This protocol allows direct identification of predicted metabolites in situ. It facilitates the determination of the type of tissues or cells that are affected by a specific metabolic process. This method is quick and straightforward.
It does not require a complicated sample preparation or compound extraction, and it allows the identification and localization of low abundant compounds. Upon acquisition of the biological sample, cool a cryo microtome sample holder and blade to minus 20 degrees Celsius. If necessary, pour embedding medium into a plastic mold placed within the cryo microtome chamber and quickly add the sample to the medium.
Cover the sample with additional embedding medium and adjust the position of the sample to the center of the mold as necessary as the matrix solidifies. When the sample is ready, use embedding medium to mount the tissue onto the cryo microtome holder and use the cooled blade to cut the tissue into appropriate sized sections, modifying the cutting thickness and temperature as necessary to obtain optimal samples. As each section is acquired, use an appropriate tool to carefully move the slice to an indium tin oxide coated glass microscope slide, and place a finger under the slide to warm and dry the sample before placing the slide into the cryo microtome chamber.
When all of the slices have been acquired, slowly remove the slides from the chamber and use an appropriate marker to indicate the exact location of each sample on the slides. When all of the slides have been marked, use a high-resolution scanner to scan the samples. After scanning, clean the matrix deposition robot with 100%methanol, and use methanol and a precision wipe to clean each slide, holding the samples without touching them and without removing the marks.
Place a clean coverslip over each slide in an area without sample and place the coverslip end of the first slide over the optical detector, then place no more than six milliliters of freshly prepared matrix solution in the matrix deposition reservoir and add two milliliters of 100%methanol to a total volume of eight milliliters. For data acquisition set up, place one microliter of matrix onto a MALDI plate and insert the plate into the source. Click load target in the device software and click on the spot in the image that represents the MALDI plate to select the position of the matrix spot.
Indicate the sample name and folder in the sample info tab. Click acquisition to start the acquisition and use the mouse pointer to move the plate slightly so that the laser points at different spots. When the acquisition is finished, open the calibration tab and select HCCA calibration list in the quadratic mode and click automatic.
The global calibration result will be indicated in the calibration plot window. If the calibration is in the appropriate range, click accept and save the method. Once the matrix deposition on the samples is finished, place the slides in the slide adapter and use a plastic cover to retrieve the position of the marks.
In the imaging software, set up a new imaging run in the first window that opens in the software and name the imaging run. Select the result directory and click next. Indicate the raster size, select the method to be used, and click next.
Load the optical image from a scanned slide and click next. The image will open in a wider window and place the plastic cover bearing the marks onto a MALDI plate to recover its position and to facilitate the teaching. Under FTMS control, place the target of the MALDI video window on the exact position of a mark and open flex imaging, then click on the exact same point on the optical image.
Use the add measurement regions tools to draw the regions of interest in the samples and save the imaging run. If several samples are to be analyzed, run the auto execute sequence and use auto execute batch runner to launch a sequence. When all of the images have been acquired, use the batch importer tool to select the raw data, indicate the target directory, and click import.
Click file and new to select the data set tool to select the type of instrument used for the acquisition and click plus to add the imported dataset. Click and drag to arrange the images and check and modify the mass range settings as necessary. Click next to see the import summary and launch the import.
Visualize the master charge ratio in different tissues within a sample or between different samples and click spectra to select the mass-to-charge ratios of interest. Export the mass-to-charge ratios of interest as a CSV file from the object tab and click export. Create a new dataset in an annotation software program and click projects and import CSV project to import the CSV file into the software.
Annotate with custom-made analyte lists, which can be derived from publicly available databases. A template is given by the software to create analyte lists. Use the prediction software to perform in silico prediction of the metabolites of the annotated compounds.
Recover the list of metabolites, create an analyte list, and use the list in the annotation software to annotate the raw data with predicted metabolites, then right-click on the predicted metabolite of interest to recover the names of the enzymes involved in the metabolic processes in the prediction software. In this example, the drug Telmisartan was determined to be distributed throughout the plant leaf tissue samples. The drug metabolites were predicted and searched for in the raw data, and the annotations revealed that one first-generation metabolite was detected in the internal tissues of the leaves and further degraded into second-generation metabolites that were localized in internal tissues or more generally distributed in all of the leaf tissues.
These results suggest an active metabolic reaction in the leaves to degrade Telmisartan. The process was then applied to several compounds of interest annotated in the leaves and the enzymes involved in the reactions were recovered to investigate their role in the plant's response to xenobiotic accumulation. Although this procedure is quite simple, a good sample preparation is key.
Be sure to take the time to find the parameters that fit your sample type during tissue sectioning. The sample size can be further used for microscopy observations. It can be stained to obtain a panel of complimentary biological observations.
