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

我们感谢查尔斯·皮诺、梅兰妮·拉加里格和雷吉斯·拉维涅在植物样本的MALDI成像样品准备方面的技巧和技巧。

1. 样品准备
<ol><li>获取生物样本，要么保持新鲜和完好无损（例如，不要强迫它进入管子），要么将其冻结。拟议的协议适用于任何类型的固体生物样本（即植物、动物或人体组织），以本地化特定组织中的化合物。</li>
	<li>冷却低温微分到-20°C。 将样品支架和刀片保持在相同的温度下。</li>
	<li>如有必要，将对象嵌入 M1 嵌入介质中，以便在切割过程中保存它。<ol>
<li>将一些基质倒入放置在低?...

<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>

此协议的关键部分是样品准备：样品必须柔软且完好无损。切割是最困难的部分，因为样品的温度和厚度可能因所研究的样品类型而异。动物组织通常是同质的，更容易切割。植物样本通常包含不同的结构，因此由于刀片遇到软、硬或空血管组织，因此更难保持完好无损。强烈建议在与植物样本一起工作时使用新鲜组织，以避免在亲水组织中形成冰并破坏冰。切片存放在 ITO 涂层滑梯上时，必须?...

由于人类活动，异种生物在世界各地广泛分布。其中一些化合物是水溶性，并被土壤1吸收，并进入食物链时，他们积累在植物组织2，3，4。这些植物被昆虫和食草动物吃掉，它们是其他生物的猎物。一些异种生物的摄入及其对植物健康的影响被描述为5，6，7，8，但最近才在组织水平9。因此，目前还不清楚异种生物代谢发生于何处或如何发生，或者特定植物代谢物是否与特定组织中的异生物积累相关。此外，大多数研究忽视了植物中异种生物及其代谢物的新陈代谢，因此对植物组织中的这些反应知之甚少。
这里建议的是一种研究生物样本中的酶反应的方法，该反应可以与基质和反应产品的组织定位相关。该方法可以在一个实验中绘制生物样本的完整代谢剖面，因为分析是无针对性的，并且可以使用感兴趣的定制分析列表进行调查。提供的是在原始数据集中跟踪的候选人列表。如果在样本中注意到一个或几个感兴趣的分析，特定的组织定位可以提供有关相关生物过程的重要信息。然后，利用相关的生物定律在硅中修改感兴趣的分析，以寻找可能的产品/代谢物。然后，通过识别所涉及的酶并本地化组织中的反应来分析原始数据，从而帮助了解发生的代谢过程。没有其他方法提供生物样本中发生的反应类型、感兴趣化合物的本地化及其相关代谢物的信息。一旦有新鲜和完整的组织，并且感兴趣的化合物可以电解，这种方法可用于任何类型的生物材料。拟议的议定书发表在《维莱特》等12 期中，详见此处供科学界使用。

<table border="0" cellpadding="0" cellspacing="0" style="width:664px;" width="664">
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	<tbody>
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			<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>
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			<td></td>
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			<td height="20" style="height:20px;width:216px;">GTX primescan</td>
			<td style="width:163px;">GX Microscopes</td>
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			<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>
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			<td height="20" style="height:20px;width:216px;">Metabolite Predict</td>
			<td style="width:163px;">Bruker Daltonics</td>
			<td style="width:119px;"></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>
			<td style="width:119px;"></td>
			<td></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

所介绍的方法使用质谱成像（MSI）来建立 S.阿尔巴 叶在接触异种生物时的代谢轮廓。使用非靶向方法，在植物组织中识别和本地化感兴趣的植物代谢物和异种生物，以发现特定的分布模式。然后，在从已识别的异种生物中预测潜在的代谢物（即代谢物和结合物）。当异生物代谢物位于组织中时，记录植物改变其所涉及的酶类型。这些结果用于描述在 S.alba 叶中发生的不同类型的生物反应，以回应叶子中的异生物积累。代谢物在两代人中预测，允许记录连续的生物反应，以改变叶组织中的异种生物。

此协议适用于从暴露于环境中的异种生物的S. alba树中采样的新鲜叶子。这个过程在图1中描述。第一步是准备兴趣样本的薄片。植物样本通常比动物样本更难切割，因为组织是异质的，可以含有水和/或空气。此困难使用嵌入介质处理，在样品周围形成均匀块。矩阵沉积通过使用机器人，避免手部操纵和确保可重复的结果而得到促进。MALDI基质层厚度在整个过程中遵循?...

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

这种方法使用质谱成像（MSI）来了解在接触异种生物时 ，S.阿尔巴 叶的代谢过程。该方法允许在特定、完整的组织内对感兴趣的化合物及其预测的代谢物进行空间定位。

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

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.

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Environment

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

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3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

利用目标筛选和非靶向筛选高分辨率质谱相结合的工作流程识别跨氟和多氟化学物种

Historical and emerging per- and polyfluoroalkyl substances (PFASs) have garnered significant interest from the public and government agencies from the ...

Investigation of Plant Interactions Across Common Mycorrhizal Networks Using Rotated Cores

旋转芯在常见菌根网络中植物相互作用的研究

Arbuscular mycorrhizal (AM) fungi influence plant mineral nutrient uptake and growth, hence, they have the potential to influence plant interactions. The ...

通过质谱成像对 萨利克斯阿尔巴 叶中异种生物代谢的调查

通过质谱成像对萨利克斯阿尔巴叶中异种生物代谢的调查