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

The overall goal of this procedure is to perform reliable, multi mass spectrometric imaging of small molecules in plant root tissue. This is accomplished by first preparing the tissue samples to the appropriate size. The second step is to optimize and apply the maldi matrix to the tissue sample.

Next, the sample is subjected to Maldi, MSI and Spectra are collected across the tissue. The final step is data processing in which biologically important analytes can be identified. Ultimately, MALDI MSI is used to detect endogenous metabolites present in legume root nodules.

This method can provide insight into the metabolites present in plant root tissue, but it can also be applied to the study of small molecules and tissues from other model systems, including mammals, crustacean insects, and more. First, remove previously prepared plant tissue samples from the minus 80 degrees Celsius freezer. Then cut away the plastic cryostat cup and trim away the excess gelatin, mount the embedded tissue to a cryostat chuck with a dime-sized amount of optimal cutting temperature or OCT media while not letting the OCT touch the tissue.

Place the cryostat chuck in a cryostat box set to minus 20 degrees Celsius until the OCT solidifies. After allowing the chuck and gelatin to AC equilibrate in the cryostat box for about 15 minutes, use the cryostat to section the tissue to approximately the thickness of one cell. Then thumb mount each slice onto the pre chilled glass slide by warming the uncoated side of the slide on the back of the hand.

Next place the ITO coated side of the warm slide near the frozen tissue slice and allow the slice to stick onto the slide for airbrush application of the MALDI matrix. Thoroughly clean the airbrush solution container and nozzle with methanol and fill the solution container with DHB matrix solution. Hold the airbrush approximately 35 centimeters from the sample and apply 10 to 15 coats of matrix on the surface of the slide with a duration of 10 seconds of spray and 30 seconds of drying time in between each coat.

When finished, thoroughly clean the airbrush with methanol to avoid clogging Due to the matrix solution, it is important to note that while airbrushes are more readily available, automatic matrix sprayer systems often produce more even and reproducible matrix coverage for sublimation application of the maldi Matrixx weigh out 300 milligrams of DHB into the bottom of the sublimation chamber. Stick the glass slide to the cold finger with the tissue sections facing down with double-sided conductive tape. Cut the slide if it is too large.

For the sublimation chamber clamp the top and bottom halves of the sublimation chamber together with the C clamp. Then place the sublimation chamber in a heating mantle that is at room temperature. Connect the vacuum and add ice in cold water to the top reservoir.

Once the vacuum pump has been turned on, wait 15 minutes and turn on the heating mantle. After 10 minutes, turn off the heat, close the valve to the vacuum and turn off the vacuum pump. Once the chamber has cooled to room temperature, open the valve releasing the vacuum pressure and remove the sample.

Next, mark a plus pattern on each corner of the sample with the correction fluid pen to be used as teach points. Place the glass slide into the Maldi slide adapter plate and take an optical image of the sample using a scanner. Check the image on the computer and rotate the picture so it appears on the screen.

In the same orientation, the sample will be placed in the instrument. Set up an image acquisition file using the software provided by the instrument company with a raster step size of 50 micrometers and a laser diameter equal to or smaller than the raster step size eyes. At this point, load the optical image into the software and align the plate with the optical image.

After calibrating the instrument, specify the areas of tissue to be analyzed with MSI, including a spot of pure matrix on the slide to be used as a blank. Following this, begin the acquisition after data acquisition, open the imaging file in the software provided by the vendor and extract the ion images by selecting a specific mass to charge ratio of interest from the mass spectrum. Using the software, create a list of specific analytes of interest for further identification.

Perform accurate mass database searching to determine putative identifications for the targeted analytes. To confirm the putative identifications from accurate mass database searching match the ms. MS from the targeted analytes to msms spectra of standards, literature and or fragmentation prediction software shown here is an optical image of a mego Tula root nodule section pictured.

Here is an optical image example of the matrix coverage and crystal sizes using airbrush automatic sprayer and sublimation respectively. The airbrush application method generates large and small crystals. While the automatic sprayer method produces small evenly sized crystals, sublimation produces one even layer of matrix.

Conventional matrices like DHB produce many ions in the lower mass range below 500 Daltons. These matrix ions can interfere with the detection of metabolites in this range. Shown here are the MS spectra of just DHB matrix compared to root nodule tissue coated with DHB matrix.

Matrix peaks can be distinguished from real metabolites using the MS images. When a peak is clicked on, the ion image is extracted and displayed overlaid with the optical image. Those peaks that generate images with distinct localization to the tissue and are not present in the matrix only area imaged are considered metabolites shown here are several representative ion images of metabolites found in root nodule tissue.

While examples of MS images corresponding to matrix related peaks are pictured Here, this image shows distinct localization to the root nodule tissue and a lack of signal in the matrix only area that was imaged. This signal shows little localization and is present over the entire tissue. The signal is also seen in the matrix only area that was imaged.

The end goal of untargeted metabolomics experiments is to detect and determine biologically important analytes and identify the compounds of interest. An example of one of the metabolites detected with MSI and LCMS is shown here. This metabolite was identified as heme based on the accurate mass collected with high resolution LCMS and the MSM MSS spectrum.

This MSMS data was compared to the MSM S spectra previously published by Shima and Sato, the two MSM S spectrum match. Therefore, the identity of the mass to charge ratio of 6 1 6 0.2 was confidently assigned as heme based on the accurate mass database searching and Ms.MS data compared to literature Ms.MS data. After watching this video, you should have a good understanding of how to use maldi, mass spectrometric imaging to detect and map the spatial distribution of biologically relevant metabolites using a method that can be applied to the study of small molecules in a variety of tissue types and model systems.