SE157:/S1/M1/D1

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.
Comment

PubMed

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
Comment

Analytical Method Information

ID	M1
Title	LC-FTICR-MS
Method Details ID	MS1
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS1
Title	LC–FTICR–MS
Instrument	LC, Agilent 1200 series; MS, Bruker Daltonics solariX 7.0 T
Instrument Type	LC-FTICR-MS
Ionization	ESI
Ion Mode	Positive
Description	Extraction of Metabolites The freeze-dried samples were extracted in a mixer mill (MM300, Retsch) with 50 μL of 80% MeOH per mg dry weight and zirconia beads. After 7 min of milling at 18 Hz and 4 °C, the extractions were centrifuged for 10 min and the supernatant was filtered through an HLB μElution plate (Waters). LC–FTICR–MS Analysis Ultrahigh-resolution metabolome data were acquired by an FTICR–MS solariX 7.0 T (Bruker Daltonics) with the ESI source. LC–FTICR–MS analysis was performed as previously described. The FTICR–MS was controlled by the software ftmsControl 2.1.0 (Bruker Daltonics). For internal calibration, lidcaine (250 μM in MeOH; Tokyo Chemical Industry Co. Ltd., Tokyo, Japan) was added to both solvent A (water with 0.1% formic acid, Wako Pure Chemical Industries Ltd.) and solvent B (acetonitrile with 0.1% formic acid, Wako Pure Chemical Industries Ltd.). The column was changed to an Xselect CSH Phenyl-Hexyl (3.5 μm, 2.1 mm × 150 mm, Waters, Milford, MA, U.S.A.). LC–FTICR–MS/MS Analysis The MS/MS boost analysis was carried out at a collision energy of 30 V as previously described.
Comment_of_details

doi: 10.1021/ac302733c

doi: 10.3389/fpls.2015.01127

Data Analysis Information

ID	D1
Title	Data Processing
Data Analysis Details ID	DS1
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS1
Title	Data processing
Description	The MS spectra were recorded using Hystar 4.0 (Bruker Daltonik GmbH, Bremen, Germany) and the data were processed by DataAnalysis 4.2 (Bruker Daltonik GmbH).
Comment_of_details