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

Summary

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.

Abstract

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.

Introduction

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-spray^5,6, is a direct ambient ionization MS technique, which requires essentially no sample preparation^5,7. 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 others^{6,8,9,10,11,12}. 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 effects^14,15. Currently, standardized extracts are used for the treatment of neuropsychiatric and neuropsychological disorders^16,17. 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.

Protocol

1. Modifications to Nanospray Source for Leaf Spray MS

1. 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.
2. 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 or may not have teeth. For small tissue, a clamp without teeth is preferred. An optional flex arm with a clamp may be added to the nanospray source to assist in positioning the plant tissue. Note that this protocol specifically describes how to perform leaf spray MS on a hybrid quadrupole ion trap mass analyzer MS system (see the Table of Materials); however, other MS systems may be altered to perform this technique⁶. Coupling leaf spray MS with a portable mass spectrometer real-time chemical analysis can be performed on-site without the need to transport the plant material to the laboratory^19,20.
3. Position an anti-static floor mat on the floor below the source to reduce the electrical discharge that may occur from the source when using high voltages.

2. Preparation of the MS System for Leaf Spray MS

1. If the system has recently been in use, allow it to cool to the touch and remove any alternative source and the sweep cone. Attach the nanospray leaf spray MS source.
2. Create a tune file with the appropriate ionization parameters set as follows: sheath, auxiliary, and sweep gas to 0; the spray voltage to 2 - 5 kV; the capillary temperature to 150 - 250 °C; and the S-lens RF level to 50. Save the tune file with the desired parameters^8,13. Optimize the voltage and temperature for the best ionization of the tissue and compound(s) of interest.
   NOTE: Good starting points are 4 kV and 200 °C.
3. Make a method file including the leaf spray MS tune file with: positive and negative full MS; a resolution of 70,000; an AGC target of 1 x 10⁶; a maximum IT of 200 ms; and the desired scan range m/z. Alternatively, use only 1 polarity.

3. Preparation of Instrument, Solvents, and Plant Tissue

NOTE: 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.

1. Bring the plant tissues for the analysis to the same room as the MS system to allow for a rapid sampling.
2. For plant tissue that does not have a naturally pointed tip, use a razor blade on a glass slide to cut a tapered point (Figure 2). Determine the amount of tissue needed for the analysis based on the instrument sensitivity, tissue type, and compound(s) of interest (e.g., a young S. tortuosum leaf that is ~5 mm in length).
   1. Cut S. tortuosum leaves at 10 weeks post-germination into thin strips, each with a tapered end to form a point.
3. 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.
   CAUTION: Do not touch the instrument source if the voltage is on.
4. Adjust the flexible arm and the wire with the clamp to position the tissue in line with the MS inlet so that the distance between the plant tissue and the ion inlet of the MS is 5 - 10 mm for the triple quadrupole (e.g., TSQ) and linear trap quadrupole (LTQ) and 10 - 50 mm for the ion trap mass analyzer (e.g., orbitrap)⁸.
   1. Plug the opposite end of the wire into the source. If the first attempts produce a low signal intensity, move the plant tissue closer to the ion inlet (refer to the Discussion for optimization).
5. 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.
6. Apply a solvent (e.g., methanol) using a pipet with a gel-loading tip to maximize the distance between the hands and the high voltage to protect the user.
   NOTE: The solvent volume required depends on the size, dryness, and texture of the tissue, typically ~2 - 20 µL. S. tortuosum leaves do not require any solvent to be added. Carefully apply the solvent and do not touch the instrument source when the voltage is on. Use LC-MS grade solvents and glassware that has been acid washed and is free of detergents. In some tissues, a signal may be observed without the addition of a solvent due to the natural water content of the plant tissue. However, a greater signal intensity and reduced S/N is typically achieved by applying a solvent to the tissue.
7. Acquire data as long as the signal persists or until the adequate spectra have been collected, typically 30 - 60 s. If needed, apply additional solvent to maintain a high signal intensity for a longer amount of time. Stop the data acquisition and pause the MS system.
8. Remove the tissue and wash the clamp with 100% methanol and a lint-free wipe. Clean the MS ion inlet after approximately 1 - 2 h of the acquisition by leaf spray MS according to the vendor's specifications. Also, clean the MS ion inlet between analyses of different tissue types.

4. Data Quality Assessment

1. Open the data file and visually inspect the base peak mass chronogram. Check that the signal intensity is ~1.0 x 10⁷ to 5.0 x 10⁸. If the signal is lower, move the tissue closer to the ion inlet. If higher, the front end of the MS system will become dirty, so move the tissue further from the ion inlet.
2. Based on the presence or absence of the ions of interest in the mass spectra produced, alter the parameters.
   NOTE: The protocol can be paused here.

5. Tandem Mass Fragmentation

1. Decide what ions are of interest for the tandem mass fragmentation (MS/MS); a mass spectra signal that is > 1.0 x 10⁵ is sufficient for the selection of ions for MS/MS.
2. Make a new method file with an inclusion list of m/z out to 4 decimal places. Click on Global lists and Inclusion. Under properties of PRM, select the fragmentation energy [e.g., normalized collision energy (NCE) of 30 - 50 is a good range to start with] and other MS/MS parameters.
   1. To obtain MS/MS data for mesembrine alkaloids, fragment the following ions, 276.1583 m/z, 290.1742 m/z, and 292.1897 m/z, at NCE 35.
      NOTE: The MS/MS data acquisition can be performed immediately after the MS or at a later time. The same tissue can often remain clamped after a full MS and can be reused to acquire MS/MS data. However, if a respraying does not provide a sufficient signal, use a new tissue.
3. Load the MS/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 - 60 s, stop the acquisition.
4. Collect fragmentation at many different energies when assigning fragment ions.
   NOTE: Since leaf spray MS lacks a chromatographic separation, the fragmentation spectra are likely to contain many ions, and fragmenting at different energies will help bring clarity.

6. Putative Identifications by Accurate Mass and Tandem Mass Fragmentation

1. Make putative identifications by referencing accurate mass measurements from publicly available metabolite databases such as Metlin²¹, Human Metabolome Database²², Mass Bank²³, Lipid Maps²⁴, National Institute of Standards and Technology MS Search²⁵, ReSpect for Phytochemicals²⁶, or GNPS²⁷.
2. As these databases are not exhaustive, perform an additional literature review on the plant species chemically characterized as necessary.
3. Match fragmentation ions from leaf spray MS/MS to the aforementioned databases when MS/MS information is available, or to the literature. Alternatively, use a manual interpretation of MS/MS fragment ions or a fragmentation of an authentic standard performed by the direct injection or LC-MS/MS.

7. Data Analysis

1. Convert the MS raw files to mzXML files with the msConvert tool from Proteowizard²⁸.
2. Use the XCMS software package implemented in R for peak picking. Use a direct infusion processing method for the leaf spray MS analysis.
   NOTE: The well-annotated scripts used for data processing can be found at https://github.com/HegemanLab/Leaf-Spray-Code.
3. To obtain semi-quantitative measurements, accounting for the experimental variability, normalize the intensity of each metabolite by the total ion current (TIC), as the leaf spray MS signal intensity can vary, in part due to slight variations in the positioning of the leaf in the source and the differences in leaf shape and size.
4. Alternatively, use vendor-provided software for the data analysis or MZmine2 (to be found at http://mzmine.github.io/)²⁹.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
Conn Pin	Digi-Key elctronics	WM2563CT-ND	pin will insert into Thermo Scientific source to provide voltage
small clamp	Digi-Key elctronics	314-1018-ND	CLIP MICRO ALLIGATOR COPPER 5A
large clamp	Digi-Key elctronics	290-1951-ND	ALLIGATOR CLIP NARROW NICKLE 5A
Heat shrink	Digi-Key elctronics	Q2Z1-KIT-ND	to cover soldering joints
NSI source Nanospray Ion Source	Thermo scientific	NA	Another brand will work if you are not using a Thermo instrument
Q Exactive- hybrid quadrupole Orbitrap	Thermo scientific	NA	Another brand will work if you are not using a Thermo instrument
Tune Software	Thermo scientific		Another brand will work if you are not using a Thermo instrument
Xcalibur Software	Thermo scientific
Plant of interest - S. tortousum