Preparation of Hard Palm Seeds for Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry Analysis

Our research has a focus on valorizing plant biomass from the tropical biodiversity. For that, it's very important to characterize well those materials so we can give the best value to them and propose the best destinations. For that, in this work, we employ the MALDI imaging mass-spectrometry analysis as a characterizing technique because it's a very precise method that can give us a molecularly-specific imaging of the biological tissue.

With the results, we can have an overview of the main components of this plant biomass, and also their localization, and this can guide our exploration of new products and processes from that material. However, to have a very good MALDI image mass-spectrometry analysis, it's very important to have a good sample preparation method, and that's exactly what we describe in this work. Advancements in technology have led to many challenges in imaging mass-spectrometry analysis.

Plant tissues in particular pose a complex challenge due to their specialized organs. It's crucial for this technique, the sample preparation. If the sample preparation is done inadequately, the signals won't be detected, or artifacts will appear, leading to inaccurate results.

These protocols allow us the preparation of a thin slice of cut-resistant palm seeds, serving as a proof-of-concept for molecular mapping for analyzing tissue. So this technique offers valuable insights into oligosaccharides present in these seed imprints. So we hope that technique will be helpful for the researchers with similar obstacles.

This technique can help on the study of other hard seeds or any other biological material that imposes a challenge to be cut into thin slices. For example, this protocol can be a tool on the study of seed development and germination if one is to study a hard seed. To begin, dip three Euterpe precatoria seeds and three Euterpe edulis seeds into deionized water for 24 hours.

After 24 hours, turn the cryostat on and let the temperature reach minus 20 degrees Celsius. Take the wet seeds out of the water and cut them in half using a microtome blade. Prepare a fresh, warm 10%gelatin solution, place half of the seeds on a mold, and fill it with the gelatin solution.

Freeze at minus 20 degrees Celsius for two hours before taking it to the cryostat. Attach the embedded seed to the cryostat support using an optimal cutting temperature compound, or OCT, and leave it for 10 minutes inside the cryostat for OCT hardening. Next, add copper double-faced adhesive tape to an indium tin oxide-coated glass slide or ITO slide.

Produce 20-micrometer thick sections from each species and place them on the adhesive tape adhered to the ITO glass slide. For matrix deposition, place the slide containing slices in a vacuum desiccator until it reaches room temperature. Make teaching marks using a correction pen in each slide corner, and scan the slide using a table scanner.

Use an analytical balance to weigh 30 milligrams of DHB. Then, prepare a one milliliter solution containing equal parts of methanol and 0.1%trifluoroacetic acid and dissolve the weighed DHB in the prepared solution. Fill a one milliliter glass syringe with the DHB solution and place it on a syringe pump with a flow rate of 0.8 milliliters per hour.

Using pick tubing, connect the syringe to an atmospheric pressure chemical ionization or APCI needle, and then connect nitrogen to the needle. Set the flow rate to 12.5 PSI. Attach the APCI needle to the XY motion platform.

Ensure that the tip of the APCI needle is four centimeters above the slide. Using the drawing software, set the XY motion platform to follow the template, and wait for the platform to repeat the template 20 times. To begin, place the slide containing the matrix-deposited seed slices in the mass spectrometer.

Load the sample image, and use correction pen marks to set teaching points on the referenced software. Then, set the 99%data reduction factor, save the FID file for the posterior data calibration, and save the method. Delimitate the area to be analyzed using the Add Polygon Measurement Region tool from the mass spectrometer software.

Set the raster width to 100 micrometers. Edit the measurement region parameters indicating the saved method, and save the imaging run. Then, start the imaging acquisition.

Use matrix cluster and known contaminants to create a mass list in the software in the Calibrant tab. Then in the Calibration tab, open the created mass list. Right click to open a dialog box and choose the set Lock Masses option.

Select Gaussian Window mode with 0.5 Gaussian broadening and 3.5 line broadening. Leave online calibration unchecked. Set mode to single, threshold to 1, 000, and mass tolerance to five PPM.

Calibrate the data with the process and save the 2D serial dataset tool. After calibration, open the mis file to a compatible software and alter the normalization from no norm to RMS or TIC. Click on the Edit tab and select Automatic Mass Filtering.

Fill start mass and end mass with the minimum and maximum number of dalton at your interest threshold and click on the OK icon. Select the highlighted peak of interest at the filtered list generated with the number corresponding to the mass value of interest in dalton. Change the percentage to better view the interested area.

Click on the intensity scale bar and the color map icons. Click on the image area and drag slide the mouse to position the area of interest. Change the transparency percentage to produce a merged signal image with the scanned section image as the background.

If the analytes to be mapped are known, plot each M by Z value for each analyte and save the generated images and the spectral average plot. The mass spectrum of Euterpe precatoria and Euterpe edulis seeds tissue obtained by MALDI-IMS in positive mode is shown here. This MALDI-IMS analysis of E precatoria seeds exhibited peaks representing adducts of hexoses oligomers without adding salt to the matrix.

Hexoses dimers, trimers, tetramers, pentamers, hexamers, and up to 14 unit oligomers were identified. The same results were also obtained for E edulis seed tissue. The box plots for both samples indicate the peak intensity of each hexose oligomer found in the seed endosperm, demonstrating their distributions, and a slightly higher content of a high degree of polymerization of oligomers.