The authors would like to acknowledge Genome Canada and Genome British Columbia for platform funding, and support. We also thank Dr. Carol E. Parker for critical review of the manuscript and editing assistance. CHL also thanks the British Columbia Proteomics Network for support.

1. Tissue Sectioning
<ol>
	<li>Flash-freeze the issue specimens, once harvested, using liquid nitrogen, ship them on dry ice (if shipping is required), and store them at -80 &#176;C until tissue sectioning. (If commercial samples are used, ensure that the samples are prepared in this manner.)</li>
	<li>Cut organs to a manageable size to fit the MALDI target. Trim off any unwanted parts of the organ. For this study described here, bovine calf lenses were decapsulated using a previously-described procedure35 before tissue sectioning.</li>
	<li>Remove whole organs from the -80 &#176;C freezer and fix them onto a cryogenic tissue cutting stage. To fix a bovine calf lens, place one or two drops of water on the tissue-cutting stage of a&#160;cryostat. Quickly place the lens in the water before it solidifies. Alternatively, optimal cutting temperature (OCT) compounds can also be used to fix a tissue onto the cutting stage. If OCT compounds are used, a minimal amount of OCT should be applied and care should be taken to ensure that the cut tissue sections will not be contaminated with OCT compounds which can interfere with ionization and detection of the analytes5,36,37.</li>
	<li>Allow the temperature inside the cryostat to equilibrate to -18 &#176;C. Colder or warmer temperatures can be used for softer or harder tissues, respectively. Then cut the tissue equatorially into 20&#160;&#956;m thick slices. Use 10-15 &#956;m thick slices for most tissues; however, because of the fragile nature of the bovine lens tissue, 20&#160;&#956;m thick slices were used. For bovine lens tissue, discard the first tissue sections and only use slices which are close to or at the equatorial plane.</li>
	<li>If an ocular lens tissue is imaged, use 1.5 &#956;l of formic acid (98% in purity, LC-MS grade) to prewet the surface of an indium tin oxide (ITO)-coated glass slide.</li>
	<li>Carefully transfer the tissue sections to the surface of the ITO-coated glass microscopic slide inside the cryostat. The tissue section will quickly thaw and will become tightly affixed to the slide surface. Usually, multiple tissue sections can be mounted onto a same ITO-coated slide in this way.</li>
	<li>Lyophilize the slide&#160;for 15 min before MALDI matrix application.</li>
	<li>For matrix testing, dissolve the individual matrices in appropriate solvents. Manually spot 1 &#956;l of each matrix solution onto the tissue section. Additionally, spot a small-molecule calibration standard onto the tissue for verification of MALDI sensitivity.</li>
	<li>Add three teaching marks to the ITO coated glass slide&#160;by writing on the nonconductive surface of the ITO coated glass slide with a correction-fluid pen. Take an optical image of the tissue slide using a flatbed scanner and save it in an appropriate format such as tiff or jpg.</li>
</ol>
2. Matrix Coating
2.1. Automated Matrix Coating
<ol>
	<li>Apply matrix solutions, which contain acetonitrile or mixed acetonitrile/water as solvents, automatically to the surfaces of the tissue sections using a Bruker ImagePrep or a similar electronic matrix sprayer.</li>
	<li>Cover the edges of the front surface of the ITO-coated glass slide&#160;with tape so that the matrix does not coat the edges of the slide. This ensures that the teaching marks on the opposite surface can be used for tissue slide alignment. Do not cover the edges of the slide with matrix as they are used as contact points to maintain the electrical conductivity of the ITO-coated slide.</li>
	<li>Coat the glass slide by using twenty cycles of matrix coating (2-sec spray, 30-sec incubation, and 60-sec drying time for each cycle).</li>
</ol>
2.2. Manual Matrix Coating
<ol>
	<li>If organic solvents (e.g.&#160;chloroform and ethyl acetate) that are incompatible with the manufacturing materials of the electronic matrix sprayer are required, use a pneumatically-assisted airbrush sprayer to apply the matrix. Add the prepared matrix solution to the solvent reservoir of the airbrush gun and apply a gentle flow of pressurized nitrogen gas to prime the spray.</li>
	<li>Cover the edges of the front surface of the ITO-coated glass slides with tape so that the matrix does not coat the edges of the slide. This ensures that the teaching marks on the opposite surface can be used for tissue slide alignment. Do not cover the edges of the slide with matrix as they are used as contact points to maintain the electrical&#160;conductivity of the ITO-coated slide.</li>
	<li>After stable and fine sprays have been observed, manually spray the matrix so that it completely coats the tissue section. Apply the minimum amount of matrix solution required to barely wet the surface during each cycle to prevent possible analyte delocalization. In general, use approximately 10 cycles of matrix spray to coat a tissue section; the number of cycles is dependent on the tissue type and matrix composition.</li>
</ol>
3. MALDI MS
<ol>
	<li>Prepare a mass calibration solution by diluting the &#34;ES Tuning Mix&#34; standard solution by a factor of 1:200 in 60:40 isopropanol:water (containing 0.1% formic acid in the final mixture).</li>
	<li>Introduce 2 &#956;l/min of the diluted &#34;ES Tuning Mix&#34; solution into the dual-mode electrospray ionization (ESI)/MALDI ion source on the FTICR mass spectrometer, from the ESI side.</li>
	<li>Operate the FTICR instrument in the positive-ion ESI mode, with broadband detection and a data acquisition size of 1,024 kb/sec. Typical ESI parameters are capillary electrospray voltage, 3,900 V; spray shield voltage,&#160;3,600 V; nebulizer gas (N2) flow, 2 L/min;&#160;dry gas (N2) flow and temperature, 4 L/min and 200 &#176;C; skimmer 1 voltage, 15 V; time of flight (TOF), 0.009 sec; collision gas (Ar) flow, 0.4 L/s; source ion accumulation time, 0.1 sec; and collision cell ion accumulation time, 0.2 sec. Tune the FTICR operation parameters in order to maximize the analytical sensitivity over the mass range from m/z 200 to 1,400, while maintaining good time-domain free-induction decay (FID) signals. Typically, the ICR operation parameters are sidekick voltage, 8 V; sidekick offset voltage, 8 V; excitation amplification of 10; excitation pulse time, 0.01-0.015 sec; front trap plate voltage, 1.5 V; back trap plate voltage, 1.6 V and analyzer entrance voltage, -4 V. After a set of FTICR operation parameters has been determined, acquire the ESI mass spectra and calibrate the instrument using the reference masses of the standard compounds in the &#34;ES Tuning Mix&#34; solution.</li>
	<li>To tune the instrument for MALDI operation, dissolve several 1&#160;&#956;l aliquots of a mixed terfenadine and reserpine standard solution in the matrix solution at a concentration of 1 &#956;M each, and spot these solutions directly onto one of the tissue sections (i.e.&#160;a test tissue section) which has been mounted on an ITO-coated slide. Place the ITO-coated slide into a tissue slide adaptor (i.e.&#160;a special MALDI target) and load the adaptor from the MALDI side into the dual ESI/MALDI ion source. Optimize the appropriate MALDI operating parameters for the laser power and the number of laser shots for MALDI signal accumulation for each mass scan, etc. Typical MALDI operation parameters are: laser shots, 50; and a MALDI plate voltage of 300 V.</li>
	<li>After tuning, calibrating, and optimizing the instrument for MALDI-MSI experiments, align the physical location of a tissue section to be imaged with its recorded optical image within the imaging software. Use the three &#34;correction-fluid&#34; marks, which had been previously put on the opposite side of the ITO-coated slide surface (step 1.9), for this alignment using a three-point triangulation method.</li>
	<li>Perform a simultaneous ESI and MALDI operation so that each mass spectrum contains the reference mass peaks of the &#34;ES Tuning Mix&#34; solution for post-acquisition internal mass calibration. This will result in the most accurate mass measurement during MALDI MS. To do this, first attenuate the ESI signal by decreasing the capillary voltage until the MALDI signals dominate the spectra while the ESI calibrant signals are still high enough for internal mass calibration.</li>
	<li>Next, set up an automated rastering method for laser irradiation. Define the tissue regions to be imaged and set the appropriate laser raster step size. Note that smaller raster step sizes provide higher resolution tissue images, but require a significantly longer mass spectral acquisition time and more data storage space. The number of the image pixels is dependent on the laser raster step size to set up and the tissue size. For a typical bovine lens which has a 1 cm2 tissue size, a tissue image is typically composed of ca. 5,000 pixels if a laser raster step size of 200 &#956;m is used on an FTICR instrument. Use a &#34;random spot&#34; analysis, as this prevents location-based bias due to gradual signal attenuation during the experiment.</li>
</ol>
4. Data Analysis
<ol>
	<li>Calibrate the MALDI mass spectra using internal calibration for the initial comparison and to select peaks for MS/MS. De-isotope and select the monoisotopic peaks as previously described, using a customized VBA script38.</li>
	<li>Export the resulting monoisotopic peak lists and input the measured m/z values into the METLIN39 and/or the HMDB40 metabolome databases for mass matching with the library entries. Consider the (M+H)+, (M+Na)+, and (M+K)+ ions during the database searches, with an allowable mass error of &#177;1 ppm.</li>
	<li>Generate MALDI images for all of the lipid entities detected across the entire tissue section using image-analysis software, with a mass filter width of 1 ppm at the peak apex.</li>
	<li>Once images have been generated for all m/z values that match database entries, generate images for all other peaks as well&#160;to look for unique distribution patterns that can be investigated later.</li>
</ol>
5. Confirmation of the Identities of the Imaged Lipids
<ol>
	<li>Confirm the identities of the high abundance lipids, which have characteristic fragment ions that can be detected using the FTICR instrument (e.g.&#160;184.073 for phospholipids), by MALDI-MS/MS. Perform MALDI-MS/MS using collision-induced dissociation (CID) directly on the tissue.</li>
	<li>For those lipid species that cannot be directly confirmed by MALDI-MS/MS, use a UPLC system coupled to a q-TOF mass spectrometer34.
	<ol>
		<li>Manually dissect aliquots containing ~10 mg of tissue from the area where the species of interest was localized. Place these tissue aliquots into 2&#160;ml centrifuge tubes.</li>
		<li>Homogenize each tissue aliquot in 250 &#956;l of water, using a mixer mill with two 5 mm stainless steel metal balls.</li>
		<li>Add 1 ml of chloroform-methanol solution (1:3, v/v), and vortex the tubes. Next, centrifuge the tubes using a microcentrifuge at 12,000 x g&#160;for 10 min.</li>
		<li>Collect the supernatants and dry them in a rotary speed-vacuum concentrator.</li>
		<li>Dissolve the residues in 100 &#956;l of 30:70 isopropanol:water. Inject a 10-&#956;l aliquot onto the UPLC column for separation using gradient elution.</li>
		<li>Use the chromatographic conditions for on-line lipid LC-MS/MS, which have been published previously34.</li>
		<li>Generate extracted ion current (XIC) chromatograms using the theoretical m/z values, with a window of &#177;50 ppm around the theoretical masses.</li>
		<li>If authentic compounds for those lipids are available, match the retention times of the authentic compounds with those of the corresponding XIC peaks from the tissue samples. If the compounds are the same, the retention times and the MS/MS spectra should match.</li>
	</ol>
	</li>
	<li>If an authentic compound is unavailable, use the fragmentation pattern of the detected lipid to match a standard MS/MS spectrum&#160;from a metabolome database such as METLIN or HMDB. Use de novo mass spectral interpretation to determine a possible structure for the lipid.</li>
</ol>

We have nothing to disclose.

The most important considerations for successful MALDI MSI are: 1) tissue preparation; 2) matrix choice; 3) matrix application; and 4) data interpretation and analysis. When the sample and the matrix are appropriately prepared, the MS data acquisition is automated. The data analysis from this type of experiment is quite labor-intensive.
Appropriate tissue preparation is crucial for successful MALDI MSI experiments. The source of the tissue and the handling can have a large impact on the final ...

Mass spectrometry imaging (MSI) is an analytical technique for determining the spatial localization and distribution patterns of compounds on the surface of a tissue section1,2. Matrix assisted laser&#160;desorption/ionization (MALDI) MSI for the analysis of peptides and proteins has been used for over a decade and there have been great improvements in methods for sample preparation, detection sensitivity, spatial resolution, reproducibility and data processing3,4. By combining information from histologically stained sections and MSI experiments, pathologists are able to correlate the distributions of specific compounds with pathophysiologically interesting features5.
The distribution patterns of small molecules, including exogenous drugs6,7 and their metabolites8-10 have also been interrogated by MALDI-MS tissue imaging11. Lipids are perhaps the most widely-studied class of compounds with MALDI imaging, both in the MS12-17 and MS/MS18 modes. The use of MALDI MSI for small molecule imaging has been limited by several factors: 1) MALDI matrices are themselves small molecules (typically m/z &#60;500), which generate abundant ion signals. These abundant signals can suppress the ionization of small-molecule analytes and interfere with their detection19,20. Solvent-free matrix coating21, matrix sublimation22, and matrix precoated MALDI MS23, among others, have been developed to improve MSI of small molecules.
New matrices that can improve the analysis of low-MW compounds are of great interest in small-molecule MSI. These matrices should provide increased analyte signals with decreased matrix signals. In the positive-ion mode, 2,5-dihydroxybenzoic acid (DHB) and &#945;-cyano-4-hydroxycinnamic acid (CHCA) are the two commonly used MALDI MS matrices for MSI24. The ideal matrix would form small crystals, so as to preserve the spatial localization of the analytes. DHB tends to form larger crystals, therefore applying the matrix using sublimation has been developed to partially overcome this problem, and has allowed the use of this matrix for sensitive imaging of phospholipids22,25. 9-Aminoacridine has been used for MSI of protic analytes in the positive-ion mode26 and for nucleotides and phospholipids in the negative-ion mode26-29. 2-Mercaptobenzothiazole has been found to give efficient MALDI detection of lipids30, and has been used for the imaging of mouse&#160;brain gangliosides31. The ultrahigh resolution of Fourier transform ion cyclotron resonance (FTICR) mass spectrometers can somewhat alleviate this problem by resolving analyte signals from matrix signals32. Another advantage of the use of FTICR-MS is that the intensities of the metastable matrix clusters are reduced33, which also reduces these interferences27.
The use of dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) as a MALDI matrix for tissue imaging has previously been reported34. In this current work, a detailed protocol is provided for the use of DT for the MSI of endogenous lipids on the surfaces of bovine lens tissue sections, in the positive-ion mode.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Rat Liver</td>
			<td>Pel-Freez Biologicals</td>
			<td>56023-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Calf Lens</td>
			<td>Pel-Freez Biologicals</td>
			<td>57114-2</td>
			<td>Sample should be decapsulated29 before use</td>
		</tr>
		<tr>
			<td>Dithranol (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>10608</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>&#945;-Cyano-4-hydroxy-cinnamic Acid (CHCA)</td>
			<td>Sigma-Aldrich</td>
			<td>70990</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>2,5-Dihydroxybenzoic Acid (DHB)</td>
			<td>Sigma-Aldrich</td>
			<td>85707</td>
			<td>MALDI Matrix</td>
		</tr>
		<tr>
			<td>Reserpine</td>
			<td>Sigma-Aldrich</td>
			<td>83580</td>
			<td></td>
		</tr>
		<tr>
			<td>Terfenadine</td>
			<td>Sigma-Aldrich</td>
			<td>T9652</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>14265</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Formate</td>
			<td>Sigma-Aldrich</td>
			<td>14266</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid (TFA)</td>
			<td>Sigma-Aldrich</td>
			<td>302031</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma-Aldrich</td>
			<td>39253</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34967</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>34972</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich</td>
			<td>34965</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>366927</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Sigma-Aldrich</td>
			<td>34850</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Commercial Alcohols</td>
			<td></td>
			<td>95%</td>
		</tr>
		<tr>
			<td>ES Tuning Mix</td>
			<td>Agilent Technologies</td>
			<td>G2431A</td>
			<td></td>
		</tr>
		<tr>
			<td>ITO Coated Glass Slides</td>
			<td>Hudson Surface Technology</td>
			<td>PSI1207000</td>
			<td>Ensure that samples are placed on the electrically conductive side</td>
		</tr>
		<tr>
			<td>Wite-Out Shake-N-Squeeze Correction Pen</td>
			<td>Bic</td>
			<td>WOSQP11</td>
			<td></td>
		</tr>
		<tr>
			<td>Airbrush Sprayer</td>
			<td>Iwata</td>
			<td>Eclipse HP-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>ImagePrep</td>
			<td>Bruker</td>
			<td>249500-LS</td>
			<td></td>
		</tr>
		<tr>
			<td>MALDI adapter</td>
			<td>Bruker</td>
			<td>235380</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Tissue samples that are sectioned and thaw mounted onto the ITO coated glass slides should be intact, without visible tearing. For many tissues, direct tissue thaw mounting onto an ITO coated glass slide is acceptable. For some specific tissues such as bovine lens, extensive tearing of the tissue is often seen when direct thaw mounting is used (Figure 1a). Precoating of the ITO glass slide with ethanol or formic acid (Figure 1b) helps to maintain the integrity of the tissue sections duri...

Watch this Scientific Journal Video about Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer at JoVE.com

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

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 ...

dithranol-as-matrix-for-matrix-assisted-laser-desorptionionization

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Biology

14.1K Views.  University of Victoria. 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 by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.