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JoVE Journal Medicine

Medicine

Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI

Published: December 16, 2022 doi: 10.3791/64912
Automatic Translation
1,4, 3, 3, 1, 1, 1,2
1Innovative Institute of Chinese Medicine and Pharmacy, Chengdu University of Traditional Chinese Medicine, 2Tianfu Chinese Medicine Innovation Harbour, Chengdu University of Traditional Chinese Medicine, 3School of Medical Technology, Chengdu University of Traditional Chinese Medicine, 4China Resources Sanjiu (Ya’an) Pharmaceutical Co., Ltd.

Summary

In this study, a series of methods are presented to prepare DESI-MSI samples from plants, and a procedure of DESI assembly installation, MSI data acquisition, and processing is described in detail. This protocol can be applied in several conditions for acquiring spatial metabolome information in plants.

Abstract

The medicinal use of traditional Chinese medicine is mainly due to its secondary metabolites. Visualization of the distribution of these metabolites has become a crucial topic in plant science. Mass spectrometry imaging can extract huge volumes of data and provide spatial distribution information about these by analyzing tissue slices. With the advantage of high throughput and higher accuracy, desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is often used in biological research and in the study of traditional Chinese medicine. However, the procedures used in this research are complicated and not affordable. In this study, we optimized sectioning and DESI imaging procedures and developed a more cost-effective method to identify the distribution of metabolites and categorize these compounds in plant tissues, with a special focus on traditional Chinese medicines. The study will promote the utilization of DESI in metabolite analysis and standardization of traditional Chinese medicine/ethnic medicine for research-related technologies.

Introduction

Visualization of metabolite distribution has become a crucial topic in plant science, especially in traditional Chinese medicine, as it unveils the formation process of specific metabolites within the plant. With reference to traditional Chinese medicine (TCM), it provides information regarding the active components and guides the application of plant parts in pharmaceutical applications. Normally, visualization of metabolites is achieved by in situ hybridization, fluorescence microscopy, or immunohistochemistry, however the number of compounds detected by these experiments conveys limited chemical information. Combined with tissue staining, mass spectrometry imaging (MSI) can provide large amount of data and supply spatial distribution information of compounds by scanning and analyzing tissue slices at micron-level1. MSI uses analytes for desorption and ionization from the sample surface, followed by mass analysis of the resulting vapor phase ions and application of imaging software to integrate the information and plot a two-dimensional image recording a specific ion abundance. This technology can determine both exogenous and endogenous molecules by detecting the characteristic distribution of drugs and their induced metabolites in target tissues and organs2,3,4,5.

Various imaging MS modalities have been developed over recent decades; the most prominent among them are desorption electrospray ionization-based MSI (DESI-MSI), matrix-assisted laser desorption/ionization (MALDI), and secondary ion mass spectrometry (SIMS)6. DESI-MSI is often used in biological research due to its atmospheric operation, high throughput, and higher accuracy7. MALDI has been applied to identify a transthyretin fragment as a potential nephrotoxic biomarker for gentamicin and to analyze the distribution of the neurotoxic metabolite 1-methyl-4-phenylpyridinium after the management of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice brains8,9. MALDI and DESI have been used to determine the composition of drug-induced crystal-like structures in the kidney of dosed rabbits; these structures are mainly composed of metabolites formed due to the demethylation and/or oxidation of the drug10. Additionally, MSI has been applied in the localization of metabolic distribution of drug toxicity in target organs. However, the cells in plant tissue vary and are different from animals and require special sectioning procedures.

In plants, by using MALDI imaging, so far, the distribution of different compounds in wheat (Triticum aestivum) stem, soya bean (Glycine max), rice (Oryza sativa) seeds, Arabidopsis thaliana flowers and roots, and barley (Hordeum vulgare) seeds have been analyzed11,12,13,14,15,16,17,18. Recent studies have reported that DESI-MSI is emerging in the metabolite analysis of natural drugs and products, especially in TCMs such as Ginkgo biloba, Fuzi, and Artemisia annua L19,20,21. In these studies, the protocols for the preparation of plant material samples differ, and some require more complex equipment, like a freezing microtome. DESI-MSI has strict requirements for the surface flatness of the detected sample. When analyzing the organ or tissue of an animal, the sample is usually made by cryo-sectioning22. However, the procedure for cryo-sectioning is complicated and more expensive, and the commonly used adhesive optimal cutting temperature (OCT) method has a strong signal when imaging. In addition, the medicinal tissues of TCM vary; for instance, the root of Salvia miltiorrhiza, known as Danshen in Chinese, is medicinally used, while in Zisu (Perilla frutescens), the leaf is used23,24. Therefore, it is necessary to improve the sample preparation procedures to promote the utilization of DESI in metabolite analysis for TCM.

As a perennial herb and a commonly used TCM, S. miltiorrhiza was initially recorded in the oldest medicine monograph, Shennong's Classic of Materia Medica (known as Shennong Bencao Jing in Chinese). In this study, we optimized sectioning and DESI imaging procedures and developed a more cost-effective method to identify the distribution and categorize the compounds in tissues of S. miltiorrhiza. This method can also overcome the disadvantages associated with dry tissues - that they usually easily fracture under the nitrogen blow - and promote the development of TCM. The study will promote the standardization of TCM/ethnic medicine for research-related technologies.

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Protocol

1. Sample preparation

  1. Collect cleaned roots and leaves from a 2-year-old Salvia miltiorrhiza plant (Figure 1A), and directly slice at a cross-sectional thickness of approximately 3-5 mm by hand. Then, stick the sample onto an adhesion microscope glass slide using double-sided tape (Figure 1B).
    NOTE: Ensure the size of the double-sided tape is bigger than the sample. If the tissues are dried, soak them in water or 4% paraformaldehyde overnight before slicing.
  2. Put another microscope glass slide above the sample and wrap the two glass slides with a sealing film like a sandwich (Figure 1C). Freeze the sandwich sample at -80 °C for at least 4 h, then subject it to an air vacuum for 2 h (Figure 1D) with the following setting parameters: trap temperature at -75 to -82 °C and vacuum gauge at 2.5 to 3.7 Pa.
    NOTE: Ensure the two glass slides are parallel when wrapping the sealing film to keep surface of the sample intact. If the plant tissues have a high moisture content, extend the time of air-vacuum to 3 h. Do not exceed 5 h, otherwise the tissues will easily fracture.
  3. Store the sandwich samples at -80 °C until analysis. Bring the samples to room temperature in a desiccator to avoid the condensation on the sample surface. Then, subject the sample to matrix application.

2. Installation of desorption electrospray ionization (DESI) unit

  1. Implement detector setup and mass calibration of the instrument in ESI mode; carry out detector setup using Leucine Enkephalin (LE) in water-acetonitrile (1:1 v/v) solution and perform mass calibration with sodium formate (NaFA) in water-isopropanol (1:1 v/v) solution.
  2. Take the ESI source out and mount the DESI unit onto the mass spectrometer. Connect the N2 gas supply to the DESI unit and adjust the gas pressure to around 0.5 MPa (Figure 2A). There is no need to vent the instrument when exchanging sources.
  3. Fill the 5 mL syringe with LE and formic acid in water-methanol (1:9 v/v) solution and attach the syringe to the high-performance syringe pump to provide solvent for ionization of the chemicals in the sample (Figure 2B).
  4. Attach a solvent providing capillary to the syringe and the DESI sprayer (Figure 2C). The solvent providing capillary is a standard 75 µm internal diameter and 375 µm outside diameter capillary; it is rather narrow and easily gets blocked by impurities, therefore solvents used in the scanning processes should be MS grade and filtered before usage to reduce the risk of blockage.
  5. Start the syringe pump and set the infuse rate at 2 µL/min to get a constant flow and spray of the solvent (Figure 2B). Turn off the N2 gas valve, then after about 15 s turn it on; a small drop of solvent will be blown out onto the stage, and spray can be seen if the solvent flow is in a constant state.
  6. Adjust the position of the sprayer in terms of the spray angle, XYZ axis, protrusion, and height (Figure 2D). Use red and black markers as references to optimize the mass spectrometry signal, to get a signal intensity above 1 x 105 in sensitivity mode (Figure 2E).
    1. Protrusion of the sprayer is the most significant factor that affects the signal intensity; adjust the protrusion by changing the N2 gas guard with a 5 mm wrench. Spray direction influences the quality of the mass image; rotate the sprayer until the spray is straight. Once the protrusion is adjusted to the best signal intensity position, try not to change it when exchanging sources.
  7. After all the steps above, the setup is ready for experiments, and the setup is normally stable for >3 weeks of usability, observed after the initial setup.

3. DESI-MS image acquisition

  1. For DESI-MSI, perform no sample pretreatment. For samples that already have pretreatment, minimize the pretreatment steps as much as possible. For instance, some samples can only be made with mounting media, so remove the excess media on the slides if possible.
  2. Take an image of the sample on the slide (Figure 3A). Do not touch the surface of the sample to avoid any impurity take-in.
  3. Place the slide on the plate position on the DESI stage. The stage has two plate positions, A and B; it is important to remember the right position. Use standard slides (75 mm x 25 mm) or a full slide, otherwise the slide will not fit in the position and cannot be stably held. A full slide (120 mm x 80mm) can accommodate up to four slides, and thus has a much larger area for experiments.
  4. Open the high-definition mass image processing software, set a new plate in the Acquire tab, and select the right plate position (A or B) and the plate type. On the image-select page, select the four corners of the slide, then the image is auto-adjusted to the correct orientation (Figure 3A).
  5. Set the MS parameters; the commonly used experiment type is DESI-MS mode, in which only the parent ion will be detected. The instrument can use only one polarity in one experiment; therefore, select the polarity as positive or negative. To get more information on chemicals in small amounts, apply the sensitivity mode (Figure 3B).
  6. Draw a rectangle to define the scanning area in the Pattern tab and set the pixel size. Generally, for DESI-MS mode, keep the X and Y sizes of the pixel equal. Set the scanning rate to no more than 5x the pixel size (Figure 3C).
  7. Save the project and export a worksheet for the mass spectrometry acquisition software.
  8. Open the mass spectrometry acquisition software, import the worksheet, and save it as a new sample list. Press Start Run to begin the MSI scanning. Multiple images can be added to the experiment queue by importing more worksheets.

4. Processing DESI-MSI data and visualization

  1. Load the data file of the sample into the mass image processing software and set the parameters for DESI image processing (Figure 3D). As Leucine Enkephalin was used for internal lock mass, and the lock mass is the only point to identify the polarity of the experiment, it is of great importance to set the correct lock mass. Set the following values: for positive mode: 556.2772; for negative mode: 554.2620.
  2. It is possible to build a list of target chemicals, in which case the processing result will focus on the chemicals in the target list. Load the processed data file to visualize the DESI image of the sample. Click the "Normalization" button to normalize the data by total ion chromatography (TIC) to get the relative intensity of a specific chemical to the reference, then different samples can be compared with each other (Figure 3E).
  3. Draw a region of interest (ROI) and copy several copies on the sample image; ROIs can be made across different images. Select all the ROIs and export multi-variate analysis (MVA) to extract MS information from all ROIs for MVA (Figure 3F).

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Representative Results

This protocol can lead to the identification and distribution of compounds in plant samples. In the MS image of a specific m/z, the color of every single pixel represents the relative intensity of the m/z, thus can be associated with the natural distribution and the abundance of the metabolite ion throughout the sample. The higher the abundance of the chemical at the collecting position, the brighter the color is. The bar in the picture (Figure 4A-D) shows the gradient of the colors. Here, we selected two compounds that are valuable in the medicinal use of S. miltiorrhiza. As shown in Figure 4A-D, the distribution of target compounds, Tanshinone IIA (m/z: 333.0893, M+H) and Rosmarinic acid (m/z: 705.1848, 2M+H-O), is visible in different areas of the root. Meanwhile, the compound Danshenol A (m/z: 297.1127, M+H; m/z: 335.0686, M+K) was detected in the leaf, as shown in Figure 4E-H. The distribution of the compounds can be used to guide the usage of the plant part in medical applications; in addition, the exported MVA data can be applied to take further metabolomics analysis.

Figure 1
Figure 1: Method of sample preparation. (A) The plant (Salvia miltiorrhiza) used in this research. The red arrow indicates the collected tissue as a sample. (B,C) Schematic showing how to make a sandwich sample. (D) Air-vacuum of samples. The temperature set is -83.1 ± 3 °C, and the vacuum range is 3-5 Pa. Please click here to view a larger version of this figure.

Figure 2
Figure 2: Equipment and apparatus in the DESI-MSI unit. (A) Front view of the DESI assembly. (B) Syringe pump. (C) Sprayer capillary. (D) Top view of the DESI assembly. (E) Optimization of the signal. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Acquisition, data analysis, and visualization by DESI-MSI. (A) Load the image into the mass image processing software and select the corners of the slide to adjust the image to the right orientation. (B) Set the MS parameters, set the m/z scanning range, and select positive or negative mode. (C) Define the scanning area, image resolution, and scanning rate. (D) Set the processing parameters: number of target masses, lock mass, sample frequency, and duration. (E) Load the outcome and normalize the data. Select the expected m/z from the mass list to display the MS image of the m/z. (F) Draw regions of interest (ROIs) on the MS image, and export MVA for metabolomics analysis. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Mass spectrometry imaging of root and whole leaf sections. (A-D) Images showing the spatial distribution of two selected compounds in the root. (E-H) Images showing the spatial distribution of two selected compounds in leaf. The color of every single pixel represents the relative intensity of the m/z and thus can be associated with the natural distribution and the abundance of the metabolite ion throughout the sample. The higher the abundance of the chemical at the collecting position, the brighter the color is. The bar in the pictures shows the gradient of the colors. Please click here to view a larger version of this figure.

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Discussion

The emergence of MS technology has opened a new insight in natural product research at the molecular level during recent years24. The MS instrument, with its high sensitivity and high throughput, enables targeted and untargeted analysis of metabolites in natural products, even with trace concentration25. Therefore, MS is currently widely used in the field of traditional Chinese medicine (TCM) chemistry. The qualitative and quantitative research on the chemical composition of TCM can provide information about the ingredients of the medicine and its associated compound, which not only provide a suitable reference for pharmacological research but also provide the basis for the construction of a quality standard system for TCM26. Besides, in natural products, metabolic signatures are usually related to the morphological and histological characteristics27; therefore, it is of great value to conduct in situ analysis to identify the mechanism and response of plants to various biotic and abiotic stress conditions28. However, as samples for traditional MS analysis are solutions of extracts from a certain natural product or its specific parts, MS does not gain information with respect to the spatial or temporal distribution of metabolites in the samples. The MSI technique, a relatively new technology developed only two decades ago, obtains metabolites from the natural product samples, analyzes the molecular information both qualitatively and quantitatively, and records the spatiotemporal information. Thereafter, with the help of mapping tools, the 2D or 3D coordinates of specific molecules can be simulated29.

The DESI-MSI technique used in this study is a novel MSI technique developed in 2004 by Cooks' group at Purdue University (USA)30. Compared to other early used MSI techniques, including secondary ion mass spectrometry (SIMS)31, matrix assisted laser desorption ionization (MALDI)32, and laser ablation electrospray ionization (LAESI)33, DESI has several advantages. SIMS and MALDI both need a high vacuum environment to ionize the samples, and for MALDI, the samples need to be mounted on a conductive surface7. Besides, the sample preparation for all these three techniques involves several complicated steps. DESI, as a novel ESI technique, is based on a soft ionization principle similar to electrospray ionization (ESI) in liquid chromatography mass spectrometry (LC-MS)30. Therefore, the detected ions are mostly quasi-molecular ions, and fragmentation can also be performed if necessary, which overcomes the drawback of hard ionization in the SIMS technique, generating secondary ions which may insult the loss of information7. DESI works in ambient conditions, so it does not need much time to reach the working condition after placing samples in the apparatus. Because of the minimized destructive ionization principle, it is possible to execute experiments repeatedly on one sample, therefore no additional samples are needed for a second mode (negative or positive).

This article mainly describes a cost-effective method of preparing plant samples and imaging using the DESI-MSI technique. In this method, the cross-sectional thickness of the sample does not play any key role; instead, the flat surface of the sample is crucial, which is guaranteed by the air-vacuum sandwich. In the case of plants, the preparation of DESI samples can be achieved in different ways and play a key role in MS imaging. Leaves are often problematic as they show an irregular, soft, and wax cuticle surface, which might result in a low signal during imaging, while the root contains high lignin content and is easy to fracture during imaging. Previous work showed that the root of S. miltiorrhiza was cryo-sectioned on a cryostat microtome when in DESI-MSI analysis, whereas the leaf was prepared by imprinting34. However, the imprinting method might induce a loss of signal intensity during MSI imaging due to the rapid dissolving of metabolites deposited on the glass surface. With this protocol (step 1.2), as expected, the sections of root (Figure 4A,B) and leaf (Figure 4E,F) stay intact during the MS imaging. Besides, the method to prepare the samples, by cyto-sectioning with a cryostat microtome, is high-cost due to the expensive machine.

Although our method has many advantages compared with other techniques, there are still a few limitations. First, the hand cutting of samples (step 1.1) requires practice to keep the thickness of the cross-section suitable. In addition, the spatial resolution and peak intensity of DESI is relatively low as compared to MALDI. Despite the imperfection, all the advantages make the DESI technique a fast and cost-effective method to investigate the spatiotemporal distribution of metabolites in plants. Furthermore, DESI-MSI has already been used in the field of medicine, microbiology, and natural product chemistry35. With the increasing popularity and rapid improvement in several dimensions of this technique, it will get more and more applications in all relative fields in the future7.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

This work was supported by the Natural Science Foundation of Sichuan province (No. 2022NSFSC0171) and the Xinglin Talent Program of Chengdu University of TCM (No. 030058042).

Materials

Name Company Catalog Number Comments
2-Propanol Fisher CAS:67-63-0 HPLC grade
Acetonitrile Sigma-aldrich Number-75-05-8 LC-MS grade
Adhesion Microscope slides Citotest scientific 80312-3161 Microscope glass slides  can adhere to  the sample 
Air cooled dry vacuum pump EYELA FDU-2110 Air-vaccum equipment at -80°C
Formic Acid ACS F1089 | 64-18-6 LC-MS grade
LE (Leucine Enkephalin) Waters 186006013-1 LC-MS grade
Methanol Sigma-aldrich Number-67-56-1 LC-MS grade
Parafilm  Bemis Company sc-200288 Laboratory Sealing Film
Paraformaldehyde Sigma-aldrich V900894 Reagent grade
Q-Tof Mass Spectrometer with DESI source Waters Synapt XS

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Tags

Visualization Metabolites Spatial Metabolome Traditional Chinese Medicine DESI-MSI Plant Samples Imaging Cost-effective Method Dry Tissues Nitrogen Blow In Cryosectioned Cryostat Microtome Imprinting Expensive Machine Signal Intensity Limitations Spatial Metabolomics Toxicants Distribution Abiotic Stress Biotic Stress Salvia Miltiorrhiza Plant Cross-sectional Thickness Adhesion Microscope Glass Slide Double-sided Tape Sealing Film Freeze Sample Air Vacuum Analysis Cold Storage Room Temperature Desiccator
Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI
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Cite this Article

Xu, B., Chen, L., Lv, F., Pan, Y.,… More

Xu, B., Chen, L., Lv, F., Pan, Y., Fu, X., Pei, Z. Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI. J. Vis. Exp. (190), e64912, doi:10.3791/64912 (2022).

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