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

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.