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.

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

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

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.

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. ...

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935 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.

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...