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.
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.
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.
1. Sample preparation
2. Installation of desorption electrospray ionization (DESI) unit
3. DESI-MS image acquisition
4. Processing DESI-MSI data and visualization
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: 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: 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: 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: 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.
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.
The authors have nothing to disclose.
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).
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 |