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 4% paraformaldehyde overnight before slicing.</li>
	<li>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 &#176;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 &#176;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.</li>
	<li>Store the sandwich samples at -80 &#176;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.</li>
</ol>
2. Installation of desorption electrospray ionization (DESI) unit
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Attach a solvent providing capillary to the syringe and the DESI sprayer (Figure 2C). The solvent providing capillary is a standard 75 &#181;m internal diameter and 375 &#181;m 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.</li>
	<li>Start the syringe pump and set the infuse rate at 2 &#181;L/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.</li>
	<li>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).
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>After all the steps above, the setup is ready for experiments, and the setup is normally stable for &#62;3 weeks of usability, observed after the initial setup.</li>
</ol>
3. DESI-MS image acquisition
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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).</li>
	<li>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).</li>
	<li>Save the project and export a worksheet for the mass spectrometry acquisition software.</li>
	<li>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.</li>
</ol>
4. Processing DESI-MSI data and visualization
<ol>
	<li>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.</li>
	<li>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 &#34;Normalization&#34; 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).</li>
	<li>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).</li>
</ol>

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The authors have nothing to disclose.

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&#39; 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.

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><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&amp;nbsp; can adhere to&amp;nbsp; the sample&amp;nbsp;</td></tr><tr><td>Air cooled dry vacuum pump</td><td>EYELA</td><td>FDU-2110</td><td>Air-vaccum equipment at -80&amp;deg;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&amp;nbsp;</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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64912/64912fig01.jpg" /> 
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 &#177; 3 &#176;C, and the vacuum range is 3-5 Pa. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64912/64912fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64912/64912fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64912/64912fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64912/64912fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64912/64912fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64912/64912fig04.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64912/64912fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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 ...

visualization-metabolites-identified-spatial-metabolome-traditional

Nanyang Technological University

Research

JoVE Journal

Medicine

1.8K Views.  Chengdu University of Traditional Chinese Medicine. Dit artikel beschrijft voornamelijk een kosteneffectieve methode voor het bereiden van plantenmonsters en beeldvorming met behulp van DESI-MSI, en kan de nadelen overwinnen die gepaard gaan met droge weefsels, die gemakkelijk breken wanneer stikstof inblaast. De wortel werd gecryosectie op het cryostaatmicrotoom, terwijl het blad werd bereid door bedrukking, wat een dure machine vereist of de signaalintensiteit verliest. Onze methode kan deze beperkingen overwinnen.Deze methode kan wor...

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

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Bioengineering

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an enormous amount of spectra, setting challenges for quality control of the mass spectrometry spectra. To reduce the false positives, false discovery rate (FDR) quality control is necessary. Recently, we developed a software for FDR control of untargeted metabolome identification that is based on a Target-Decoy strategy named XY-Meta. Here, we demonstrated a complete analysis pipeline that integrates XY-Meta and metaX together. This protocol shows how to use XY-meta to generate a decoy database from an existing reference database and perform FDR control using the Target-Decoy strategy for large-scale metabolome identification on an open-access dataset. The differential analysis and metabolites annotation were performed after running metaX for metabolites peaks detection and quantitation. In order to help more researchers, we also developed a user-friendly cloud-based analysis platform for these analyses, without the need for bioinformatics skills or any computer languages.

We constructed an untargeted metabolomic workflow that integrated XY-Meta and metaX together. In this protocol, we displayed how to use XY-Meta to generate a decoy spectral library from open access spectra reference, and then performed FDR control and used the metaX to quantitate the metabolites after identifying the metabolomics spectra.

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an ...

Biochemistry

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

Chemistry

Ultra High-Performance Liquid Chromatography-Mass Spectrometry and Self-established Database Analysis of Chinese Herbal Medicine Components

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) is the most widely used method in qualitative analysis of compounds. The method includes standardized and programmed protocols for sample pretreatment, MS tune, MS acquisition, and data processing. The sample pretreatments include collection, pulverization, solvent extraction, ultrasound, centrifugation, and filtration. Data post-processing was described in detail and includes data importing, self-established database construction, method establishment, data processing, and other manual operations. The above-ground part of the alpine yarrow herb, Achillea millefolium L., is used to treat inflammation, gastrointestinal disturbances, and pain and its 3-oxa-guaianolides could be useful leads for anti-inflammatory drug development. Three representative compounds in AML were identified, combining TOF-MS with a self-established database. Moreover, the differences from existing literature, liquid-phase parameter optimization, scan mode selection, ion source suitability, collision energy adjustment, isomer screening, method limitation, and possible solutions were discussed. This standardized analysis method is universal and can be applied to identify complex compounds in Chinese herbal medicine.

We describe a general protocol and systematic design that could be applied to separate and recognize complex components in alpine yarrow herb, Achillea millefolium L., a Chinese herbal medicine.

Chinese herbal medicine is complex and has numerous unknown compounds, making qualitative research crucial. Ultra-high-performance liquid chromatography ...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Biology

 Advanced MALDI-MSI Mapping of Metabolites in Brain Organoids

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry 

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry

An advanced method was developed for mass spectrometry imaging (MSI) of brain organoids that allows mapping metabolite distributions within these models. This technology offers insights into brain metabolic pathways and metabolite signatures during early development and in disease, promising a deeper understanding of the human brain function.

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge ...

Neuroscience

Analyzing Tibetan Medicinal Plant APB Using 2D-HPLC-MS with Molecular Networking

2D-HPLC-MS Technology Combined with Molecular Network for the Identification of Components in Tibetan Medicine Aconitum pendulum

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate chemical composition of the Tibetan medicinal plant APB. Through the implementation of 2D-HPLC, enhanced separation of complex mixtures was achieved, enabling the isolation of individual compounds for subsequent analysis. The molecular network approach further aided in elucidating structural relationships among these compounds, contributing to the determination of potential bioactive molecules. This integrated strategy efficiently identified a wide array of chemical components present within the plant. The findings revealed a diverse spectrum of chemical constituents within APB, including alkaloids, among others. This research not only advances understanding of the phytochemical profile of this traditional Tibetan medicine but also provides valuable insights into its potential therapeutic properties. The integration of 2D-HPLC-MS and molecular network proves to be a powerful tool for systematically exploring and identifying complex chemical compositions in herbal medicines, paving the way for further research and development in the field of natural product discovery.

Author Spotlight: Integrating 2D-HPLC-MS and Molecular Networking in Natural Medicine Analysis

This study utilizes two-dimensional high-performance liquid chromatography-mass spectrometry (2D-HPLC-MS) technology in conjunction with molecular networking to unravel the intricate chemical composition of the Tibetan medicinal plant Aconitum pendulum Busch (APB). The article provides a detailed protocol for the systematic exploration and identification of complex chemical components of herbal medicines.

In this study, a comprehensive approach was employed, utilizing 2D-HPLC-MS technology in conjunction with the molecular network to unravel the intricate ...

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

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