A minimalist approach to MALDI imaging of glycerophospholipids and sphingolipids in rat brain sections

https://doi.org/10.1016/j.ijms.2008.04.005Get rights and content

Abstract

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that has allowed researchers to directly probe tissue molecular structure and drug content with minimal manipulations, while maintaining anatomical integrity. In the present work glycerophospholipids and sphingolipids images were acquired from 16-μm thick coronal rat brain sections using MALDI-MS. Images of phosphatidylinositol 38:4 (PI 38:4), sulfatide 24:1 (ST 24:1), and hydroxyl sulfatide 24:1 (ST 24:1 (OH)) were acquired in negative ion mode, while the images of phosphatidylcholine 34:1 (PC 34:1), potassiated phosphatidylcholines 32:0 (PC 32:0 + K+) and 36:1 (PC 36:1 + K+) were acquired in positive ion mode. The images of PI 38:4 and PC 36:1 + K+ show the preferential distribution of these two lipids in gray matter; and the images of two sulfatides and PC 32:0 + K+ show their preferential distribution in white matter. In addition, the gray cortical band and its adjacent anatomical structures were also identified by contrasting their lipid makeup. The resulting images were compared to lipid images acquired by secondary ion mass spectrometry (SIMS). The suitability of TLC sprayers, Collison Nebulizer, and artistic airbrush were also evaluated as means for matrix deposition.

Introduction

Mass spectrometry has opened new vistas into the probing of biological problems. Because of its sensitivity and accuracy it has had a major impact on qualitative and quantitative detection and identification of biological molecules. Direct tissue imaging is a powerful tool, and at the same time a “minimalist approach” as with a modicum of manipulations, it gives a complete and accurate picture that permits the localizaton of drugs and endogenous molecules in tissue while preserving their anatomical structure. This approach also provides a highly efficient tool for the better understanding of physiological processes as well as the pathophysiology of disease while significantly shortening sample processing and turnaround time.

MALDI-MS has certain advantages over other MS techniques. To just mention a few, MALDI instruments have a wide mass range, are easy to use, require little maintenance have a higher tolerance to contaminants, their ion source design allows the adaptation of sample plates for direct analysis of biological molecules and pharmaceutical compounds; and sample preparation is fairly straight forward. The above-mentioned advantages result in the gathering of a wealth of data from complex biological matrices, within a short sample turn around time [1], [2], [3], [4], [5], [6]. The initial success of these applications has promoted the development of MALDI imaging mass spectrometry (MALDI-IMS) for visualizing endogenous and exogenous molecules in tissue sections that would otherwise require elaborate, time-consuming, and costly (immuno-)histochemistry and autoradiography techniques.

Several investigators have applied MALDI-IMS for direct tissue profiling and imaging of proteins and peptides with various level of success [2], [3], [7], [8]. Others applied this technique for visualizing drug distribution in rat brains, [9] throughout the whole animal body [10] to image agrochemicals accumulated in soy bean plants, [11] and to detect protein and peptide in the ganglia of invertebrates [12]. All these examples support the notion that MALDI-IMS is a versatile analytical technique that has wide application possibilities. In spite of its promising potential, MALDI-IMS is nonetheless confronted by several technical issues in detection sensitivity and sample preparation. However, to date only a few studies devoted their attention to the optimization of sample preparation and handling for MALDI-IMS [13], [14].

In addition to MALDI-MS, other mass spectrometric techniques, such as desorption electrospray ionization mass spectrometry (DESI), [15] and secondary ion mass spectrometry (SIMS) [16], [17] have also been adapted for tissue imaging studies. The lateral resolution of SIMS is far superior to what MALDI-IMS can offer. However, the sample preparation for SIMS imaging [16] and the profile depth of the samples [18] are very different from those of MALDI-IMS technique. Hence the differences between SIMS and MALDI-IMS imaging come as no surprise.

Although most MALDI-IMS applications focus on imaging of proteins and small organic molecules, a recent study has shifted the focus to brain lipids [19]. The authors utilized new matrix deposition approaches and obtained fairly interesting results. Nevertheless, some aspect of their matrix application methods deserves further evaluation and investigation for brain lipid imaging studies.

In this study, we further investigated and optimized one of the previously reported MALDI-IMS matrix deposition methods [19] and compared it with other available matrix deposition methods. We acquired ion density maps of such as phosphatidylinositol and phosphatidylcholines (glycerophospholipids) and sulfatides (sphingolipids) in a rat brain section.

Section snippets

Matrix

2,5-Dihydroxy benzoic acid solution (DHB, Aldrich, Milwaukee, WI) 100 mg/mL in 90% ethanol.

Animals

Male Sprague–Dawley rats (Harlan Industries, IN) between 280 g and 420 g. All animal use and handling procedures were approved by the Animal Care and Use Committee (ACUC) of NIDA-IRP, NIH. Rats were euthanized with over dose isoflurane and decapitated upon cessation of breathing. Rat brains were rapidly removed from the skull and frozen in isopentane pre-chilled on dry ice for 10–15 s, then wrapped in

Results

Different techniques were used by various groups to deposit matrix onto tissue sections for MALDI-IMS studies. In this work we evaluated three matrix deposition methods: spraying with a TLC sprayer, a Collision Nebulizer, or an artistic airbrush to compare their suitability for brain lipid imaging.

Although widely accepted as a matrix deposition method for MALDI-IMS application, the TLC sprayer appeared to generate coarse and unevenly sized droplets in spite of careful adjustment of the air flow

Discussion

One of the most critical steps in tissue MALDI-IMS studies is a well-executed matrix deposition on the tissue section, which permits homogeneous mixing and re-crystallization between analytes and matrix, without causing any noticeable displacement or redistribution of biomolecules. Several investigators have demonstrated various approaches addressing this issue. Airbrush deposition, among the commonly accessible matrix deposition methods, appears to carry significant advantages over others that

Acknowledgements

This research was supported by the Intramural Research Program of National Institute on Drug Abuse, NIH. The authors thank the Office of National Drug Control Policy (ONDCP) for instrumentation funding, without which this and other projects could not have been accomplished. We also would like to thank Dr. Patricia Tagliaferro and Dr. Marisela Morales for their assistance in light microscope photography.

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