The authors thank Professor H. Troy Ghashghaei from NCSU Department of Molecular Biomedical Sciences for providing the whole mouse tissue. The authors also gratefully acknowledge the financial assistance received from National Institutes of Health (R01GM087964), the W.M. Keck foundation, and North Carolina State University.

Note: The following protocol describes all the necessary steps for performing IR-MALDESI MSI experiments. In-depth details about the optimized geometry of the IR-MALDESI source and its synchronization with the laser, stage, and mass spectrometer can be found elsewhere5,6. Animal tissue samples used in this protocol were obtained according to Institutional Animal Care and Use Committee (IACUC) and North Carolina State University regulations.
1. Tissue Preparation
<ol>
	<li>Prepare an isopentane/dry ice bath by placing ~200 ml of isopentane in a clean beaker inside a secondary container of dry ice in a fume hood. Use protective gloves and safety goggles at all times when handling the isopentane/dry ice bath and the tissue.
	<ol>
		<li>Euthanize 2-day-old whole neonatal mouse pup by Avertin overdose (7.5 mg/g body weight), and then freeze the tissue in the isopentane/dry ice bath to preserve tissue structure. Use a pre-cleaned pair of forceps to place and remove the mouse pup in cryomold from the isopentane/dry ice bath. Store the flash-frozen tissue at -80 &#176;C until analysis.</li>
	</ol>
	</li>
	<li>Using protective gloves, apply a layer of optimal cutting temperature (OCT) mounting medium to a 40 mm cryostat specimen disc. Gently place the frozen whole mouse onto the OCT coated specimen holder to adhere mouse to the disc in the desired orientation for sectioning. 
	Note: OCT is used solely to adhere the tissue to the specimen disc for sectioning. Do not completely embed tissue in OCT and avoid excess as OCT is known to have detrimental effects on MSI analysis. Other media, such as gelatin, can be used to cryo-embed the tissue before slicing it9.</li>
	<li>Place the disc (with OCT and tissue section) on Peltier sample holder in cryostat and press Peltier power button. Wait 10 min for sample to come to thermal equilibrium.</li>
	<li>Place the specimens disc (with the tissue mounted on it) inside the disc holder, and face the tissue down to the desired plane for analysis. Slice the tissue into sections of the desired thickness at -20 &#176;C using the rotary microtome housed inside the cryostat10. 
	Note: For whole-body analyses, slice the tissue into 25-&#956;m-thick sections to maintain the tissue integrity. For smaller regions (e.g., liver, brain, kidney), slice the tissue into 10-&#956;m-thick sections.
	<ol>
		<li>Use the anti-roll glass plate to prevent the sliced tissue section from rolling. Use the cryostat vacuum hose and brushes to remove unwanted tissue debris. Once the tissue is sectioned, use the blade guard to cover the sharp microtome blade to avoid personal injuries.</li>
	</ol>
	</li>
	<li>Orient mouse section on the specimen plate and thaw-mount onto a pre-cleaned glass microscope slide by bringing the slide as close as possible to the tissue section, without touching it10. 
	Note: For quantitative MSI experiments, coat the slide with an internal standard using an automated pneumatic sprayer prior to mounting the tissue, and thaw-mount the tissue section on the coated slide11.</li>
	<li>Remove the microtome blade used to sectioning the tissue using the blade ejector, and safely discard the blade in the appropriate &#34;sharp waste&#34; container.</li>
	<li>Keep the glass slide inside the cryostat until Step 3.2 to maintain cryopreservation of the tissue.</li>
</ol>
2. IR-MALDESI Preparation/Calibration
<ol>
	<li>Turn on the mid-IR laser and launch the laser control application on the computer. Choose the desired wavelength using the laser control application supplied by the manufacturer. IR-MALDESI experiments are typically performed at 2,940 nm. 
	Note: Recent experiments have investigated the dependence of mid-IR laser wavelengths and the ice matrix with noted differences which appear to be analyte specific8.</li>
	<li>Turn on the stage controller, launch the custom user control program (RASTIR), and calibrate the stage position using the home button.</li>
	<li>Prepare electrospray solution. Typically, use a 50:50 (v/v) methanol/water with 0.2% formic acid for the electrospray solvent in positive-ion mode. Select the electrospray solvent composition according to the experiment. For instance, use 5 mM ammonium hydroxide as electrospray modifier for imaging applications in negative-ion mode. 
	Note: For polarity switching IR-MALDESI MSI, use 1 mM acetic acid as the modifier. This modifier was found optimum for obtaining stable and reproducible signal in both positive- and negative-ion modes12.</li>
	<li>Fill a 1 ml syringe with electrospray solvent, and flush the silica capillary with new solvent.</li>
	<li>Align ESI emitter on axis with MS inlet using the source parameters such as ESI-spot distance, spot-inlet distance, sample height, and solvent flow rate that have been optimized using statistical DOE for analysis of tissue sections6. Note: See Figure 1 for a schematic depicting these parameters and optimized values for MSI of tissue sections.</li>
	<li>Start the electrospray and evaluate its stability (&#62;10 min) by monitoring the total ion current (TIC) which, at this point, consists solely of ambient compounds. Typically, a TIC variation of &#60;10% over 10-15 min is a good indication of stability.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53942/53942fig1.jpg" /> 
Figure 1. IR-MALDESI schematic and parameters. (A) Schematic of IR-MALDESI source setup (not to scale) and the adjustable parameters. (B) Optimized parameter values for imaging of tissue sections. <a href="https://www.jove.com/files/ftp_upload/53942/53942fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Deposition of Ice Matrix
<ol>
	<li>Caution: Turn off the electrospray before placing sample on the stage.</li>
	<li>Place the thaw-mounted tissue onto the IR-MALDESI sample plate within the camera view.</li>
	<li>Make sure hands are clear of the ESI emitter and restart the electrospray.</li>
	<li>Close the access door of the IR-MALDESI source and purge enclosure with dry nitrogen to prevent condensation of water on the surface of the tissue. Once the relative humidity inside the enclosure reaches &#60;3%, turn on the DC power supply (~12 V) to the Peltier plate, which will cool the sample plate. Note: The cooling process in the Peltier-cooled stage is discussed in detail elsewhere13.</li>
	<li>Cool the stage to -9 &#176;C, and allow the mounted tissue to come to thermal equilibrium (~5-10 min).</li>
	<li>Stop nitrogen purge and expose the tissue to the ambient relative humidity by opening the source access door. A thin layer of ice will be deposited onto the cold surface of the Peltier stage and mounted tissue section due to desublimation of water from the air. 
	Note: If the relative humidity in the lab is below 10-15%, place a beaker of warm water inside the enclosure to facilitate the formation of the ice matrix layer.</li>
	<li>After the ice matrix layer is formed, close the access door and restart the flow of dry nitrogen to reduce the relative humidity to 10&#177;2%. Adjust the nitrogen flow rate to keep the relative humidity at this level, empirically found to be the balance of ice formation and sublimation to maintain a constant layer of ice throughout the experiment6.</li>
</ol>
4. Mass Spectrometry Imaging Data Acquisition
<ol>
	<li>Using the RASTIR GUI (Figure 2), move the stage to the analysis position by clicking the &#34;LASER position&#34; button. Use the adjustment diode of the laser to ensure an off-tissue region will be ablated. Fire a laser shot in an off-tissue region to calibrate the laser offset with respect to camera view. Click the &#34;Laser Test Fire&#34; button to update the laser position.</li>
	<li>Return to &#34;CAMERA position&#34; and update the laser position in RASTIR by placing the laser alignment reticle on the laser ablation spot (Figure 2-1).</li>
	<li>Click the region of interest (ROI) button and adjust the size and position of ROI for the tissue section being analyzed (Figure 2-2). Input appropriate experimental parameters such as the step size in X and Y directions (in mm) (Figure 2-3), and name the MSI file in the &#34;Project Name&#34; box (Figure 2-4).
	<ol>
		<li>When drawing an ROI, include a portion of off-tissue that serves as control. Use this off-tissue section for peak picking (Step 5.4). 
		Note: The desorption diameter (spot size) of the laser on tissue is approximately 150 &#181;m; however, higher spatial resolutions can be obtained by using the oversampling method14,15.</li>
	</ol>
	</li>
	<li>Select the appropriate laser frequency from the dropdown box, number of laser pulses per voxel (integer values), and the delay between the laser trigger and mass spectrometer signal acquisition (Figure 2-5). Typically use two laser pulses per voxel at 20 Hz to completely desorb material in an IR-MALDESI MSI experiments, with a 10 msec delay time to allow ions generated to reach the mass analyzer for measurement5. 
	Note: Choose the highest repetition rate that the laser is capable of operating at. It was recently shown that using higher repetition rate will improve analyte detectability16.</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53942/53942fig2.jpg" /> 
Figure 2. User interface for IR-MALDESI MSI operation. Screenshot of the RASTIR Scan Control program is presented. The steps for performing a MSI experiment are (1) locating the laser spot, (2) drawing an ROI, (3) choosing the stage step size (in mm), (4) giving a name to the file, (5) choosing the correct number of pulses per voxel along with the desired repetition rate, and (6) checking the list for imaging and MS setup. <a href="https://www.jove.com/files/ftp_upload/53942/53942fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Choose mass spectrometer parameters such as ionization mode, electrospray voltage, solvent flow rate, capillary temperature, and the injection time in the mass spectrometer software. Refer to Table 1 for an example of the parameters used in whole-body IR-MALDESI MSI. 
	Note: Due to the complexity of biological samples and lack of separation methods in MSI analyses, the IR-MALDESI source is coupled to a high resolving power and high mass accuracy instrument. An acquisition method can be loaded for analyses such as parallel reaction monitoring (PRM)7 or polarity switching MSI12.</li>
	<li>Once all parameters (laser, stage, and mass spectrometer) have been chosen, place the MS in &#34;handshake&#34; mode for synchronization, and start MS acquisition.</li>
	<li>Using RASTIR imaging software, verify all steps in checklist (Figure 2-6) are completed by checking boxes, and load the program. Once the program has been loaded, the &#34;Run&#34; button will be available. Press Run to start MSI signal acquisition.</li>
	<li>Upon completion of the imaging experiment, stop mass spectrometer acquisition and place the instrument in standby mode. Turn off nitrogen purge to the enclosure, turn off power supply to Peltier plate, and turn off the stage controller and laser.</li>
	<li>Open the access door, remove the electrospray emitter tip from inside the source enclosure, and remove the extended heated metal MS inlet using protective gloves. Caution: The extended metal MS inlet will be very hot.</li>
	<li>Wearing gloves and safety goggles, clean the extended metal capillary inlet by ultrasonic bath in 15% nitric acid for 10 min, then in HPLC-grade water for 10 min, and finally in HPLC-grade methanol for 10 min. Dry the metal capillary inlet under a stream of nitrogen, and reinsert into MS. Note: Dispose solvent in the appropriate waste containers afterward.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0" width="432">
	<colgroup>
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:216px;">Parameter</td>
			<td dir="LTR" style="width:216px;">Value</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Ionization Mode</td>
			<td dir="LTR" style="width:216px;">Positive</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Electrospray Voltage</td>
			<td dir="LTR" style="width:216px;">3.8-4.2 kV</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Solvent Flow Rate</td>
			<td dir="LTR" style="width:216px;">2 &#956;l/min</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Capillary Temperature</td>
			<td dir="LTR" style="width:216px;">275 &#176;C</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Scan Range</td>
			<td dir="LTR" style="width:216px;">m/z 250-1,000</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Scan Type&#160;</td>
			<td dir="LTR" style="width:216px;">Full Scan</td>
		</tr>
		<tr height="21">
			<td dir="LTR" height="21" style="height:21px;width:216px;">Injection Time</td>
			<td dir="LTR" style="width:216px;">110 msec</td>
		</tr>
		<tr height="22">
			<td dir="LTR" height="22" style="height:22px;width:216px;">Resolving Power</td>
			<td dir="LTR" style="width:216px;">140,000</td>
		</tr>
	</tbody>
</table>
Table 1. Instrument parameters used in whole-body IR-MALDESI MSI.
5. Data Analysis
<ol>
	<li>Convert the raw data files generated by the instrument to data formats such as mzXML17 or imzML18 using free software such as MSConvert17 and imzML converter19.</li>
	<li>Start MSiReader v1.0, an open-source software developed for analysis of high-resolving power MSI data20, on a dedicated processing computer. Load the imaging file onto MSiReader. For user interface of MSiReader and some of its built-in features, see Figure 3.</li>
	<li>Generate ion maps of analytes of interest by inputting their m/z and choose an appropriate m/z window in parts per million (ppm) or Thomson (Th) (Figure 3-1). For high resolving power (RP) instruments choose a window in the low-ppm range. 
	Note: The appropriate m/z window depends on the RP and mass measurement accuracy (MMA) of the instrument that IR-MALDESI source is coupled to.</li>
	<li>Further interpret the data using the built-in features such as interpolation (Figure 3-2), normalization (Figure 3-3), optical image overlay (Figure 3-4), and peak picking (Figure 3-5)20.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/53942/53942fig3.jpg" /> 
Figure 3. User interface of MSiReader; v1.020. Once a file is loaded into the software, ion maps of analytes of interest are displayed by (1) inputting the m/z and tolerance in ppm or Th. Further analysis such as (2) interpolation or (3) normalization can also be performed. An optical image of the tissue can also be imported and superimposed with the ion maps (4) for better visualization. For untargeted analyses the peak picking function (5) can be used to extract tissue-specific peaks by choosing the area of tissue (magenta line) and a reference area off-tissue (green box). <a href="https://www.jove.com/files/ftp_upload/53942/53942fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Use the peak picking function to generate a list of tissue-specific m/z values using user-defined criteria. Note: The PeakFinder algorithm uses differences in average mass spectrum of two regions. These m/z values can then be searched against metabolite databases such as METLIN21 or LIPID MAPS.22
	<ol>
		<li>Refer to Figure 3 for an example of choosing an interrogated tissue region (magenta polygon ROI) and off-tissue reference region (green polygon ROI).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Mcdonnell, L. A., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Mass+Spectrometry.">Imaging Mass Spectrometry.</a> Mass Spectrom. Rev. 26, 606-643 (2007).</li><li>Chughtai, K., Heeren, R. M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometric+imaging+for+biomedical+tissue+analysis.">Mass spectrometric imaging for biomedical tissue analysis.</a> Chem. Rev. 110 (5), 3237-3277 (2010).</li><li>Robichaud, G., 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=Atmospheric+Pressure+Mass+Spectrometry+Imaging.">Atmospheric Pressure Mass Spectrometry Imaging.</a> Encycl. Anal. Chem. , (2014).</li><li>Sampson, J. S., Hawkridge, A. M., 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=Generation+and+detection+of+multiply-charged+peptides+and+proteins+by+matrix-assisted+laser+desorption+electrospray+ionization+(MALDESI)+Fourier+transform+ion+cyclotron+resonance+mass+spectrometry.">Generation and detection of multiply-charged peptides and proteins by matrix-assisted laser desorption electrospray ionization (MALDESI) Fourier transform ion cyclotron resonance mass spectrometry.</a> J. Am. Soc. Mass Spectrom. 17 (12), 1712-1716 (2006).</li><li>Robichaud, G., Barry, J. A., Garrard, K. P., 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=Infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+imaging+source+coupled+to+a+FT-ICR+mass+spectrometer.">Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging source coupled to a FT-ICR mass spectrometer.</a> J. Am. Soc. Mass Spectrom. 24 (1), 92-100 (2013).</li><li>Robichaud, G., 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=IR-MALDESI+Mass+Spectrometry+Imaging+of+Biological+Tissue+Sections+Using+Ice+as+a+Matrix.">IR-MALDESI Mass Spectrometry Imaging of Biological Tissue Sections Using Ice as a Matrix.</a> J. Am. Soc. Mass Spectrom. 25 (3), 319-328 (2014).</li><li>Barry, J. 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=Mapping+Antiretroviral+Drugs+in+Tissue+by+IR-MALDESI+MSI+Coupled+to+the+Q+Exactive+and+Comparison+with+LC-MS/MS+SRM+Assay.">Mapping Antiretroviral Drugs in Tissue by IR-MALDESI MSI Coupled to the Q Exactive and Comparison with LC-MS/MS SRM Assay.</a> J. Am. Soc. Mass Spectrom. 25 (12), 2038-2047 (2014).</li><li>Rosen, E. P., Bokhart, M. T., Ghashghaei, H. T., 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=Influence+of+Desorption+Conditions+on+Analyte+Sensitivity+and+Internal+Energy+in+Discrete+Tissue+or+Whole+Body+Imaging+by+IR-MALDESI.">Influence of Desorption Conditions on Analyte Sensitivity and Internal Energy in Discrete Tissue or Whole Body Imaging by IR-MALDESI.</a> J. Am. Soc. Mass Spectrom. 26, 899-910 (2015).</li><li>Nelson, K. A., Daniels, G. J., Fournie, J. W., Hemmer, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+whole-body+zebrafish+sectioning+methods+for+mass+spectrometry+imaging.">Optimization of whole-body zebrafish sectioning methods for mass spectrometry imaging.</a> J. Biomol. Tech. 24 (3), 119-127 (2013).</li><li>Park, J. J., Cunningham, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thin+sectioning+of+slice+preparations+for+immunohistochemistry.">Thin sectioning of slice preparations for immunohistochemistry.</a> J. Vis. Exp. (3), e194 (2007).</li><li>Bokhart, M. T., Rosen, E., Thompson, C., Sykes, C., Kashuba, A. D. M., 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=Quantitative+mass+spectrometry+imaging+of+emtricitabine+in+cervical+tissue+model+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization.">Quantitative mass spectrometry imaging of emtricitabine in cervical tissue model using infrared matrix-assisted laser desorption electrospray ionization.</a> Anal. Bioanal. Chem. 407 (8), 2073-2084 (2015).</li><li>Nazari, M., 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=Polarity+Switching+Mass+Spectrometry+Imaging+of+Healthy+and+Cancerous+Hen+Ovarian+Tissue+Sections+by+Infrared+Matrix-Assisted+Laser+Desorption+Electrospray+Ionization+(IR-MALDESI).">Polarity Switching Mass Spectrometry Imaging of Healthy and Cancerous Hen Ovarian Tissue Sections by Infrared Matrix-Assisted Laser Desorption Electrospray Ionization (IR-MALDESI).</a> Analyst. 141, 595-605 (2016).</li><li>Hsu, C. 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=Design+and+Application+of+a+Low-Temperature+Peltier-Cooling+Microscope.">Design and Application of a Low-Temperature Peltier-Cooling Microscope.</a> J. Pharm. Sci. 85 (1), 70-74 (1996).</li><li>Jurchen, J. C., Rubakhin, S. S., Sweedler, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MALDI-MS+imaging+of+features+smaller+than+the+size+of+the+laser+beam.">MALDI-MS imaging of features smaller than the size of the laser beam.</a> J. Am. Soc.Mass Spectrom. 16 (10), 1654-1659 (2005).</li><li>Nazari, M., 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=Cellular-level+mass+spectrometry+imaging+using+infrared+matrix-assisted+laser+desorption+electrospray+ionization+(IR-MALDESI)+by+oversampling.">Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling.</a> Anal. Bioanal. Chem. 407 (8), 2265-2271 (2015).</li><li>Rosen, E. P., Bokhart, M. T., Nazari, M., 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=Influence+of+C-Trap+Ion+Accumulation+Time+on+the+Detectability+of+Analytes+in+IR-MALDESI+MSI.">Influence of C-Trap Ion Accumulation Time on the Detectability of Analytes in IR-MALDESI MSI.</a> Anal. Chem. 87, 10483-10490 (2015).</li><li>Kessner, D., Chambers, M., Burke, R., Agus, D., Mallick, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProteoWizard:+open+source+software+for+rapid+proteomics+tools+development.">ProteoWizard: open source software for rapid proteomics tools development.</a> Bioinformatics. 24 (21), 2534-2536 (2008).</li><li>Schramm, 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=ImzML+-+A+common+data+format+for+the+flexible+exchange+and+processing+of+mass+spectrometry+imaging+data.">ImzML - A common data format for the flexible exchange and processing of mass spectrometry imaging data.</a> J. Proteomics. 75 (16), 5106-5110 (2012).</li><li>Race, A. M., Styles, I. B., Bunch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusive+sharing+of+mass+spectrometry+imaging+data+requires+a+converter+for+all.">Inclusive sharing of mass spectrometry imaging data requires a converter for all.</a> J. Proteomics. 75 (16), 5111-5112 (2012).</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 (5), 718-721 (2013).</li><li>Smith, C. A., O'Maille, G., 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=METLIN:+a+metabolite+mass+spectral+database.">METLIN: a metabolite mass spectral database.</a> Ther. Drug. Monit. 27 (6), 747-751 (2005).</li><li>Sud, M., 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=LMSD:+LIPID+MAPS+structure+database.">LMSD: LIPID MAPS structure database.</a> Nucleic Acids Res. 35, D527-D532 (2007).</li><li>Schwartz, S. A., Reyzer, M. L., 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+tissue+analysis+using+matrix-assisted+laser+desorption/ionization+mass+spectrometry:+practical+aspects+of+sample+preparation.">Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation.</a> J. Mass Spectrom. 38 (7), 699-708 (2003).</li><li>Takai, N., Tanaka, Y., Inazawa, K., Saji, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+pharmaceutical+drug+distribution+in+multiple+organs+by+imaging+mass+spectrometry.">Quantitative analysis of pharmaceutical drug distribution in multiple organs by imaging mass spectrometry.</a> Rapid Commun. Mass Spectrom. 26 (13), 1549-1556 (2012).</li><li>Liu, J., Gingras, J., Ganley, K. P., Vismeh, R., Teffera, Y., Zhao, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-body+tissue+distribution+study+of+drugs+in+neonate+mice+using+desorption+electrospray+ionization+mass+spectrometry+imaging.">Whole-body tissue distribution study of drugs in neonate mice using desorption electrospray ionization mass spectrometry imaging.</a> Rapid Commun. Mass Spectrom. 28 (2), 185-190 (2014).</li></ol>

The authors declare no competing financial interests.

The protocol above describes the key steps for performing an IR-MALDESI MSI experiment. The matrix application process (Section 3) takes approximately 20 min, which is similar to a typical matrix application process for MALDI MSI experiments by sublimation or spray-coating using a robotic sprayer. Furthermore, IR-MALDESI does not rely on partitioning of analytes into the matrix crystals6, and the ice matrix can be universally used for all analytes regardless of their mass, size, or chemical properties. In addi...

Mass spectrometry imaging (MSI) in microprobe mode involves desorption of the sample from a surface by a beam (laser or ions) at discrete locations over the surface of a sample. At each raster point, a mass spectrum is generated and the acquired spectra, along with the spatial location from which they were collected, can be used to simultaneously map numerous analytes within the sample. This label-free manner of imaging coupled to the sensitivity and specificity of mass spectrometry have helped MSI become one of the most rapidly evolving fields in mass spectrometry1,2.
Matrix-assisted laser desorption/ionization (MALDI) is the most common ionization method used for MSI analyses. However, the need for an organic matrix and the vacuum requirements of MALDI pose significant limitations on reproducibility, sample throughput, and the types of samples that can be analyzed using the method. A number of atmospheric pressure (AP) ionization methods have been developed in recent years to circumvent these restrictions3. These ambient ionization methods allow for analysis of biological samples in an environment that is much closer to their natural state and simplify sample preparation steps prior to analysis. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such an ionization method4,5.
In IR-MALDESI experiments, a thin layer of ice is deposited on the tissue surface as the energy-absorbing matrix. A mid-IR laser pulse is absorbed by the ice matrix, and facilitates desorption of neutral materials from the surface by resonantly exciting the O-H stretching mode of water. The desorbed neutrals partition into the charged droplets of an orthogonal electrospray and are post-ionized in an ESI-like fashion4-6. The addition of exogenous ice matrix is preferred over relying solely on the endogenous water in tissue since it helps account for variations in water content in different tissue compartments, and has been shown to enhance desorption6 and improve ion abundance by ~15-fold7,8 in tissue imaging experiments.
In this work, we utilize IR-MALDESI MSI to elicit the distribution of metabolites across different organs in a neonatal mouse whole body. An overview of adjustable parameters of the IR-MALDESI source is given, and the necessary steps for successful imaging of tissue sections are demonstrated.

<table><tbody><tr><td>IR-MALDESI Source</td><td>Custom-made</td><td>N/A</td><td>Please refer to references 4 and 12 for an in-depth discussion of IR-MALDESI source development.</td></tr><tr><td>Q Exactive Plus&amp;nbsp;</td><td>Thermo Scientific</td><td></td><td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer</td></tr><tr><td>Water, HPLC Grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH365-4</td><td></td></tr><tr><td>Methanol, HPLC Grade</td><td>Burdick &amp;amp; Jackson&amp;nbsp;</td><td>AH230-4</td><td></td></tr><tr><td>Formic Acid</td><td>Sigma Aldrich&amp;nbsp;</td><td>56302</td><td></td></tr><tr><td>Tunable mid-IR Laser</td><td>Opotek Inc.</td><td>IR Opolette</td><td>Tunable 2,700-3,100&amp;nbsp;nm IR OPO laser</td></tr><tr><td>Nitrogen Gas</td><td>Arc3 Gases</td><td>AG S-NI300-5.0</td><td>Grade 5.0 high purity nitrogen gas cylinder (300)</td></tr><tr><td>Cryostat</td><td>Leica Biosystems</td><td>CM 1950</td><td>Cryomicrotome</td></tr><tr><td>High Profile Microtome Blades</td><td>Leica Biosystems</td><td>3802123</td><td>Leica DB80HS</td></tr><tr><td>Mounting Medium (OCT)</td><td>Leica Biosystems</td><td>3801480</td><td>Surgipath FSC 22 mounting medium</td></tr><tr><td>Cryostat Specimen Disc</td><td>Leica Biosystems</td><td>14047740045</td><td>40 mm diameter</td></tr><tr><td>Glass Microscope Slides</td><td>VWR</td><td>48312-003</td><td>Frosted, selected, pre-cleaned</td></tr></tbody></table>

whole-body mass spectrometry imaging by infrared matrix-assisted laser desorption electrospray ionization (ir-maldesi)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

The images presented in Figure 4 show the spatial distribution of metabolites in different organs in the whole-body tissue section. Unique m/z values to specific regions of the body were found using MSiReader PeakFinder, followed by batch processing for image generation. The image overlay tool (Figure 3-4) was used to align the optical image taken before ice matrix deposition with the resulting ion maps. Cholesterol is observed across all tissue ...

Watch this Scientific Journal Video about Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI at JoVE.com

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

whole-body-mass-spectrometry-imaging-infrared-matrix-assisted-laser

Research

JoVE Journal

Bioengineering

9.4K Views.  North Carolina State University. A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse. 

Video: Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

Chemistry

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Biochemistry

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample types have been analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to visualize the distribution of proteins, lipids, and metabolites across the biological sample of interest. We have developed a novel sample preparation method that combines the strengths of the three previous applications to address an underexplored approach for identifying chemical communication in cancer, by seeding mammalian cell cultures into agarose in coculture with healthy tissues followed by desiccation of the sample. Mammalian tissue and cells are cocultured in close proximity allowing chemical communication via diffusion between the tissue and cells. At specific time points, the agarose-based sample is dried in the same manner as microbial colonies prepared for IMS analysis. Our method was developed to model the communication between high grade serous ovarian cancer derived from the fallopian tube as it interacts with the ovary during metastasis. Optimization of the sample preparation resulted in the identification of norepinephrine as a key chemical component in the ovarian microenvironment. This newly developed method can be applied to other biological systems that require an understanding of chemical communication between adjacent cells or tissues.

A novel method of sample preparation was developed to accommodate cell and tissue coculture to detect small molecule exchange using imaging mass spectrometry.

Imaging mass spectrometry (IMS) has routinely been applied to three types of samples: tissue sections, spheroids, and microbial colonies. These sample ...

Cancer Research

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Biology

Visualization of Amyloid &#946; Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into extracellular plaques of the brain. The A&#946; peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by &#946;- and &#947;-secretases. A&#946; is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of A&#946; peptides were identified, the detailed production and distribution of individual A&#946; peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 &#181;m. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various A&#946; species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 &#181;m) imaging of an AD brain and severe CAA sample clearly shows that A&#946;1-36 to A&#946;1-41 were deposited into leptomeningeal vessels, while A&#946;1-42 and A&#946;1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

Molecular imaging with matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) allows the simultaneous mapping of multiple analytes in biological samples. Here, we present a protocol for the detection and visualization of amyloid &#946; protein on brain tissues of Alzheimer&#39;s disease and cerebral amyloid angiopathy samples using MALDI-IMS.

The neuropathology of Alzheimer&#39;s disease (AD) is characterized by the accumulation and aggregation of amyloid &#946; (A&#946;) peptides into ...

Neuroscience

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...

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 ...

MALDI-TOF Mass Spectrometry

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing compounds in the gaseous state, samples are embedded in a matrix, which is struck by a laser. The matrix absorbs the majority of the energy; some of this energy is then transferred to the sample, which ionizes as a result. Sample ions can then be identified using a time-of-flight analyzer (TOF).
This video covers principles of MALDI-TOF, including matrix selection and how TOF is used to elucidate mass-to-charge ratios. This procedure shows the preparation of a MALDI plate, the loading of samples onto the plate, and the operation of the TOF-mass spectrometer. In the final section, applications and variations are shown, including whole-cell analysis, characterization of complex biological samples, and electron spray ionization.
<div class="chapter_text" id="script_0">
Matrix-assisted laser desorption ionization, or MALDI, is a mass spectrometry ion source ideal for the analysis of biomolecules. Most ion sources remove structural information from large, fragile biomolecules. MALDI maintains structural integrity, and therefore information, while accelerating the molecules into the mass analyzer, which separates the compounds based on size and charge. The most commonly coupled with MALDI is the time of flight, or TOF, mass analyzer. This video will show the concepts of MALDI ionization, a general procedure, and some of its uses in biochemistry.
</div>
<div class="chapter_text" id="script_1">
For mass spectrometry to function, molecules must be ionized into the gaseous state. In MALDI, the sample is embedded in a matrix, typically an organic compound containing aromatic and conjugated double bonds.
When a laser pulse strikes this mixture the matrix absorbs the majority of the energy, rapidly heats, and is desorbed, or released, from the surface. The energized matrix transfers some of its energy to the biomolecules, desorbing and then ionizing them.
MALDI is typically paired with a time of flight, or TOF, mass analyzer. An electric field applies kinetic energy to the ions, moving them into a field-free region called a drift tube. The velocity of the ions as they move through the tube is related to their mass-to-charge ratio, so heavier particles travel slower through instrument. A detector at the end of the tube measures each ion&#39;s flight time. With this knowledge, as well as the tube length and applied field strength, the mass-to-charge ratio of each ion can be elucidated.
This plot of signal intensity to mass-to-charge-ratio, known as a mass spectrum, can be compared to a library of collected spectra. If no matches are found, it can molecules can be identified by further techniques, such as tandem mass spectrometry. For more information, see this collection&#39;s video on the topic.
Now that the basics of MALDI-TOF have been discussed, let&#39;s look at the process in the laboratory.
</div>
<div class="chapter_text" id="script_2">
Before beginning an experiment, it&#39;s important to consider the choice of matrix from which samples will be desorbed. It must absorb the laser energy, be stable in a vacuum, not react with the target molecules, and be able to desorb. Depending on the sample, different matrices are preferred. For a large protein, a combination of CHCA and DHB has shown better separation of the peaks, called resolution, than the individual matrices.
There are a number of ways to prepare samples. We&#39;ll show what is known as the &#34;double-layer&#34;, or &#34;sandwich,&#34; method. To begin, clean the MALDI plate with ultra-pure reagents, as mass spectrometry is very sensitive to contamination. Dry the plate with a stream of inert gas.
Next, a saturated matrix solution is made, typically with an organic solvent . The solution is streaked onto the MALDI plate and dried. A second saturated solution of matrix containing trifluoroacetic acid, or TFA, is prepared. TFA helps ions into the gaseous phase.
Next, the sample solution is added on top of the dried matrix spot. Add the matrix solution containing TFA on top of the sample, thereby completing the matrix &#34;sandwich&#34;. Homogeneity of the spot can be verified under a low-powered microscope.
Plate a calibration standard, which is a mixture with a wide range of known masses and is used to correlate the time-of-flight to m/z. Finally, plate the matrix alone as a negative control.
</div>
<div class="chapter_text" id="script_3">
To analyze the spots, place the target plate into the instrument. Ensure there&#39;s no debris present, allowing for the formation of a tight vacuum. In the software, select the standard, negative control, and samples of interest. Label the spots with the correct identification.
The ion source and lens voltages can be manipulated to improve performance of the analysis. This will depend on the specifics of the instrument and sample. Focus on the standard spot and calibrate the instrument with the software.
Next, collect spectra from each of the sample spots. Try a few different locations on the spot to maximize the quality of the collected data. Once finished, the MALDI plate can be collected and reused after cleaning.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve reviewed a procedure, let&#39;s look at some of the ways MALDI is utilized, and a different ionization technique.
In addition to biomolecules, MALDI can be used to analyze living cells. Macrophages are immune cells that take on one of several different forms, based on their microenvironment. After exposing the cells to various signaling molecules, or cytokines, they can be added directly to the plate, and analyzed. The MALDI spectra can be used as unique &#34;fingerprints&#34;, depending on the cytokine used.
Complex biological samples like mammalian sebaceous secretions require a step of purification before MALDI analysis. Thin layer chromatography is one such technique that relies on the components&#39; polarity. The compounds are collected from the TLC pate, purified, and transferred to a MALDI matrix. The resulting spectra verify the identity and purity of the separated biomolecules from the mammalian sebaceous secretions.
Another common ion source for biomolecules is electrospray ionization, or ESI. In this method, the sample is injected into the instrument, where a high voltage is applied, creating an aerosol of charged droplets. As the solvent in the droplet evaporates, the charge is moved to the sample molecules, till they are completely gaseous. ESI doesn&#39;t require the spotting procedure, and the sample can be injected directly into the instrument. On the other hand, ESI is more sensitive to the presence of buffer components and other contaminants, meaning MALDI is more robust.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on MALDI mass spectrometry. This video described the theory behind the instrument, went over a general procedure, and covered some of the uses of the technique. Thanks for watching!
</div>

MALDI-TOF Mass Spectrometry-Principle, Operation and Uses

Matrix-assisted laser desorption ionization (MALDI) is a mass spectrometry ion source ideal for the analysis of biomolecules. Instead of ionizing ...

Education

JoVE Science Education

Introduction to Mass Spectrometry

Source: Laboratory of Dr. Khuloud Al-Jamal - King&#39;s College London

Mass spectrometry is an analytical chemistry technique that enables the identification of unknown compounds within a sample, the quantification of known materials, the determination of the structure, and chemical properties of different molecules.
A mass spectrometer is composed of an ionization source, an analyzer,&#160;and a detector. The process involves the ionization of chemical compounds to generate ions. When using inductively coupled plasma (ICP), samples containing elements of interest are introduced into argon plasma as aerosol droplets. The plasma dries the aerosol, dissociates the molecules, and then removes an electron from the components to be detected by the mass spectrometer. Other ionization methods such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) are used to analyze biological samples. Following the ionization procedure, ions are separated in the mass spectrometer according to their mass-to-charge ratio (m/z), and the relative abundance of each ion type is measured. Finally, the detector commonly consists in an electron multiplier where the collision of ions with a charged anode leads to a cascade of increasing number of electrons, which can be detected by an electrical circuit connected to a computer.
In this video, the procedure of ICP-MS analysis will be described by the detection of 56Fe as an example.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Summary

Explore More Videos

Chapters in this video

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Capturing Small Molecule Communication Between Tissues and Cells Using Imaging Mass Spectrometry

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

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

MALDI-TOF Mass Spectrometry

Introduction to Mass Spectrometry