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Keywords:

  • carbohydrates;
  • imaging;
  • mass spectrometry;
  • matrix-assisted laser desorption ionization (MALDI);
  • oligosaccharide;
  • stem;
  • wheat (Triticum aestivum)

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The pool of endogenous water-soluble oligosaccharides found in the stems of wheat (Triticum aestivum) is being investigated as a potential indicator of grain yield. Techniques such as liquid chromatography with mass spectrometry (LC-MS) can profile these analytes but provide no spatial information regarding their distribution in the wheat stem. The imaging matrix-assisted laser desorption ionization (MALDI) mass spectrometry technique has not been utilized for the analysis of oligosaccharides in plant systems previously.
  • • 
    Imaging MALDI mass spectrometry was used to analyse cross and longitudinal sections from the stems of Triticum aestivum.
  • • 
    A range of oligosaccharides up to Hex11 were observed. Water-soluble oligosaccharides were ionized as potassiated molecules, and found to be located in the stem pith that is retained predominantly around the inner stem wall.
  • • 
    Imaging MALDI analyses provided spatial information on endogenous oligosaccharides present in wheat stems. The technique was found to offer comparable sensitivities for oligosaccharide detection to those of our established LC-MS method, and has potential for broad application in studying the in situ localization of other compound types in plant material.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Wheat is the world's major crop in terms of food production, and bread wheat (Triticum aestivum) is the most widely cultivated variety of all of the Triticum species. The world's growing population is placing increasing demands on available agricultural land to produce greater yields from crops. Both traditional cross-breeding methods and genetic modification offer potential routes to increasing crop yields. It is known that the stems of wheat contain pith which varies in quantity between different cultivars and at different stages of the growth cycle. It is also known that stored in the pith is a pool of endogenous water-soluble carbohydrate metabolites, and these carbohydrates are translocated into plant grains (Wardlaw & Porter, 1967). It has further been suggested that some wheat genotypes that contain high concentrations of soluble carbohydrates in their stems may be able to deposit more carbohydrates into plant grains and significantly increase grain yield (Ford et al., 1979).

Both electrospray (Fenn & Yamashita, 1984) and matrix-assisted laser desorption ionization (MALDI) (Karas et al., 1987; Karas & Hillenkamp, 1988) are soft ionization techniques that have been successfully applied to the analysis of native carbohydrates (Harvey et al., 1996; Stahl et al., 1997). However, because of the low proton affinity of neutral and acidic native oligosaccharides, they do not ionize efficiently and often form cationized molecules (Reinhold et al., 1995). These yield a particularly low mass spectrometric response, especially when compared with, for example, peptides. We have utilized the electrospray technique in an on-line coupling of liquid chromatography with mass spectrometry (LC-MS) and then applied this method to the separation and analysis of the native water-soluble carbohydrates extracted from the pith of a range of wheat cultivars (Edmond et al., 2004). On-line LC-MS analysis of these analytes has provided valuable insights into the range, structure and relative quantities of water-soluble carbohydrates present in the stems of a range of wheat cultivars at anthesis and other growth stages. However, because of the nature of the extraction process (extraction into ethanol/water) (Kerepesi et al., 1996), information about the location of these carbohydrates within the stem tissue is lost. Therefore, in order to examine the spatial distribution of carbohydrates, we have investigated a technique known as imaging MALDI mass spectrometry, which was developed (Caprioli et al., 1997) and validated (Stoeckli et al., 1999) by Caproli's group, for the analysis of a range of water-soluble oligosaccharides found in the stems of wheat.

Imaging MALDI mass spectrometry

In ‘standard’ MALDI analyses the matrix is usually mixed with the analyte, and the matrix and analyte then cocrystallize, allowing dispersion of the analyte in the matrix. In imaging MALDI the tissue surface is instead coated with a homogenous thin layer of matrix. In this instance, the technique is carried out using a MALDI ionization source coupled with a quadrupole orthogonal time of flight mass spectrometer. Previously, and still most commonly, linear time of flight (ToF) mass analysers have been used (Caprioli et al., 1997); however, the orthogonal time of flight arrangement compensates well for the topographical effects of an uneven surface of the analyte tissue, which may compromise mass spectrometric resolution. In addition to the mass spectrometer, specific software is required that allows ion images of the surface to be reconstructed. The software is used to preselect an area of the MALDI target that contains the tissue. The laser is then rastered across the tissue surface in an ordered pattern acquiring a mass spectrum at each point analysed. After acquiring all the spectra, mass/charge (m/z) values may be selected to build up an ion image which depicts the distribution of ions within the tissue and produces spatially resolved data (Fig. 1). This technique has been used previously in many mammalian systems, particularly for the localization of drugs in tissue (Bunch et al., 2004a) and for the identification of diagnostic proteins in diseased tissue (Chaurand et al., 2003). A study has been carried out in a plant system, initially to look for primary metabolites, namely glucose-6-phosphate, in potato (Solanum tuberosum) tubers (Bunch et al., 2004b) and more recently to determine agrochemical compounds in soybean (Glycine max) plants (Mullen et al., 2005). Here we describe our further novel application of the imaging MALDI technique in a plant system to study a range of water-soluble oligosaccharides found in the stems of wheat.

image

Figure 1. Abstract representation of the imaging matrix-assisted laser desorption ionization (MALDI) mass spectrometry technique.

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Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Materials

‘Tissue-Tek’ optimum cutting temperature (OCT) compound was purchased from Sakura Finetek Europe B.V. (Newbury, UK). α-Cyano-4-hydroxycinnamic acid (CHCA) and formic acid (AR grade) were purchased from Sigma-Aldrich (Poole, UK). Methanol and water (high-performance liquid chromatography (HPLC) grade) were purchased from Fisher Scientific (Loughborough, UK). Stems of wheat (Triticum aestivum L.) cv. Spark were provided by Syngenta (Bracknell, UK) (growth conditions: grown under glass; 16°C day, 12°C night; 16 h daylight; relative humidity approx. 70–85%), having been harvested 22 days postanthesis.

Instrumentation

Wheat sections were cut using a Leica CM1900 cryostat (Nussloch, Germany). A Badger 150 suction-feed air brush (The Air Brush Company, Lancing, UK) was used in the application of matrix to the tissue surface. Mass spectrometric analyses were carried out on a hybrid quadrupole orthogonal time-of-flight (QqToF) Applied Biosystems API QSTAR mass spectrometer (Foster City, CA, USA). The ionization was via an orthogonal MALDI ion source with a nitrogen laser (337 nm); the laser has an elliptical spot size of 100 × 150 m and power range of 0–58.4 J. Instrument software was the oMALDI server data processor, version 4.0, including imaging option, in addition to analyst QSTM (Applied Biosystems, Foster City, CA, USA). Data were processed using biomap version 3.72. This is a software package developed by Novartis (Basel, Switzerland) for the display and manipulation of MALDI imaging data acquired from a variety of instrument platforms. It is available as a free download from http://maldi-msi.org/ (note: before processing, the data were converted to the required data format using a macro kindly supplied by Applied Biosystems/MDS Sciex).

Sectioning of tissue slices

The ear and sheath leaf were removed from the stems of wheat cv. Spark (harvested 22 days postanthesis) and then lyophilized. Cross and longitudinal sections, 50 m thick, were cut using a cryostat at −20°C. Some cross sections were cut manually using a razor blade.

Coating tissue with matrix

Three coats of a saturated matrix solution of CHCA in 50 : 50 (volume/volume (v/v)) MeOH:H2O plus 0.1% formic acid were sprayed over the stem tissue, using an air brush, before the tissue was mounted on the target. Double-sided adhesive tape was added to the MALDI target and pieces of stem tissue were pressed gently onto the tape for adherence and support. No matrix was applied to the adhesive tape.

Mass spectrometric analyses

Areas of the target where stem tissue was mounted were selected using the software. Each area was then imaged by rastering the nitrogen laser (intensity 52%) in a serpentine pattern over the tissue; the magnitudes of the x and y increments used in the ‘snake’ pattern were 0.2 mm and 0.2 mm, respectively, with a laser repetition rate of 20 Hz. Thus, 50 laser shots every 2.5 s per spot and 598 spots were acquired in total.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Both cross and longitudinal sections of a wheat stem, cv. Spark (harvested 22 days postanthesis), were analysed using MALDI-QqToF-MS; photographic examples of wheat stem tissue slices are presented in Fig. 2. Ion images of the stem section surface were constructed using the imaging software. It was noted that the relative intensities of the potassiated molecule signals from both the matrix and the carbohydrate were higher than those of the sodiated or protonated forms (data not shown). In light of this observation, the mass spectra from each sample (cross and longitudinal sections) were processed with the software so that all ions were normalized to the potassiated matrix peak.

image

Figure 2. Example photographs of (a) a longitudinal section and (b) a cross section through a piece of wheat (Triticum aestivum) stem before positioning in the matrix-assisted laser desorption ionization (MALDI) source of the mass spectrometer.

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In the preparation of the stem tissue for MALDI imaging, the sample was evenly coated with matrix before being mounted, and the mounting tape was not sprayed with matrix. Therefore, the selected ion image of m/z 228 (potassiated matrix molecule) (Fig. 3a) shows that the stem longitudinal section had complete matrix coverage, because the image displays the outline of the piece of stem tissue. The selected ion image of m/z 228 can therefore be considered as a representative image of the whole tissue surface with which to compare other selected ion images. All further constructed ion images were normalized to the matrix ion so that intensity variations across the surface were accounted for. Normalization to the matrix ion as an internal standard has been successfully applied to other analyses using the imaging MALDI technique (Bunch et al., 2004a; Mullen et al., 2005). (Note that in MALDI images each pixel corresponds to one laser firing position and in this case the relatively coarse raster pattern employed leads to the production of the relatively ‘pixelated’ images shown in Figs 3a–d and 5a–c.)

image

Figure 3. Matrix-assisted laser desorption ionization quadrupole orthogonal time of flight mass spectrometry (MALDI-qoToF-MS) extracted ion images of a longitudinal section of a stem from wheat (Triticum aestivum) cv. Spark, harvested 22 days postanthesis. (a) mass/charge (m/z) 228, [M + K]+ for α-cyano-4-hydroxycinnamic acid (matrix); (b) m/z 381, [M + K]+ for hexose disaccharide; (c) m/z 705, [M + K]+ for hexose tetrasaccharide; (d) m/z 1353, [M + K]+ for hexose octasaccharide. In (b–d), data are normalized to m/z 228 (matrix ion) and smoothed (nine points).

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image

Figure 5. Matrix-assisted laser desorption ionization quadrupole orthogonal time of flight mass spectrometry (MALDI-qoToF-MS) extracted ion images of a cross section of a stem from wheat (Triticum aestivum) cv. Spark, harvested 22 days postanthesis. (a) m/z 381, [M + K]+ for hexose disaccharide; (b) m/z 543, [M + K]+ for hexose trisaccharide; (c) m/z 705, [M + K]+ for hexose tetrasaccharide. In (a–c), data are normalized to m/z 228 (matrix ion) and smoothed (nine points).

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The hexose oligosaccharide molecules that were observed as sodiated ions on LC-MS analyses of these analytes following extraction from wheat stems (Fig. 4) were selected (as potassiated molecules in MALDI) to produce extracted ion images. It was observed that the oligosaccharides were predominantly located in the stem pith which, in most of the cross sections we cut, did not survive intact but was present around the inner stem wall; an example is shown in Fig. 5(a), for the potassiated hexose disaccharide at m/z 381. It was also observed that the ion intensity, in the selected ion images, decreased as the size of the oligosaccharide increased; for example, the potassiated hexose trisaccharide (m/z 543) and potassiated hexose tetrasaccharide (m/z 705) presented in Fig. 5(b) and (c), respectively, gave less intense images than did the disaccharide. This may suggest that the smaller oligosaccharides are present in greater quantities than the larger oligosaccharides.

image

Figure 4. Porous graphitized carbon liquid chromatography electrospray ion trap mass spectrometry summed mass spectra from the analysis of stem extract from wheat (Triticum aestivum) cv. Spark, harvested 22 days postanthesis. All ions annotated correspond to sodiated molecules and Hex11 is a doubly charged ion.

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Extracted ion images for potassiated hexose oligosaccharide molecules from the longitudinal section of stem were also plotted. Oligosaccharide ions were observed along the entire section of stem, thus showing that the pith, which in these sections was more or less intact, contains the carbohydrates; examples are presented in Fig. 3(b–d). For all tissue sections the mass spectra from the analysis were summed across the duration of the acquisition, and oligosaccharides up to Hex11 were observed (Fig. 6). For this cultivar at this growth stage, these data are consistent with those obtained using the LC-MS method developed in this laboratory (Fig. 4).

image

Figure 6. Matrix-assisted laser desorption ionization quadrupole orthogonal time of flight mass spectrometry (MALDI-qoToF-MS) summed mass spectra from the analysis of a cross section of a stem from wheat (Triticum aestivum) cv. Spark, harvested 22 days postanthesis. All ions annotated correspond to potassiated molecules.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We have successfully applied the imaging MALDI mass spectrometry technique to the novel analysis of the water-soluble oligosaccharides that are found in wheat stem tissue. We have shown that a range of oligosaccharides, up to Hex11, are ionized and that these oligosaccharides are present in the stem pith. We also noted that the relative intensity of the signals obtained for smaller oligosaccharides (Hex2 and Hex3) were greater than those for the larger oligosaccharides. By comparison of these data with LC-MS data for the same analytes after extraction, we have shown that the same range of oligosaccharides was observed using both techniques. Thus, the imaging MALDI mass spectrometry technique can be as sensitive as hyphenated techniques such as LC-MS. The sensitivity and ease of use of the imaging MALDI mass spectrometry approach therefore show that it has the potential for much broader application in plant research; many other classes of compound are equally amenable to in situ analysis using similar protocols. In conclusion, imaging MALDI mass spectrometry has been shown to be a useful technique if information about the spatial distribution of carbohydrate metabolites in plant tissue is required. Furthermore, the imaging MALDI technique demonstrates high potential for the in situ localization of other target molecules that are of research interest in the molecular plant sciences.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We gratefully acknowledge the studentship for SR, provided by Syngenta and the Engineering and Physical Sciences Research Council (EPSRC). JTO gratefully acknowledges funding from the Analytical Chemistry Trust Fund, the RSC Analytical Division and EPSRC. We are grateful to Susan Crosland at Syngenta for encouraging this collaboration. The authors would also like to acknowledge Meg Stark at the University of York for training in the use of the cryostat instrumentation. The authors gratefully acknowledge Professor John Snape at the John Innes Centre, Norwich for providing the seeds of Spark.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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