Analysis of Secondary Plant Metabolites by Indirect Desorption

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Analysis of Secondary Plant Metabolites by Indirect Desorption Electrospray Ionization Imaging Mass Spectrometry Janina Thunig, Steen H. Hansen, and Christian Janfelt* Department of Pharmaceutics and Analytical Chemistry, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark

bS Supporting Information ABSTRACT: Secondary metabolites in plant material can be imaged in a simple and robust way by creating an imprint of the plant material on a porous Teflon surface. The Teflon surface serves to extract compounds from the plant material for enhanced desorption electrospray ionization imaging analysis, while maintaining the spatial information of the sample. The method, which remedies for limitations in mass spectrometry imaging of compounds embedded in plant material, was demonstrated on leaves and petals of Hypericum perforatum and leaves of Datura stramonium.

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nowing the distribution of secondary metabolites in plants is important for understanding their function, biosynthesis, and possible transport within the plant. The enzymes involved in the biosynthesis of compounds can be imaged quite specifically using, e.g., immunolocalization,1 whereas for smaller molecules, such as secondary metabolites, imaging by staining and microscopy is not very specific. However, with the recent advances in imaging mass spectrometry, highly specific imaging of small molecules in a variety of biomaterials is now possible. Imaging mass spectrometry2 has been available for many years, initially with secondary-ion mass spectrometry (SIMS)3 and later with matrix assisted laser desorption ionization (MALDI).4,5 An alternative technique, desorption electrospray ionization (DESI) imaging,6,7 was presented by Cooks and co-workers in 2006 where the analysis takes place under ambient conditions without applying any matrix. Although the spatial resolution in DESI imaging is limited to around 100 μm, DESI has the advantage that it is simpler in use and instrumentation compared to SIMS and MALDI imaging. It is, thus, in general easier to build (or more affordable to purchase) and can be implemented on most existing electrospray mass spectrometers. In the recent years, several examples of imaging of animal tissue has been presented, using DESI as well as MALDI, revealing, for example, the distribution of lipids in rat brain tissue,7 the distribution of neuropeptides in a mouse pituitary gland,8 or the distribution of a drug dosed to a rat.9 However, until now, relative little mass spectrometry imaging of plant tissue has been reported, in spite of the fact that plants form the basis of the whole area of natural medicines and serve as a vast source of inspiration in the development of new drugs. MALDI and laser desorption ionization (LDI) imaging of plants has been reported,10,11 but reproducibility is often a challenge to MALDI imaging on natural products,12 one of the challenges, for example, being that the laser should be able to penetrate the r 2011 American Chemical Society

cuticle of a plant.12,13 Indeed, in the case of GALDI imaging of Arabidopsis leaves, ions from flavonoids were only detected from the portion of the leaves that were damaged during attachment to the MS plate or after removal of the cuticular waxes by chloroform.13 Solutions to this have been found with the use of infrared lasers in MALDI14 and the presentation of novel techniques such as electrospray-assisted laser desorption/ionization (ELDI)15 and laser ablation electrospray ionization (LAESI),16 capable of disintegrating the plant tissue and thereby enabling direct analysis of plant metabolites. Infrared (IR)-MALDI was, thus, applied to image the petals of the lilly flower (Lilium candidum),14 and LAESI was applied to image the leaves of the Zebra plant (Aphelandra squarrosa),17 even allowing 3D imaging to be performed, due to the stepwise removal of tissue by the laser ablation.18 The IR-MALDI imaging was performed with 200 μm spatial resolution, and the 2D and 3D LAESI imaging was performed with 400 and 500 μm spatial resolution, respectively. The use of an IR laser, thus, in general implies a decrease in spatial resolution compared to traditional UV-MALDI, although this is likely to improve in the future.18 Initial results of desorption atmospheric pressure photoionization (DAPPI) imaging of dried plant material were recently presented,19 however only offering a quite modest spatial resolution of 1 mm. Nonimaging DESI work on plants has been published,20 and DESI imaging was recently used in the study of a red alga to elucidate its defense mechanisms.21 One of the problems in DESI imaging on plant surfaces such as flowers and leaves is to obtain an MS signal stable enough for imaging. The insufficient signal intensity and stability is partly Received: February 25, 2011 Accepted: April 7, 2011 Published: April 07, 2011 3256

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Analytical Chemistry

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Figure 1. Leaf of Hypericum perforatum. (a) Optical image. (b) Optical image of the Teflon imprint taken after recording of the DESI images. (c) Indirect DESI image of hyperforin (m/z 535). (d) Indirect DESI image of hypericin (m/z 503). The pixel size is 100 μm, and the acquisition time is 80 min.

related to the problems of penetrating the cuticle of plant material and partly to the nature of most plant material being relatively soft and absorbing, in contrast to the hard and nonabsorbing surfaces usually preferred in DESI experiments.22,23 Here, we present a further development of imaging mass spectrometry which enables simple and highly reproducible imaging of secondary metabolites in plant tissue. It is based on an indirect approach where an imprint of the sample is made on a microporous Teflon surface, thereby extracting the compounds from their matrix while maintaining the spatial integrity of the sample. In previous DESI studies, porous Teflon was found to be ideal for DESI analysis of liquids deposited on the surface.24 We have found that the porous Teflon surface can also be used in DESI imaging work on plant material by pressing the plant material directly on the surface. An imprint of the plant material containing the compounds is obtained on the surface, providing a very clear, intense, and stable signal, compared to that obtained in a direct DESI analysis of the same plant material. A similar approach was recently taken in imaging of bacterial metabolites from agar plates via a mixed cellulose ester filter membrane.25 Using this indirect imaging method, we have recorded DESI images of Hypericum perforatum L. (St. John’s wort, leaves and petals), Datura stramonium L. (thorn apple, leaves), and Papaver somniferum L. (opium poppy, capsule). The indirect DESI images were recorded with high signal intensities and good reproducibility, providing higher success ratios compared to most other imaging mass spectrometry methods (Figure S-1, Supporting Information).

’ EXPERIMENTAL SECTION The imprints were made of fresh plant material which was subjected to hard pressure against a 1.5 mm thick porous Teflon surface in a vice for 5 min. More details on the procedure are found in the Supporting Information. DESI imaging of the Teflon imprints was performed on a Thermo LTQ XL linear ion trap mass spectrometer, equipped with a DESI imaging source based on a M€arzh€auser-Wetzlar microscope stage controlled by software written in-house. Data conversion was made with an imzML26 converter (available from

www.maldi-msi.org), and Data Cube Explorer (AMOLF, Amsterdam) was used for image generation. MATLAB was used to create colored overlaid images. H. perforatum was analyzed in negative ion mode with a spray voltage of 5 kV and a 50:50 mixture of MeOH/H20 with 1% of ammonia as solvent, and D. stramonium was analyzed in positive ion mode with a spray voltage of 5 kV and a 50:50 mixture of MeOH/H20 with 1% of formic acid as solvent.

’ RESULTS AND DISCUSSION Hypericum perforatum. Extracts of H. perforatum are used as a drug against depression and show antiviral and antibacterial activities.27,28 The green leaves of the plant have translucent glands randomly distributed on the leaves as well as dark glands along the edge of the leaves, while the petals appear only to have the dark but no translucent glands. We analyzed a number of leaves, petals, and whole flowers of H. perforatum using indirect DESI imaging in negative ion mode. Figure 1a shows a photo of a leaf, and Figure 1b shows the corresponding imprint on the Teflon surface. It is observed that hyperforin (m/z 535, Figure 1c) is located in the translucent glands while hypericin (m/z 503, Figure 1d) is located in the dark glands. The hyperforin distribution confirms the findings of a recent study in which the translucent glands were punched out and analyzed with traditional methods based on extraction and LC-MS, showing an increased abundance of hyperforin in the translucent glands.29 Correspondingly, the distribution of hypericin in the dark glands confirms another recent study which by means of extraction and LC-UV showed that hypericin was synthesized and accumulated in these glands.30 Besides hyperforin and hypericin several other secondary metabolites known from H. perforatum were detected and identified by MS/MS. MS/MS identification was performed on the imprints on the translucent glands for hyperforin and adhyperforin and on the dark glands for hypericin, pseudohypericin, protopseudohypericin, and protohypericin, thus confirming the distributions found from the images recorded in full scan mode. The images of these secondary metabolites as well as their MS/MS spectra are found in Figure S-2, Supporting Information. 3257

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Analytical Chemistry

Figure 2. Petals of Hypericum perforatum. (a) Optical image. (b) Optical image of the Teflon imprint. (cf) Indirect DESI images of four different petals of Hypericum perforatum; c is the image of the petal of the optical images in a and b. An unidentified ion at m/z 367 reveals the shape of the petals (green). Hypericin (red, m/z 503) is found in the black glands along the edges, and hyperforin (blue, m/z 535) is found in spots (c þ d) and broad bands (e þ f), respectively. The pixel size is 100 μm, and the acquisition times were approximately 80 min each.

In the case of hyperforin, the distribution in the translucent glands was also confirmed by recording an MS/MS image based on the fragmentation of the m/z 535 peak of hyperforin. Images of the three characteristic fragments at m/z 466, m/z 397, and m/z 383 are seen in Figure S-3, Supporting Information. Since the petals of the H. perforatum flower also have black glands, the distribution of hypericin can be visually assessed, whereas the absence of translucent glands makes assessment of the hyperforin distribution in the petals impossible with traditional methods. We imaged several petals and found that the hyperforin distribution was varying significantly from petal to petal as seen in Figure 2, showing the distributions of hyperforin and hypericin in four different petals. The shape of the petals is seen in the distribution of an unidentified compound at m/z 367 which is present everywhere in the petal where hyperforin in not present. In many petals, hyperforin (shown in blue) is only present in small randomly distributed spots (Figure 2c,d), while in others it seems to be located in broad bands parallel to the veins (Figure 2e,f). Using this approach, it will be elucidated in future studies whether these bands are related to the secretory transport canals described by Cicarelli and co-workers31 or an effect of the asymmetry of the petals originating from their packing in the bud. Datura stramonium. Datura stramonium is known for its contents of anticholinergic secondary metabolites.32 Figure 3 shows indirect DESI images in positive ion mode of a small leaf of the plant, revealing the distributions of the tropane alkaloids atropine (m/z 290, Figure 3c) and scopolamine (m/z 304, Figure 3d) as well as hexose sugars (m/z 219, Figure 3e) and sucrose (m/z 381, Figure 3f). A spectrum from the images is found in Figure S-4, Supporting Information. The two alkaloids appear to be more abundant in the ribs and veins of the leaf which could suggest where the metabolites are being transported within the plant. The imaged distributions are not due to uneven

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Figure 3. Leaf of Datura stramonium. (a) Optical image. (b) Optical image of the Teflon imprint. (c) Indirect DESI image of atropin (m/z 290). (d) Indirect DESI image of scopolamin (m/z 304). (e) Indirect DESI image of hexose sugars (Kþ adduct, m/z 219). (f) Indirect DESI image of sucrose (Kþ adduct, m/z 381). The pixel size is 125 μm, and the acquisition time was 4 h and 10 min.

pressing of the leaf nor to incomplete extraction over the leaf surface, as evidenced by the images of the two sugars, showing quite homogeneous distributions throughout the leaf surface. Likewise, the photo of the imprint (Figure 3b) does not indicate any incomplete pressing of the leaf surface. Traditional direct DESI imaging of the plant material was attempted without any success, mainly due to the signal being too low and fluctuating for use with imaging. In some cases, e.g., cyano glucosides in barley, direct DESI analysis was impossible, while indirect DESI analysis via the porous Teflon surface was quite straightforward once the compounds were released from the plant material to the Teflon surface. In other cases, e.g., the leaves of H. perforatum, direct DESI analysis was possible (i.e., spectra could be obtained) but only of a quality which was inadequate for imaging. The main advantages of the imprinting method are the elimination of the problems of penetrating the cuticle, as well as a general decrease in matrix effects in the analysis due to extraction of compounds from the plant material while preserving the spatial information. The extraction of analytes to the porous Teflon appears to be quite nonselective with regard to polarity. In similar work on the opium poppy (Papaver somniferum), both quite nonpolar alkaloids such as papaverine as well as relatively polar alkaloids such as morphine were successfully extracted to the surface. The mutual intensities in the resulting indirect DESI spectra are, thus, comparable with ESI spectra of methanol extract of the sample (Figure S-5, Supporting Information). The explanation is likely that the majority of the liquid phase from the fresh plant material with all its contents (regardless of polarity) is pressed into the pores of the Teflon surface, in contrast to the traditional, more selective solid phase extraction experiment where the liquid is 3258

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Analytical Chemistry passed through a nonpolar sorbent material and all polar compounds are washed off the column.

’ CONCLUSION The ability to generate reproducible DESI images of plant material in a simple and robust manner opens the way for analyzing a large number of plants of interest. DESI imaging of plant material is relevant in the study of enzymatic activities in plants, since conventional immunostaining followed by microscopy can be used to localize enzymes, while DESI imaging can complement the picture with images of substrate and product distributions. Furthermore, images of secondary metabolites in plants provide new information about biosynthesis and subsequent transport of metabolites in plants, e.g., by comparing images of different secondary metabolites, revealing which compounds are formed together. The function of secondary metabolites could be revealed by correlating their distributions with the feeding pattern of insects.11 Imaging mass spectrometry could also be used as a supplementary tool in the dereplication of natural products in the search for bioactive compounds. As for most imaging methods, the obtained results are only of qualitative nature; the method, thus serves, as an initial screening method for distributions, providing a good starting point for subsequent quantitative analysis with extraction and LC-MS. It is likely that the extra sensitivity obtained with the method presented here would apply to other objects of interest, such as animal tissue sections where higher sensitivity is desired in imaging of drug and metabolite distributions. Furthermore, the method may also be modified for use in MALDI imaging in order to circumvent the problem of penetrating the cuticle of plants and other materials. ’ 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 Author

*E-mail: [email protected]. Phone: þ45 35 33 65 57. Fax: þ45 35 33 60 30.

’ ACKNOWLEDGMENT The authors thank Nanna Bjarnholt for useful discussions and Ralf Sack for his work on the construction of the DESI imaging source. Support from the Carlsberg Foundation, The Danish Council for Independent Research|Natural Sciences, and LEO Pharma A/S is gratefully acknowledged. ’ REFERENCES

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1994; The American Society for Mass Spectrometry: Santa Fe, NM, 1994; p 1041. (5) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69 4751–4760. (6) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (7) Wiseman, J. M.; Ifa, D. R.; Song, Q. Y.; Cooks, R. G. Angew. Chem., Int. Ed. 2006, 45, 7188–7192. (8) Rompp, A.; Guenther, S.; Schober, Y.; Schulz, O.; Takats, Z.; Kummer, W.; Spengler, B. Angew. Chem., Int. Ed. 2010, 49, 3834–3838. (9) Kertesz, V.; Van Berkel, G. J.; Vavrek, M.; Koeplinger, K. A.; Schneider, B. B.; Covey, T. R. Anal. Chem. 2008, 80, 5168–5177. (10) Holscher, D.; Shroff, R.; Knop, K.; Gottschaldt, M.; Crecelius, A.; Schneider, B.; Heckel, D. G.; Schubert, U. S.; Svatos, A. Plant J. 2009, 60, 907–918. (11) Shroff, R.; Vergara, F.; Muck, A.; Svatos, A.; Gershenzon, J. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 6196–6201. (12) Esquenazi, E.; Yang, Y. L.; Watrous, J.; Gerwick, W. H.; Dorrestein, P. C. Nat. Prod. Rep. 2009, 26, 1521–1534. (13) Cha, S. W.; Zhang, H.; Ilarslan, H. I.; Wurtele, E. S.; Brachova, L.; Nikolau, B. J.; Yeung, E. S. Plant J. 2008, 55, 348–360. (14) Li, Y.; Shrestha, B.; Vertes, A. Anal. Chem. 2008, 80, 407–420. (15) Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. Rapid Commun. Mass Spectrom. 2005, 19, 3701–3704. (16) Nemes, P.; Vertes, A. Anal. Chem. 2007, 79, 8098–8106. (17) Nemes, P.; Barton, A. A.; Li, Y.; Vertes, A. Anal. Chem. 2008, 80 4575–4582. (18) Nemes, P.; Barton, A. A.; Vertes, A. Anal. Chem. 2009, 81 6668–6675. (19) Pol, J.; Vidova, V.; Kruppa, G.; Kobliha, V.; Novak, P.; Lemr, K.; Kotiaho, T.; Kostiainen, R.; Havlicek, V.; Volny, M. Anal. Chem. 2009, 81, 8479–8487. (20) Jackson, A. U.; Tata, A.; Wu, C. P.; Perry, R. H.; Haas, G.; West, L.; Cooks, R. G. Analyst 2009, 134, 867–874. (21) 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. (22) Zivolic, F.; Zancanaro, F.; Favretto, D.; Ferrara, S. D.; Seraglia, R.; Traldi, P. J. Mass Spectrom. 2010, 45, 411–420. (23) Costa, A. B.; Cooks, R. G. Chem. Phys. Lett. 2008, 464, 1–8. (24) Ifa, D. R.; Manicke, N. E.; Rusine, A. L.; Cooks, R. G. Rapid Commun. Mass Spectrom. 2008, 22, 503–510. (25) Watrous, J.; Hendricks, N.; Meehan, M.; Dorrestein, P. C. Anal. Chem. 2010, 82, 1598–1600. (26) Roempp, A.; Schramm, T.; Hester, A.; Klinkert, I.; Both, J.-P.; Laprevote, O.; Brunelle, A.; Heeren, R. M. A.; Stoeckli, M.; Spengler, B. In 57th ASMS conference on mass spectrometry and allied topics; Philadelphia, PA, May 31June 4, 2009; The American Society for Mass Spectrometry: Santa Fe, NM, 2009. (27) Barnes, J.; Anderson, L. A.; Phillipson, J. D. J. Pharm. Pharmacol. 2001, 53, 583–600. (28) Nahrstedt, A.; Butterweck, V. J. Nat. Prod. 2010, 73, 1015–1021. (29) Soelberg, J.; Jorgensen, L. B.; Jager, A. K. Ann. Bot. 2007, 99 1097–1100. (30) Zobayed, S. M. A.; Afreen, F.; Goto, E.; Kozai, T. Ann. Bot. 2006 98, 793–804. (31) Ciccarelli, D.; Andreucci, A. C.; Pagni, A. M. Ann. Bot. 2001, 88 637–644. (32) Miraldi, E.; Masti, A.; Ferri, S.; Comparini, I. B. Fitoterapia 2001, 72, 644–648.

(1) Sauer, M.; Friml, J. In Plant Developmental Biology; Hennig, L.; K€ohler, C., Eds.; Humana Press: New York, 2010, Vol. 655, pp 253263. (2) McDonnell, L. A.; Heeren, R. M. A. Mass Spectrom. Rev. 2007, 26 606–643. (3) Liebl, H. J. Appl. Phys. 1967, 38, 5277–5283. (4) Spengler, B.; Hubert, M.; Kaufmann, R. In 42nd Annual Conference on Mass Spectrometry and Allied Topics; Chicago, IL, May 29June 3, 3259

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