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JXB Advance Access originally published online on September 27, 2006
Journal of Experimental Botany 2007 58(4):757-763; doi:10.1093/jxb/erl139
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© The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Approaches to Cellular Imaging

Imaging Matrix Assisted Laser Desorption Ionization Mass Spectrometry: a technique to map plant metabolites within tissues at high spatial resolution

MM Burrell1,*, CJ Earnshaw2 and MR Clench2

1Department of Animal and Plant Sciences, Sheffield University, Western Bank, Sheffield S10 2TN, UK
2Biomedical Research Centre, Sheffield Hallam University, Howard Street, Sheffield S1 1WB, UK

* To whom correspondence should be addressed. E-mail: m.burrell{at}sheffield.ac.uk

Received 17 May 2006; Accepted 2 August 2006


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results and discussion
 Conclusions
 Supplementary data
 References
 
Imaging Matrix Assisted Laser Desorption Ionization Mass Spectrometry provides a new and powerful tool to analyse the distribution of metabolites within plant tissues. The two matrices {alpha}-cyano-4-hydroxycinnamic acid ({alpha}-CHCA) and 9-aminoacridine provide a useful combination that allows the measurement of amino acids, sugars, and phosphorylated metabolites. Results are presented showing that representatives of these metabolites are unevenly distributed in wheat seeds at different stages of development and under temperature stress.

Key words: 9-aminoacridine, CHCA, I-MALDI, mass spectrometry, metabolites, phosphorylated metabolites, plants, sugars, wheat


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results and discussion
 Conclusions
 Supplementary data
 References
 
The aim of the work reported in this paper was to develop a method which would allow the cellular distribution of metabolites in plant tissues to be determined. To understand the relationships between cells in a tissue, and how metabolism is controlled in cells, and between cells, it is necessary to determine the amounts of metabolites present in the cell and the amounts of enzyme present (Fell, 1997). Many recent studies with techniques such as immunolocalization and in situ hybridization have shown that cells that are, based on anatomy, apparently similar in an apparently homogeneous tissue, are in fact metabolically different. For example, the galactinol synthase promoter from melon directs expression in only some of the companion cells of minor veins but not all (Haritatos et al., 2000). In developing maize kernels, sucrose metabolizing enzymes and those involved in starch synthesis are not evenly distributed (Wittich and Vreugdenhil, 1998). Thus there is a clear need for a technique that will demonstrate the amounts of metabolites at the cellular level. However, the task of identifying plant metabolites is huge since there are in excess of 100 000 metabolites that can occur (Oksman-Caldentey and Inzé, 2004; Weckwerth and Fiehn, 2002).

Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) offers potential as an approach to solving this problem. MALDI is a very versatile technique since it will ionize compounds of a wide range of chemical classes (Karas et al., 1987). One of the difficulties with MALDI is the selection of the chemical matrix required for the ionization of many compounds. Clearly, to obtain good ionization the matrix has to absorb strongly at the wavelength of the laser and have good vacuum stability. At present, the choice of matrix for a particular tissue preparation and metabolite spectrum is rather empirical (Kinumi et al., 2000). The most widely used matrices tend either to be cinnamic acid derivitives such as {alpha}-cyano-4-hydroxycinnamic acid ({alpha}-CHCA) or aromatic compounds such 2,5-dihydroxybenzoic acid (2,5-DHB). The latter has been shown to be particularly useful for oligosaccharides from plant tissues. One of the disadvantages of these organic matrices is the number of mass peaks in the low molecular weight range. This has led some researchers to examine inorganic matrices such as titanium oxide (Kinumi et al., 2000).

Imaging MALDI is a derivation of this technique where the sample on the MALDI stage is moved on the x/y axis and each sample position is assayed (Bunch et al., 2004). Therefore, by recording the mass spectrum at each position a two-dimensional image of the metabolic profile of a sample may be obtained. The diameter of the laser in modern mass spectrometers is in the order of 100 µm or less. Thus at a spatial resolution of 100 µm it is possible to obtain a mass profile of those compounds present on the surface of a tissue. With modern high resolution mass spectrometers and careful selection of the matrix, MALDI has the potential to allow analysis of metabolites and avoid too many interfering peaks. In recent imaging MALDI (I-MALDI) experiments {alpha}-CHCA has found favour as the matrix of choice (Bunch et al., 2003), but many compounds of primary metabolism do not ionize well with this matrix. In this paper, the development and application of a method involving either {alpha}-CHCA or 9-aminoacridine is reported.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results and discussion
 Conclusions
 Supplementary data
 References
 
Wheat (Triticum aestivum L. var. Axona) plants were grown in a growth room under a day/night regime of 20/16 °C or at 25/20 °C, a daylength of 16 h and light intensity of 400 µE. The plants were grown in Levingtons M3 compost supplemented with Osmacote.

Developing wheat ears were tagged when the anthers appeared and developing seed harvested at the specified time post-anthesis. The developing seed was flash frozen in liquid nitrogen. Seed was embedded in ice and then cryosectioned with a Leica CM1900 with the sample at –10 °C and the knife at –20 °C. Sections were attached to double-sided adhesive carbon tape and dried in a freeze drier. Once dried the sections were coated using an airspray with either a solution of 25 mg ml–1 {alpha}-CHCA in methanol containing 0.1% (v/v) trifluoracetic acid or a solution of 10 mg ml–1 9-aminoacridine dissolved in acetone containing 0.1% TFA.

MALDI-MS spectra were acquired with an Applied Biosystems/MDS Sciex hybrid quadrapole time-of-flight (Q-Star Pulsar-i), fitted with an orthogonal MALDI ion source and an Nd:YAG laser. The instrument conditions were repetition rate: 1000 Hz, laser energy for CHCA –20% (2.3 µJ), acridine 25% (2.8 µJ) and analysis time of 5 s per position. At a resolution of 100 µm it takes approximately 6 h to complete an imaging run of one wheat section. After analysis the masses of interest are normalized against the appropriate matrix peak before the intensity is plotted.


   Results and discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
Conclusions
 Supplementary data
 References
 
A key step in any analytical system is to determine the relationship between signal strength and the concentration of the analyte compound. Dilution curves for arginine, phenylalanine and asparagine were therefore prepared as shown in Fig. 1 which would reflect the concentrations that occur in vivo (Burrell, 1981). Back calculation indicates that amino acids present in tissues in submicromolar concentrations could be detected with ease. There is a good relationship between concentration and intensity. Analysis of variance of the slope of curves fitted to the points gave F values of 0.4538 and 0.0004 indicating that the curves have the same profile. The ionization pattern reflects the basicity of the amino acid as indicated by capillary electrophoresis (Soga and Heiger, 2000).


Figure 1
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  		Fig. 1. Calibration curve. One µl of sample was mixed with 1 µl of {alpha}CHCA solution and 1 µl spotted onto the MALDI target plate. Each spot was replicated three times. Results are plotted as the mean ±sem after the intensity had been normalized to the intensity of the {alpha}-CHCA-H+ ion. Where error bars are not visible the error is less than the size of the symbol. Phenylalanine, open squares; asparagine, open triangles; arginine, open circles.

 
The usefulness of {alpha}-CHCA to examine the distribution of metabolites in sections of immature wheat seeds was examined next. An example for part of the mass spectrum detected between m/z 230 and 500 from one 100 µm laser ionized spot is shown in Fig. 2. Most metabolic intermediates of interest have masses between 50 and 700 which is easily in the mass range for the Q-star. Over this range there will be several hundred masses detected above background (data not shown). Images from the section in Fig. 3a for m/z 175 and m/z 381 are shown in Fig. 3b and c, respectively. The two plots are obtained from the same scan. The mass of 175 corresponds to arginine and that for 381.0799 corresponds to the potassium adduct of sucrose. Clearly, the localization of these two compounds are quite different, with arginine being concentrated more in the region where the vascular tissue enters the grain and in the region of the developing embryo. Unfortunately, the m/z value for the H+ ion of arginine is close to several other potential ions. It may be positively identified by using an MS/MS experiment (Fig. 4) on the same section. There is a good match between the standard and the sample indicating that the majority of the 175 mass is arginine. Similarly, in the examination of sucrose distribution, there is an intense ion matrix ion at m/z 379.0925. The second isotope peak of this component at 381.0925 could potentially interfere with this analysis. However, in this case the high resolution of the hybrid quadrupole time of flight mass spectrometer used in these experiments allows the peak at 381.0799 to be designated as sucrose from its accurate mass and, as for the example of arginine, this can be confirmed by an MS/MS experiment.


Figure 2
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  		Fig. 2. Part of mass spectrum from one laser spot. Part of the mass spectrum is plotted for one x/y position of the target.

 

Figure 3
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  		Fig. 3. Distribution of arginine and sucrose in a crysosection of 10 dpa developing wheat grain. (a) Section of wheat grain imaged. (b). The distribution of arginine m/z 175.1195 plotted as intensity after normalization against any variations in matrix intensity. The darker the colour the more signal. (c) Corresponding image for sucrose, m/z 381.0799.

 

Figure 4
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  		Fig. 4. MS/MS analysis of mass 175. An MS/MS analysis of the mass 175 was done for the scan shown in Fig 3. The mass spectrum (b) is compared with a standard (a) of arginine applied onto a spot target.

 
It was not possible to get strong signals for metabolites such as sucrose and glucose-6-phosphate in negative ion mode with {alpha}-CHCA-coated sections. Therefore wheat sections were coated with 9-aminoacridine. Figure 5 shows the distribution (as the H+) of sucrose product and glucose-6-phosphate for an older seed (16 dpa) than shown above. Further images of other masses detected with aminoacridine are provided as supplementary information (see supplementary data at JXB online). These masses have been selected on the basis that they were tentatively identified as metabolites by Edwards and Kennedy (2005). The sensitivity for sucrose and glucose-6-phosphate is different. The images (Fig. 5) have only been normalized against the matrix peak and thus cannot be compared in terms of absolute concentration of metabolite. Within the section, sucrose shows a greater variation in concentration than glucose-6-phosphate. In this older seed it is interesting to note that the relative distribution of sucrose and glucose-6-phosphate is different. One of the advantages of using a quadrapole time of flight instrument is the ability to distinguish some isomers. The fragmentation pattern of glucose-1-phosphate and glucose-6-phosphate are different (Fig. 6) thus allowing the separation of these two compounds during analysis.


Figure 5
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  		Fig. 5. MS analysis of glucose-6-phosphate and sucrose in 15 dpa developing wheat seeds. (a) Glucose 6-phosphate, (b) sucrose. Intensity is plotted from blue to red with red being the greatest signal.

 

Figure 6
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  		Fig. 6. MS/MS analysis of hexose phosphates. Fragmentation pattern for glucose-1-phosphate (a) and glucose-6-phosphate (b) obtained by MS/MS analysis in negative ion mode from a mixture of the two compounds.

 
To determine the usefulness of this approach for metabolite mapping, the distribution of sucrose was compared when the seed develops at different temperatures (Fig. 7). There is good reproducibility between two developing seed at the lower temperature. Sucrose is much more evenly distributed throughout the developing seed at the higher temperature.


Figure 7
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  		Fig. 7. Comparison of the distribution of sucrose in wheat seed developing at different temperatures. Seed was harvested at 9 dpa when grown at 20 °C or 8 dpa when grown at 25 °C. Sucrose (m/z 381) is plotted for the 25 °C seed (a) and the 20 °C seed (b, c). The darker the colour the more signal.

 

   Conclusions
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
Supplementary data
 References
 
The sensitivity of MALDI allows the detection of metabolites at the micromolar concentrations present in plant tissues, and the distribution may be correlated with the anatomy of the tissue or organ studied. In the work presented here, cryosections were used but where it is difficult to obtain good sections of material it is possible to blot the freshly cut surfaces of plant material directly onto precoated cellulose membranes. For metabolites that are fairly stable this approach is more rapid and can offer improved sensitivity. This procedure was used to good effect with potato tuber material.

A common problem in mass spectrometric analysis of plant tissues is assigning a mass to a metabolite. Plants contain over 100 000 metabolites (Fiehn et al., 2000; Harborne et al., 1999) and there is no knowledge of how many will occur in one cell. Edwards and Kennedy (2005) used MALDI tentatively to identify 105 metabolites in mammalian tissue by assigning mass to a metabolite, but acknowledged that this approach was not sufficient in itself. In plant tissues with the large number of possible metabolites this approach is unlikely to be sufficiently robust. In a typical MALDI image of a plant section hundreds of masses can be identified above the background signal. There is no simple solution to allocation of a mass to a specific metabolite other than detailed MS/MS work or the use of NMR when there is sufficient sample. One of the advantages of using MALDI is that it is quite tolerant of the salt concentrations found in living tissues. However, this does mean that to determine the distribution of, for example, sucrose, one has first to determine whether it is being detected as the protonated ion or as alkali metal (Na or K) adduct ions. In our case the only detectable ion with {alpha}-CHCA in positive mode is the potassium adduct. This is a simpler case than when using electrospray-MS on an extract where all three are present.

Several workers have developed methods of mapping metabolites by enzyme linked assay. The most sensitive method for this is where the assay can be linked to a fluorophore (Borisjuk et al., 1998, 2002; Walenta et al., 2002). Such methods offer sensitivities of less than 1 µg g–1 FW. A detailed comparison of sensitivities of fluorescence versus I-MALDI has not been done, but the methods certainly offer comparable sensitivities and, at the present stage of development, where it is possible to use a fluorometric-based assay for a specific metabolite it may be more sensitive than I-MALDI. However, MALDI offers an analytical method for many compounds which are not easily assayed. The advantage of I-MALDI is that it offers the potential to assay many metabolites simultaneously in the same section rather than the need to use serial sections for different metabolites.

At present, this approach can only be considered as a semi-quantitative method for determining the distribution of metabolites, but it has the distinct advantage of simple and rapid sample handling under conditions where losses can be avoided. In the work presented here it offers a method of understanding how temperature alters metabolite distribution during development. In wheat grown at temperatures above 25 °C there is a decrease in yield which is, in the main, due to reduced starch synthesis (Denyer et al., 1994). I-MALDI offers a simple way of examining the other changes that occur as a consequence of higher temperature.


   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 Supplementary data
References
 
Supplementary data are available at JXB online.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 Conclusions
 Supplementary data
 References
 
Borisjuk L, Walenta S, Rolletschek H, Mueller-Klieser W, Wobus U, Weber H. (2002) Spatial analysis of plant metabolism: sucrose imaging within Vicia faba cotyledons reveals specific developmental patterns. The Plant Journal 29 521–530.[CrossRef][Web of Science][Medline]

Borisjuk L, Walenta S, Weber H, Mueller-Klieser W, Wobus U. (1998) High-resolution histographical mapping of glucose concentrations in developing cotyledons of Vicia faba in relation to mitotic activity and storage processes: glucose as a possible developmental trigger. The Plant Journal 15 583–591.[CrossRef]

Bunch J, Clench MR, Richards DS. (2003) The analysis of pharmaceutical compounds in skin by matrix assisted laser desorption ionization mass spectrometry. Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topic sCanada Montreal, Quebec pp. 8–12 June 2003.

Bunch J, Clench MR, Richards DS. (2004) Determination of pharmaceutical compounds in skin by imaging matrix-assisted laser desorption/ionization mass spectrometry. Rapid Communications in Mass Spectrometry 18 3051–3060.[CrossRef][Web of Science][Medline]

Burrell MM. (1981) The mode of action of ethionine foliar sprays against potato common scab (Streptomyces scabies). Physiological Plant Pathology 18 369–378.

Denyer K, Hylton CM, Smith AM. (1994) The effect of high temperature on starch synthesis and the activity of starch synthase. Australian Journal of Plant Physiology 21 783–789.

Edwards JL and Kennedy RT. (2005) Metabolomic analysis of eukaryotic tissue and prokaryotes using negative mode MALDI Time-of-Flight Mass Spectrometry. Analytical Chemistry 77 2201–2209.[Medline]

Fell D. (1997) Understanding the control of metabolismLondon, Miami Portland Press.

Fiehn O, Kopka J, Dormann P, Altmann T, Trethewey RN, Willmitzer L. (2000) Metabolite profiling for plant functional genomics. Nature Biotechnology 18 1157–1161.[CrossRef][Web of Science][Medline]

Harborne JB, Baxter H, Moss GP. (1999) Phytochemical dictionary: a handbook of bioactive compounds from plants 2nd edn London Taylor and Francis.

Haritatos E, Ayre BG, Turgeon R. (2000) Identification of phloem involved in assimilate loading in leaves by the activity of the galactinol synthase promoter. Plant Physiology 123 929–937.[Abstract/Free Full Text]

Karas M, Bachmann D, Bahr U, Hillenkamp F. (1987) Matrix-assisted ultraviolet-laser desorption of non-volatile compounds. International Journal of Mass Spectrometry and Ion Processes 78 53–68.[CrossRef]

Kinumi T, Saisu T, Takayama M, Niwa H. (2000) Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using an inorganic particle matrix for small molecule analysis. Journal of Mass Spectrometry 35 417–422.[CrossRef][Web of Science][Medline]

Oksman-Caldentey KM and Inzé D. (2004) Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. Trends in Plant Science 9 433–440.[CrossRef][Web of Science][Medline]

Soga T and Heiger DN. (2000) Amino acid analysis by capillary electrophoresis electrospray ionization mass spectrometry. Analytical Chemistry 72 1236–1241.[Medline]

Walenta S, Schroeder T, Mueller-Klieser W. (2002) Metabolic mapping with bioluminescence: basic and clinical relevance. Biomolecular Engineering 18 249–262.[CrossRef][Web of Science][Medline]

Weckwerth W and Fiehn O. (2002) Can we discover novel pathways using metabolomic analysis? Current Opinion in Biotechnology 13 156–160.[CrossRef][Web of Science][Medline]

Wittich PE and Vreugdenhil D. (1998) Localization of sucrose synthase activity in developing maize kernels by in situ enzyme histochemistry. Journal of Experimental Botany 49 1163–1171.[Abstract/Free Full Text]


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This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
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