Leaf Spray: Direct Chemical Analysis of Plant Material and Living

Sep 14, 2011 - were simply cut to a point, and a copper clip was used to hold the plant tissue (in ..... ionized from a wide range of plant tissues wi...
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Leaf Spray: Direct Chemical Analysis of Plant Material and Living Plants by Mass Spectrometry Jiangjiang Liu,† He Wang,† R. Graham Cooks,*,‡,§ and Zheng Ouyang*,†,§ †

Weldon School of Biomedical Engineering, ‡Department of Chemistry, and §Center for Analytical Instrumentation Development, Purdue University, West Lafayette, Indiana 47907, United States

bS Supporting Information ABSTRACT: The chemical constituents of intact plant material, including living plants, are examined by a simple spray method that provides real-time information on sugars, amino acids, fatty acids, lipids, and alkaloids. The experiment is applicable to various plant parts and is demonstrated for a wide variety of species. An electrical potential is applied to the plant and its natural sap, or an applied solvent generates an electrospray that carries endogenous chemicals into an adjacent benchtop or miniature mass spectrometer. The sharp tip needed to create a high electric field can be either natural (e.g., bean sprout) or a small nick can be cut in a leaf, fruit, bark, etc. Stress-induced changes in glucosinolates can be followed on the minute time scale in several plants, including potted vegetables. Differences in spatial distributions and the possibility of studying plant metabolism are demonstrated.

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lants are complex systems that contain a wide range of chemicals from small molecules to biopolymers in a state of flux. Characterization of these compounds has been dominated by traditional natural product methods, in which most temporal and spatial phytochemical information is lost. Mass spectrometry (MS) is attractive because of its high selectivity and sensitivity, although sample preparation and/or chromatographic separation are normally required for MS measurements on plant tissue.1 6 Instances of direct analysis of plant tissue by tandem mass spectrometry (MS/MS) also exist7 with the ambient ionization methods8 10 like desorption electrospray ionization (DESI),11 15 laser ablation electrospray ionization (LAESI),16 18 laser electrospray mass spectrometry (LEMS),19 extractive electrospray ionization (EESI),20,21 contactless atmospheric pressure ionization,22 and atmospheric pressure femtosecond laser imaging,23 allowing endogenous compounds to be ionized directly from the native plant tissue with little or no sample preparation. In this Letter, we describe leaf spray, a new ambient ionization method applicable to the direct analysis of living plants. Leaf spray is derived from paper spray,24,25 a method in which ionized analytes are released into an electrospray when an electrical potential is applied to a solvent-wetted paper triangle bearing the sample. In the simpler leaf spray experiment,26 the plant itself serves as both substrate and sample. Field emission of droplets containing endogenous compounds is achieved by cutting a tip in the plant material and applying an electrical potential. If plants have natural tips (e.g., sprouts or plants with needles), cutting is unnecessary. The spray of charged droplets carries endogenous compounds from the plant to an adjacent mass spectrometer. Leaf spray operates continuously for minutes, which is adequate to perform tandem mass spectrometry (MS/MS) and exact mass measurements. No visible change is observed on the tested plant r 2011 American Chemical Society

tissue even after some minutes of spraying. Amino acids, carbohydrates, fatty acids, alkaloids, and lipids are identified with high abundance in many tested plant tissues.

’ EXPERIMENTAL SECTION Figure 1 illustrates the leaf spray experiment. Plant tissues were simply cut to a point, and a copper clip was used to hold the plant tissue (in some cases the living plant). A high voltage (4.5 kV, negative or positive) was applied to the tissue, with or without wetting with a spray solvent, to generate a spray of charged droplets carrying endogenous chemicals toward the inlet of a mass spectrometer. MS and MS/MS measurements of leaf spray were carried out using Thermo TSQ and LTQ mass spectrometers (Thermo-Fisher, San Jose, CA). The exact mass measurements were performed by the same procedure with a Thermo-Fisher Exactive Orbitrap to facilitate the compound identification. The distance between the plant tissue and the inlet to the mass spectrometer is 5 10 mm for TSQ and LTQ and 10 30 mm for Exactive Orbitrap. Mass spectra of both positive and negative ions can be recorded. A hand-held Mini 10.5 mass spectrometer was used for the in vivo chemical analysis. It used a 2-D linear version of Paul trap mass analyzer, constructed using four planar electrodes (a rectilinear ion trap) and coupled via a discontinuous atmospheric pressure interface (DAPI), which allowed the introduction of ions from ambient air into the manifold without compromising the vacuum inside the ion trap.27 Received: August 3, 2011 Accepted: September 14, 2011 Published: September 14, 2011 7608

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Figure 1. (a) Photograph of leaf spray ionization of green onion leaf cut to a point and held by a high voltage connector in front of the atmospheric inlet of a mass spectrometer. (b) Leaf spray spectrum acquired from green onion leaf in positive ion mode, showing sucrose and glucose ions. (c) Photograph of leaf spray ionization of spinach leaf in negative ion mode. The spinach leaf was cut into a triangle, and methanol was applied on the leaf to achieve leaf spray ionization. (d) Leaf spray spectrum acquired from spinach leaf, showing amino acids and organic acids. (e) Leaf spray spectrum acquired from peanut seed in negative ion mode, showing three fatty acids. (f) Leaf spray spectrum acquired from cranberry fruit in positive ion mode, showing a series of phytochemicals. Assignments given are based on exact mass and/or MS/MS data.

The ejection frequency was set to 350 kHz, and the supplemental AC voltage was set to 0.6 1.5 V.

’ RESULTS AND DISCUSSION Green onion was tested without added spray solvent (Figure 1a). The mass spectrum (Figure 1b) is dominated by cationized sugars (m/z 203.1 [glucose + Na]+, m/z 219.0 [glucose + K]+, m/z 365.1 [sucrose + Na]+, and m/z 381.1 [sucrose + K]+). Plants such as green onion, with high water content, do not require addition of solvent, and this was the case for most juicy fruits and some fresh plants such as grapefruit, tomato, pepper, cucumber, corn kernels, and onion (see Supporting Information, Figure S-1). Spray solvents were applied to plant tissues when the water content was too low to generate a spray directly. For example, spinach leaf did not give a mass spectrum directly; however, when methanol was added and a high voltage was applied to the plant tissue, a Taylor cone formed in the solution and endogenous phytochemicals were extracted within

ca. 1 s (Figure 1c). The spectrum of spinach leaf (Figure 1d) shows that a number of amino acids (m/z 116.1 [valine H] , m/z 130.1 [Ile/Leu H] ) and organic acids (m/z 133.0 [malic acid H] , m/z 175.0 [ascorbic acid H] , m/z 191.0 [citric acid H] , and m/z 337.1 [2-O-acetyl-trans-coutaric acid H] ) were detected as deprotonated ions. The methanol volume and extraction time were not critical to the performance of leaf spray. No obvious differences in spectra were observed when changing the methanol volume from 10 to 50 μL or the voltage application time from 1 s to several minutes. Leaf spray spectra obtained from a green onion leaf with and without spray solvent, respectively, are compared in Figure S-2, Supporting Information. There are some differences, but in the lower mass range, both spectra are dominated by the Na+ and K+ adducts of sucrose and glucose. In a test of the reliability of the leaf spray method, a number of leaf triangles were cut from a cabbage leaf, with some of them from the lamina part but some including veins (Figure S-3, Supporting Information). The spectra acquired using methanol 7609

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Figure 2. Leaf spray spectra of peanut seed acquired with added spray solvents: (a) methanol, (b) dichloromethane, (c) hexanes, (d) acetonitrile, (e) chloroform, and (f) acetone. Data recorded in positive ion mode.

in the positive and negative ion modes show that similar chemicals were observed for the different parts of the leaf, with some variation in the relative intensities over the wide mass range examined (Figure S-3, Supporting Information). The use of spray solvent not only realizes the direct ionization of chemicals from drier samples, but the choice of solvent also is expected to offer an opportunity to selectively extract different compounds from the same sample. We tested a series of organic solvents with different polarity and dielectric constants. Methanol, dichloromethane, hexanes, acetonitrile, acetone, and chloroform gave significantly different spectra from peanuts when different spray solvents are used (Figure 2). The spectra acquired using methanol, acetone, and acetonitrile look similar, but different solvents polarities cause some differences in relative efficiencies of extraction and ionization. Because leaf spray works well with methanol for all plant tissues tested, a standard protocol was adopted in which methanol (10 50 μL) was applied to the plant tissue to perform leaf spray whether or not the experiment was successful without added solvent. The leaf spray protocol is not limited to leaf material but gave characteristic spectra for other parts of plants, including root, stem, flower, fruit, and seed. A wide variety of vegetable, fruit, and other species have been tested, including potato, onion, cabbage, ginger, cranberry, gingko, Arabidopsis thaliana, etc. (see Supporting Information, Table S-1). Chemicals could be analyzed directly from these plant parts without sample preparation, except that small points (mm dimensions) were cut into bulk materials when necessary. Methanol is a good solvent for a wide range of

phytochemicals, including amino acids, alkaloids, flavonols, carbohydrates, organic acids, fatty acids, and phospholipids. Common phytochemicals, such as amino acids, carbohydrates, and fatty acids, were detected in all tested plant materials, although the relative abundance of each chemical (such as amino acids and fatty acids) may vary 2 or 3 orders of magnitude between different tissues (Figures S-4, Supporting Information). These variations are likely due to the differences in the intrinsic concentrations of the chemical as well as the effects of the plant tissue matrix on sampling and ionization efficiency. Identification of particular compounds was based on positive and negative ion mass spectra supported by MS/MS and exact mass measurements. For example, peanut seed and cranberry fruit were tested with added spray solvent in the negative and positive ion modes, respectively. Three fatty acids (m/z 255.2 [palmitic acid H] , m/z 279.2 [linoleic acid H] , and m/z 281.3 [oleic acid H] ) were identified by exact mass measurement in peanut seed (Figure 1e) and a set of phytochemicals (m/z 419.1 [cyandin-arabinoside]+, m/z 433.1 [peonidin-arabinoside]+, m/z 435.1 [delphinidin-arabinoside]+, m/z 449.1 [cyanidin-glucoside]+, and m/z 463.1 [peonidingalactoside/peo-glucoside]+) were identified with exact mass (Figure 1f) and MS/MS measurements in cranberry fruit (Figure S-5, Supporting Information). The identification of alkaloids in potato and tomato was performed to demonstrate the capability of leaf spray not only for ex vivo but also for in vivo analysis. A piece of green skin (∼0.3 cm  0.3 cm) from a budding potato was tested ex vivo, and alkaloids were the main species detected in the leaf spray 7610

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Figure 3. Alkaloid analysis in potato and tomato with leaf spray ionization. Leaf spray spectra were acquired from green potato peel (a) and potato tuber (b) using added methanol spray solvent, respectively. The insets show MS/MS spectra of doubly charged potassium adducts of chaconine and solanine (m/z 445.7, m/z 453.7) in potato peel. (c) Leaf spray spectra acquired from the leaf of potted tomato. The inset shows MS/MS spectrum of protonated tomatine (m/z 1034.6). (d) Leaf spray spectra acquired from the leaf of potted tomato with miniature mass spectrometer (Mini 10.5). The inset shows molecular structure of tomatine.

mass spectrum (Figure 3a). The glycoalkaloids, chaconine and solanine, were observed in the protonated form (m/z 852.5 and m/z 868.5, respectively), while the corresponding doubly charged potassium adducts (m/z 445.7, m/z 453.7) were identified by MS/ MS (Figure 3b, inset) and by exact mass measurements. Leaf spray is not suited to high spatial resolution imaging analysis on plant tissues as are probe based MS methods.28 33 However, it does give information on the distribution of chemicals in different parts of a plant. Figure 3b shows that the glycoalkaloids in potato just discussed were observed in the tuber too, but the relative abundance of MS signals suggests that the concentrations in the tuber were much lower than those in the green skin. In vivo experiments were done readily by (i) bringing the plant to the mass spectrometer (e.g., potted plants) or (ii) bringing the mass spectrometer to the plant. The former experiment was performed using tomato plants. A small cut was made at the tip of a leaf; a 4.5 kV high voltage was applied, and about 20 μL of methanol was added to the leaf to generate spray at the cut. Abundant ions due to the well-known steroidal glycoside alkaloid tomatine (Figure 3c, m/z 536.8 [tomatine + H + K]2+, m/z 1034.6 [tomatine + H]+) were observed. These spectra could be recorded for long periods and were highly reproducible. Tested tomato plants were not affected by the experiment; even the tested leaf was unaffected except for the tiny portion where the point was cut and solvent applied. A living tomato plant was tested with a Mini 10.5 mass spectrometer to investigate the protocol of bringing the mass spectrometer to the plant for in vivo experiments. The mass spectrum was acquired using a standard leaf spray protocol on tomato leaf by cutting a triangle and using methanol as spray solvent. Figure 3d shows that the

base peak due to the doubly charged potassium adduct of tomatine (m/z 537, [tomatine + H + K]2+). The concentrations of many phytochemicals show seasonal variations as well as changes with developmental stage and part of the plant. Insights into these changes are accessible by leaf spray as demonstrated by a study on mung bean during germination. Variations in chemical components in the seed, sprout, and cotyledon were monitored. The sprout was analyzed directly since the root has a sharp tip (Figure S-6a, Supporting Information), while the cotyledon was cut to a point to generate the high electric fields needed for the leaf spray measurement. Ten simple organic acids and 14 amino acids were identified in the negative ion mode from each of the tested plant tissues (see Supporting Information, Figure S-6, note that 12 of the amino acids are labeled). The leaf spray MS spectra acquired from the seed and sprout are similar; that of the cotyledon shows large differences in the relative abundances of the organic acids and amino acids. Among the species identified, the signal due to ascorbic acid increases dramatically from trace level in the seed to become the base peak in cotyledon. Relative, approximate quantitative analysis can be achieved even though it is impracticable to introduce internal standards into plant tissue; the relative concentrations of each amino acid in bean sprouts can be calculated on the basis of their relative ion abundances. The data on amino acids measured with leaf spray using a set of 5-day mung bean sprouts are shown in Table S-2 (see Supporting Information). The relatively low standard deviation (RSD < 20%) of the measurements for each amino acid indicates that the spray solvent extraction and ionization process is reliable and reproducible. 7611

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Figure 4. Glucosinolate cleavage under mechanical stress. (a) Leaf spray spectra acquired from cauliflower leaves in negative ion mode with added spray solvent. Data was recorded from cut cauliflower leaves immediately after the cutting and 5 min after punching. The relative ion abundances of glucoiberin, glucobrassicin, and neoglucobrassicin (m/z 422.0, m/z 447.1, and m/z 477.1) dramatically decrease within 5 min after applying the mechanical stress on cauliflower leaves. Assignments given are based on exact mass and/or MS/MS data. (b) Chemical response to mechanical stress on cabbage leaf seen in glucosinolate signals. Diamond plots represent the relative abundances of sinigrin that are expressed as the ratio of the ion abundance of sinigrin to that of disaccharide. Inset shows typical MS, intact sinigrin, m/z 358.0, deprotonated disaccharide, m/z 359.1. Error bars are based on five independent measurements.

Besides fresh and living plant tissue, leaf spray also works very well on dehydrated plant tissues provided spray solvent is applied. The phytochemicals are more concentrated in the dehydrated plant tissues, and the low water content in dehydrated tissues makes it more efficient in generating charged droplets with spray solvents of low surface tension. As a demonstration, green tea leaf was tested. The leaves were produced in dehydrated form for long-term storage and transportation and used directly without cutting. Methanol was used as spray solvent, and a relatively large volume was applied to fully wet the leaves rather than just wetting the surface as was done with fresh plant tissues. Four species were identified in the positive ion mode for green tea leaf (see Supporting Information, Figure S-7a), choline (m/z 104), protonated caffeine (m/z 195), and the potassium adducts

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of glucose (m/z 219) and sucrose (m/z 381). The negative ion mode spectrum shows a series of phytochemicals, most of which are derivatives of flavan-3-ols (see Supporting Information, Figure S-7b). Besides the speed, simplicity, and sensitivity achieved in identifying endogenous compounds in plants, direct monitoring of metabolic processes in plants using MS may become possible by leaf spray. As a test of this possibility, we used leaf spray to monitor glucosinolate cleavage in cabbage leaves under mechanical stress. Glucosinolates are a group of secondary metabolites that are rapidly hydrolyzed by myrosinase in leaves when damaged by herbivores (see Supporting Information, Scheme S-1).34 A series of intact glucosinolates was identified in the leaves of cabbage, Brussels sprout, cauliflower, and Arabidopsis thaliana using leaf spray; they include sinigrin, progoitrin, glucoiberin, glucobrassicin, glucoraphanin, neoglucobrassicin, and glucohirsutin (Figure S-12 in the Supporting Information). Similar results were obtained for in vivo experiments on potted plants using cauliflower leaves. In these experiments, the glucosinolates monitored were glucoiberin, glucobrassicin, and neoglucobrassicin (m/z 422.0, m/z 447.1, and m/z 477.1, respectively). As shown in Figure 4a, the relative abundances of these three glucosinolates decrease dramatically within 5 min of applying mechanical stress to the cauliflower leaves. In another experiment, the cabbage leaf was cut into triangles for leaf spray, and the cutting action itself triggered the hydrolysis of glucosinolates in cabbage leaf. For each test, five leaf triangles (1 cm long and 5 mm wide at the base) were cut from the same cabbage leaf at the same time and each of them was analyzed using leaf spray in the negative ion mode at a different time, viz., after 0, 3, 10, 20, and 40 min, respectively (Table S-3, Supporting Information). Methanol (20 μL) was used as spray solvent and applied to the center of leaf triangles for all tests. The leaf triangles were not reused in the analysis at different times to avoid the loss of the chemicals due to respraying. The relative abundance of sinigrin at each time point is reported in Figure 4b using the intensity of the deprotonated disaccharide (m/z 359.1) as an internal standard. The concentration of the disaccharide can change due to mechanical stress, but only over a much longer time period (>24 h).35 The rapid cleavage (less than 3 min) of sinigrin leads to a relatively larger error in the plot at short time. After the initial drop, the relative abundance of sinigrin increases slightly over a 40 min period. The rise of sinigrin appears to have been induced by the enhanced production of glucosinolates in cabbage leaf which is a chemical response to the mechanical stress.

’ CONCLUSION Leaf spray mass spectrometry allows in vivo analysis of plants and characterization of plant materials. Chemicals are directly ionized from a wide range of plant tissues with minimum sample treatment, and the measurement itself minimally perturbs the intrinsic levels of phytochemicals. The general approach used is likely applicable to many other types of materials as already evidenced by the many applications of paper spray and related methods.36 Many of the compounds in plant tissues are of great interest for pharmaceutical drug development, natural product research, and understanding of primary and secondary metabolism, and this new tool should enhance these efforts.37 40 7612

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’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Authors

*Phone: (765) 494-5262 (R.G.C.); (765) 494-2214 (Z.O.). Fax: (765) 494-9421 (R.G.C.); (765) 496-1912 (Z.O.). E-mail: [email protected] (R.G.C.); [email protected] (Z.O.).

’ ACKNOWLEDGMENT This research was supported by the National Science Foundation (Projects CHE-0847205, CHE-0848650, and DBI-0852740). Helpful discussions with David Rhodes and Angus Murphy are gratefully acknowledged. ’ REFERENCES (1) Baloglu, E.; Kingston, D. G. I. J. Nat. Prod. 1999, 62, 1448–1472. (2) Wood, K. V.; Bonham, C. C.; Miles, D.; Rothwell, A. P.; Peel, G.; Wood, B. C.; Rhodes, D. Phytochemistry 2002, 59, 759–765. (3) Cooks, R. G.; Warren, F. L.; Williams, D. H. J. Chem. Soc. C 1967, 286–288. (4) Lin, L.-Z.; Harnly, J. M. J. Agric. Food Chem. 2007, 55, 1084–1096. (5) Wu, W.; Liang, Z.; Zhao, Z.; Cai, Z. J. Mass Spectrom. 2007, 42, 58–69. (6) Ng, K.-M.; Liang, Z.; Lu, W.; Tang, H.-W.; Zhao, Z.; Che, C.-M.; Cheng, Y.-C. Anal. Chem. 2007, 79, 2745–2755. (7) Kondrat, R. W.; Cooks, R. G.; Laughlin, J. L. M. Science 1978, 199, 978–980. (8) Weston, D. J. Analyst 2010, 135, 661–668. (9) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566–1570. (10) Harris, G. A.; Galhena, A. S.; Fernandez, F. M. Anal. Chem. 2011, 83, 4508–4538. (11) Lane, A. L.; Nyadong, L.; Galhena, A. S.; Shearer, T. L.; Stout, E. P.; Parry, R. M.; Kwasnik, M.; Wang, M. D.; Hay, M. E.; Fernandez, F. M.; Kubanek, J. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 7314–7319. (12) Talaty, N.; Takats, Z.; Cooks, R. G. Analyst 2005, 130, 1624–1633. (13) Thunig, J.; Hansen, S. H.; Janfelt, C. Anal. Chem. 2011, 83, 3256–3259. (14) Jackson, A. U.; Tata, A.; Wu, C.; Perry, R. H.; Haas, G.; West, L.; Cooks, R. G. Analyst 2009, 134, 867–874. (15) Nyadong, L.; Hohenstein, E. G.; Galhena, A.; Lane, A. L.; Kubanek, J.; Sherrill, C. D.; Fernandez, F. M. Anal. Bioanal. Chem. 2009, 394, 245–254. (16) Nemes, P.; Barton, A. A.; Li, Y.; Vertes, A. Anal. Chem. 2008, 80, 4575–4582. (17) Nemes, P.; Barton, A. A.; Vertes, A. Anal. Chem. 2009, 81, 6668–6675. (18) Nemes, P.; Vertes, A. Anal. Chem. 2007, 79, 8098–8106. (19) Judge, E. J.; Brady, J. J.; Barbano, P. E.; Levis, R. J. Anal. Chem. 2011, 83, 2145–2151. (20) Chen, H.; Sun, Y.; Wortmann, A.; Gu, H.; Zenobi, R. Anal. Chem. 2007, 79, 1447–1455. (21) Chen, H.; Yang, S.; Wortmann, A.; Zenobi, R. Angew. Chem., Int. Ed. 2007, 46, 7591–7594. (22) Hsieh, C.-H.; Chang, C.-H.; Urban, P. L.; Chen, Y.-C. Anal. Chem. 2011, 83, 2866–2869. (23) Coello, Y.; Jones, A. D.; Gunaratne, T. C.; Dantus, M. Anal. Chem. 2010, 82, 2753–2758.

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