Ionization Mass Spectrometric

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Direct Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging of Cellulose and Hemicellulose in Populus Tissue Kyle Ann Lunsford,† Gary F. Peter,‡ and Richard A. Yost*,† † ‡

Department of Chemistry, University of Florida, Gainesville, Florida 32611, United States School of Forest Resources and Conservation, University of Florida, Gainesville, Florida 32611, United States ABSTRACT: Imaging applied toward lignocellulosic materials requires high molecular specificity to map specific compounds within intact tissue. Although secondary ionization mass spectrometry (SIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) with a single stage of MS have been used to image lignocellulosic biomass, the complexity of the plant tissue requires tandem MS, which limits the interpretation of simple MS. MALDI linear ion trap (LIT) tandem MS offers the high molecular specificity needed for lignocellulosic analyses. MALDI-LIT MS analyses of cellulose and xylan (hemicellulose) standards were performed to determine mass-to-charge ratios and fragmentation pathways for identification of these compounds in intact tissue. The MALDI-LIT-MS images of young Populus wood stem showed even distribution of both cellulose and hemicellulose ions; in contrast, the tandem MS images of cellulose and hemicellulose generated by plotting characteristic fragment ions resulted in drastically different images. This demonstrates that isobaric ions are present during MALDI-LIT-MS analyses of wood tissue and tandem MS is necessary to distinguish between isobaric species for selective imaging of carbohydrates in biomass.

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n recent years, alternative energy research has garnered significant attention, particularly research focused on biofuel produced from plant “waste” or lignocellulosic biomass.1 4 Unlike the simple sugars and starch of sugar cane and corn grain, the complex arrangement of cellulose, hemicellulose, and lignin in plants naturally resists enzymatic digestion and limits bioconversion of biomass into biofuel.1,5 In bioconversion methods, a pretreatment step is performed to overcome some of this natural recalcitrance by increasing accessibility of the cellulose to cellulase digestion, particularly at high enzyme loadings.1 Despite the success of current pretreatment methods, most are conducted with size-reduced materials to remove anatomical differences. Because of high energies (and cost) required for size-reduction, particularly for woody biomass, the use of larger fragments (e.g., wood chips) is preferred for commercial processes.2,3 However, because of the limitations of current analytical methods, the spatial changes in chemical composition in pretreated wood chips are not well characterized.4 The ability to map spatial changes in chemical compositions within wood tissue should provide valuable information that can improve the understanding of lignocellulosic bioconversion to biofuel.5 A variety of techniques are used to image lignocellulosic material, including optical microscopy with stains, fluorescence microscopy,6 magnetic resonance imaging (MRI),7 micro X-ray computed tomography (μX-ray CT),8 confocal Raman microscopy,9,10 and scanning electron and transmission electron microscopy. Although r 2011 American Chemical Society

these techniques provide structural information, their molecular selectivity is quite limited. One approach that helps to overcome this lack of molecular specificity is mass spectrometry, specifically, high-resolution mass spectrometry, as employed in the developing field of petroleomics,11 time-of-flight-secondary ionization mass spectrometry (TOF-SIMS)12 and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS).13 In addition to molecular selectivity, TOF-SIMS and MALDI-MS are capable of MS imaging experiments.12,14 16 Despite widespread applications reported for these techniques in animal studies,17 TOF-SIMS and MALDI-MS have only recently been used to analyze lignocellulosic compounds.18 24 Developing new MS methods will help to improve the characterization of lignocellulosic compounds SIMS analyzes secondary ions emitted from a surface, which are characteristic of compounds within a sample.12,16 TOF-SIMS images of Miscanthus giganteus have obtained spatial resolution down to 1 μm, the highest offered by MS imaging.22 Although TOF-SIMS imaging provides direct surface analysis and high spatial resolution, extensive analyte fragmentation during ionization limits the chemical specificity.17 Because many plant cell wall carbohydrates are complex and composed of closely related Received: May 27, 2011 Accepted: July 18, 2011 Published: July 18, 2011 6722

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Analytical Chemistry

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Table 1. Mass-to-Charge (m/z) Values of Cellulose and Hemicellulose Ions Observed in the MS (m/z 500 2000) Spectra of Microcrystalline Cellulose Standard and Birch Xylan Standardsa number of monomers n

[Glcn

cellulose ions H2O + Na]+ m/z

hemicellulose ions [Xyln H2O + Na]+ m/z

3

509

4

671

551

5

833

683

6

995

815

7 8

1157 1319

947 1079

9

1481

1211

10

1643

1243

11

1805

1375

12

1967

1507

13

1639

14

1771

15

1903

a

The positive ions were identified as sodiated, dehydrated, singly charged in the form [Glcn H2O + Na]+ and [Xyln H2O + Na]+ for cellulose and hemicellulose, respectively.

building blocks, e.g., glucose (C6H12O6), xylose (C5H10O5), glucuronic acid (C6H10O7), and methyl glucuronic acid (C7H12O7), SIMS ionization of different carbohydrates often results in nonspecific fragmentation, complicating interpretation. MALDI is a soft ionization method used to generate biomolecular ions for mass spectrometry25 and MALDI-TOF-MS has been applied toward lignocellulosic biomass analyses;22,24 however, the molecular specificity of TOF-MS is limited. Because of chemical similarities of the various sugars and monolignols that compose lignocellulosic tissue, ions from different analytes are observed at the same nominal mass-to-charge ratio (m/z) (isobaric ions). Tandem mass spectrometry (MS/MS) can overcome this difficulty by dissociating precursor ions and using fragmentation to distinguish between isobaric ions, thereby increasing confidence in ion identification.15,26 Herein, we report direct imaging of cellulose and hemicellulose in intact wood tissue using MALDI-LIT tandem MS.

’ EXPERIMENTAL SECTION Instrumentation and Data Analysis. All MS experiments were performed using a Thermo MALDI-LTQ XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a nitrogen laser (LTB Lasertechnik Berlin, 337 nm, 60 Hz) with an approximately 100 μm diameter laser spot size. The samples were analyzed at intermediate pressure (70 mTorr), and all spectra were recorded in positive ion mode. Mass spectra were analyzed using Qual Browser v.2.0.7 (Thermo Fisher Scientific, San Jose, CA), and MS images were generated with Image Quest v.1.0.1. Preparation of MALDI Matrix and Standards. Three matrixes were tested with cellulose and xylan standards, R-cyannohydroxycinnamic acid (CHCA), trihydroxyacetophenone (THAP), and dihydroxybenzoic acid (DHB). DHB yielded the highest analyte-to-background ion signal and was used for the remainder of the experiments. DHB (99% pure) was purchased from Acros

Organics (Geel, Belgium). Optimal analyte signal was obtained with a MALDI matrix composed of 25 mg/mL DHB dissolved in 0.05 mM aqueous sodium acetate (NaOAc). Microcrystalline cellulose (MCC) (∼20 μm) and Birchwood Xylan extract were purchased from Sigma-Aldrich (St. Louis, MO) and suspended in water (4 mg/mL) for MALDI-MS analyses. Standard Analyses. Standards were analyzed by pipetting 1 μL of 4 mg/mL standard suspension onto a 384-well stainless steel MALDI sample plate, immediately followed by 1 μL of prepared DHB matrix. A heat gun was used to increase the rate of solvent evaporation and consequently reduce crystal size. The MALDI sample plate was spiraled outward from the center of each sample well underneath the laser, and the laser was fired three times at each laser step using 35 μJ laser energy. An average of 50 scans of m/z 500 2000 was recorded for one mass spectrum. The laser energy was increased from the typical 35 μJ/pulse for single-stage MS experiments to 40 μJ/pulse for tandem MS experiments. The precursor ion was isolated with a 1.2 amu isolation width in the LIT (∼0.20 mTorr), and the instrumental parameter for collision induced dissociation (CID) energy was set from 80 100 (arbitrary units). Wood Tissue Analysis. Small wood blocks, 1 cm 1 cm 1 cm, were cut from a field-grown, three year-old stem of Populus deltoidies, and 50 μm thick radial sections were cut on a sliding microtome (Leica, SM2010R). In a subset of 50 μm sections, the lignin was removed with sodium hypochlorite27 to produce holocellulose tissue. For tissue imaging, the lower stem regions from 10 week-old greenhouse-grown Populus deltoidies X Populus trichocarpa X Populus deltoides hybrids were sectioned with a vibratome (Leica, VT100S) to obtain 50 μm thick transverse sections. After sectioning, the samples were washed in acetone and mounted on CryoJane tape (Instrumedics, Inc., Richmond, IL, no. 475214) for MS analysis. The MALDI matrix (DHB) was applied to the tissue with a Meinhard nebulizer; the tissue was sprayed for approximately 30 s, followed by a 4 min drying time aided with a warm air stream. This process was repeated (typically 10 times) until a white crystal layer was observed atop the wood tissue (∼8 mL of matrix solution sprayed). Little to no analyte migration was observed (when compared with fluorescence microscopy) using this method of MALDI matrix deposition with wood sections. MS and MS2 images were generated at a rate of 130 scans per minute by rastering the tissue beneath the laser in 50 μm step sizes, which was observed to provide optimal analyte ion signal. Three laser shots were used per spot with 35 and 40 μJ laser energy/pulse for MS and MS2 analyses, respectively. The MS2 experiments were performed on tissue sections using the same CID energy and isolation width as the standard experiments.

’ RESULTS AND DISCUSSION MS of Microcrystalline Cellulose and Hemicellulose Standards. Analyses of purified cellulose and birch xylan extract

showed the capability of MALDI-LIT-MS to characterize cellulosic materials as well as to determine analyte ion m/z values to monitor in wood tissue. MCC contains semicrystalline polymers of β-1,4 linked glucose monomers (Glc, C6H10O5). The neutral molecular weight of cellulose was determined by multiplying the mass of the glucose repeating unit, 162 Da, by the number of glucose monomers and adding 18 to account for the OH and H terminal groups. The mass spectrum (m/z 500 2000) of MCC displayed ions 162 Da (Glc molecular weight) apart and 6723

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Figure 1. Comparison of MALDI MS spectra of intact wood tissue (a) and holocellulose (b). Ions labeled were identified as singly charged, sodiated, dehydrated cellulose ions. Removing the lignin from the wood tissue removes much of the background ion signal (leaving behind the cellulose), illustrating that many of the ions observed in wood tissue are due to the ionization of lignin.

were identified as sodiated, dehydrated (singly charged) ions, differing by the number of glucose monomers, n, expressed as [Glcn H2O + Na]+, where n = 3 12. The m/z of MCC ions observed in the MS spectrum are reported in Table 1. Hemicellulose encompasses a variety of different compounds, but 4-O-methylglucronoxylans are the most abundant in wood tissue of angiosperm trees, such as birch and Populus. Xylans are composed of a linear β-1,4 linked xylose (Xyl, C5H8O4) polymer backbone substituted most commonly with glucuronic acid (GlcA) and 4-O-methylglucuronic acid (MeGlcA) sugars and acetyl groups. The neutral molecular weight of linear xylan polymers was determined by multiplying the mass of the xylose repeating unit, 132 Da, by the number of monomers and adding 18 to account for the OH and H terminal groups. In the case of substituted xylans, 176 and 190 Da are added for GlcA and MeGlcA, respectively. In the mass spectrum (m/z 500 2000) of purified Birch xylan extract, ions 132 Da (Xyl residue) apart were observed above the background ions. These ions were identified as sodiated, dehydrated (singly charged) ions of a linear xylan differing by the number of xylose residues, n, in the form [Xyln H2O + Na]+, where n = 4 15. The m/z values of the most intense xylan ions observed from m/z 500 2000 are reported in Table 1. In addition to the linear xylans, substituted xylans were observed and further analyzed with MS2 experiments. MS2 spectra of the substituted xylans showed a characteristic neutral loss (NL) of 190, which corresponds to the molecular weight of MeGlcA and successive NLs of 132, indicative of xylose residues (data not shown). These ions were identified as a MeGlcAsubstituted xylan and illustrated the ability of MS2 to differentiate between different xylan ions.

MS and MS2 of Intact Wood Tissue. Compared to the mass

spectrum (m/z 500 2000) of cellulose and xylan standards, the mass spectrum from Populus wood tissue coated with DHB matrix contains substantially more ions at nearly every m/z value. Many of these ions are hypothesized to result from the ionization of lignin. Lignin is a complex polymer of polyphenol compounds found primarily in secondary cell walls of gymnosperms, woody angiosperms, and grasses and is abundant in the secondary xylem (wood) of the Populus tissue section.28 To determine the lignin ion signal in intact tissue, lignin was removed from Populus wood sections using sodium hypochlorite extraction,27 leaving behind delignified wood or holocellulose (composed of cellulose and hemicelluloses), and the sections were analyzed with MALDILIT-MS. In comparison of the average spectra of 50 MS scans (m/z 500 2000) between untreated and delignified wood from serial sections coating with DHB (Figure 1), the total ion current (TIC) was reduced by more than 50% (from 3.08 108 to 1.26 108) and the intensity of the cellulose (analyte) ion signal remained comparable (2.84 105 compared to 2.55 105). This suggests that the ionization of lignin results in abundant background ion signal in wood tissue; thus, the signal-to-background can be improved by removing lignin from wood tissue. Despite the benefit of improved signal-to-background, removing lignin from wood tissue will alter the wood structure and/or leave behind chemical contaminants, compromising both spatial and chemical information. Instead, the intrinsically high lignin ion signal was effectively reduced by using tandem MS. Tandem MS decreases background signal by isolating ions of interest (removing the remainder of the background ions from the LIT) 6724



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Figure 2. MS2 spectra from MCC standard (a) and intact wood tissue (b) of m/z 1319, identified as [Glc7 H2O + Na]+. Both spectra show abundant fragment ions, arising from successive NLs of 162 resulting from cleavages along the glycosidic bonds. The wood tissue spectrum shows additional fragment ions at m/z 1301 and 1275, (NLs of 18 and 44) resulting from loss of water and an an acetyl group, respectively. This suggests at least two isobaric ions are observed from wood tissue at m/z 1319. Tandem MS decreases the background compound ion signal (compared with MS in Figure 1), improving the signal-to-background ratio without removing lignin.

Figure 3. MS2 spectra from Birch Xylan standard (a) and intact wood tissue (b) of m/z 1079, identified as a linear xylan, [Xyl7 H2O + Na]+. Both spectra show abundant fragment ions at m/z 1019 and 947, due to cross ring and glycosidic bond cleavages, respectively. The spectrum from wood tissue also shows abundant fragment ions at m/z 917 and 875 (NLs of 162 and 204), corresponding to a glucose and glucuronic acid residue, suggesting at least two isobaric species at the m/z value 1079. 6725



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Figure 4. Comparison of an average of 25 spectra averages in four different regions of wood tissue, inner pith, around the pith, secondary xylem, and secondary phloem. Ions above the background, consistent with m/z values of MCC, are observed in the spectra of the center pith and secondary xylem. Intense ions at nearly every m/z value are observed in the pith periphery and secondary phloem spectra. The differences in spectra are consistent with the composition of the regions: center pith and secondary xylem are composed of mostly dead cells, and the pith periphery and secondary phloem are composed of mostly living cells.

and increases the signal-to-background ion signal and selectivity of MALDI-LIT-MS imaging experiments. The m/z values of the ions 162 Da apart observed in the MS of the Populus stem were consistent with the ions observed in the MS analysis of the MCC standard, suggesting cellulose ions were observed from the MS analysis of wood tissue. MS2 analyses of the four most abundant ions observed from wood tissue, m/z 833, 995, 1157, and 1319 ([Glc4 7 H2O + Na]+), were compared to MS2 of the same ions of the MCC standard (data not shown). The MS2 spectra from both wood tissue and MCC standard displayed major fragments resulting from glycosidc bond cleavages (sequential neutral losses, NLs, 162), which confirmed cellulose was observed from wood tissue. The MS2 spectra of m/z 833, 995, and 1157 also displayed an NL of 154 (molecular weight of DHB), suggesting isobaric DHB cluster ions at the same nominal m/z value; thus, m/z 1319 was used for further analysis of cellulose in wood tissue. Figure 2 compares the MS2 of m/z 1319 of the MCC standard with the MS2 spectrum from untreated wood. As expected, both spectra show similar fragmentation, the major fragments (m/z 1157, 995, 833, 671, and 509) result from successive NLs of 162, due to glycosidic bond cleavages. Although the major fragment ions of MCC and untreated wood tissue are similar, m/z 1319 f 1301 and 1319 f 1275 (NLs of 18 and 44, respectively) are more abundant in the MS2 spectra from wood tissue. The NL of 18 is likely due to a nonspecific water loss since most ions with an OH group can lose water during collision-induced dissociation in the ion trap. The NL of 44 from m/z 1319 suggests the

presence of a acetyl or carboxylic acid functional group, which is common for carbohydrates other than cellulose present in wood tissue. The greater abundance of these fragments observed in the MS2 spectrum of untreated wood tissue strongly suggests the presence of at least two isobaric ions at m/z 1319. Figure 3 displays the MS2 spectra of m/z 1079 from Birch xylan extract and Populus wood tissue. In the xylan standard, sequential NLs of 132 are observed in the MS2 of m/z 1079, resulting from glycosidic bond cleavages between xylose monomers; thus, this ion was identified as [Xyl8 H2O + Na]+. The MS2 spectrum of m/z 1079 from Populus tissue shows m/z 1079 f 917 (NL of 162) and 1079 f 875 (NL of 204), in addition to m/z 1079 f 847 (NL of 132). Since these fragments are not observed in the standard, at least two isobaric ions are likely present at m/z 1079. Further stages of MS (MS3 and MS4) determined that, in addition to the linear xylan, another ion is present at m/z 1079 of the form [(162)4(204)2 + Na]+ and is preliminarily identified as an O-acetylgalactoglucomannan (another classification of hemicellulose). However, analyses of additional standards such as galactoglucomannan are needed to confirm this preliminary identification. MS Imaging of Young Populus Stem Tissue. MALDI-LITMS imaging of intact wood tissue was performed on a quarter section of the Populus stem to ensure all tissue types were examined. Optical, MS, and MS2 images of a transverse section of young Populus stem are displayed in Figures 5 and 6. The white dotted line in the optical images illustrates the region of MS analysis. The pith is located in the center of the stem and is 6726



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Figure 5. (a) Optical image of Populus wood tissue showing the pith (1), secondary xylem (2), vascular cambium (3), and secondary phloem (4). The white outline shows the area of MS and MS2 analyses. (b) MS image of m/z 1319 ([Glc7 H2O + Na]+) shows ion signal over the whole tissue. (c) The MS2 image of m/z 1319 f 1275 (NL of 44) displays more abundant ion signal in the region around the pith and the secondary phloem, which is consistent with tissue compositions. (d) MS2 image of m/z 1319 f 995, resulting from two NLs of 162, shows localization in the secondary xylem, closest to the pith, and reduced ion signal in the center of the pith, vascular cambium and secondary phloem.

composed of thin, nonlignified, primary-walled cells. The secondary xylem extends from the pith to the vascular cambium and is composed of living ray parenchyma as well as nonliving lignified fiber and vessel cells with thickened secondary-walls. The vascular cambium is a thin layer (∼50 μm) of living tissue between the secondary xylem and secondary phloem where new cells are produced for secondary, or radial, growth. The secondary phloem is composed of living, primary walled parenchyma and companion cells as well as dead sieve and fiber cells with thickened secondary walls. In contrast to the sieve cells, the fiber walls are lignified. The mass spectra (displayed in Figure 1) displays abundant ion signal at nearly every m/z value; however, different tissue regions displayed characteristic spectra, displayed in Figure 4. For example the periphery of the pith and the secondary phloem,

where most live tissue is found, had ions at every m/z that were more than 50% relative abundance compared with the secondary xylem and middle of the pith where more dead cells are found. On the other hand, abundant ions 162 Da apart (corresponding to ions observed in the MCC standard) were observed above the background ion signal in the secondary xylem and the center of the phloem, corresponding to the thickened secondary walls of the fiber cells. These general differences in the observed spectra were attributed to differences in chemical composition of the various tissues and cell types present in the young Populus stem. Although MS analyses are capable of discerning general differences in regions of wood tissue, tandem MS increases the molecular specificity of the experiment to analyze specific analytes in the different regions of wood tissues, as shown in Figures 5 and 6. 6727



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Figure 6. (a) Optical image of Populus wood tissue with the region imaged outlined in white. (b) MS image of m/z 1079 (identified as [Xyl7 H2O + Na]+) shows ion signal over the whole tissue section, with more intensity in the secondary phloem and around the pith. (c) MS2 image of m/z 1079 f 947 (NL of 132) displays increased ion intensity in the secondary phloem. (d) The MS2 image of m/z 1079 f 947 (NL of 162) displayed increased ion signal in the secondary xylem closest to the pith. Comparing the MS2 images of two different fragment ions illustrates that two isobaric compounds are present and that tandem MS can distinguish between them.

The extracted ion MS image of m/z 1319, identified with H2O + Na]+, displays standards as a cellulose ion [Glc7 uniform ion signal over all tissue regions of the Populus wood stem (Figure 5b), as expected because cellulose is present in the walls of all cells. Interestingly, the thin, primary walled cells of the pith and intact vascular cambium (arrow) have similar intensities as the secondary xylem and phloem tissues, even though they are known to contain relatively lower proportions of cellulose.28 In the MCC standard spectrum of MS2 of 1319 f ..., the most intense fragment ion observed was m/z 995 (Figure 2); thus, it was used to identify cellulose in wood tissue sections. The MS2 images of m/z 1319 f 1275 and of 1319 f 995, resulting from NL of 44 and two sequential NLs of 162 (Glc monomers) from the precursor ion, (m/z 1319) are displayed in parts c and d of Figure 5, respectively. The MS2 image of m/z 1319 f 1275

shows the greatest ion signal in the periphery of the pith and secondary phloem. In contrast, the localized ion signal intensity of the MS2 image of m/z 1319 f 995 is nearly the inverse, with greater signal in the center region of the pith, the secondary xylem, and small localized regions in the secondary phloem, likely corresponding to fiber bundles known to have greater proportions of cellulose than the vascular cambium and secondary phloem without fiber bundles, which display less abundant ion signal. Furthermore, the m/z 1319 f 995 ion signal is greater in the older xylem tissue closet to the pith, suggesting this region could have increased cellulose abundance relative to the younger xylem closer to the secondary phloem. However, the difference in cellulose ion signal variation due to inhomogeneous MALDI matrix crystal formation atop the wood tissue needs to be ruled out first. As previously discussed, the MS2 spectrum of m/z 1319 6728


Analytical Chemistry in Populus wood tissue (Figure 1b) suggests at least two isobaric ions at m/z 1319. This was confirmed by comparing the MS image of m/z 1319 with the drastically different MS2 images for m/z 1319 f 1247 and m/z 1319 f 995. Comparison of m/z 1079 (standard analysis identified as a linear xylan ion, [Xyl8 H2O + Na]+) MS with the MS2 images also exemplifies the necessity of tandem MS for wood tissue analyses. Specifically, tandem MS is needed to distinguish between different hemicellulose ions or other interfering ions at the same nominal m/z. The extracted MS image of m/z 1079 shows nearly even ion signal over the entire tissue section (Figure 6b). In contrast, MS2 images of two fragment ions of m/z 1079, m/z 947 (NL of 132, 5-carbon sugar), and m/z 917 (NL of 162, 6-carbon sugar) have different ion intensities in different tissues and regions. The MS2 image of m/z 1079 f 947 (Figure 6c) shows higher ion signal localized in the secondary xylem closest to the pith (similar to the cellulose ion intensity), consistent with the hypothesis that this region contains thicker cell walls. The m/z 1079 f 947 ion signal is also observed in the secondary phloem and vascular cambium, which is different than the m/z 1319 f 995 cellulose ion signal. Although no glucuronoxylan is located in the vascular cambium, this region is thinner than the spatial resolution of the imaging experiment. The MS2 image of m/z 1079 f 917 (Figure 6d) shows localized ion intensity in the region of the secondary xylem closest to the pith, but less intense ion signal is observed in the secondary phloem compared to the MS2 image of m/z 1079 f 947. Moreover less signal is observed around the pith, which further demonstrates the need for tandem MS to obtain accurate spatial distributions of ions at a single m/z. Although a complete chemical composition analysis of all the regions of wood tissue was not attempted, these experiments show the viability of MALDI-MSn imaging of lignocellulosic tissue. More specifically, MS alone is incapable of providing accurate spatial distributions of different ions at a single m/z. Instead, the necessity and advantages of tandem MS analyses of wood tissue are evident after comparing MS2 images with the MS images. Specifically, characteristic fragment ions resulting from collision-induced dissociation of a precursor ion are needed to differentiate between isobaric ions, which are inherent when analyzing carbohydrate compositions in complex tissue such as wood. The MS2 images provided more selectivity for the cellulose and hemicellulose ion signals, differentiate between analytes and interfering ions at a nominal m/z value, as well as significantly reduce background ion signal compared to the MS spectrum.

’ CONCLUSIONS Successful direct analysis of cellulose and hemicellulose in Populus tissue using MALDI-tandem MS is reported here. Imaging of multiple fragment ions from MS2 experiments resulted in different ion signal localization; thus, tandem MS is necessary for separating isobaric species for accurately annotating wood tissue MS images. In addition to elucidating isobaric species, tandem MS effectively reduces the background ion signal of wood tissue, improving the signal-to-background of imaging experiments. The combination of MS2 and imaging provides specific chemical mapping in intact wood tissue; this MALDILIT-tandem MS method can be used to image other intact lignocellulosic tissues, such as switch grass or sugar cane. Imaging specific compounds within cellulosic tissue will provide further

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insight into their spatial distribution changes throughout pretreatment for the conversion to ethanol. Future experiments will focus on comparing MALDI-LIT-MS images to complementary techniques to improve the understanding of chemical changes that occur during the pretreatment process. Furthermore, future experiments will focus on developing a MALDI-LIT-MS technique to characterize lignin in wood tissue.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]fl.edu.

’ ACKNOWLEDGMENT The research was funded by an award from the Department of Energy Grant ER64499-1030816-0013824. Graduate research was supported by the University of Florida Alumni Fellowship from the University of Florida Foundation. The authors thank Jianxing Zhang for preparing the holocellulose. ’ REFERENCES (1) Ragauskas, A. J.; Williams, C. K.; Davison, B. H.; Britovsek, G.; Cairney, J.; Eckert, C. A.; Frederick, W. J.; Hallett, J. P.; Leak, D. J.; Liotta, C. L.; Mielenz, J. R.; Murphy, R.; Templer, R.; Tschaplinski, T. Science 2006, 311, 484–489. (2) Lynd, L. R.; Wyman, C. E.; Gerngross, T. U. Biotechnol. Prog. 1999, 15, 777–793. (3) Wooley, R.; Ruth, M.; Glassner, D.; Sheehan, J. Biotechnol. Prog. 1999, 15, 794–803. (4) Wyman, C. E.; Dale, B. E.; Elander, R. T.; Holtzapple, M.; Ladisch, M. R.; Lee, Y. Y. Bioresour. Technol. 2005, 96, 1959–1966. (5) McCann, M. C.; Carpita, N. C. Curr. Opin. Plant Biol. 2008, 11, 314–320. (6) Ding, S.; Xu, Q.; Ali, M. K.; Baker, J. O.; Bayer, E. A.; Barak, Y.; Lamed, R.; Sugiyama, J.; Rumbles, G.; Himmel, M. E. BioTechniques 2006, 41, 435–443. (7) Ishida, N.; Koizumi, M.; Kano, H. Ann. Bot. 2000, 86, 259–278. (8) Bulcke, J. v. d.; Masschaele, B.; Dierick, M.; Acker, J. v.; Stevens, M.; Hoorebeke, L. v. Int. Biodeterior. Biodegrad. 2008, 61, 278–286. (9) Agarwal, U. P. Planta 2006, 224, 1141–1153. (10) Gierlinger, N.; Schwanninger, M. Plant Physiol. 2006, 140, 1246–1254. (11) Marshall, A. G.; Rodgers, R. P. Acc. Chem. Res. 2004, 37, 53–59. (12) Sodhi, R. N. S. Analyst 2004, 129, 483–487. (13) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. (14) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751–4760. (15) Garrett, T. J.; Prieto-Conaway, M.; Kovtoun, V.; Bui, H.; Izgarian, N.; Stafford, G.; Yost, R. A. Int. J. Mass Spectrom. 2007, 260, 166–176. (16) Belu, A. M.; Graham, D. J.; Castner, D. G. Biomaterials 2003, 24, 3635–3653. (17) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom. Rev. 2007, 26, 606–643. (18) Cha, S. W.; Song, Z. H.; Nikolau, B. J.; Yeung, E. S. Anal. Chem. 2009, 81, 2991–3000. (19) Jung, S.; Foston, M.; Sullards, M. C.; Ragauskas, A. J. Energy Fuels 2010, 24, 1347–1357. (20) Jung, S.; Chen, Y.; Sullards, M. C.; Ragauskas, A. J. Rapid Commun. Mass Spectrom. 2010, 24, 3230–3236. (21) Tokareva, E. N.; Fardim, P.; Pranovich, A. V.; Fagerholm, H. P.; Daniel, G.; Holmbom, B. Appl. Surf. Sci. 2007, 253, 7569–7577. 6729



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(22) Li, Z.; Chu, L.; Sweedler, J. V.; Bohn, P. W. Anal. Chem. 2010, 82, 2608–2611. (23) Zaia, J. Mass Spectrom. Rev. 2004, 23, 161–227. (24) Li, Z.; Bohn, P. W.; Sweedler, J. V. Bioresour. Technol. 2010, 101, 5578–5585. (25) Karas, M.; Bahr, U.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1989, 92, 231–242. (26) Landgraf, R. R.; Conaway, M. C. P.; Garrett, T. J.; Stacpoole, P. W.; Yost, R. A. Anal. Chem. 2009, 81, 8488–8495. (27) Yokoyama, T.; Kadla, J. F.; Chang, H. M. J. Agric. Food Chem. 2002, 50, 1040–1044. (28) Evert, R. F.; Esau, K. Esau’s Plant Anatomy Meristems, Cells, and Tissues of the Plant Body: Their Structure, Function, and Development; John Wiley & Sons, Inc.: Hoboken, NJ, 2006.

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