SE157:/S1/M2/D2

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	M2
Title	MALDI
Method Details ID	MS2
Sample Amount
Comment

Analytical Method Details Information

ID	MS2
Title	MALDI
Instrument	MS FTICR–MS solariX 7.0 T (Bruker Daltonics)
Instrument Type
Ionization	ESI
Ion Mode	Positive
Description	The extract solutions of the non- and 15N-labeled Catharanthus plants (100 μL each) were evaporated and completely dried. The extracts were redissolved in 10 μL of 80% MeOH. Aliquots of the concentrated extract solutions (0.2 μL) were dispensed into 384-well plates and mixed with a CHCA matrix reagent solution [0.2 μL, 70 mg/mL 80% MeOH including 0.2% trifluoroacetic acid (TFA)]. The crystals obtained on the plate were analyzed by an FTICR–MS solariX 7.0 T (Bruker Daltonics) operated with the MALDI source. Analytical conditions were as follows: Mass range m/z 100.32–600.00; Average scan, 1; accumulation, 0.100 s; polarity, positive; Source Quench, on; resolving power, 66000 at m/z 400; transient length, 0.4893 s; mode (data storage: save reduced profile spectrum, on; reduced profile spectrum peak list, on; data reduction, 95%; auto calibration: online calibration, on; mode, single; threshold (abs), 1 × 105; mass tolerance, 50 ppm; reference mass m/z 337.191054); API Source (API source: source, ESI; capillary, 4500 V, end plate offset, −500; source gas tune: nebulizer, 1.0 bar; dry gas, 2.0 L/min; dry temperature, 180 °C); ion transfer (Source Optics: capillary exit, 220 V; detector plate, 200 V; funnel 1, 150 V; skimmer 1, 55 V; funnel RF amplitude, 200 Vpp; octopole: frequency, 5 MHz; RF amplitude, 500 Vpp; quadrupole: Q1Mass, m/z 100; collision cell: collision voltage, −2.0 V; DC extract bias, 0.0 V; RF frequency, 2 MHz; collision RF amplitude, 1500.0 Vpp; transfer optics: time of flight, 1800 ms; frequency, 2 MHz; RF amplitude, 400.0 Vpp); analyzer (infinity cell: transfer exit lens, −20.0 V; analyzer entrance, −10.0 V; side kick, 8.0 V; side kick offset, −1.5 V; front trap plate, 0.500 V; back trap plate, 0.450 V; sweep excitation power, 12.0%; multiple cell accumulations: ICR cell fills).
Comment_of_details

Data Analysis Information

ID	D2
Title	Data Analysis (MALDI)
Data Analysis Details ID	DS3
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS3
Title	Data Analysis (MALDI)
Description	Elemental composition candidates were estimated in DataAnalysis under the following conditions: adduct type, [M + H]+; tolerance, 1 mDa; number of nitrogen atoms in formula, ≥1; charge, +1; check rings plus double bonds, ≤80; filter H/C ration, ≤3. Finally, the molecular formulas of the 64 N-ions were determined from the mass shifts of the target N-ions from the non-labeled data to the 15N-labeled data. To assign the molecular formulas, the metabolite information was extracted by searching the word “Catharanthus” in the databases KNApSAcK WEB: Keyword Search Web Version 1.000.01 and Dictionary of Natural Products 25.1.
Comment_of_details