Sublimation of DAN Matrix for the Detection and Visualization of Gangliosides in Rat Brain Tissue for MALDI Imaging Mass Spectrometry

Summary

A protocol for the sublimation of DAN matrix onto rat brain tissue for the detection of gangliosides using MALDI Imaging Mass Spectrometry is presented.

Abstract

Sample preparation is key for optimal detection and visualization of analytes in Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) experiments. Determining the appropriate protocol to follow throughout the sample preparation process can be difficult as each step must be optimized to comply with the unique characteristics of the analytes of interest. This process involves not only finding a compatible matrix that can desorb and ionize the molecules of interest efficiently, but also selecting the appropriate matrix deposition technique. For example, a wet matrix deposition technique, which entails dissolving a matrix in solvent, is superior for desorption of most proteins and peptides, whereas dry matrix deposition techniques are particularly effective for ionization of lipids. Sublimation has been reported as a highly efficient method of dry matrix deposition for the detection of lipids in tissue by MALDI IMS due to the homogeneity of matrix crystal deposition and minimal analyte delocalization as compared to many wet deposition methods ^1,2. Broadly, it involves placing a sample and powdered matrix in a vacuum-sealed chamber with the samples pressed against a cold surface. The apparatus is then lowered into a heated bath (sand or oil), resulting in sublimation of the powdered matrix onto the cooled tissue sample surface. Here we describe a sublimation protocol using 1,5-diaminonaphthalene (DAN) matrix for the detection and visualization of gangliosides in the rat brain using MALDI IMS.

Introduction

Matrix-assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) is becoming a highly sought after technique for visualization of the spatial distribution of lipids, peptides and proteins across intact sample surfaces. MALDI IMS was previously known as an analytical technique for pre-purified analytes, but in recent years, it has been drawing attention in many other disciplines because of the ability to combine the accuracy of mass spectrometry with high resolution visual/anatomical reference points without the need for any external labelling. As the scientific pool of researchers utilizing this technique continues to grow, there is increased need f....

Protocol

All animal handling procedures described below adhere to the University of Western Ontario's animal care committee (2016-014).

1. Tissue Preparation and Sectioning

1. Extract brain tissue from the rat through a process known as "Fresh Frozen Extraction" (FFE).
   1. Euthanize the rat with an overdose of pentobarbital sodium and monitor reflexes in the limbs. Once all reflexes have ceased, sever the head of the rat using a guillotine.
   2. Carefully separate the muscles and other tissues from the skull using a scalpel. Use bone-cutting forceps to break the skull to expose the brain, starting from the base of ....

Results

Upon completion of the sublimation experiment, the two halves of the glass apparatus are separated in the fume hood and the slide, taped to the condenser, can be removed (Figure 3A). At this point, the slide should be examined for uneven matrix distribution and the time, temperature, or placement of the apparatus in the sand bath may have to be adjusted for the next slide. A successful DAN sublimation experiment will result in an even coating of grey/brown matrix along th.......

Discussion

This work details a standardized protocol of matrix sublimation on tissue for the detection of negatively charged lipids, such as gangliosides, in MALDI IMS experiments. Sample preparation for MALDI IMS is highly variable and must be customized to suit the unique properties of the analyte of interest. The application of matrix onto the tissue sample surface is a crucial aspect of the sample preparation process with regards to the quality of results in IMS. Particular care should be taken when selecting matrices, ensuring.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sublimator	Chemglass Life Sciences	CG3038-01
1,5-Diaminonapthalene (DAN) matrix	Sigma-Aldrich	D21200	100 G
Cryostat	Thermo-Fisher Scientific		CryoStar NX50
Hot plate with temperature feedback	Thermo-Fisher Scientific	HP88857290	Isotemp ADVD 7x7 HP 100 - 120 V
Stainless Steel Jack	Thermo-Fisher Scientific	2216479	10x10
Cold Trap			Custom built on site
Vacuum Pump	Franklin Electric	1102180403	Savant VP100 Two Stage
Indium-tin-oxide (ITO) Slides	Hudson Surface Technology	PSI 1111000	type II, 1.1 mm/25 each
MALDI TOF/TOF 5800 Instrument	AB Sciex
Desiccator	Sigma-Aldrich	D2797	tabletop desiccator