Name: investigation of xenobiotics metabolism in salix alba leaves via mass spectrometry imaging
Uploaded: 2020-06-15T12:30:00
Description: Watch this Scientific Journal Video about Investigation of Xenobiotics Metabolism In Salix alba Leaves via Mass Spectrometry Imaging at JoVE.com

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 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.

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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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			<td height="20" style="height:20px;width:216px;">Plastic molds</td>
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			<td>No specific reference needed</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">SCiLS Lab</td>
			<td style="width:163px;">Bruker Daltonics</td>
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		<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 ...

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