The authors would like to acknowledge Dr. Jean-Michel An&#233; in the Department of Agronomy at UW-Madison for providing Medicago truncatula samples. This work was supported in part by funding from the National Science Foundation (NSF) grant CHE-0957784, the University of Wisconsin Graduate School and the Wisconsin Alumni Research Foundation (WARF) and Romnes Faculty Research Fellowship program (to L.L.). E.G. acknowledges an NSF Graduate Research Fellowship (DGE-1256259).

1. Instrumentation
<ol>
	<li>MALDI-TOF/TOF MSI. Use a mass spectrometer equipped with a MALDI source for analysis of small molecules (see Table of Materials/Equipment). Perform acquisitions in positive or negative ion mode depending on the analytes of interest. Specify a mass range of interest and collect 500 laser shots/spot at 50 &#181;m intervals in both the x and y dimensions across the surface of the sample to generate ion images. The raster width and number of laser shots can be adjusted to obta...

<ol>
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B., Gemperline, E., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+quantitative+mass+spectrometry+imaging+of+drugs+and+metabolites.">Qualitative and quantitative mass spectrometry imaging of drugs and metabolites.</a> Adv. Drug Deliv. Rev. 65, 1074-1085 (2013).</li><li>Ye, H., 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=MALDI+mass+spectrometry-assisted+molecular+imaging+of+metabolites+during+nitrogen+fixation+in+the+Medicago+truncatula-Sinorhizobium+meliloti+symbiosis.">MALDI mass spectrometry-assisted molecular imaging of metabolites during nitrogen fixation in the Medicago truncatula-Sinorhizobium meliloti symbiosis.</a> Plant J. 75, 130-145 (2013).</li><li>Ye, H., Gemperline, E., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vision+for+better+health:+mass+spectrometry+imaging+for+clinical+diagnostics.">A vision for better health: mass spectrometry imaging for clinical diagnostics.</a> Clin. Chim. Acta. 420, 11-22 (2013).</li><li>Wei, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolomics+and+Its+Practical+Value+in+Pharmaceutical+Industry.">Metabolomics and Its Practical Value in Pharmaceutical Industry.</a> Curr. Drug Metab. 12, 345-358 (2011).</li><li>Kobayashi, T., 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=A+Novel+Serum+Metabolomics-Based+Diagnostic+Approach+to+Pancreatic+Cancer.">A Novel Serum Metabolomics-Based Diagnostic Approach to Pancreatic Cancer.</a> Cancer Epidem. Biomar. 22, 571-579 (2013).</li><li>West, P. R., Weir, A. M., Smith, A. M., Donley, E. L. R., Cezar, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+human+developmental+toxicity+of+pharmaceuticals+using+human+embryonic+stem+cells+and+metabolomics.">Predicting human developmental toxicity of pharmaceuticals using human embryonic stem cells and metabolomics.</a> Toxicol. Appl. Pharm. 247, 18-27 (2010).</li><li>Spegel, 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=Time-resolved+metabolomics+analysis+of+beta-cells+implicates+the+pentose+phosphate+pathway+in+the+control+of+insulin+release.">Time-resolved metabolomics analysis of beta-cells implicates the pentose phosphate pathway in the control of insulin release.</a> Biochem. J. 450, 595-605 (2013).</li><li>Pendyala, G., Want, E. J., Webb, W., Siuzdak, G., Fox, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+for+neuroAIDS:+The+widening+scope+of+metabolomics.">Biomarkers for neuroAIDS: The widening scope of metabolomics.</a> J. Neuroimmune. Pharm. 2, 72-80 (2007).</li><li>Prell, J., Poole, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+changes+of+rhizobia+in+legume+nodules.">Metabolic changes of rhizobia in legume nodules.</a> Trends Microbiol. 14, 161-168 (2006).</li><li>Kutz, K. K., Schmidt, J. J., Li, L. J. <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+tissue+analysis+of+neuropeptides+by+MALDI+FTMS+in-cell+accumulation.">In situ tissue analysis of neuropeptides by MALDI FTMS in-cell accumulation.</a> Anal. Chem. 76, 5630-5640 (1021).</li><li>Stemmler, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-mass-resolution+direct-tissue+MALDI-FTMS+reveals+broad+conservation+of+three+neuropeptides+(APSGFLGMRamide,+GYRKPPFNGSIFamide+and+pQDLDHVFLRFamide)+across+members+of+seven+decapod+crustaean+infraorders.">High-mass-resolution direct-tissue MALDI-FTMS reveals broad conservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapod crustaean infraorders.</a> Peptides. 28, 2104-2115 (2007).</li><li>Rubakhin, S. S., Churchill, J. D., Greenough, W. T., Sweedler, J. 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B., Gile, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+of+biological+samples:+localization+of+peptides+and+proteins+using.">Molecular imaging of biological samples: localization of peptides and proteins using.</a> MALDI-TOF MS. Anal. Chem. 69, 4751-4760 (1997).</li><li>Baluya, D. L., Garrett, T. J., Yost, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+MALDI+matrix+deposition+method+with+inkjet+printing+for+imaging+mass+spectrometry.">Automated MALDI matrix deposition method with inkjet printing for imaging mass spectrometry.</a> Anal. Chem. 79, 6862-6867 (2007).</li><li>Hankin, J. A., Barkley, R. M., Murphy, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublimation+as+a+method+of+matrix+application+for+mass+spectrometric+imaging.">Sublimation as a method of matrix application for mass spectrometric imaging.</a> J. Am. Soc. Mass Spectrom. 18, 1646-1652 (2007).</li><li>Robichaud, G., Garrard, K. P., Barry, J. A., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSiReader:+an+open-source+interface+to+view+and+analyze+high+resolving+power+MS+imaging+files+on+Matlab+platform.">MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.</a> J. Am. Soc. Mass Spectrom. 24, 718-721 (2013).</li><li>Northen, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrate+nanostructures+for+mass+spectrometry.">Clathrate nanostructures for mass spectrometry.</a> Nature. 449, (2007).</li><li>Shrivas, K., Hayasaka, T., Sugiura, Y., Setou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+simultaneous+imaging+of+endogenous+low+molecular+weight+metabolites+in+mouse+brain+using+TiO2+nanoparticles+in+nanoparticle-assisted+laser+desorption/ionization-imaging+mass+spectrometry.">Method for simultaneous imaging of endogenous low molecular weight metabolites in mouse brain using TiO2 nanoparticles in nanoparticle-assisted laser desorption/ionization-imaging mass spectrometry.</a> Anal. Chem. 83, 7283-7289 (2011).</li><li>Thomas, A., Charbonneau, J. L., Fournaise, E., Chaurand, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sublimation+of+new+matrix+candidates+for+high+spatial+resolution+imaging+mass+spectrometry+of+lipids:+enhanced+information+in+both+positive+and+negative+polarities+after+1,5-diaminonapthalene+deposition.">Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.</a> Anal. Chem. 84, 2048-2054 (2012).</li><li>Chen, S., 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=2,3,4,5-Tetrakis(3',4'-dihydroxylphenyl)thiophene:+a+new+matrix+for+the+selective+analysis+of+low+molecular+weight+amines+and+direct+determination+of+creatinine+in+urine+by+MALDI-TOF+MS.">2,3,4,5-Tetrakis(3',4'-dihydroxylphenyl)thiophene: a new matrix for the selective analysis of low molecular weight amines and direct determination of creatinine in urine by MALDI-TOF MS.</a> Anal. Chem. 84, 10291-10297 (2012).</li><li>Shroff, R., Rulisek, L., Doubsky, J., Svatos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acid-base-driven+matrix-assisted+mass+spectrometry+for+targeted+metabolomics.">Acid-base-driven matrix-assisted mass spectrometry for targeted metabolomics.</a> Proc. Natl. Acad. Sci. 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The authors declare that they have no competing financial interests.

As discussed above, sample preparation is the most critical step in the MSI workflow. Embedding the tissue unevenly will cause sectioning to be difficult or not possible in some cases. The section size and adequate equilibration time are crucial to maintaining the tissue integrity and avoiding folding and tears. Selection of matrix and application technique will play a role in determining the types of analytes to be detected, the spatial resolution, and reproducibility of the results. Using a combination of matrices or a...

The growing field of metabolomics has many important biological applications including biomarker discovery, deciphering metabolic pathways in plants and other biological systems, and toxicology profiling4,6-10. A major technical challenge when studying biological systems is to study metabolomic pathways without disrupting them11. MALDI-MSI allows for direct analysis of intact tissues that enables sensitive detection of analytes in single organs12,13 and even single cells14,15.
Sample preparation is a crucial step in producing reproducible and reliable mass spectral images. The quality of the images greatly depends upon factors such as tissue embedding medium, slice thickness, MALDI matrix, and matrix application technique. For imaging applications, ideal section thickness is the width of one cell (8-20 &#181;m depending on the sample type). MALDI requires deposition of an organic, crystalline matrix compound, typically a weak acid, on the sample to assist analyte ablation and ionization.16 Different matrices provide different signal intensities, interfering ions, and ionization efficiencies of different classes of compounds.
The matrix application technique also plays a role in the quality of mass spectral images and different techniques are appropriate for different classes of analytes. Three matrix application methods are presented in this protocol: airbrush, automatic sprayer, and sublimation. Airbrush matrix application has been widely used in MALDI imaging. The advantage of airbrush matrix application is that it is relatively fast and easy. However, the quality of the airbrush matrix application greatly depends on the skill of the user and tends to be less reproducible and cause diffusion of analytes, especially small molecules17. Automatic sprayer systems have similar mechanics to airbrush matrix application, but have been developed to remove the variability seen with manual airbrush application, making the spray more reproducible. This method can sometimes be more time-consuming than traditional airbrush matrix application. Both manual airbrush and automatic sprayer systems are solvent-based matrix application methods. Sublimation is a dry matrix application technique that is becoming more and more popular for mass spectral imaging of metabolites and small molecules because it reduces analyte diffusion; however, it lacks the solvent necessary to extract and observe higher mass compounds18.
Confident identification of metabolites typically requires accurate mass measurements to obtain putative identifications followed by tandem mass (MS/MS) experiments for validation, with MS/MS spectra being compared to standards, literature, or theoretical spectra. In this protocol high resolution (mass resolving power of 60,000 at m/z 400), liquid chromatography (LC)-MS is coupled to MALDI-MSI to obtain both spatial information and confident identifications of endogenous metabolites, using Medicago truncatula roots and root nodules as the biological system. MS/MS experiments can be performed directly on the tissue with MALDI-MSI or on tissue extracts with LC-MS and used for the validation of metabolite identifications.
This protocol provides a simple method to map endogenous metabolites in M. truncatula, which can be adapted and applied to MSI of small molecules in various tissue types and biological systems.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gelatin</td>
			<td style="width:181px;">Difco</td>
			<td style="width:163px;"><a href="http://catalog.bd.com/bdCat/viewProduct.doCustomer?productNumber=214340">214340</a></td>
			<td style="width:611px;">heat to dissolve</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostat- HM 550</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td style="width:163px;">956564A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">indium tin oxide (ITO)-coated glass slides&#160;</td>
			<td>Delta Technologies</td>
			<td>CB-90IN-S107</td>
			<td>25-75-0.8 mm (width-length-thickness)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2,5-Dihydroxybenzoic acid (DHB) matrix&#160;</td>
			<td>ICN Biomedicals</td>
			<td><a href="http://www.labsource.com/Catalog/Item.aspx?ItemID=1787195">PI90033</a></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Airbrush</td>
			<td>Paasche Airbrush Company</td>
			<td>TG-100D</td>
			<td>coupled with 75 ml steel container</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;">Automatic matrix sprayer system- TM-Sprayer</td>
			<td>HTX Technologies, LLC</td>
			<td style="width:163px;">HTX.TMSP.H021-U</td>
			<td>Specific start-up and shut-down instructions will be given when the instrument is installed</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sublimation apparatus&#160;</td>
			<td>Chemglass Life Science</td>
			<td style="width:163px;"><a href="http://www.chemglass.com/product_view.asp?pnr=CG-3038">CG-3038-01</a></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Vaccum pump- Alcatel 2008 A</td>
			<td>Ideal Vacuum Products</td>
			<td>P10976</td>
			<td>Ultimate Pressure = 1x10-4 Torr</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ultrafleXtreme MALDI-TOF/TOF</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td style="width:163px;">276601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">FlexImaging</td>
			<td style="width:181px;">Bruker Daltonics</td>
			<td>269841</td>
			<td>One example of &#34;vender specific software&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">MALDI LTQ Orbitrap</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFADBMASZ</td>
			<td>High resolution MALDI instrument for accurate mass measurements</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Q Exactive</td>
			<td style="width:181px;">Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMAZR</td>
			<td>High resolution LC-MS instrument for accurate mass measurements</td>
		</tr>
	</tbody>
</table>

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.

An experimental overview of MSI is shown in Figure 1. At the very beginning of the experiment, sample preparation is a critical step. Nodules are trimmed from the plant root and embedded in gelatin. The tissue must be pressed flat against the cryostat cup, with no bubbles, while it is being frozen; this will ensure easier and proper alignment of the tissue while it is being sectioned. When the tissue is being sliced, it is important to cut the tissue at the proper thickness; too thin of sections will tea...

Watch this Scientific Journal Video about MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules at JoVE.com

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;

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 ...

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18.8K ビュー.  University of Wisconsin- Madison. 質量分析イメージング（MSI）は、生物学的プロセスに新たな洞察を提供することができ、ネイティブ環境で化合物を保持し、無傷の組織内の様々な化学種を発見し、識別するために使用することができる強力なツールです。本明細小分子の分析のために開発MSI方法が記載されている。 