and 196Da were observed in NALDI- and MALDI-TOF mass spectra of the synthetic and isolated lignin. The mass intervals indicated that radical coupling forming β-o-4 bonds is the major pathway. Peaks in the low molecular mass region between m/z 500 and 800 were observed more extensively using NALDI-TOF MS than MALDI-TOF MS, which enabled detailed analysis of the interunit linkages in lignin. Conclusion Owing to the ionisation profile differentiation from MALDI-TOF MS, NALDI-TOF MS is useful for the structural analysis of lignin. Copyright 2011 John Wiley & Sons, Ltd. Keywords: NALDI-TOF MS; MALDI-TOF MS; lignin; biomass; dehydrogenation polymer 248 Introduction Lignin, the second most abundant biopolymer next to cellulose, has been receiving increased interest as a resource for biofuels and biomass-based chemicals. To expand the role of lignin from an energy source in the pulp and paper industry to a feedstock for lignocellulosic biorefineries, an understanding of the molecular structure of lignin is essential. Lignin is a heterogeneous aromatic polymer biosynthesised by oxidative radical coupling of monolignols, p-hydroxycinnamyl alcohol, coniferyl alcohol and sinapyl alcohol (see Fig. 1). Thus far, a number of studies have reported on the chemical and spectroscopic analyses of lignin, but knowledge of the macromolecular structure of lignin remains limited. Two-dimensional NMR (Fukagawa et al., 1991; Ralph et al., 2004) and chemical degradation are powerful methods to analyse the primary structure of lignin, but these analyses give limited information on the networks of phenylpropanoid units. Lignin has been described as a three-dimensional polymer, consisting of phenylpropanoid interunits mainly linked by aryl and alkyl ether bonds, but physicochemical studies have suggested that lignin is not a highly cross-linked three-dimensional polymer but a linear chain-based polymer with a limited number of branching points (Goring, 1989). Mass spectrometry of lignin provides important information on the molecular architecture of this biopolymer, but available mass spectrometric data remains limited. With regard to the mass spectrometry of lignin, pyrolysis GC-MS or GC-MS have been applied after chemical degradation (Reale et al., 2004). In these methods, lignin is degraded into small fragments, which constitute a fingerprint of the starting sample and give some structural information on the phenylpropane structure. In recent years, electrospray ionisation mass spectrometry (ESI-MS; Angelis et al., 1999; Evtuguin et al., 1999; Evtuguin and Amado, 2003; Önnerud et al., 2003; Morreel et al., 2004, 2010a, b; Reale et al., 2010) and secondary ion mass spectrometry (SIMS) combined with TOF have been applied to the structural analyses of lignin (Saito et al., 2006). These methods may expand mass spectrometry to the analysis of non-volatile phenylpropanoids and surface lignin molecules on plant tissues (Saito et al., 2006). However, it is difficult to obtain direct information on the polymeric structure of lignin with these methods. * Correspondence to: T. Watanabe, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto , Japan. twatanab@rish.kyoto-u.ac.jp Laboratory of Biomass Conversion Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto , Japan Phytochem. Anal. 2011, 23, Copyright 2011 John Wiley & Sons, Ltd.

2 NALDI- and MALDI-TOF MS of Lignin buffer (ph 6.4). A solution containing 2.5mg of peroxidase (130 U/mg) in 10mL of 20mM sodium phosphate buffer (ph6.4) was mixed with solutions A and B at a rate of 0.6mL/min, and the solutions were stirred for 24h. The reaction mixture was then centrifuged. We washed the precipitate with H 2 O. After being lyophilised, the residue was dissolved in a solution of 1,2-dichloroethane and ethanol (2:1, v/v) and then filtered. The supernatant (2mg/mL) was used for the mass spectrometric analysis. Figure 1. Structures of monolignnols: (a) p-hydroxycinnamyl alcohol, (b) coniferyl alcohol and (c) sinapyl alcohol. Preparation of milled wood lignin Milled wood lignin (MWL) was prepared from Pinus densiflora as described (Björkman, 1956; Lundquist and Simonson, 1975). We dissolved MWL in 1,2-dichloroethane and ethanol (2:1, v/v) (10mg/mL) and used it for the mass spectrometric analysis. Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) (Karas and Hilllenkamp, 1988) yields direct structural information in a wide range of molecular mass regions. However, reports on the structural analysis of lignin by MALDI-TOF MS are still limited because of the low efficiency of ionisation (Metzger et al., 1992; Srzić et al., 1995; Bocchini et al., 1996; Angelis et al., 1996). Surface-assisted laser desorption/ionisation (SALDI) is a matrixfree mass spectrometric approach that utilises the unique properties of a nano-structured surface to promote desorption and ionisation (Sunner et al., 1995). Desorption/ionisation on silicon mass spectrometry (DIOS-MS), a matrix-less laser vaporisation method for generating gas-phase ions, has been receiving considerable attention. The physical properties of the silicon surfaces are crucial to DIOS performance and are controlled by the selection of the silicon type and the silicon etching conditions (Wei et al., 1999). Recently, a new matrix-free method, named nano-assisted laser desorption/ionisation time-of-flight mass spectrometry (NALDI-TOF MS), using a nanostructured silicon-based target plate with an LDI-TOF instrument, was reported (Guenin et al., 2009; Shener et al., 2009). The matrix-free approaches are attractive when ionising lignin without interference from the matrix. In this paper, we comparably applied NALDI-TOF MS and MALDI- TOF MS to the study of synthetic (Freudenberg, 1956, 1965) and isolated (Björkman, 1956; Lundquist and Simonson, 1975) lignin. To our knowledge, this is the first report on the NALDI-TOF MS of lignin. Experimental Materials We purchased peroxidase from horseradish from Toyobo Co. Ltd (Osaka, Japan). 2,5-Dihydroxybenzoic acid (DHB) and peptide calibration standard were obtained from Bruker Daltonics (Bremen, Germany). Reagents and solvents were of analytical grade and purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan) and Nacalai Tesque Inc. (Kyoto, Japan). Preparation of dehydrogenation polymer Dehydrogenation polymer (DHP) was synthesised by polymerisation of coniferyl alcohol with peroxidase by gradually supplying monomers to the site of polymerisation (Zutropfverfahren DHP). The following two solutions were prepared. For solution A, we dissolved 50mg (0.278 mmol) of coniferyl alcohol in 500mL of acetone and added the solution to 10mL of 20mM sodium phosphate buffer (ph6.4). For solution B, we added 100mL of 30% H 2 O 2 to 10mL of 20mM sodium phosphate MALDI- and NALDI-TOF MS Mass spectra were acquired on an Autoflex III mass spectrometer (Bruker Daltonics, Bremen, Germany) using a Smartbeam TM laser as the ionisation source. The acceleration voltage was 20 and 19kV in the linear and reflector modes, respectively. For all spectra, we accumulated shots in the positive mode. The detector gating was Da in linear mode, and the detector deflection was Da in reflector mode. 2,5-Dihydroxybenzoic acid (10mg/mL in 40% aq. acetonitrile (v/v) containing 0.1% trifluoroacetic acid (TFA)) was used as a matrix. A peptide calibration standard or polyethylene glycol was used as the external standard. A typical sample preparation for MALDI was as follows. We dropped 0.5mL of a matrix solution to the target. After the target was air dried at room temperature, 0.5mL of the sample solution was then dropped and dried. Finally, 0.5mL of a matrix solution was dropped and air dried. We measured NALDI-TOF MS using the same instruments and conditions as those for the MALDI-MS measurements. A NALDI TM target (Bruker Daltonics, Bremen, Germany) was used. We directly placed 1mL of the sample solution (10mg/mL) on the target and subjected it to the measurement. Results and Discussion We examined the matrices, DHB, sinapinic acid and a-cyano-4- hydroxycinnamic acid for the analysis of MALDI-TOF MS and selected DHB as the best matrix as described in a previous study. Because the matrix was immiscible with the sample solution, the sample was sandwiched between matrix layers. Through this sample loading method, crystals suitable for the measurements of MALDI-TOF MS were formed. In the measurement of NALDI- TOF MS, the same sample solution was distributed onto a NALDI TM target and air dried. Synthetic lignin (DHP) was prepared by polymerisation of coniferyl alcohol with horseradish peroxidase (HRP), according to the Zutropfverfahren method, which favours end-wise polymerisation. This polymerisation system produces larger numbers of β O 4 bonds and more significantly represses condensation reactions such as β β coupling than do those prepared by introducing coniferyl alcohol all at once into the dehydropolymerising enzyme solution (Zulaufverfahren DHP). In the linear and reflector modes of MALDI-TOF MS, we observed mass peaks of Zutropfverfahren DHP mainly at intervals of 178Da and 196Da, which correspond to the mass number of the monomeric unit of DHP and its H 2 O adduct. The DHP from coniferyl alcohol polymerised with peroxidase yielded mass increments of 178Da through the radical intermediate (Angelis et al., 1996). Because the polymerisation proceeds by oxidative radical couplings, a hydrogen atom is removed from a lignin monomer. 249 Phytochem. Anal. 2011, 23, Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca

3 K. Yoshioka et al. Figure 2. Formation of lignin substructures by dehydrogenation of coniferyl alcohol via radical intermediates. For example, a dimer produced by oxidative radical coupling of coniferyl alcohols has a molecular weight of (see Fig. 2). The radical mechanism gives a mass increment of 178.2Da corresponding to the production of β β and β 5 bonds. Furthermore, a mass increment of 196Da indicated the existence of a β O 4 bond, which was produced via the quinone methide intermediate followed by the addition of a H 2 O molecule. 250 Figure 3. MALDI-TOF mass spectra of synthetic lignin obtained by dehydrogenative polymerisation of coniferyl alcohol: (a) linear-positive ion mode and (b) reflector-positive ion mode. wileyonlinelibrary.com/journal/pca Copyright 2011 John Wiley & Sons, Ltd. Phytochem. Anal. 2011, 23,

4 NALDI- and MALDI-TOF MS of Lignin Figure 4. NALDI-TOF mass spectra of synthetic lignin obtained by dehydrogenative polymerisation of coniferyl alcohol: (a) linear-positive ion mode and (b) reflector-positive ion mode. On the other hand, the ionic mechanism, driven by the addition of a coniferyl alcohol or its oligomers to a quinone methide intermediate, gives a mass increment of Da. The results of MALDI-TOF MS indicated that DHP was mainly formed by the radical coupling mechanism (see Fig. 3). The radical coupling of monolignol has been reported as a principal mode of lignin polymerisation in plant tissues (Boerjan et al., 2003; Ralph et al., 2004). In the enzymatic synthesis of Zutropfverfahren DHP, the Figure 5. Mass spectra of isolated lignin obtained from Pinus densiflora (linear-positive ion mode). Measurement by (a) MALDI-TOF MS and (b) NALDI- TOF MS. 251 Phytochem. Anal. 2011, 23, Copyright 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pca

5 K. Yoshioka et al. 252 polymerisation proceeds through a radical dehydrogenation process rather than an ionic mechanism (Angelis et al., 1996). Two-dimensional heteronuclear single quantum coherence (HSQC) correlation NMR spectra of DHP from coniferin showed that a O 4 was considerably minor (Terashima et al., 2009). In the ionic mechanism, nucleophilic addition of phenols to a quinone methide forms benzyl aryl ether bonds in lignin, and the addition of hydroxyl or carboxylic groups of cell wall polysaccharides to the quinone methide forms benzyl ether and ester bonds between lignin and carbohydrates in lignin carbohydrate complexes (LCCs; Koshijima and Watanabe, 2003). In the present research, we first applied NALDI-TOF MS to isolated and synthetic lignin. In NALDI-TOF MS of DHP, we clearly observed increments between peaks of 178 and 196Da (Fig. 4). Intensities of the peaks in a high molecular mass range were smaller than those of MALDI-TOF MS, but the spectral pattern was similar. Figure 5 shows MALDI- and NALDI-TOF mass spectra of MWL from Pinus densiflora. In preliminary experiments, mass spectra were measured at concentrations of 1mg/mL (data not shown), but the low concentration was insufficient for the NALDI-TOF MS experiments. Therefore, MALDI- and NALDI-TOF mass spectra of MWL were measured at 10mg/mL throughout this study. We detected mass peaks at intervals of 178 and 196Da in both the MALDI- and NALDI-TOF mass spectra of MWL. Close similarities were observed between the spectra of MWL from P. densiflora and DHP from coniferyl alcohol. In a comparison between the MALDI- and NALDI-TOF mass spectra, distinct differences were found in low molecular mass regions between m/z 500 and 800. Intensive peaks were found in NALDI-TOF mass spectra, which enabled detailed analysis of mass increase due to addition of monolignol units (Fig. 5). Thus we found an initial advantage for using NALDI-TOF MS in the structural analysis of lignin. We have provided mass spectral evidence that radical coupling forming β O 4 bonds is the major pathway in polymerisation of DHP and softwood lignin. To interpret more detailed polymeric structures of lignin including minor branching structures such as benzyl aryl ether (a O 4), diaryl ether (4 O 5), biphenyl (5 5) and dibenzodioxocin (5 5 O 4), application of NALDI and MALDI ultrahigh resolution multiple-stage mass spectrometry (MS n ) would be useful. Research is in progress to analyse lignins from various plant origins, aiming at elucidating differences in the linkage structures of the natural polymer. CONCLUSIONS MALDI is an important technique for mass spectrometric analysis of compounds with a wide range of molecular mass ranges, but its applications to lignin is still limited due to its low efficiency of ionisation. In the present paper, we first applied NALDI-TOF MS to synthetic and isolated lignin, and found peaks with molecular mass range between m/z 500 and 800 were more intensively observed by NALDI than those by MALDI-TOF MS. The ionisation profile of NALDI expands the potential of mass spectrometry for detailed analysis of interunit linkages of lignin. NALDI-TOF MS is attractive for the structural analysis of lignin owing to the unique ionisation profile and noninterference from matrices. Acknowledgements A part of this work was financially supported by the New Energy and Industrial Technology Development Organization (NEDO). 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