Name: leaf spray mass spectrometry: a rapid ambient ionization technique to directly assess metabolites from plant tissues
Uploaded: 2018-06-21T15:00:00
Description: Watch this Scientific Journal Video about Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues at JoVE.com

This work was funded by the NSF Plant Genome Research Program grant IOS-1238812 and the Postdoctoral Fellowship in Biology IOS-1400818. The work was also funded by a Monsanto Graduate Student Fellowship to Katherine A. Sammons. The Fulbright African Researcher Scholars Program (2017-2018) is thanked for funding awarded to Nokwanda P. Makunga. We greatly appreciate the donation of a nanospray source from Jessica Prenni and the Proteomics and Metabolomics facility at Colorado State University.

1. Modifications to Nanospray Source for Leaf Spray MS
<ol>
	<li>Use a modified nanospray source for leaf spray MS. As no fluidic components are necessary for leaf spray MS, modify the source by removing the LC probe from the source.</li>
	<li>Assemble the leaf spray MS wire that will apply the voltage to the plant tissue with the appropriate pin for plugging into the source. Solder the pin to one end of an insulated wire; solder a clamp to the opposite end of the wire. 
	NOTE: The clamp (alligator clip type) may ...

<ol>
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G., Ouyang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+spray:+direct+chemical+analysis+of+plant+material+and+living+plants+by+mass+spectrometry.">Leaf spray: direct chemical analysis of plant material and living plants by mass spectrometry.</a> Analytical Chemistry. 83 (20), 7608-7613 (2011).</li><li>Chan, S. L. -. F., Wong, M. Y. -. M., Tang, H. -. W., Che, C. -. M., Ng, K. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-spray+ionization+mass+spectrometry+for+raw+herb+analysis.">Tissue-spray ionization mass spectrometry for raw herb analysis.</a> Rapid Communications in Mass Spectrometry. 25 (19), 2837-2843 (2011).</li><li>Wang, H., Liu, J., Cooks, R. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sceletium-a+review+update.">Sceletium-a review update.</a> Journal of Ethnopharmacology. 119 (3), 653-663 (2008).</li><li>Terburg, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+effects+of+Sceletium+tortuosum+(Zembrin),+a+dual+5-HT+reuptake+and+PDE4+inhibitor,+in+the+human+amygdala+and+its+connection+to+the+hypothalamus.">Acute effects of Sceletium tortuosum (Zembrin), a dual 5-HT reuptake and PDE4 inhibitor, in the human amygdala and its connection to the hypothalamus.</a> Neuropsychopharmacology. 38 (13), 2708-2716 (2013).</li><li>Coetzee, D. D., L&#243;pez, V., Smith, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-mesembrine+Sceletium+extract+(TrimesemineTM)+is+a+monoamine+releasing+agent,+rather+than+only+a+selective+serotonin+reuptake+inhibitor.">High-mesembrine Sceletium extract (TrimesemineTM) is a monoamine releasing agent, rather than only a selective serotonin reuptake inhibitor.</a> Journal of Ethnopharmacology. 177, 111-116 (2016).</li><li>Shikanga, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+permeation+of+mesembrine+alkaloids+from+Sceletium+tortuosum+across+porcine+buccal,+sublingual,+and+intestinal+mucosa.">In vitro permeation of mesembrine alkaloids from Sceletium tortuosum across porcine buccal, sublingual, and intestinal mucosa.</a> Planta Medica. 78 (3), 260-268 (2012).</li><li>Pulliam, C. J., Bain, R. M., Wiley, J. S., Ouyang, Z., Cooks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+in+the+home+and+garden.">Mass spectrometry in the home and garden.</a> Journal of The American Society for Mass Spectrometry. 26 (2), 224-230 (2015).</li><li>Lawton, Z. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+validation+of+a+portable+mass+spectrometer+featuring+interchangeable,+ambient+ionization+sources.">Analytical validation of a portable mass spectrometer featuring interchangeable, ambient ionization sources.</a> Journal of the American Society for Mass Spectrometry. 28 (6), 1048-1059 (2017).</li><li>. GNPS Available from: <a target="_blank" href="https://gnps.ucsd.edu/">https://gnps.ucsd.edu/</a> (2018)</li><li>Chambers, M. 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The successful use of this protocol relies on the optimization of various steps for the plant species, tissue type, and target compound(s) of interest. The parameters described in the protocol provide a good starting point. The following experimental decisions need to be made and tested: whether or not to use (1) cut or uncut tissue and (2) solvent or no solvent, (3) what solvent to use and in what volume, (4) what the distance of tissue from the ion inlet should be, and (5) the voltage amplitude. The goal of optimizatio...

Plants contain a wide range of small molecules with diverse chemical properties. MS is a powerful technique for analyzing plant compounds because it can provide elemental compositions with a high sensitivity and specificity for the detection and identification of metabolites1. Most commonly, MS is performed on solvent-extracted samples, which are separated by chromatography prior to the MS analysis1. However, the use of liquid chromatography (LC) requires lengthy analysis times and is often associated with an extensive sample preparation1. In contrast, direct chemical analysis of intact tissues that circumvents chromatography is a very rapid technique, requiring minimal sample preparation2. Thus, in instances where chromatographic steps can be forgone, a direct chemical analysis can be highly advantageous.
Typical LC-MS for natural products and metabolomics research relies on lengthy bulk extractions of dried or frozen plant materials containing multiple tissues and cell types3. Alternatively, direct chemical analysis, such as the MS detection of metabolites from plant tissue, can isolate cell types and avoid preparation artifacts4. Leaf spray MS, also referred to as tissue-spray5,6, is a direct ambient ionization MS technique, which requires essentially no sample preparation5,7. Leaf spray MS is closely related to paper spray MS, an ambient ionization technique with characteristics of electrospray ionization that allows for the detection of analytes that are deposited onto paper7. In spite of the name, leaf spray MS is applicable to various types of plant tissues, not just leaves, and has been demonstrated on fruit, seeds, roots, floral tissues, and tubers, among others6,8,9,10,11,12. The technique facilitates the ionization of endogenous phytochemicals directly from the plant materials into the mass spectrometer for detection8. Leaf spray MS can also provide information about the spatial distribution of chemicals in different tissue types in plants13. When leaf spray MS is compared with solvent extraction and LC-MS, the results suggest leaf spray MS allows for the rapid detection of surface metabolites from unique cell types such as trichomes13. Figure 1 illustrates the leaf spray MS experimental set-up. Direct electrospray ionization occurs after only minor source modifications. A high voltage is applied to the plant tissue via a metal clamp, producing a spray of highly charged droplets forming a Taylor cone that carries the ions to the ion inlet of the MS. Electrospray ionization occurs from the natural liquid of the plant or from the solvent applied to the plant surface. A pointed tip on the tissue facilitates the electrospray and can be naturally occurring or created by cutting.
Leaf spray MS is a fast method for the qualitative and semi-quantitative analysis of intact plant tissues that have found utility for a wide variety of applications. For example, the technique has been used to detect endogenous compounds to distinguish related species, and even to evaluate changes in the same species grown under different conditions. Previous studies have shown this approach by measuring metabolites in beautyberry (Callicarpa L.)12 and American ginseng (Panax quinquefolium L.)6. In the latter example, ginsenosides, amino acids, and oligosaccharides could be detected after wetting raw ginseng tissue. Wild and cultivated American ginseng were differentiated from tuber slices6. Ginseng tuber integrity was preserved succeeding leaf spray MS, which allowed for a subsequent morphological and microscopic inspection6. Furthermore, exogenous compounds on plant samples can also be detected. A number of pesticides (acetamiprid, diphenylamine, imazalil, linuron, and thiabendazole) have been detected on peel or pulp of fruits and vegetables9. While these studies and many others have shown the utility of leaf spray MS for various specific purposes, a detailed protocol has not been previously reported.
Here, the protocol description will not focus on the optimization of the method for a specific tissue or compound. Rather, the detection of mesembrine alkaloids from Sceletium tortuosum (L.) N.E.Br. (Aizoaceae) is used as an example to discuss the necessary optimization measures that should be taken when setting up a leaf spray MS experiment for a species, tissue, or compound(s) for the first time. S. tortuosum is a succulent endemic to the semi-arid Karroo region of South Africa. A traditional medicine of the San and Khoi Khoi peoples, it was used for appetite and thirst suppression as well as for its psychotropic and analgesic effects14,15. Currently, standardized extracts are used for the treatment of neuropsychiatric and neuropsychological disorders16,17. The primary compounds of interest include the alkaloid mesembrine and its derivatives, many of which are also found in related Sceletium species15. Both wild and cultivated populations of S. tortuosum have variable concentrations of mesembrine alkaloids, thus presenting a quality control challenge18. A method for the rapid detection of mesembrine alkaloids, such as leaf spray MS, may be useful in monitoring Sceletium products. Because previously, there had been no detailed visual experimental protocol for the leaf spray MS technique, we will illustrate the method using the example of S. tortuosum, and the following is described: the modification of a nanospray source, the selection and preparation of the plant tissues, the acquisition of the data, the interpretation of the results, and the optimization of the MS parameters.

<table border="0" cellpadding="0" cellspacing="0" width="880">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Conn Pin</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td style="width:119px;">WM2563CT-ND</td>
			<td style="width:385px;">pin will insert into Thermo Scientific&#160; source to provide voltage&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">small clamp</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td>314-1018-ND</td>
			<td>CLIP MICRO ALLIGATOR COPPER 5A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">large clamp</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td>290-1951-ND</td>
			<td>ALLIGATOR CLIP NARROW NICKLE 5A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heat shrink</td>
			<td style="width:139px;">Digi-Key elctronics&#160;</td>
			<td style="width:119px;">Q2Z1-KIT-ND</td>
			<td>to cover soldering joints&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">NSI source Nanospray Ion Source</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;">NA</td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:237px;">Q Exactive- hybrid quadrupole Orbitrap</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;">NA</td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Tune Software</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td>Another brand will work if you are not using a Thermo instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Xcalibur Software</td>
			<td style="width:139px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:237px;">Plant of interest - S. tortousum</td>
			<td style="width:139px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

leaf spray mass spectrometry: a rapid ambient ionization technique to directly assess metabolites from plant tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

At 10 weeks post-germination, freshly collected greenhouse-grown S. tortuosum leaves were analyzed by leaf spray MS. The experimental workflow for detecting metabolites from S. tortuosum leaves using leaf spray MS is illustrated in Figure 2. A leaf was selected, cut into a thin strip with a tapered end to form a point, and clamped with the leaf spray MS wire clamp apparatus. The plant tissue was positioned ~30 mm from the ion inlet and in li...

Watch this Scientific Journal Video about Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues at JoVE.com

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Leaf spray MS is a fast method for qualitative and semi-quantitative analysis of intact plant tissues that has a wide variety of applications. The main advantage of this technique is plant tissue can be analyzed directly with essentially no sample preparation. Although this method provides good insights into mesembrine alkaloids from Sceletium, it can also be used for analysis of various other plant species and exogenous chemicals such as pesticides.Individuals new to this method may find it challenging because it is necessary to optimize the protocol for each plant species, tissue type, and compound of interest. Use a modified nano spray source for leaf spray MS.As no fluidic components are necessary for leaf spray MS, modify the source by removing the liquid chromatography probe from the source. The leaf spray mass spectrometry wire that will apply voltage to the plant tissue has a pin that has been soldered to the wire on one end and a clamp that has been soldered on the other end.If the system has recently been in use, allow it to cool to the touch before removing any alternative source in the sweep cone. Then attach the nano spray leaf spray MS source. Finally, create a tune file and a method file as described in the text protocol.Always wear gloves and do not use plant tissue that has been handled with bare hands. Otherwise, contaminant ions such as polyethylene glycol will dominate the spectra. Bring the plant tissues for the analysis to the same room as the mass spectrometry system to allow for a rapid sampling.For plant tissue that does not have a naturally pointed tip, use a razor blade on a glass slide to cut a tapered point. Cut Sceletium tortuosum leaves at 10 weeks post germination into thin strips each with a tapered end to form a point. Use forceps to gently select the plant tissue at the end that will be clamped.Holding the tissue with forceps, carefully transfer it to the clamp. Adjust the flexible arm and the wire with the clamp to position the tissue in line with the MS inlet. Refer to the text for the optimal distance from the ion inlet for each specific mass spectrometer.If the first attempts produce a low signal intensity, the plant tissue can be moved closer to the ion inlet. Load the method file. Name the data file and set the file storage location.Then turn on the MS system by clicking play and click on start to begin acquiring data. Apply a solvent using a pipette with a gel loading tip to maximize the distance between the high voltage and the user's hands for protection. Acquire data as long as the signal persists or until the adequate spectra have been collected, typically 30 to 60 seconds.Stop the data acquisition and pause the MS system. Following acquisition, remove the tissue and wash the clamp with 100%methanol in a lint-free wipe. Open the data file and visually inspect the base peak mass chronogram.Check that the signal intensity is approximately 10 million to 500 million. Make a new method file with an inclusion list of M over Z out to four decimal places. Click on global lists and inclusion.Under properties of PRM, select the fragmentation energy and other tandem MS parameters. To obtain the tandem MS data for mesembrine alkaloids, fragment the ions at 276.1583 M over Z, 290.1742 M over Z, and 292.1897 M over Z at a normalized collision energy of 35. Next, load the tandem MS method file and a named data file.Turn on the MS system and start acquiring data adding a solvent if necessary. When adequate spectra have been collected, typically after 30 to 60 seconds, stop the acquisition. Perform data analysis as described in the text protocol.Representative results of metabolic profiling of Sceletium tortuosum by leaf spray MS and positive ionization are shown with the inset displaying 260 to 295 M over Z.In this study, a total of nine mesembrine alkaloids were detected by leaf spray MS and puditive identifications were made. The leaf spray MS total ion count mass chronogram represents a successful leaf spray MS experiment where a stable signal was maintained for the full duration of the acquisition. For each peak, the top number is the time in minutes and the bottom is the M over Z.Tandem mass spectra were collected in positive ionization made from a Sceletium tortuosum cut leaf with leaf spray MS.Puditive identifications of the alkaloids were made from accurate mass and mass fragmentation for mesembrine M and mesembrenone M isomers, mesembrine and mesembrine M dihydro.After watching this video, you should have a good understanding of how to use leaf spray MS to detect plant metabolites. Leaf spray MS has been used to detect endogenous compounds as well as exogenous compounds such as pesticides.

Research

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Biochemistry

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