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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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. 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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><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&amp;nbsp;</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&quot;X18YD</td><td></td></tr><tr><td>Cryostat CM 1860 UV</td><td>Leica&amp;nbsp; 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&amp;nbsp; 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&amp;Omega;.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 &quot;M&quot; 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&amp;nbsp;</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. ...

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

Research

JoVE Journal

Biochemistry

976 Views.  Instituto Nacional de Tecnologia. Ons onderzoek richt zich op het valoriseren van plantaardige biomassa uit de tropische biodiversiteit. Daarvoor is het erg belangrijk om die materialen goed te karakteriseren, zodat we er de beste waarde aan kunnen geven en de beste bestemmingen kunnen voorstellen. Daarvoor gebruiken we in dit werk de MALDI-beeldvormingsmassaspectrometrie-analyse als karakteriseringstechniek, omdat het een zeer nauwkeurige methode is die ons een moleculair-specifieke beeldvorming van het biologische weefsel k...

Video: Auteur in de schijnwerpers: een op maat gemaakte aanpak voor monstervoorbereiding voor verbeterde MALDI-IMS-analyse van harde palmzaden

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Chemistry

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 ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Bioengineering

Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods 1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass ...

Neuroscience

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

Biology

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

Lipid profiling, or lipidomics, is a well-established technique used to study the entire lipid content of a cell or tissue. Information acquired from&#160;lipidomics is valuable in studying the pathways involved in development, disease, and cellular metabolism. Many tools and instrumentations have aided lipidomics projects, most notably various combinations of mass spectrometry and liquid chromatography techniques. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has recently emerged as a powerful imaging technique that complements conventional approaches. This novel technique provides unique information on the spatial distribution of lipids within tissue compartments, which was previously unattainable without the use of excessive modifications. The sample preparation of the MALDI MSI approach is critical and, therefore, is the focus of this paper. This paper presents a rapid lipid analysis of a large number of Drosophila brains embedded in optimal cutting temperature compound (OCT) to provide a detailed protocol for the preparation of small tissues for lipid analysis or metabolite and small molecule analysis through MALDI MSI.

Sample Preparation for Rapid Lipid Analysis in Drosophila Brain Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging

The aim of this protocol is to provide detailed guidance on the proper sample preparation for lipid and metabolite analysis in small tissues, such as the Drosophila brain, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging.

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

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