This work was financed by Serrapilheira Institute (Serra-1708-15009), and Carlos Chagas Filho Foundation for Supporting Research in the State of Rio de Janeiro (FAPERJ-JCNE-SEI-260003/004754/2021). Serrapilheira Institute and the National Council for Scientific and Technological Development (CNPq) granted scholarships for Dr. Felipe Lopes Brum and Dr. Gabriel R. Martins (Institutional Capacity Building Program/INT/MCTI). The Coordination for the Improvement of Higher Education Personnel (CAPES) is acknowledged for granting a Master&#39;s scholarship for Mr. Davi M. M. C. da Silva. Centro de Espectrometria de Massas de Biomol&#233;culas (CEMBIO-UFRJ) is recognized for the services provided with MALDI-IMS analyses, and Mr. Alan Menezes do Nascimento and the Centro de Caracteriza&#231;&#227;o em Nanotecnologia para Materiais e Cat&#225;lise (CENANO-INT), funded by MCTI/SISNANO/INT-CENANO-CNPQ grant N&#186; 442604/2019, are thanked for the elementary composition analysis.

Euterpe precatoria seeds were kindly donated by the Instituto Nacional de Pesquisas da Amaz&#244;nia (Manaus, Brazil), and Euterpe edulis seeds were kindly donated by the Silo - Arte e Latitude Rural (Resende, Brazil) after the industrial depulping process. The seeds were maintained in sealed plastic boxes at room temperature.
1. Matrix-assisted laser desorption/ionization-imaging mass spectroscopy (MALDI-IMS)
<ol>
	<li>Seed sectioning protocol
	<ol>
		<li>Allow three seeds from each species to sit in deionized water for 24 h.</li>
		<li>The next day, turn the cryostat (see Table of Materials) on and let it reach -20 &#176;C.</li>
		<li>Take the wet seeds out of the water. Cut the seeds in half using a microtome blade (see Table of Materials).</li>
		<li>Prepare a fresh, warm (10%) gelatin solution (see Table of Materials).</li>
		<li>Place half of the seed on a mold and fill it with fresh-made gelatin. Freeze at -80 &#176;C for 2 h before taking to the cryostat.</li>
		<li>Attach the embedded seed to the cryostat support using an optimal cutting temperature compound (OCT, see Table of Materials) and leave for 10 min inside the cryostat for OCT hardening.</li>
		<li>Add copper double-faced adhesive tape (see Table of Materials) to an indium tin oxide-coated glass slide (ITO slide; see Table of Materials).</li>
		<li>Produce 20 &#181;m thick sections from each species and place them on the copper double-faced adhesive tape adhered to the ITO glass slide. 
		&#8203;NOTE: At this point, the slices collected on the slides can be stored in a -80 &#176;C freezer; alternatively, proceed to matrix deposition. The slides must be placed in a holder slide box, flushed with gaseous N2, and sealed with film to prevent sample oxidation (see Table of Materials).</li>
	</ol>
	</li>
	<li>Matrix deposition
	<ol>
		<li>Place the slide containing slices in a vacuum desiccator until it reaches room temperature.</li>
		<li>Make teaching marks using a correction pen in each slide corner. Scan the slide using a table scanner. Set to the resolution of 4,800 ppi.</li>
		<li>Use an analytical balance to weigh 30 mg of 2,5-dihydroxybenzoic acid (DHB; see Table of Materials) and prepare 1 mL of 1:1 methanol:0.1% trifluoroacetic acid (TFA; Table of Materials) solution to dissolve the DHB.</li>
		<li>Fill a 1 mL glass syringe (see Table of Materials) with DHB solution and place it in a syringe pump (see Table of Materials) set to a flow rate of 0.8 mL/h.</li>
		<li>Using PEEK tubing, connect the syringe to an atmospheric pressure chemical ionization (APCI) needle (see Table of Materials).</li>
		<li>Connect N2 to the APCI needle and set it to a 12.5 psi flow rate.</li>
		<li>Attach the APCI needle to the xy motion platform (see Table of Materials). Ensure that the tip of the APCI needle is 4 cm above the slide.</li>
		<li>Using the drawing software (see Table of Materials and Supplementary Figure 1), set the xy motion platform to follow the template. The template consists of horizontal parallel lines spaced by 1 mm.</li>
		<li>To achieve matrix deposition, wait for the xy motion platform to repeat the template 20 times. 
		&#8203;CAUTION: Matrix deposition must be carried out in a chemical fume hood.</li>
	</ol>
	</li>
	<li>Imaging acquisition
	<ol>
		<li>Place the slide in the mass spectrometer (see Table of Materials).</li>
		<li>Use correction pen marks to set teaching points on the referenced software (see Table of Materials).</li>
		<li>Set the laser power (60%), laser focus (medium), number of shots (100), and polarity in the software (see Table of Materials). Set 99% data reduction factor and save the FID file for posterior data calibration. Save the method.</li>
		<li>Delimitate the area to be analyzed using the add polygon measurement region tool from the mass spectrometer software. Edit the measurement region parameters indicating the method saved in the previous step and the raster width to 100 &#181;m (Supplementary Figure S4A).</li>
		<li>Start imaging acquisition.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Use matrix cluster and known contaminants to create a mass list in the referenced software (see Table of Materials) in the calibrant tab (Supplementary Figure S4B).
		<ol>
			<li>Open the data to be calibrated in the referenced software (see Table of Materials). In the calibration tab, open the mass list created and open a dialog box by right-clicking and choose the set lock masses option (Supplementary Figure S4C).</li>
			<li>Select Gaussian window mode with 0.5 Gaussian broadening and 3.5 Line broadening. Leave online calibration unchecked. Select mode (single), threshold (1,000), and mass tolerance (5 ppm). Calibrate data with process and save the 2D data tool (Supplementary Figure S4C).</li>
		</ol>
		</li>
		<li>After calibration, export the data to SCiLS lab or other compatible software and set the desired m/z value threshold (range chosen: 150 to 2,500). 
		NOTE: Depending on the file size or the computer&#39;s characteristics, this might take some time.</li>
		<li>Choose a normalization method between total ion count (TIC) or root mean square (RMS). 
		NOTE: For this analysis, TIC was chosen.</li>
		<li>If the analytes to be mapped are known, plot each m/z value for each analyte and save the generated images and the spectral average plot. 
		NOTE: In this work, m/z values from hexose oligomers were chosen by considering the potassium adduct.</li>
	</ol>
	</li>
</ol>
2. Energy-dispersive spectroscopy (EDS)
<ol>
	<li>Seed sectioning protocol
	<ol>
		<li>Acquire a thin seed slice in a sectioning saw machine (see Table of Materials).</li>
		<li>Cut the seeds with the following parameters: 500 RPM cutting speed, 100 N load charge, and 15 HC Diamond Wafering Blade (see Table of Materials). 
		NOTE: Handle the manual removal of fibers from the seeds due to the necessary adhesion to the hold in the precision vises attached to the goniometer. Clean the blade before use, cutting a section of the seed to prevent adding undesirable minerals during the analysis.</li>
		<li>Clear the samples with compressed air to remove all the particle residues from the cut (see Table of Materials).</li>
		<li>Fix a seed section in carbon double-sided conductive tape (see Table of Materials) in the support.</li>
	</ol>
	</li>
	<li>Analysis conditions
	<ol>
		<li>Attach the support with a seed section inside the vacuum chamber of a scanning electron microscope (SEM) coupled with EDS (see Table of Materials).</li>
		<li>Acquire electron micrographs on a model microscope with 20 kV acceleration and a spot size of 4.0, using secondary electron signals (see Table of Materials), backscattered electrons (solid state detector fixed on the polar piece), and mixtures of these two types of signals (MIX), building artificially colored images.</li>
		<li>For the acquisition of EDS spectra, use a spectrometer (see Table of Materials) coupled with the aforementioned SEM. The condition configuration of the image acquisition from EDS is the same as the SEM.</li>
		<li>Set tilt degrees, elevation, and azimuth at 0.0, 35.0, and 0.0, respectively.</li>
	</ol>
	</li>
	<li>Data acquisition
	<ol>
		<li>Set three different areas with 91x magnification of the seed section to acquire data.</li>
		<li>Identify the peaks in the spectrum upon termination of acquisition or during acquisition. Use the Confirm Elements step button to identify the peaks manually.</li>
		<li>Set the acquisition time to 60 s for each selected area. Set the process time to five; the parameters are available for a process time of one to six. 
		NOTE: The signs of the elements are constant and add up, while the noises are random and deleterious. The longer the processing time, the lower the noise.</li>
		<li>The default settings to display significant quantitative results are above two sigmas (standard deviation). Set two sigmas to zero to reduce undesirable results.</li>
		<li>Normalize each intensity of the elements; this is a standardized parameter in the software (see Table of Materials).</li>
		<li>To analyze the data, export it in a DOC file.</li>
	</ol>
	</li>
	<li>Data analysis
	<ol>
		<li>Ensure the accurate measurement of peak intensities for quantitative elemental analysis.</li>
		<li>Overlapping peaks require deconvolution for better peak separation; subtract a noisy background when required.</li>
		<li>When there is no overlapping, increase the gain to amplify the signals and improve the spectrum quality.</li>
	</ol>
	</li>
</ol>

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

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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging,+an+emerging+technology+in+neuropsychopharmacology.">Mass spectrometry imaging, an emerging technology in neuropsychopharmacology.</a> Neuropsychopharmacology. 39 (1), 34-49 (2014).</li><li>Zaima, N., Hayasaka, T., Goto-Inoue, N., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix-assisted+laser+desorption/ionization+imaging+mass+spectrometry.">Matrix-assisted laser desorption/ionization imaging mass spectrometry.</a> International Journal of Molecular Sciences. 11 (12), 5040-5055 (2010).</li><li>Qin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+matrix-assisted+laser+desorption/ionisation+mass+spectrometry+imaging+(MALDI-MSI)+for+in+Situ+analysis+of+endogenous+molecules+in+plants.">Recent advances in matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) for in Situ analysis of endogenous molecules in plants.</a> Phytochemical Analysis. 29 (4), 351-364 (2018).</li><li>Bhandari, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+mass+spectrometry+imaging+of+plant+tissues:+Towards+a+plant+metabolite+atlas.">High resolution mass spectrometry imaging of plant tissues: Towards a plant metabolite atlas.</a> Analyst. 140 (22), 7696-7709 (2015).</li><li>Boughton, B. A., Thinagaran, D., Sarabia, D., Bacic, A., Roessner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+imaging+for+plant+biology:+a+review.">Mass spectrometry imaging for plant biology: a review.</a> Phytochemistry Reviews. 15 (3), 445-488 (2016).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+preparation+for+mass+spectrometry+imaging+of+plant+tissues:+a+review.">Sample preparation for mass spectrometry imaging of plant tissues: a review.</a> Frontiers in Plant Science. 7, 60 (2016).</li><li>Zhang, Y. X., Zhang, Y., Shi, Y. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+salt+doping+and+matrix+sublimation+for+high+spatial+resolution+MALDI+imaging+mass+spectrometry+of+neutral+lipids.">Combining salt doping and matrix sublimation for high spatial resolution MALDI imaging mass spectrometry of neutral lipids.</a> Analytical Chemistry. 91 (20), 12928-12934 (2019).</li><li>Aguiar, M. O., de Mendon&#231;a, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morfo-anatomia+da+semente+de+Euterpe+precatoria+Mart+(Palmae).">Morfo-anatomia da semente de Euterpe precatoria Mart (Palmae).</a> Revista Brasileira de Sementes. 25, 37-42 (2003).</li><li>Panza, V., L&#225;inez, V., Maldonado, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seed+structure+and+histochemistry+in+the+palm+Euterpe+edulis.">Seed structure and histochemistry in the palm Euterpe edulis.</a> Botanical Journal of the Linnean Society. 145 (4), 445-453 (2004).</li><li>Alves, V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Provenient+residues+from+industrial+processing+of+a&#231;a&#237;+berries+(Euterpe+precatoria+Mart):+nutritional+and+antinutritional+contents,+phenolic+profile,+and+pigments.">Provenient residues from industrial processing of a&#231;a&#237; berries (Euterpe precatoria Mart): nutritional and antinutritional contents, phenolic profile, and pigments.</a> Food Science and Technology. 42, (2022).</li><li>Inada, K. O. 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The authors declare no conflicts of interest.

Plants are composed of specialized tissues for specific biochemical functions. Therefore, the sample preparation protocol for MALDI-IMS must be designed according to various plant tissues with specific physicochemical properties, as samples must maintain their original analyte distribution and abundance for high-quality signal and spatial resolution8.
Prior to MALDI-IMS analysis, the primary consideration is collecting and storing samples properly. However, in plants, s...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Gastight Syringe Model 1001 TLL, PTFE Luer Lock</td>
			<td>Hamilton Company</td>
			<td>81320</td>
			<td></td>
		</tr>
		<tr>
			<td>2,5-Dihydroxybenzoic acid</td>
			<td>Sigma Aldrich Co, MO, USA</td>
			<td>149357</td>
			<td></td>
		</tr>
		<tr>
			<td>APCI needle</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>602193</td>
			<td></td>
		</tr>
		<tr>
			<td>AxiDraw V3 xy motion platform</td>
			<td>Evil Mad Scientist, CA, USA</td>
			<td>2510</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon double-sided conductive tape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Compass Data Analysis software&#160;</td>
			<td></td>
			<td></td>
			<td>creation of mass list</td>
		</tr>
		<tr>
			<td>Compressed air</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>copper double-faced adhesive tape</td>
			<td>3M, USA</td>
			<td>1182-3/4&#34;X18YD</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat CM 1860 UV</td>
			<td>Leica&#160; Biosystems, Nussloch, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Wafering Blade 15 HC</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Everhart-Thornley detector</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlexImaging</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td></td>
			<td>image acquisition</td>
		</tr>
		<tr>
			<td>FTMS Processing</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td></td>
			<td>data calibration</td>
		</tr>
		<tr>
			<td>Gelatin from bovine skin</td>
			<td>Sigma Aldrich Co, MO, USA</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr>
			<td>High Profile Microtome Blades Leica 818</td>
			<td>Leica&#160; Biosystems, Nussloch, Germany</td>
			<td>0358 38926</td>
			<td></td>
		</tr>
		<tr>
			<td>indium tin oxide coated glass slide</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td>8237001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inkscape</td>
			<td>Inkscape Project c/o Software Freedom Conservancy, NY, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IsoMet 1000 precision cutter</td>
			<td>Buehler, Illinois, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>J.T.Baker</td>
			<td>9093-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Mili-Q water</td>
			<td></td>
			<td></td>
			<td>18.2 M&#937;.cm</td>
		</tr>
		<tr>
			<td>Oil vacuum pump</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal Cutting Temperature Compound</td>
			<td>Fisher HealthCare, Texas, USA</td>
			<td>4585</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm &#34;M&#34; Sealing Film</td>
			<td>Amcor</td>
			<td>HS234526B</td>
			<td></td>
		</tr>
		<tr>
			<td>Quanta 450 FEG</td>
			<td>FEI Co, Hillsboro, OR, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SCiLS Lab (Multi-vendor support) MS Software&#160;</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software INCA Suite 4.14 V</td>
			<td>Oxford Instruments, Ableton, UK</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solarix 7T</td>
			<td>Bruker Daltonik, Bremen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>kdScientific, MA, USA</td>
			<td>78-9100K</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma Aldrich Co, MO, USA</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>X-Max spectrometer</td>
			<td>Oxford Instruments, Ableton, UK</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of hard palm seeds for matrix-assisted laser desorption/ionization-imaging mass spectrometry analysis

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.

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.

The devised protocol enabled MALDI-IMS analysis of E. precatoria and E. edulis seeds. As a result, we could confirm carbohydrates&#39; molecular weight and degree of polymerization (DP) as a partial structural elucidation. The molecular information provided by the MALDI-IMS analysis (Figure 1 and Figure 2) exhibited peaks representing [M+K]+ adducts of hexose oligomers (&#916; = 162 Da) without adding salt to the mat...

Watch this Scientific Journal Video about Preparation of Hard Palm Seeds for Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry Analysis at JoVE.com

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. ...

preparation-hard-palm-seeds-for-matrix-assisted-laser

Research

JoVE Journal

Biochemistry

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.

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.

936 Views.  Instituto Nacional de Tecnologia.  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.

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

Summary