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).

1. Sample preparation
<ol>
	<li>Collect cleaned roots and leaves from a 2-year-old Salvia miltiorrhiza plant&#160;(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...

<ol>
	<li>Buchberger, A. R., DeLaney, K., Johnson, J., Li, L. <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+imaging:+a+review+of+emerging+advancements+and+future+insights.">Mass spectrometry imaging: a review of emerging advancements and future insights.</a> Analytical Chemistry. 90 (1), 240-265 (2018).</li><li>Karlsson, O., Hanrieder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mass+spectrometry+in+drug+development+and+toxicology.">Imaging mass spectrometry in drug development and toxicology.</a> Archives of Toxicology. 91 (6), 2283-2294 (2016).</li><li>Qiu, Z. -. 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=Real-time+toxicity+prediction+of+Aconitum+stewing+system+using+extractive+electrospray+ionization+mass+spectrometry.">Real-time toxicity prediction of Aconitum stewing system using extractive electrospray ionization mass spectrometry.</a> Acta Pharmaceutica Sinica B. 10 (5), 903-912 (2020).</li><li>Wang, Z., 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+situ+metabolomics+in+nephrotoxicity+of+aristolochic+acids+based+on+air+flow-assisted+desorption+electrospray+ionization+mass+spectrometry+imaging.">In situ metabolomics in nephrotoxicity of aristolochic acids based on air flow-assisted desorption electrospray ionization mass spectrometry imaging.</a> Acta Pharmaceutica Sinica B. 10 (6), 1083-1093 (2020).</li><li>Jiang, H., Gao, S., Hu, G., He, J., Jin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovation+in+drug+toxicology:+Application+of+mass+spectrometry+imaging+technology.">Innovation in drug toxicology: Application of mass spectrometry imaging technology.</a> Toxicology. 464, 153000 (2021).</li><li>Unsihuay, D., Mesa Sanchez, D., Laskin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry+imaging+of+biological+systems.">Quantitative mass spectrometry imaging of biological systems.</a> Annual Review of Physical Chemistry. 72, 307-329 (2021).</li><li>Parrot, D., Papazian, S., Foil, D., Tasdemir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+the+unimaginable:+desorption+electrospray+ionization-imaging+mass+spectrometry+(DESI-IMS)+in+natural+product+research.">Imaging the unimaginable: desorption electrospray ionization-imaging mass spectrometry (DESI-IMS) in natural product research.</a> Planta Medica. 84 (9-10), 584-593 (2018).</li><li>Meistermann, H., 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=Biomarker+discovery+by+imaging+mass+spectrometry:+transthyretin+is+a+biomarker+for+gentamicin-induced+nephrotoxicity+in+rat.">Biomarker discovery by imaging mass spectrometry: transthyretin is a biomarker for gentamicin-induced nephrotoxicity in rat.</a> Molecular &amp; Cellular Proteomics. 5 (10), 1876-1886 (2006).</li><li>Kadar, H., 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=MALDI+mass+spectrometry+imaging+of+1-methyl-4-phenylpyridinium+(MPP+)+in+mouse+brain.">MALDI mass spectrometry imaging of 1-methyl-4-phenylpyridinium (MPP+) in mouse brain.</a> Neurotoxicity Research. 25 (1), 135-145 (2014).</li><li>Bruinen, A. L., 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=Mass+spectrometry+imaging+of+drug+related+crystal-like+structures+in+formalin-fixed+frozen+and+paraffin-embedded+rabbit+kidney+tissue+sections.">Mass spectrometry imaging of drug related crystal-like structures in formalin-fixed frozen and paraffin-embedded rabbit kidney tissue sections.</a> Journal of the American Society for Mass Spectrometry. 27 (1), 117-123 (2016).</li><li>Mullen, A. K., Clench, M. R., Crosland, S., Sharples, K. 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E., Gao, P., Smith, C. D., Norris, J. S., Drake, R. 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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 o...

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&#160;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&#39;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td>Fisher</td>
			<td>CAS:67-63-0</td>
			<td>HPLC grade</td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-aldrich</td>
			<td>Number-75-05-8</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>Adhesion Microscope slides</td>
			<td>Citotest scientific</td>
			<td>80312-3161</td>
			<td>Microscope glass slides&#160; can adhere to&#160; the sample&#160;</td>
		</tr>
		<tr>
			<td>Air cooled dry vacuum pump</td>
			<td>EYELA</td>
			<td>FDU-2110</td>
			<td>Air-vaccum equipment at -80&#176;C</td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>ACS</td>
			<td>F1089 | 64-18-6</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>LE (Leucine Enkephalin)</td>
			<td>Waters</td>
			<td>186006013-1</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-aldrich</td>
			<td>Number-67-56-1</td>
			<td>LC-MS grade</td>
		</tr>
		<tr>
			<td>Parafilm&#160;</td>
			<td>Bemis Company</td>
			<td>sc-200288</td>
			<td>Laboratory Sealing Film</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-aldrich</td>
			<td>V900894</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Q-Tof Mass Spectrometer with DESI source</td>
			<td>Waters</td>
			<td>Synapt XS</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of metabolites identified in the spatial metabolome of traditional chinese medicine using desi-msi

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.

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 t...

Watch this Scientific Journal Video about Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI at JoVE.com

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

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 ...

This article mainly describes a cost-effective method of preparing plant samples and imaging using DESI-MSI, and can overcome the disadvantages associated with dry tissues, which easily fracture when nitrogen blow in. The root was cryosectioned on the cryostat microtome, whereas the leaf was prepared by imprinting, which requires expensive machine or loses signal intensity. Our method can overcome these limitations.This method can be used in spatial metabolomics of plants such as distribution and movement of toxicants in plants under abiotic and biotic stress. To begin, take cleaned roots and leaves from a two-year-old salvia miltiorrhiza plant and directly slice at a cross-sectional thickness of approximately three to five millimeters. Then, stick the sample onto an adhesion microscope glass slide using double-sided tape.Place another microscope glass slide above the sample and wrap them with a sealing film like a sandwich. Next, freeze the sandwich sample at minus 80 degrees Celsius for at least four hours, then subject it to an air vacuum for two hours with the trapped temperature at minus 75 to minus 82 degrees Celsius and vacuum gauge at 2.5 to 3.7 pascal. For analysis, after removing the samples from the cold storage, bring them to room temperature in a desiccator to avoid condensation on the sample surface before subjecting the sample to matrix application.Implement the instruments detector setup and mass calibration in electrospray ionization, or ESI mode. Carry out detector setup using leucine and cephalin in water acetonitrile at a ratio of one to one, and perform mass calibration with sodium formate and water isopropanol at a ratio of one to one. Take the ESI source out and mount the DESI unit onto the mass spectrometer.Connect the nitrogen gas supply to the DESI unit and adjust the gas pressure to around 0.5 megapascal. Fill a five milliliter syringe with leucine and cephalin and formic acid in water methanol at a ratio of one to nine, and attach the syringe to the high performance syringe pump to provide solvent for ionization of the chemicals in the sample. Next, attach a solvent-providing capillary to the syringe and the DESI sprayer.The solvent providing capillary is a standard capillary of dimensions with a 75 micrometer internal diameter and 375 micrometer outside diameter. Start the syringe pump and set the infuse rate to two microliters per minute to get a constant flow and spray of the solvent. Turn off the nitrogen gas valve and then turn it on after about 15 seconds.A small drop of solvent blows out onto the stage and spray can be seen if the solvent flow is in a constant state. Next, adjust the position of the sprayer in terms of the spray angle, XYZ axis, protrusion, and height. Use red and black markers as references to optimize the mass spectrometry signal to get a signal intensity of around one times 10 to the fifth in sensitivity mode.As the protrusion of the sprayer is the most significant factor that affects the signal intensity, adjust the protrusion by changing the nitrogen gas guard using a five millimeter wrench. Spray direction influences the quality of the mass image. Hence, rotate the sprayer until the spray is straight.After all these steps, the setup is ready for experiments and is normally stable for approximately three weeks of usability after the initial setup. For DESI-MS imaging, no sample pre-treatment is required. However, remove the excess media on the slides if possible.Take an image of the sample on the slide. Without touching the sample's surface to avoid any impurity, place the slide on the plate position on the DESI stage, which has two plate positions, A and B.Use standard slides or a full slide to fit in the position. A full slide can accommodate up to four slides and thus has a much larger area for experiments.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, and then the image is auto adjusted to the correct orientation.To set the MS parameters, select the experiment type as DESI-MS mode that is commonly used in which only the parent ion will be detected. Next, select the polarity as either positive or negative as the instrument can use only one polarity in an experiment. Then apply the sensitivity mode to get more information on chemicals in small amounts.Next, draw a rectangle to define the scanning area in the Pattern tab, and set the pixel size by keeping the X and Y sizes of the pixel equal. Set the scanning rate to no more than five times the pixel size. Save the project and export a worksheet for the MS acquisition software.Next, import the worksheet into the MS acquisition software and save it as a new sample list. Press Start Run to begin the MS image scanning, and multiple images can be added to the experiment queue by importing more worksheets. Load the data file of the sample into the mass image processing software and set the parameters for DESI image processing.As leucine and cephalin was used for internal lock mass, and the lock mass is the only point to identify the polarity of the experiment, it is crucial to set the correct lock mass. Set 556.2772 for positive mode and 554.2620 for negative mode. To build a list of target chemicals, 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 to get the relative intensity of a specific chemical to the reference, and then different samples can be compared with each other. Draw a region of interest, or ROI, and copy several copies on the sample image. Then select all the ROIs and export multivariate analysis to extract MS information from all ROIs.Mass spectrometry imaging of the root and whole leaf sections shows the spatial distribution of the selected compounds. The color of every single pixel represents the relative intensity of the mass to charge ratio, and thus can be associated with the natural distribution and the abundance of the metabolite ion throughout the sample. The distribution of the target compounds, tanshinone IIA and rosmarinic acid, is visible in different areas of the root.The compound tanshinol A was detected in the leaf sections As a protocol aiming for reproducibility, the most important thing is to optimize the signal intensity by adjusting the position of the sprayer in several dimensions.

visualization-metabolites-identified-spatial-metabolome-traditional

Research

JoVE Journal

Medicine

1.7K visualizações.  Chengdu University of Traditional Chinese Medicine. Este artigo descreve principalmente um método custo-efetivo de preparação de amostras de plantas e imagens usando DESI-MSI, e pode superar as desvantagens associadas aos tecidos secos, que facilmente fraturam quando o nitrogênio sopra. A raiz foi criossecada no micrótomo criostato, enquanto a folha foi preparada por imprinting, o que requer máquina cara ou perde intensidade de sinal. Nosso método pode superar essas limitações.Este método pode ser utilizado na metabolômica es...