SE157:/S1/M3

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Sample Set Information

ID TSE1313
Title Top-down Metabolomic Approaches for Nitrogen-Containing Metabolites.
Description Streamlining the processes that reveal heteroatom-containing metabolites and their biosynthetic genes is essential in integrated metabolomics studies. These metabolites are especially targeted for their potential pharmaceutical activities. By using a Fourier-transform ion cyclotron resonance-mass spectrometry (FTICR-MS) instrument, we provide top-down targeted metabolomic analyses using ultrahigh-resolution liquid chromatography-mass spectrometry (LC-MS), high-resolution matrix-assisted laser desorption/ionization (MALDI), and high-resolution imaging mass spectrometry (IMS) with 15N labeling of nitrogen-containing metabolites. In this study, we efficiently extract known and unknown chemicals and spatial information from the medicinal plant Catharanthus roseus, which sources several cancer drugs. The ultrahigh-resolution LC-MS analysis showed that the molecular formula of 65 N-metabolites were identified using the petals, peduncles, leaves, petioles, stems, and roots of the non- and 15N-labeled Catharanthus plants. The high resolution MALDI analysis showed the molecular formula of 64 N-metabolites using the petals, leaves, and stems of the non- and 15N-labeled Catharanthus. The chemical assignments using molecular formulas stored in databases identified known and unknown metabolites. The comparative analyses using the assigned metabolites revealed that most of the organ-specific ions are derived from unknown N-metabolites. The high-resolution IMS analysis characterized the spatial accumulation patterns of 32 N-metabolites using the buds, leaves, stems, and roots in Catharanthus. The comparative analysis using the non- and 15N-labeled IMS data showed the same spatial accumulation patterns of a non- and 15N-labeled metabolite in the organs, showing that top-down analysis can be performed even in IMS analysis.
Authors Nakabayashi R, Hashimoto K, Toyooka K, Saito K.
Reference Anal Chem. 2017 Mar 7;89(5):2698-2703. doi: 10.1021/acs.analchem.6b04163. Epub 2017 Feb 22.
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Sample Information

ID S1
Title Catharanthus roseus
Organism - Scientific Name Catharanthus roseus
Organism - ID NCBI taxonomy:4058
Compound - ID
Compound - Source
Preparation All analyses were performed on Catharanthus roseus (Equator White Eye, Sakata Seed Corporation). Non- and 15N-labeled Catharanthus plants were purchased from Shoko Science Co., Ltd.. The plants were individually grown in pots filled with vermiculite. The pots were placed in a plant growth room under a 16/8 h light/dark cycle with an illuminance of 252–420 μm olm–2 s–1 during the light period. The temperature was maintained at 20–25 °C. The plants were fed daily with a non- or 15N-labeled liquid fertilizer (Table S1 in the Supporting Information), and watered every 2–3 days. After 8 weeks of growth, the flowers, petals, peduncles, leaves, petioles, stems, and roots of the non- and 15N-labeled Catharanthus plants were harvested and immediately lyophilized at −55 °C. The lyophilized materials were stored at room temperature with silica gel. The labeling rate of 15N was approximately 95.3%.
Sample Preparation Details ID
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Analytical Method Information

ID M3
Title IMS (MALDI)
Method Details ID MS3
Sample Amount
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Analytical Method Details Information

ID MS3
Title IMS (MALDI)
Instrument MS FTICR–MS solariX 7.0 T (Bruker Daltonics)
Instrument Type
Ionization ESI
Ion Mode Positive
Description Preparation of Frozen Section

Fresh tissues were cut about 5 mm with a razor, embedded with a compound (Surgipath FSC22: Leica Microsystems, Germany) and frozen in a −75 °C acetone bath (Histo-Tek Pino: Sakura Finetek Japan Co.,Ltd., Tokyo, Japan). The frozen samples were placed on the cryostat specimen disk and cut with the knife blade until the desired tissue is exposed. The face of the frozen sample was put on the adhesive film (Kawamoto’s film method) at 16μm thickness in a cryostat (CM3050S, Leica Microsystems, Germany). The section on the film was freeze-dried overnight at −30 °C in the cryostat (Figure S9 in the Supporting Information). For light microscopy, the frozen sections were stained with 0.05% toluidine-blue O solution for a minute and then washed with distilled water.

IMS Analysis
The freeze-dried section on the film was attached to a glass slide (ITO coating, Bruker Daltonik GmbH) with cellophane tape. The CHCA matrix solution (7 mg/mL 80% MeOH including 0.2% TFA) was sprayed onto the glass slide using ImagePrep (Bruker Daltonik GmbH) set to the default parameters. The freeze-dried section with the matrix was analyzed in the FTICR–MS instrument using the MALDI source. The analytical conditions were as follows. MALDI control: geometry, MTP 384 ground steel; plate offset, 100.0 V; deflector plate, 200.0 V; laser power, 30.00%; laser shots, 200; frequency, 2000 Hz; laser focus, small; raster width, 30 μm. The analytical conditions of MALDI in the IMS analysis were identical to those described in the MALDI analysis.

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