Journal of Experimental Botany, Vol. 58, No. 4, pp.

757763, 2007
Imaging Stress Responses in Plants Special Issue
doi:10.1093/jxb/erl139 Advance Access publication 27 September, 2006

SPECIAL ISSUE PAPER

Imaging Matrix Assisted Laser Desorption Ionization Mass

Spectrometry: a technique to map plant metabolites
within tissues at high spatial resolution
M. M. Burrell1,*, C. J. Earnshaw2 and M. R. Clench2
Department of Animal and Plant Sciences, Shefeld University, Western Bank, Shefeld S10 2TN, UK

Biomedical Research Centre, Shefeld Hallam University, Howard Street, Shefeld S1 1WB, UK

Received 17 May 2006; Accepted 2 August 2006

Abstract
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 a-cyano-4-hydroxycinnamic
acid (a-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
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 (OksmanCaldentey and Inze, 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 a-cyano-4-hydroxycinnamic
acid (a-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

* To whom correspondence should be addressed. E-mail: m.burrell@shefeld.ac.uk

The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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758 Burrell et al.

of a sample may be obtained. The diameter of the laser

in modern mass spectrometers is in the order of 100 lm or
less. Thus at a spatial resolution of 100 lm 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 a-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 a-CHCA or 9-aminoacridine is reported.

Materials and methods

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.

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Fig. 1. Calibration curve. One ll of sample was mixed with 1 ll of

aCHCA solution and 1 ll spotted onto the MALDI target plate. Each spot
was replicated three times. Results are plotted as the mean 6sem after
the intensity had been normalized to the intensity of the a-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.

Wheat (Triticum aestivum L. var. Axona) plants were grown in

a growth room under a day/night regime of 20/16 8C or at 25/20 8C,
a daylength of 16 h and light intensity of 400 lE. 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 8C and the knife at 20 8C. 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 ml1 a-CHCA in methanol containing 0.1% (v/v) trifluoracetic acid or a solution of 10 mg ml1 9-aminoacridine dissolved
in acetone containing 0.1% TFA.

Imaging MALDI-MS

759

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 lJ), acridine 25% (2.8 lJ) and analysis time of
5 s per position. At a resolution of 100 lm 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

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.

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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).
The usefulness of a-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 lm 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

760 Burrell et al.

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.

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.

It was not possible to get strong signals for metabolites

such as sucrose and glucose-6-phosphate in negative ion
mode with a-CHCA-coated sections. Therefore wheat
sections were coated with 9-aminoacridine. Figure 5 shows
the distribution (as the H+) of sucrose product and glucose6-phosphate for an older seed (16 dpa) than shown above.

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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.

Imaging MALDI-MS

761

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 glucose6-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.
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.

Conclusions
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

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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.

762 Burrell et al.

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 8C or 8 dpa when grown at 25 8C. Sucrose (m/z 381) is plotted
for the 25 8C seed (a) and the 20 8C seed (b, c). The darker the colour
the more signal.

Supplementary data
Supplementary data are available at JXB online.

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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
a-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 lg
g1 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 8C 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.

Imaging MALDI-MS

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