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 b...

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

Research

JoVE Journal

Biology

14.1K visualizações.  University of Victoria.  Ditranol (DT; 1,8-di-hidroxi-9 ,10-dihydroanthracen-9-ona) foi previamente relatada como uma matriz de DLAMI para a imagiologia de tecido de pequenas moléculas; protocolos para a utilização de DT para a imagiologia de MALDI de lípidos endógenos na superfície de cortes de tecido por positivo-ion MALDI-MS em um ultra-alta resolução quadrupolo-FTICR instrumento são fornecidos aqui. 