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.
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Method Article
Visualization of Metabolites Identified in the Spatial Metabolome of Traditional Chinese Medicine Using DESI-MSI
In This Article
Summary
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....
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Protocol
1. Sample preparation
- 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. - Put another microscope glass slide above the sample and wrap the two glass slides with a seal....
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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 t.......
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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 o.......
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Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
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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 |
References
- Buchberger, A. R., DeLaney, K., Johnson, J., Li, L. Mass spectrometry imaging: a review of emerging advancements and future insights. Analytical Chemistry. 90 (1), 240-265 (2018).
- Karlsson, O., Hanrieder, J. Imaging mass spectrometry in drug development and....
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