We thank Charles Pineau, M&#233;lanie Lagarrigue and R&#233;gis Lavigne for their tips and tricks regarding sample preparation for MALDI imaging of plant samples.

1. Sample preparation
<ol>
	<li>Obtain the biological sample and either keep it fresh and intact (e.g., do not force it into a tube) or freeze it. The proposed protocol applies to any type of solid biological sample (i.e., plant, animal, or human tissues) to localize compounds in specific tissues.</li>
	<li>Cool down a cryomicrotome to -20 &#176;C. Keep the sample holder and the blade at the same temperature.</li>
	<li>If necessary, embed the object in M1 embedding medium to preserve it during cutting.
	<ol>
		<li>Pour...

<ol>
	<li>Zhang, D., Gersberg, R. M., Ng, W. J., Tan, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Removal+of+pharmaceuticals+and+personal+care+products+in+aquatic+plant-based+systems:+A+review.">Removal of pharmaceuticals and personal care products in aquatic plant-based systems: A review.</a> Environmental Pollution. 184, 620-639 (2014).</li><li>Adeel, M., Song, X., Wang, Y., Francis, D., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+impact+of+estrogens+on+human,+animal+and+plant+life:+A+critical+review.">Environmental impact of estrogens on human, animal and plant life: A critical review.</a> Environment International. 99, 107-119 (2017).</li><li>Prosser, R. S., Sibley, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+health+risk+assessment+of+pharmaceuticals+and+personal+care+products+in+plant+tissue+due+to+biosolids+and+manure+amendments,+and+wastewater+irrigation.">Human health risk assessment of pharmaceuticals and personal care products in plant tissue due to biosolids and manure amendments, and wastewater irrigation.</a> Environment International. 75, 223-233 (2015).</li><li>Wang, J., 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=Application+of+biochar+to+soils+may+result+in+plant+contamination+and+human+cancer+risk+due+to+exposure+of+polycyclic+aromatic+hydrocarbons.">Application of biochar to soils may result in plant contamination and human cancer risk due to exposure of polycyclic aromatic hydrocarbons.</a> Environment International. 121, 169-177 (2018).</li><li>Marsik, 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=Metabolism+of+ibuprofen+in+higher+plants:+A+model+Arabidopsis+thaliana+cell+suspension+culture+system.">Metabolism of ibuprofen in higher plants: A model Arabidopsis thaliana cell suspension culture system.</a> Environmental Pollution. 220, 383-392 (2017).</li><li>He, 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=Metabolism+of+ibuprofen+by+Phragmites+australis:+uptake+and+phytodegradation.">Metabolism of ibuprofen by Phragmites australis: uptake and phytodegradation.</a> Environmental Science and Technology. 51 (8), 4576-4584 (2017).</li><li>Huber, C., Bartha, B., Harpaintner, R., Schr&#246;der, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolism+of+acetaminophen+(paracetamol)+in+plants-two+independent+pathways+result+in+the+formation+of+a+glutathione+and+a+glucose+conjugate.">Metabolism of acetaminophen (paracetamol) in plants-two independent pathways result in the formation of a glutathione and a glucose conjugate.</a> Environmental Science and Pollution Research. 16 (2), 206-213 (2009).</li><li>Thomas, F., C&#233;bron, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+rhizosphere+effect+on+available+carbon+sources,+phenanthrene+degradation,+and+active+microbiome+in+an+aged-contaminated+industrial+soil.">Short-term rhizosphere effect on available carbon sources, phenanthrene degradation, and active microbiome in an aged-contaminated industrial soil.</a> Frontiers in Microbiology. 7, 1-15 (2016).</li><li>Villette, C., 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=In+situ+localization+of+micropollutants+and+associated+stress+response+in+Populus+nigra+leaves.">In situ localization of micropollutants and associated stress response in Populus nigra leaves.</a> Environment International. 126, 523-532 (2019).</li><li>Sandermann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+metabolism+of+organic+xenobiotics.+Status+and+prospects+of+the+'Green+Liver'+concept.">Plant metabolism of organic xenobiotics. Status and prospects of the 'Green Liver' concept.</a> Plant Biotechnology and In Vitro Biology in the 21st Century. , 321-328 (1999).</li><li>Sula, B., Deveci, E., &#214;zevren, H., Ekinci, C., Elbey, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+histopathological+changes+in+the+skin+of+rats+after+administration+of+lead+acetate.">Immunohistochemical and histopathological changes in the skin of rats after administration of lead acetate.</a> International Journal of Morphology. 34 (3), 918-922 (2016).</li><li>Villette, C., Maurer, L., Wanko, A., Heintz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenobiotics+metabolization+in+Salix+alba+leaves+uncovered+by+mass+spectrometry+imaging.">Xenobiotics metabolization in Salix alba leaves uncovered by mass spectrometry imaging.</a> Metabolomics. 15, 122 (2019).</li><li>Khatib-Shahidi, S., Andersson, M., Herman, J. L., Gillespie, T. A., 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=Direct+Molecular+Analysis+of+Whole-Body+Animal+Tissue+Sections+by+Imaging+MALDI+Mass+Spectrometry.">Direct Molecular Analysis of Whole-Body Animal Tissue Sections by Imaging MALDI Mass Spectrometry.</a> Analytical Chemistry. 78 (18), 6448-6456 (2006).</li></ol>

The authors have nothing to disclose.

The critical part of this protocol is the sample preparation: the sample must be soft and intact. Cutting is the most difficult part, as the temperature and thickness of the sample can vary depending on the type of sample studied. Animal tissues are usually homogeneous and easier to cut. Plant samples often incorporate different structures and therefore are more difficult to keep intact as the blade encounters soft, hard, or empty vascular tissues. It is highly recommended to use fresh tissues when working with plant sam...

Xenobiotics are widely distributed around the world due to human activities. Some of these compounds are water-soluble and absorbed by soil1, and enter the food chain when they accumulate in plant tissues2,3,4. The plants are eaten by insects and herbivores, which are prey to other organisms. The intake of some xenobiotics and their impact on a plant&#8217;s health have been described5,6,7,8, but only recently at a tissue level9. Therefore, it is still unclear where or how the metabolism of xenobiotics occurs, or if specific plant metabolites are correlated to xenobiotic accumulation in specific tissues10. Moreover, most research has overlooked the metabolism of xenobiotics and their metabolites in plants, so little is known about these reactions in plant tissues.
Proposed here is a method to investigate enzymatic reactions in biological samples that can be associated to the tissue localization of substrates and products of the reactions. The method can draw the complete metabolic profile of a biological sample in one experiment, as the analysis is non-targeted and can be investigated using custom lists of analytes of interest. Provided is a list of candidates tracked in the original dataset. If one or several analytes of interest are noted in the sample, the specific tissue localization can provide important information on the related biological processes. The analytes of interest can then be modified in silico using relevant biological laws to search for possible products/metabolites. The list of metabolites obtained is then used to analyze the original data by identifying the enzymes involved and localizing the reactions in the tissues, thus helping to understand the occurring metabolic processes. No other method provides information on the types of reactions occurring in the biological samples, the localization of the compounds of interest, and their related metabolites. This method can be used on any type of biological material once fresh and intact tissues are available and the compounds of interest can be ionized. The proposed protocol was published in Villette et al.12 and is detailed here for use by the scientific community.

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="664">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cover slips</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">267942</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Cryomicrotome</td>
			<td style="width:163px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Excel</td>
			<td style="width:163px;">Microsoft corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">flexImaging</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ftmsControl</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">GTX primescan</td>
			<td style="width:163px;">GX Microscopes</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HCCA MALDI matrix</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">8201344</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ImagePrep</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">ITO-coated slides</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">237001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">M1-embedding matrix</td>
			<td style="width:163px;">ThermoScientific</td>
			<td align="right" style="width:119px;">1310</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Metabolite Predict</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Metaboscape</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Methanol</td>
			<td style="width:163px;">Fisher Chemicals</td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">MX 35 Ultra blades</td>
			<td style="width:163px;">Thermo Scientific</td>
			<td align="right" style="width:119px;">15835682</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Plastic molds</td>
			<td style="width:163px;"></td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SCiLS Lab</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SolariX XR 7Tesla</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></td>
			<td>The method proposed is not limited to this instrument</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Spray sheets for ImagePrep</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td align="right" style="width:119px;">8261614</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">TFA</td>
			<td style="width:163px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td>No specific reference needed</td>
		</tr>
	</tbody>
</table>

investigation of xenobiotics metabolism in salix alba leaves via mass spectrometry imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a non-targeted approach, plant metabolites and xenobiotics of interest are identified and localized in plant tissues to uncover&#160;specific distribution patterns. Then, in silico prediction of potential metabolites (i.e., catabolites and conjugates) from the identified xenobiotics is performed. When a xenobiotic metabolite is located in the tissue, the type of enzyme involved in its alteration by the plant is recorded. These results were used to describe different types of biological reactions occurring in S. alba leaves in response to xenobiotic accumulation in the leaves. The metabolites were predicted in two generations, allowing the documentation of successive biological reactions to transform xenobiotics in the leaf tissues.

This protocol was applied to fresh leaves sampled from a S. alba tree exposed to xenobiotics in the environment. The process is depicted in Figure 1. The first step is to prepare thin slices of the sample of interest. Plant samples are often more difficult to cut than animal samples, as the tissues are heterogeneous and can contain water and/or air. This difficulty is handled using embedding medium, which forms a homogeneous block around the sample. The matrix deposition is facilita...

Watch this Scientific Journal Video about Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging at JoVE.com

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

This method uses mass spectrometry imaging (MSI) to understand metabolic processes in S. alba leaves when exposed to xenobiotics. The method allows the spatial localization of compounds of interest and their predicted metabolites within specific, intact tissues.

Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging

The method presented uses mass spectrometry imaging (MSI) to establish the metabolic profile of S. alba leaves when exposed to xenobiotics. Using a ...

This protocol allows direct identification of predicted metabolites in situ. It facilitates the determination of the type of tissues or cells that are affected by a specific metabolic process. This method is quick and straightforward.It does not require a complicated sample preparation or compound extraction, and it allows the identification and localization of low abundant compounds. Upon acquisition of the biological sample, cool a cryo microtome sample holder and blade to minus 20 degrees Celsius. If necessary, pour embedding medium into a plastic mold placed within the cryo microtome chamber and quickly add the sample to the medium.Cover the sample with additional embedding medium and adjust the position of the sample to the center of the mold as necessary as the matrix solidifies. When the sample is ready, use embedding medium to mount the tissue onto the cryo microtome holder and use the cooled blade to cut the tissue into appropriate sized sections, modifying the cutting thickness and temperature as necessary to obtain optimal samples. As each section is acquired, use an appropriate tool to carefully move the slice to an indium tin oxide coated glass microscope slide, and place a finger under the slide to warm and dry the sample before placing the slide into the cryo microtome chamber.When all of the slices have been acquired, slowly remove the slides from the chamber and use an appropriate marker to indicate the exact location of each sample on the slides. When all of the slides have been marked, use a high-resolution scanner to scan the samples. After scanning, clean the matrix deposition robot with 100%methanol, and use methanol and a precision wipe to clean each slide, holding the samples without touching them and without removing the marks.Place a clean coverslip over each slide in an area without sample and place the coverslip end of the first slide over the optical detector, then place no more than six milliliters of freshly prepared matrix solution in the matrix deposition reservoir and add two milliliters of 100%methanol to a total volume of eight milliliters. For data acquisition set up, place one microliter of matrix onto a MALDI plate and insert the plate into the source. Click load target in the device software and click on the spot in the image that represents the MALDI plate to select the position of the matrix spot.Indicate the sample name and folder in the sample info tab. Click acquisition to start the acquisition and use the mouse pointer to move the plate slightly so that the laser points at different spots. When the acquisition is finished, open the calibration tab and select HCCA calibration list in the quadratic mode and click automatic.The global calibration result will be indicated in the calibration plot window. If the calibration is in the appropriate range, click accept and save the method. Once the matrix deposition on the samples is finished, place the slides in the slide adapter and use a plastic cover to retrieve the position of the marks.In the imaging software, set up a new imaging run in the first window that opens in the software and name the imaging run. Select the result directory and click next. Indicate the raster size, select the method to be used, and click next.Load the optical image from a scanned slide and click next. The image will open in a wider window and place the plastic cover bearing the marks onto a MALDI plate to recover its position and to facilitate the teaching. Under FTMS control, place the target of the MALDI video window on the exact position of a mark and open flex imaging, then click on the exact same point on the optical image.Use the add measurement regions tools to draw the regions of interest in the samples and save the imaging run. If several samples are to be analyzed, run the auto execute sequence and use auto execute batch runner to launch a sequence. When all of the images have been acquired, use the batch importer tool to select the raw data, indicate the target directory, and click import.Click file and new to select the data set tool to select the type of instrument used for the acquisition and click plus to add the imported dataset. Click and drag to arrange the images and check and modify the mass range settings as necessary. Click next to see the import summary and launch the import.Visualize the master charge ratio in different tissues within a sample or between different samples and click spectra to select the mass-to-charge ratios of interest. Export the mass-to-charge ratios of interest as a CSV file from the object tab and click export. Create a new dataset in an annotation software program and click projects and import CSV project to import the CSV file into the software.Annotate with custom-made analyte lists, which can be derived from publicly available databases. A template is given by the software to create analyte lists. Use the prediction software to perform in silico prediction of the metabolites of the annotated compounds.Recover the list of metabolites, create an analyte list, and use the list in the annotation software to annotate the raw data with predicted metabolites, then right-click on the predicted metabolite of interest to recover the names of the enzymes involved in the metabolic processes in the prediction software. In this example, the drug Telmisartan was determined to be distributed throughout the plant leaf tissue samples. The drug metabolites were predicted and searched for in the raw data, and the annotations revealed that one first-generation metabolite was detected in the internal tissues of the leaves and further degraded into second-generation metabolites that were localized in internal tissues or more generally distributed in all of the leaf tissues.These results suggest an active metabolic reaction in the leaves to degrade Telmisartan. The process was then applied to several compounds of interest annotated in the leaves and the enzymes involved in the reactions were recovered to investigate their role in the plant's response to xenobiotic accumulation. Although this procedure is quite simple, a good sample preparation is key.Be sure to take the time to find the parameters that fit your sample type during tissue sectioning. The sample size can be further used for microscopy observations. It can be stained to obtain a panel of complimentary biological observations.

investigation-xenobiotics-metabolism-salix-alba-leaves-via-mass

Research

JoVE Journal

Environment

3.0K Views.  Université de Strasbourg. This protocol allows direct identification of predicted metabolites in situ. It facilitates the determination of the type of tissues or cells that are affected by a specific metabolic process. This method is quick and straightforward.It does not require a complicated sample preparation or compound extraction, and it allows the identification and localization of low abundant compounds. Upon acquisition of the biological sample, cool a cryo microtome sample holder and blade to minus 20 d...