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

Summary

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.

Abstract

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

Introduction

Mass spectrometry imaging (MSI) is an analytical technique for determining the spatial localization and distribution patterns of compounds on the surface of a tissue section^1,2. Matrix assisted laser 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 processing^3,4. By combining information from histologically stained sections and MSI experiments, pathologists are able to correlate the distributions of specific compounds with pathophysiologically ....

Protocol

1. Tissue Sectioning

1. Flash-freeze the issue specimens, once harvested, using liquid nitrogen, ship them on dry ice (if shipping is required), and store them at -80 °C until tissue sectioning. (If commercial samples are used, ensure that the samples are prepared in this manner.)
2. 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.
3. Remove whole organs from the -80 °C freezer and fix them onto a cryogenic tissue cutting stage. To fix a bovin....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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