2009年度に採択された、杉浦悠毅さん(浜松医科大学)が58th ASMS Conference on Mass Spectrometry and Allied Topics（ソルトレークシティ、ユタ）にて研究結果を発表されます。
A combined approach of imaging mass spectrometry (IMS) and capillary electrophoresis mass spectrometry (CE−MS) accomplishes region-specific metabolomics during epileptic seizure.
Yuki Sugiura， Mitsutoshi Setou
Eight-weeks-old male C57BL/6J mice were intraperitoneally injected with kainate (25 mg/kg) and then sacrificed at 0.5, 3 and 24h post-administration. Frozen brain sections were cut on a cryostat and mounted onto ITO glasses. 9-aminoacridine matrix solution (10 mg/mL in 70% ethanol) was sprayed with an air-brush. IMS was performed with Ultraflex TOF/TOF (Bruker Daltonics) and data were acquired in the negative-ion refrectron mode. The interval of data points was 20 µm. CE-MS quantified the about 900 metabolites in the mouse brain extracts, by using Agilent Capillary Electrophoresis System, an Agilent 1100 series MSD mass spectrometer and an Agilent1100 series isocratic HPLC pump, a G1603A Agilent CE-MS adapter kit and a G1607A Agilent CE-ESI-MS sprayer kit (Agilent Technologies).
We developed a combination technique of IMS and CE-MS for metabolic mapping of both abundant and trace amount of metabolite species. In our approach, (1) IMS is used to determine the tissue region where dynamic changes of abundant-metabolites were observed, (2) and then by CE-MS, wide varieties of metabolites were precisely quantified in the dissected tissue samples. Here we uncovered the region specific dynamics (i.e., how their expression fluctuates within time) of numerous metabolites during on kainate-stimulation, which is widely used as a model of limbic seizures, in the mouse hippocampus. Among metabolite species, we focused region specific metabolism of adenosine-nucleotides; adenosine nucleotides, especially adenosine-tri-phosphate (ATP) is well known as the “molecular unit of currency” of intracellular energy transfer. Cellular metabolic processes that use ATP as an energy source convert it into ADP and AMP, therefore, simultaneous imaging of these molecules by IMS provides valuable information regarding tissue metabolic activity. By IMS, we could successfully visualize the distribution pattern of ions for AMP, ADP and ATP in the mouse brain, and furthermore, MS/MS analysis validated these molecular assignments. We found that the ATP was selectively decreased in the cell layer regions of mouse hippocampus, while AMP increased in the wide regions of hippocampus during epileptic seizure. Since the results suggest the enhanced metabolism of adenosine nucleotides in the mouse hippocampus, we quantified amount of all adenosine-nucleotides and even their precursor metabolites. The results verified the rapid reduction of ATP and corresponding increases of AMP in the mouse hippocampus. This combination approach established a comprehensive profiling of metabolites in the specific tissue location. Such spatio-temporal metabolomic information provides a novel interpretation of excitatory neural network via metabolic-molecules.
A combined approach of IMS and CE−MS accomplished quantitative metabolomics in the small tissue regions of brain during epileptic seizure.