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In situ Identification and Imaging of Metabolites using High Resolution Mass Spectrometry
Gregory Hamm, David Bonnel, Raphael Legouffe, Guillaume Hochart, Fabien Pamelard, Joachim Trouverie and
Jonathan Stauber
ImaBiotech, MALDI Imaging Department, Parc Eurasanté, Loos, France
By definition, metabolites are intermediates and products from different metabolisms. More commonly,
they are small compounds (between 100 and 1000 Da) found in organisms that have an important part in cells live
and survival. They can be identified with different technics such as Nuclear Magnetic Resonance or Mass
Spectrometry. Each of them is different at a biological level and can have a specific function. They can be
nucleotides, amino acids or vitamins. The glucose-6-phosphate and the couple ATP (Adenosine TriPhosphate)/ADP
(Adenosine DiPhosphate) are examples of metabolites. Their identification permitted the decomposition of major
biological pathways: the Glucose-6-Phosphate is implicated in glycolysis that converts glucose to pyruvate. This
metabolism permits the synthesis of ATP, which one has a critical role in energy transfer and metabolism. ATP is
also implicated in the Krebs Cycle (or Citrate Cycle), which is another example of important biological pathway.
The purpose of this study was the identification and the localization of these metabolites using high
resolution mass spectrometry. This study was done using two different organs (liver and brain) from rat. Slices of
about 10µm thickness of each organ were put on ITO slides. For MALDI experiments, different matrixes were tested
for best results and various polarities (2,5-dihydroxy benzoic acid, α-Cyano-4-hydroxycinnamic acid or 9Aminoacridine). Matrixes were deposited using a SunCollect Automated Sprayer (SunChrome, Friedrichsdorf,
Germany). For LESA experiments, slices were let without preparation. All the data were obtained using FTICR
Solarix 7.0T (Bruker Daltonics, Bremen, Germany). In order to perform at a higher spectral resolution, spectra were
performed using Full Scan and CASI (Continuous Accumulation of Selected Ions) mode between 100 and 600 Da,
each windows corresponding to 100 Da. After data treatments, a list of metabolites was obtained. In order to
localize these compounds, MALDI Images were performed on the different organs at a special resolution of 100µm.
These images show the distribution of different metabolites such as ATP of Glucose-6-Sulfate in specific
histological regions of the tissue. These results are correlated with previous studies of metabolites such as the
citrate cycle composition. Consequently, mass spectrometry imaging is successfully applied to study in-situ
metabolism in tissue and to follow the process of biological pathways.
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