seed sectioning protocol and matrix deposition technique for maldi-ims analysis of hard palm seeds

A Detailed Guide for Preparing Hard Palm Seeds for MALDI-IMS Analysis

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is a powerful method that allows two-dimensional biomolecule assignment, provides untargeted investigation of ionizable compounds, and determines their spatial distribution, especially in biological samples1,2. For two decades, this technique has enabled the simultaneous detection and identification of lipids, peptides, carbohydrates, proteins, other metabolites, and synthetic molecules such as therapeutic drugs3,4. MALDI-IMS facilitates chemical analysis in a tissue sample surface without extraction, purification, separation, labeling, or staining agents of biological samples. However, for successful analysis, a pivotal step in this technique is the sample preparation, particularly in plant tissues, which are specialized and modified to widespread complex organs due to environmental acclimatization5.
Because of the inherent plant tissue physicochemical properties, there is a need for an adapted protocol to suit the requirements of MALDI-IMS analysis and preserve the tissue&#39;s original shape during sectioning preparation6,7. In the case of unconventional samples, such as seeds, established protocols8 are not applicable because these tissues have rigid cell walls and low water content, which can easily cause section fragmentation and lead to compound delocalization9.
Our research group has published experimental data on molecular mapping and an adapted protocol for MALDI-IMS analysis of a&#231;a&#237; (Euterpe oleracea Mart.) seed10,11,12, which is a byproduct generated in high amounts during the production of the rentable a&#231;a&#237; pulp13. The idea was to develop a protocol for in situ mapping of different metabolites in a&#231;a&#237; seeds, helping to suggest possible uses for this agricultural waste that are currently not being explored commercially. However, due to the resistance of the a&#231;a&#237; seed, it was necessary to tailor-make a protocol to obtain proper sample sectioning from MALDI-IMS analysis.
In this context, the economically important a&#231;a&#237; pulp has motivated the increasing commercialization of other fruits from Euterpe genus palm trees with similar sensory characteristics. The two emerging palm trees&#39; fruits that have been produced on an industrial scale as an alternative to a&#231;a&#237;14,15 are E. precatoria (known as a&#231;a&#237;-do-amazonas), which grows in the Amazon dryland, and E. edulis (known as ju&#231;ara), which is typical from the Atlantic Forest. Nevertheless, the consumption of a&#231;a&#237;-do-amazonas and ju&#231;ara leads to the same accumulation of resistant and inedible seeds that are not availed and have not been studied so far regarding their detailed chemical composition.
Thus, we demonstrate here that the previously devised protocol can be used, with few adaptations, to analyze E. precatoria and E. edulis seeds for molecular mapping by MALDI-IMS, proving to be a powerful tool that can be used for analysis of the composition of these resources and can help to determine their potential biotechnological uses. Moreover, the detailed description provided here can aid others with similar difficulties in preparing resistant materials for MALDI-IMS analysis.

Author Spotlight: A Tailor-Made Sample Preparation Approach for Enhanced MALDI-IMS Analysis of Hard Palm Seeds

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 molecular-specific imaging of the biological tissue.With the results, we can have a 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, this 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 thin slice of gut-resistant palm seeds, serving as a proof of a concept for molecular mapping within a live seed tissue.So this technique offers valuable insights into oligosaccharides present in these seeds. 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 fin slices.For example, this protocol can be a tool on the study of seed development and germination if one is studying a hard seed.

Seed Sectioning Protocol and Matrix Deposition Technique for MALDI-IMS Analysis of Hard Palm Seeds

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 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 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%trichloroacetic 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.

seed-sectioning-protocol-matrix-deposition-technique-for-maldi-ims

Research

JoVE Journal

Biochemistry

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is applied to identify compounds in their native environments. Currently, MALDI-IMS is frequently used in clinical analysis. Still, an excellent perspective exists for better applying this technique to understand chemical compounds&#39; physiological information in plant tissues. However, preparation may be challenging for specific samples from botanical materials, as MALDI-IMS requires thin slices (12-20 &#181;m) for appropriate data acquisition and successful analysis. In this sense, previously, we developed a sample preparation protocol to obtain thin sections of Euterpe oleracea (a&#231;a&#237; palm) hard seeds, enabling their molecular mapping by MALDI-IMS.
Here, we show that the developed protocol is suitable for preparing other seeds from the same genus. Briefly, the protocol was based on submerging the seeds in deionized water for 24 h, embedding samples with gelatin, and sectioning them in an acclimatized cryostat. Then, for matrix deposition, an xy motion platform was coupled to an electrospray ionization&#160;(ESI) needle spray using a 1:1 (v/v) 2,5-dihydroxybenzoic acid (DHB) and methanol solution with 0.1% trifluoroacetic acid at 30 mg/mL. E. precatoria and E. edulis seed data were processed using software to map their metabolite patterns.
Hexose oligomers were mapped within sample slices to prove the adequacy of the protocol for those samples, as it is known that those seeds contain large amounts of mannan, a polymer of the hexose mannose. As a result, peaks of hexose oligomers, represented by [M + K]+ adducts of (&#916; = 162 Da), were identified. Thus, the sample preparation protocol, previously developed tailor-made for E. oleracea seeds, also enabled MALDI-IMS analysis of two other hard palm seeds. In short, the method could constitute a valuable tool for research in the morpho-anatomy and physiology of botanical materials, especially from cut-resistant samples.

This protocol aimed to describe detailed guidance on the preparation of hard seed sample sections with low water content for MALDI-IMS analysis, maintaining analytes' original distribution and abundance and providing high-quality signal and spatial resolution.

Matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS) is applied to identify compounds in their native environments. ...

Image Acquisition and MALDI-IMS Analysis of Hard Palm Seeds

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 dialogue 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 1000, and mass tolerance to five PPM. Calibrate the data with the process and save a 2D serial dataset tool. After calibration, open the dot 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 okay 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 scan 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.