Name: Author Spotlight: A Tailor-Made Sample Preparation Approach for Enhanced MALDI-IMS Analysis of Hard Palm Seeds
Uploaded: 2023-06-30T13:50:00
Description: 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 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>...

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

<ol>
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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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reliable+and+effective+sample+preparation+protocol+of+MALDI-TOF-MSI+for+lipids+imaging+analysis+in+hard+and+dry+cereals.">A reliable and effective sample preparation protocol of MALDI-TOF-MSI for lipids imaging analysis in hard and dry cereals.</a> Food Chemistry. 398, 133911 (2023).</li><li>Brum, F. L., Martins, G. R., Mohana-Borges, R., da Silva, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acquisition+of+thin+sections+of+a&#231;a&#237;+(Euterpe+oleracea+Mart.)+seed+with+elevated+potassium+content+for+molecular+mapping+by+mass+spectrometry+imaging.">The acquisition of thin sections of a&#231;a&#237; (Euterpe oleracea Mart.) seed with elevated potassium content for molecular mapping by mass spectrometry imaging.</a> Rapid Communications in Mass Spectrometry. , e9474 (2023).</li><li>Martins, G. 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=Chemical+characterization,+antioxidant+and+antimicrobial+activities+of+a&#231;a&#237;+seed+(Euterpe+oleracea+Mart.)+extracts+containing+A-+and+B-type+procyanidins.">Chemical characterization, antioxidant and antimicrobial activities of a&#231;a&#237; seed (Euterpe oleracea Mart.) extracts containing A- and B-type procyanidins.</a> LWT. 132, 109830 (2020).</li><li>Martins, G. 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=Phenolic+profile+and+antioxidant+properties+in+extracts+of+developing+a&#231;a&#237;+(Euterpe+oleracea+Mart.)+seeds.">Phenolic profile and antioxidant properties in extracts of developing a&#231;a&#237; (Euterpe oleracea Mart.) seeds.</a> Journal of Agricultural and Food Chemistry. 70 (51), 16218-16228 (2022).</li><li>Jorge, F. T. A., Silva, A. S. A., Brigag&#227;o, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A&#231;a&#237;+waste+valorization+via+mannose+and+polyphenols+production:+techno-economic+and+environmental+assessment.">A&#231;a&#237; waste valorization via mannose and polyphenols production: techno-economic and environmental assessment.</a> Biomass Conversion and Biorefinery. , (2022).</li><li>Carvalho, L. M. J., Esmerino, A. A., Carvalho, J. L. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Jussa&#237;+(Euterpe+edulis):+a+review.">Jussa&#237; (Euterpe edulis): a review.</a> Food Science and Technology. 42, (2022).</li><li>Yamaguchi, K. K. d. L., Pereira, L. F. R., Lamar&#227;o, C. V., Lima, E. S., Veiga-Junior, V. F. d. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amazon+acai:+chemistry+and+biological+activities:+A+Review.">Amazon acai: chemistry and biological activities: A Review.</a> Food Chemistry. 179, 137-151 (2015).</li><li>Wu, 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=Copper+adhesive+tape+attached+to+the+reverse+side+of+a+non-conductive+glass+slide+to+achieve+protein+MALDI-imaging+in+FFPE-tissue+sections.">Copper adhesive tape attached to the reverse side of a non-conductive glass slide to achieve protein MALDI-imaging in FFPE-tissue sections.</a> Chemical Communications. 57 (82), 10707-10710 (2021).</li><li>Dufresne, M., Patterson, N. H., Norris, J. L., Caprioli, R. 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. P., 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=Screening+of+the+chemical+composition+and+occurring+antioxidants+in+jabuticaba+(Myrciaria+jaboticaba)+and+jussara+(Euterpe+edulis)+fruits+and+their+fractions.">Screening of the chemical composition and occurring antioxidants in jabuticaba (Myrciaria jaboticaba) and jussara (Euterpe edulis) fruits and their fractions.</a> Journal of FunctionalFoods. 17, 422-433 (2015).</li><li>Monteiro, A. F., Miguez, I. S., Silva, J. P. R. B., Silva, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+concentration+and+yield+production+of+mannose+from+a&#231;a&#237;+(Euterpe+oleracea+Mart.)+seeds+via+mannanase-catalyzed+hydrolysis.">High concentration and yield production of mannose from a&#231;a&#237; (Euterpe oleracea Mart.) seeds via mannanase-catalyzed hydrolysis.</a> Scientific Reports. 9 (1), 10939 (2019).</li></ol>

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.