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 some matrix in a plastic mold placed in the cryomicrotome chamber. Rapidly add the sample and pour some more embedding medium to cover it. Maintain the sample in the center of the mold as the matrix solidifies while cooling down. 
		NOTE: Embedding is not necessary for all biological objects. Homogenous objects such as mouse brains do not need embedding medium and can be cut frozen.</li>
	</ol>
	</li>
	<li>Place the embedded or frozen sample on the cryomicrotome holder and cut it with a sharp blade. A thickness of 5&#8211;30 &#181;m is good for plant samples, which are difficult to cut due to their heterogeneity. Adapt the cutting thickness and temperature to the sample, making several cuts to find the best conditions. In the example provided, the sample was cut at -20 &#176;C. Consider keeping the sample under 0 &#176;C to avoid sample degradation. 
	NOTE: The use of a binocular microscope placed next to the cryomicrotome can help determine the quality of the slices. The tissues must stay intact.</li>
	<li>Carefully move the slice to an ITO-coated slide using forceps or a small paintbrush, then place a finger under the slide to warm up and dry the sample. Keep the slide in the cryomicrotome chamber until all samples are ready. Take the slide out of the chamber slowly to avoid heat shock. 
	NOTE: To check which side of the slide is ITO-coated, place a drop of water on the slide at room temperature (RT). The drop will stay round on the ITO-coated side or flatten on the uncoated side.</li>
	<li>Draw marks on the slide using a thin marker pen or correction fluid and scan it with a high-resolution scanner before matrix deposition. The marks will be used to determine the sample&#39;s exact position on the slide when it is in the mass spectrometer. The best way to obtain precise points of reference is to draw a cross.</li>
</ol>
2. Matrix deposition
<ol>
	<li>Prepare the MALDI matrix: weigh 70 mg of &#945;-cyano-4-hydroxycinnamic&#160;acid (HCCA) matrix and dilute it in 10 mL of a water and methanol solution (50:50) with 0.2% TFA. Sonicate the matrix for 10 mins at RT. There may be extra solid matrix after sonication. 
	NOTE: Different types of MALDI matrices can be used depending on the analytes of interest.</li>
	<li>Clean the matrix deposition robot with 100% methanol.</li>
	<li>Using methanol and a precision wipe, clean the slide, holding the samples without touching them and without removing the marks. Add a clean coverslip over the slide in an area without sample. That part of the slide is then placed over the optical detector to track matrix deposition. 
	NOTE: To optically track matrix thickness, the slide and coverslip must be very clean.</li>
	<li>Use the robot for matrix deposition: place only 6 mL of the matrix solution in the reservoir, and add 2 mL of 100% methanol for a total volume of 8 mL. 
	NOTE: Recording of matrix thickness along deposition is automatic and can be recovered using a USB stick. The development of a spraying method is not detailed here.</li>
</ol>
3. Data acquisition
<ol>
	<li>Place 1 &#181;L of the matrix on a MALDI plate to calibrate the mass spectrometer using the matrix peaks as references. The development of an acquisition method on the mass spectrometer is not detailed here.
	<ol>
		<li>Insert the MALDI plate in the source, click &#8220;Load Target&#8221; in ftmsControl software, and wait for the plate to be loaded.</li>
		<li>Choose the position of the matrix spot by clicking on the spot in the image representing the MALDI plate.</li>
		<li>Indicate the sample name and the folder in the &#8220;Sample Info&#8221; tab of the software.</li>
		<li>Start acquisition by clicking &#8220;Acquisition&#8221;.</li>
		<li>Move the plate slightly during the acquisition using the mouse pointer on the &#8220;MALDI video&#8221; tab so that the laser points at different spots.</li>
		<li>When the acquisition is finished, go to the &#8220;Calibration&#8221; tab, choose HCCA calibration list in Quadratic Mode and click Automatic. The global calibration result is indicated in the &#8220;Calibration Plot&#8221; window and should be less than 0.2 ppm for a SolariX XR 7T.</li>
		<li>If the calibration is good, click &#8220;Accept&#8221; and save the method.</li>
	</ol>
	</li>
	<li>Once the matrix deposition on the samples is finished, place the slide in a slide adapter and retrieve the position of the marks using a plastic cover.</li>
	<li>In flexImaging software, set up a new imaging run using the first window that opens with the software.
	<ol>
		<li>Name the imaging run, select the Result Directory, and click Next.</li>
		<li>Indicate the raster size (i.e., user-defined measurement region), choose the method to be used (calibrated at section 3.1), and click Next.</li>
		<li>Load the optical image of the slide obtained at step 1.6 after scanning the slide and click Next.</li>
		<li>The image opens in a wider window. Use the marks on the slide to teach the position of the slides: in ftmsControl, place the target of the MALDI video window on the exact position of a mark, then come back to felxImaging and click on the exact same point on the optical image. Repeat this for three independent points. The plastic cover bearing the marks is placed on a MALDI plate to recover its position and facilitate teaching. 
		NOTE: If the marks were made with a marker, adding white correction fluid at the back of the slide can make them stand out during teaching.</li>
	</ol>
	</li>
	<li>Draw the regions of interest in the samples in the flexImaging software using the &#8220;Add Measurement Regions&#8221; tools.</li>
	<li>Save the imaging run and an &#8220;AutoXecute Sequence&#8221; if several samples are to be analyzed.</li>
	<li>Launch a sequence using &#8220;AutoXecute Batch Runner&#8221; if several samples are analyzed. 
	NOTE: Synchronize the data regularly to a secure location.</li>
</ol>
4. Data processing
<ol>
	<li>Import the raw data to the visualization software (SCiLS Lab) and create a dataset. This is done in two separate steps in this software.
	<ol>
		<li>Use the &#8220;Batch Importer&#8221; tool and select the raw data, indicate the target directory, and click on &#8220;Import&#8221;.</li>
		<li>After import, several datasets can be combined for further analysis. To combine datasets, use the &#8220;File|New|Dataset&#8221; tool.</li>
		<li>Select the type of instrument used for acquisition, add the imported datasets by clicking &#8220;+&#8221;, and arrange the images by clicking and dragging the objects.</li>
		<li>Check the mass range settings or modify if needed by indicating the range of interest. Click Next to see the import summary and launch the import.</li>
	</ol>
	</li>
	<li>Visualize the m/z of interest in the different tissues of a sample and/or in different samples. Simply click on the spectra to select the m/z of interest or type the value in the m/z box.
	<ol>
		<li>Perform statistical tests if needed to search for colocalized or discriminative values between different tissues and/or different samples to determine the m/z of interest. The tools are available in the &#8220;Tool&#8221; menu and will not be described in this protocol.</li>
	</ol>
	</li>
	<li>Export the m/z of interest (e.g., colocalized, discriminative compounds) as a .csv file from the Object tab. The whole dataset can also be exported if it is not too big (i.e., maximum 60,000 lines). Click on the &#8220;Export&#8221; icon on the right side of the region of interest indicated by a red arrow.</li>
	<li>Import the .csv file to create a new dataset in the annotation software (Metaboscape). Click on &#8220;Projects | Import CSV project&#8221;. Be aware that only the exact m/z will be considered, and the isotopic profile is lost. 
	NOTE: The 5.0 software version allows direct import of data from the visualization software, which enables a more accurate annotation, because the isotopic profile can be considered.</li>
	<li>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.</li>
	<li>Use the prediction software (Metabolite Predict) to perform in silico prediction of the metabolites of the annotated compounds. The developed formula of the compound of interest is needed, either drawn by the user in the software or imported as a .mol file. Then the method is a simple step-by-step protocol.</li>
	<li>Recover the list of metabolites, create an analyte list, and use it in the annotation software to annotate the raw data with predicted metabolites. Alternatively, if the list of metabolites is short, manually search for it in step 4.8.</li>
	<li>Visualize the tissue distribution of the metabolites in the raw data in the visualization software.</li>
	<li>Recover the names of the enzymes involved in the metabolic processes in the prediction software by right-clicking on the predicted metabolite of interest.</li>
</ol>

<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. 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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><tbody><tr><td>Cover slips</td><td>Bruker Daltonics</td><td>267942</td><td></td></tr><tr><td>Cryomicrotome</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>Excel</td><td>Microsoft corporation</td><td></td><td></td></tr><tr><td>flexImaging</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>ftmsControl</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>GTX primescan</td><td>GX Microscopes</td><td></td><td></td></tr><tr><td>HCCA MALDI matrix</td><td>Bruker Daltonics</td><td>8201344</td><td></td></tr><tr><td>ImagePrep</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>ITO-coated slides</td><td>Bruker Daltonics</td><td>237001</td><td></td></tr><tr><td>M1-embedding matrix</td><td>ThermoScientific</td><td>1310</td><td></td></tr><tr><td>Metabolite Predict</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>Metaboscape</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>Methanol</td><td>Fisher Chemicals</td><td></td><td>No specific reference needed</td></tr><tr><td>MX 35 Ultra blades</td><td>Thermo Scientific</td><td>15835682</td><td></td></tr><tr><td>Plastic molds</td><td></td><td></td><td>No specific reference needed</td></tr><tr><td>SCiLS Lab</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>SolariX XR 7Tesla</td><td>Bruker Daltonics</td><td></td><td>The method proposed is not limited to this instrument</td></tr><tr><td>Spray sheets for ImagePrep</td><td>Bruker Daltonics</td><td>8261614</td><td></td></tr><tr><td>TFA</td><td>Sigma Aldrich</td><td></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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3.0K Views.  Université de Strasbourg. 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.

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

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

Chemistry

Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

Plant biomass offers a renewable resource for multiple products, including fuel, feed, food, and a variety of materials. In this paper we investigate the properties of tobacco tree (Nicotiana glauca) and poplar as suitable sources for a biorefinery pipeline.

The global demand for food, feed, energy, and water poses extraordinary challenges for future generations. It is evident that robust platforms for the ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Biochemistry

Elucidating the Metabolism of 2,4-Dibromophenol in Plants

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential human exposure through the consumption of pollutant-accumulated foods. Elucidating the uptake and metabolism of xenobiotics in crops is essential for the assessment of dietary exposure risk in humans. However, for such experiments, the use of intact plants requires long-term experiments and complex sample preparation protocols that can be affected by various factors. Plant callus cultures combined with high-resolution mass spectrometry (HRMS) may provide a solution for the accurate and time-saving identification of metabolites of xenobiotics in plants, as it can avoid interference from the microbial or fungal microenvironment, shorten the treatment duration, and simplify the matrix effect of intact plants. 2,4-dibromophenol, a typical flame retardant and endocrine disrupter, was chosen as the model substance due to its widespread occurrence in soil and its uptake potential by plants. Herein, plant callus was generated from asepsis seeds and exposed to sterile 2,4-dibromophenol-containing culture medium. The results showed that eight metabolites of 2,4-dibromophenol were identified in the plant callus tissues after 120 h of incubation. This indicates that 2,4-dibromophenol was rapidly metabolized in&#160;the plant callus tissues. Thus, the plant callus culture platform is an effective method to evaluate the uptake and metabolism of xenobiotics in plants.

The present protocol describes a simple and efficient method for the identification of 2,4-dibromophenol metabolites in plants.

Crops can be extensively exposed to organic pollutants, since soil is a major sink for pollutants discarded into the environment. This creates potential ...

Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss of muscle mass). Plasma samples were taken at the beginning of the intervention and after 30 days of consuming the functional meals. A semi-targeted ultra-high-performance chromatography coupled with tandem mass (UPLC-MS/MS) analysis was carried out to identify phenolic compounds and their metabolites. Plasma proteins were precipitated with ethanol and the samples were concentrated and resuspended in the mobile phase (1:1 acetonitrile: water) before injection into the UPLC-MS/MS instrument. Separation was carried out with a C18 reverse-phase column, and compounds were identified using their experimental mass, isotopic distribution, and fragment pattern. Compounds of interest were compared to those of data banks and the internal semi-targeted library. Preliminary results showed that the major metabolites identified after the intervention were phenylacetic acid, glycitin, 3-hydroxyphenylvaleric acid, and gomisin M2.

The goal of this protocol is to detect phenolic metabolites in plasma using a semi-targeted chromatography-mass spectrometry method.

A group of 23 elderly persons was given functional meals (a beverage and a muffin) specially formulated for the prevention of sarcopenia (age-related loss ...

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) is commonly used to extract Tibetan medicine; however, many unpredictable unknown compounds remain after using the spectrum database. The present article developed a universal method for identifying components in Tibetan medicine using ion trap mass spectrometry (IT-MS). The method includes standardized and programmed protocols for sample preparation, MS setting, LC prerun, method establishment, MS acquisition, multiple-stage MS operation, and manual data analysis. Two representative compounds in the Tibetan medicine Abelmoschus manihot seeds were identified using multiple-stage fragmentation, with a detailed analysis of typical compound structures. In addition, the article discusses aspects such as ion mode selection, mobile phase adjustment, scanning range optimization, collision energy control, collision mode switchover, fragmentation factors, and limitations of the method. The developed standardized analysis method is universal and can be applied to unknown compounds in Tibetan medicine.

Here, we describe a general protocol and design that could be applied to identify trace amounts and minor constituents in the complex natural product formulations (matrixes) in Tibetan medicine.

Tibetan medicines are complex and contain numerous unknown compounds, making in-depth research on their molecular structures crucial. Liquid ...

Identifying Chemical Composition of Gynura bicolor by UPLC-TOF Mass Spectrometry

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). Ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) has gradually become a leading technology for the identification of TCM ingredients. Gynura bicolor DC. (BFH), a perennial stemless herb used for medicine and food in China has medicinal effects such as clearing heat, moistening the lung, relieving cough, dispersing stasis, and relieving swelling. Polyphenols and flavonoids contain numerous isomers, which hinder the identification of the complex compounds in BFH. This paper presents a systematic protocol for studying chemical constituents of BFH based on solvent extraction and integrated data via UPLC-Q-TOF-MS.
The method described here includes systematic protocols for sample pretreatment, MS calibration, MS acquisition, data processing, and analysis of results. Sample pretreatment includes collection, cleaning, drying, crushing, and extraction. MS calibration consists of multipoint and single-point correction. Data processing includes data importing, method establishment, analysis processing, and result presentation. Representative results of the typical fragmentation pattern of phenolic acids, esters, and glycosides in Gynura bicolor DC. (BFH) are presented in this paper. In addition, organic solvent selection, extraction, data integration, collision energy selection, and method improvement are discussed in detail. This universal protocol can be widely used to identify complex compounds in TCM.

Preparation of Gynura bicolor DC samples for High-Resolution Tandem Mass Spectrometry

We describe a general protocol that integrates high-resolution mass spectrometry analysis and molecular docking in traditional Chinese medicine research.

The separation and analysis of the desired chemical components are important subjects for the fundamental research of traditional Chinese medicine (TCM). ...

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

Biology

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

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

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Semi-Targeted Ultra-High-Performance Chromatography Coupled to Mass Spectrometry Analysis of Phenolic Metabolites in Plasma of Elderly Adults

Standardized Identification of Compound Structure in Tibetan Medicine Using Ion Trap Mass Spectrometry and Multiple-Stage Fragmentation Analysis

High-resolution Tandem Mass Spectrometry for Studying Chemical Constituents of Gynura bicolor DC

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions