Top-down Metabolomic Approaches for Nitrogen-Containing

Feb 22, 2017 - The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04163. Data S1: I...
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Top-down Metabolomic Approaches for Nitrogen-Containing Metabolites Ryo Nakabayashi,*,† Kei Hashimoto,† Kiminori Toyooka,† and Kazuki Saito†,‡ †

RIKEN Center for Sustainable Resource Science, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan

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S Supporting Information *

ABSTRACT: Streamlining the processes that reveal heteroatomcontaining 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 15Nlabeled 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.

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common feature in N-omics. Exploiting the fact that 15N labeling enhances the signal intensity of natural 15N isotopic ions,10 we here establish three different N-omics analyses: (1) ultrahigh-resolution liquid chromatography−mass spectrometry (LC−MS) analysis, (2) high-resolution matrix-assisted laser desorption/Ionization (MALDI) analysis, and (3) highresolution imaging mass spectrometry (IMS) analysis. By combining these top-down analyses, we can assign the structures, molecular formulas, and spatial accumulation patterns of N-metabolites.

ass spectrometry (MS)-based metabolomics are central to synthetic biology,1 phytochemical genomics,2 natural product chemistry,3 and drug discovery research.4 N-metabolites (i.e., alkaloids) are frequently targeted as pharmaceutical candidates because of their potential biological activities.5 However, discovering N-metabolites by general methods is extremely slow. To resolve this situation, metabolomics methods that specifically target N-metabolites (so-called Nomics) are required. N-omics acquires the precise metabolic information on N-metabolites, which is then confirmed and refined by general methods. In MS-based targeted analysis, specialized/secondary metabolites are profiled by their common molecular features (e.g., ultraviolet spectrum or product ions).6−9 However, as N-metabolites are structurally diverse, focusing on the common molecular features is impractical in N-omics. Therefore, N-omics development requires other important features of N-metabolites. In nature, nitrogen exists as two stable isotopes: 14N (exact mass, 14.003 Da; natural abundance, 99.63%) and 15N (15.0001 Da, 0.036%). In the MS spectrum, 15N isotopic ions appear as the counterpart of 14N-containing monoisotopic ions (N-ions). Therefore, 15N isotopic ions are presumed as a © XXXX American Chemical Society

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RESULTS AND DISCUSSION The workflow of these three analyses is illustrated in Figure S1 in the Supporting Information. First, we analyze the Nmetabolites by ultrahigh-resolution LC−MS, which combines reverse-phase LC and electrospray ionization (ESI). The metabolomic MS data of non- and 15N-labeled samples are acquired with an ultrahigh-resolution instrument that separates Received: October 25, 2016 Accepted: February 10, 2017

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DOI: 10.1021/acs.analchem.6b04163 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

Figure 1. Chemical assignment of N-metabolites using the ultrahigh-resolution LC−MS analysis, high-resolution MALDI analysis, and highresolution IMS analysis. (a) Heat map of the signal intensities of N-ions identified in the ultrahigh-resolution LC−MS analysis. Each N-ion was detected at a different retention time (Data S1 in the Supporting Information). ∥, ajmalicine; †, perivine; ‡, tabersonine; §, vindoline; *, assigned Nmetabolites using the extracted molecular formulas from the databases KNApSAcK and Dictionary of Natural Products. (b) Identification of ajmalicine by LC−FTICR−MS/MS. Upper, MS/MS spectrum of the authentic standard compound; Lower, observed MS/MS spectrum. (c) Identification of perivine by LC−FTICR−MS/MS. Upper, MS/MS spectrum of the authentic standard compound; Lower, observed MS/MS spectrum. (d) Chemical assignment of N-ions in the MALDI analysis. Yellow marks indicate the assigned N-ions. Diagrams indicate the sites of nonlabeled MS data (upper) and 15N-labeled data (lower) in the Catharanthus leaf. (f) Comparative analysis of the molecular formulas identified in the petal, leaf, and root of the non- and 15N-labeled Catharanthus. Numbers in parentheses indicate the number of unknown N-metabolites. (g) Comparative analysis of the molecular formulas (nonoverlapping) in the ultrahigh-resolution LC−MS analysis and high-resolution MALDI analysis. (h) Visualization of an N-metabolite, showing the non-labeled (left) and 15N-labeled (right) data in the IMS analysis. Left and right diagrams of each result visualize C21H2114N2O3 (m/z 349.155 [M + H]+) and C21H2115N2O3 (m/z 351.149 [M + H]+), respectively. White bar indicates 1 mm (bud, leaf, and stem) and 500 μm (root).

the 15N isotopic ions from the isotopic ions. From the exact mass and signal intensity differences between the N-ions and 15 N isotopic ions, we can theoretically isolate the N-ions among the metabolomic data. After extracting the N-ions, we check the mass accuracy and pattern of isotopic ions (including15N, 13C, 18 O, and 34S),11 which narrows the range of candidates by their elemental composition. By comparing the non- and 15N-labeled data in the N-ion extraction, we can identify the mass shift due to 15N-labeling, which indicates the number of N atoms. The molecular formula of the N-ion is unambiguously identified from the N atom counts and the limited number of candidates (Figure S1a in the Supporting Information). Next, the Nmetabolites that cannot be analyzed by LC or ESI are identified through high-resolution MALDI analysis. In the MALDI process, the extract solutions of the non- and 15N-labeled samples are mixed with a matrix solution on a plate and left to dry. The MALDI analysis then acquires high-resolution MS

data of the mixed samples. The molecular formula of the Nions is again unambiguously identified from the N-ion counts and the limited number of candidates (Figure S1b in the Supporting Information). Finally, the MALDI analysis is followed by high-resolution IMS analysis. The non- and 15Nlabeled samples are sectioned and freeze-dried in a cryostat. The freeze-dried non- and 15N-labeled samples on the film are placed on a glass slide and sprayed with the matrix solution. The IMS data of the non- and 15N-labeled samples are acquired under with a high-resolution. A comparative analysis of the non- and 15 N-labeled m/z values clearly reveals the accumulation patterns of the non- and 15N-labeled Nmetabolites (Figure S1c in the Supporting Information). The above sequence of the metabolomic analyses was applied to the medicinal plant Catharanthus roseus (Apocyanaceae), which produces monoterpene indole alkaloids.5 For ultrahighresolution LC−MS analysis, we employed a Fourier transform B

DOI: 10.1021/acs.analchem.6b04163 Anal. Chem. XXXX, XXX, XXX−XXX

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Figure 2. Spatial accumulation patterns of N-metabolites. (a) Representative N-metabolites accumulated at different sites in the bud (i and ii), leaf (iii−vi), and stem (vii−x), and at the same site in the leaf (xi−xv). These metabolites were visualized at m/z value [M + H]+. White bar indicates 1 mm. (b) MS/MS spectrum of m/z 427 characterized as demethoxyvindoline. (c) MS/MS spectrum of m/z 457 identified as vindoline. (d) MS/MS spectrum of m/z 511.

from the KNApSAcK12 and Dictionary of Natural Products 25.1 revealed that some of these N-metabolites are known, whereas others have not been reported (Figure 1a and Data S2 in the Supporting Information). Among the assigned metabolites, ajmalicine (retention time (Rt), 10.7 min), perivine (Rt, 8.0 min), tabersonine (Rt, 11.1 min), and vindoline (Rt, 12.1 min) were identified by liquid chromatography−Fouriertransform ion cyclotron resonance−tandem mass spectrometry (LC−FTICR−MS/MS) with authentic standard compounds. Identification of ajmalicine and perivine was shown in Figure 1b and c, respectively. The result showed that most of the metabolites that have the redundancy of accumulation at an organ are still unknown in this plant. On the basis of this result, further research targeting the organ-redundant metabolites enables to increase efficiency to isolate those and reveal their structure. As the FTICR−MS instrument contains a MALDI source in addition to the ESI source, the same instrument was employed in the high-resolution MALDI analysis. As representative organs, we selected the petals, leaves, and stems of non- and 15 N-labeled Catharanthus. The extract solutions of the non- and

ion cyclotron resonance−mass spectrometry (FTICR−MS) instrument with an ESI source. This system clearly separates the 15N and 13C isotopic ions in the region of monoisotopic ion (M) + 1 (Figure S2 in the Supporting Information). The mass shift pattern in the leaves of non- and 15N-labeled Catharanthus plants was evaluated using non- and 15N-labeled tryptophan. The mass shift of the two N atoms was confirmed in the 15Nlabeled leaf (Figure S3 in the Supporting Information). Ultrahigh-resolution metabolome data (resolving power 260000 at m/z 400) were then acquired from the petals, peduncles, leaves, petioles, stems, and roots of the non- and 15 N-labeled Catharanthus plants. After extracting the N-ions, estimating the elemental compositions and identifying the number of N atoms in each metabolite, we obtained the molecular formula of 65 N-metabolites in the various Catharanthus organs (Data S1 in the Supporting Information). From the differences in the N- and 15N-ions, we approximated the average N-ion counts as 5 × 108, suggesting that the N-ions dominate in this analysis. The heat map of the signal intensities revealed the accumulation pattern of the 65 N-metabolites in the plant organs. Chemical assignment to the identified molecular formulas and the molecular formulas extracted C

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Analytical Chemistry N-labeled samples were mixed with the matrix solution αcyano-4-hydroxycinnamic acid (CHCA), dried on the plate, and then subjected to high-resolution MS acquisition. Candidates of N-ions were estimated among the non-labeled data. In the MALDI analysis, the N-ions were detected as [M + H]+. The mass shift indicating the number of N-ions was confirmed for each N-ion candidate and the molecular formulas of the candidates were identified (Figure 1d, Data S3 in the Supporting Information). By this analysis, we obtained the molecular formula of 64 N-metabolites in the petals, leaves, and stems of Catharanthus. The known and unknown molecular formulas were identified in comparisons with the molecular formulas stored in databases (Data S4 in the Supporting Information). The comparative analysis revealed that most of the organ-specific ions are derived from unknown Nmetabolites (Figure 1f). The LC−MS and MALDI analyses narrowed the range of formulas in the comparative analysis to the target-specific formulas (Figure 1g). The FTICR−MS instrument was again employed in the high-resolution IMS analysis. The matrix solution CHCA was sprayed onto freeze-dried sections of the buds, leaves, stems, and roots of the non- and 15N-labeled Catharanthus prepared on a glass slide. High-resolution IMS data (resolving power 66000 at m/z 400) were then acquired. In the IMS analysis, the target N-ions were detected as [M + H]+. The IMS analysis revealed the same accumulation patterns of non- and 15Nlabeled N-metabolites in all samples (Figure 1h, Figure S4 in the Supporting Information). It also characterized the spatial accumulation patterns of 32 N-metabolites (Figures S5−8 and Data S5 in the Supporting Information). The accumulation patterns differed among some of the N-metabolites (Figure 2ai−x); in others, they were identical (Figure 2axi−xv). Several of the N-metabolites presented as [M + H]+. These metabolites, C24H29N2O4 (m/z 409.212), C24H29N2O5 (m/z 425.207), C24H31N2O5 (m/z 427.223), and C25H33N2O6 (m/z 457.233), indicate reduction, oxidation, and methylation reactions of the N-metabolite presented as C24H29N2O4 (m/z 409.212) at the same site. The LC−FTICR−MS/MS analysis in the petioles showed that m/z 427 and m/z 457 was derived from demethoxyvindoline (putatively characterized) and vindoline (identified using the authentic standard compound), respectively. The difference of the main three product ions which indicates methoxy moiety led to the putative characterization of demethoxyvindoline (Figure 2b,c). It is proposed that the biosynthetic reaction goes forward from vindoline to demethoxyvindoline.13 These analyses suggest the presence of biosynthetic gene(s) regarding demethoxy reaction of vindoline at the petiole. Another N-metabolite, C27H31N2O8 (m/z 511.208), also accumulated at the same site. The MS/MS spectrum on m/z 511 showed that the N-metabolite is not an analogue of demethoxyvindoline and vindoline. The loss of m/z 162 in the MS/MS spectrum suggested that the N-metabolite has a hexose moiety (Figure 2d). The biosynthetic genes for glycosylation in N-metabolites are largely unknown in this plant. Structure elucidation of this glycosylated N-metabolite contribute to identification of biosynthetic gene encoding glycosyltransferase. 15

omics. The combination of product ion analyses in MS/MS provides the precise information on substructures, which can assist structural elucidation. Data enhancing of known Nmetabolites through authentic standard compounds improves the accuracy of finding novel N-metabolites. These analyses might also identify the biosynthetic genes regarding the N-metabolites. In Catharanthus, the N-metabolites are translocated into multicellular compartments.14 Various intermediates in the main biosynthetic pathway to vinscistine were recently found to be localized in the stem.13 However, localization of other N-metabolites in this plant has not been attempted. Biosynthetic genes are specifically expressed in several Catharanthus cells.15 By collating the spatial information on metabolites and genes through these Nomics methods and gene expression analysis, we could elucidate biosynthetic mechanisms.16

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METHODS Chemicals. Ajmalicine [Sigma-Aldrich Japan (Tokyo, Japan)], peverine [Apin Chemicals (Abingdon, U.K.)], nonlabeled tryptophan [Wako Pure Chemical Industries, Ltd. (Osaka, Japan)], 15N-labeled tryptophan [Shoko Science Co., Ltd. (Tokyo, Japan)], tabersonine [Sigma-Aldrich Japan (Tokyo, Japan)], and vindoline [Sigma-Aldrich Japan (Tokyo, Japan)] were used in this study. Plant Materials. 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%. 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.11 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.17

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CONCLUSIONS In summary, we have established three different analyses for Nomics. By using the chemical and spatial information as a marker, we can extract and isolate N-metabolites of interest from biological organs, greatly improving the efficiency of ND

DOI: 10.1021/acs.analchem.6b04163 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry MALDI Analysis. The extract solutions of the non- and 15Nlabeled 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, 1). 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 method18) at 16 μm 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. Data Analysis. 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). LC−MS Analysis. All data were calibrated with m/z 235.1804 (lidcaine, [M + H]+). After noise reduction (