Analysis of Tissue Specimens by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry in Biological and Clinical Research

Human beings are adept at discerning relevant information from complex systems by processing visual information. Similarly, as scientists labor to understand the fundamental nature of complex biological systems, they have continued to rely on visual information in the form of images to characterize and classify natural phenomena. New technologies designed to produce images of biological specimens have played a key role in the development of our modern understanding of biology. One of the earliest technological examples, the application of light microscopy to the analysis of biological tissue in the 17th century, ultimately led to the discovery of the cell as a key component of biology.1 Fortunately, the ways in which scientists now visualize biological systems have significantly matured. Currently, the methods for imaging biological specimens encompass an extraordinarily large range of technologies, capitalizing on many different measurable physical phenomena to produce images that provide insight into the underlying biology within the specimen. During the previous century, many imaging technologies including microscopy, radiography, ultrasonography, and magnetic resonance imaging have contributed greatly to the visualization of biological processes and to the practice of medicine.2 Each imaging modality has unique advantages and disadvantages that enable them to make contributions to research and clinical practice. One key aspect of imaging that remains a challenge is the effective integration of molecularly specific information as part of the image. Many of the commonly used in vivo imaging technologies produce high quality images, but these cannot be expressed as individual molecular images. Although immunostaining can be used to localize specific molecules within a biological sample, this method depends upon the use of a surrogate marker of the molecule such as an antibody or other specialized reagent and is usually performed on one or at most only a few molecules of interest in a single experiment. Mass spectrometry (MS) is unique among analytical technologies in its ability to directly measure individual molecular species in complex samples, allowing it to make significant contributions to our understanding of biological molecules. Indeed, the fundamental basis of the dynamic state of living systems was discovered by Rittenberg and Schoenheimer in the 1930's and 1940's through the use of MS and stable isotope tracers.3–5 With the introduction of ionization techniques such as electrospray ionization (ESI)6 and matrix-assisted laser desorption/ionization (MALDI),7 the field of mass spectrometry has grown exponentially in the past 20 years due to the application of MS to biological molecules. These capabilities ushered in a new era of biological research wherein a systems approach can be used to analyze the molecules in living systems in the wake of information provided by the Human Genome Project.8 With the drive to discover new biology has come a concomitant drive for the development of new mass spectrometry instrumentation. The primary benefit of this technology innovation is the ability to measure specific molecular compounds at high structural fidelity with high speed of acquisition, making it possible to perform experiments on biological systems that have not been possible before. Even single experiments have shown near comprehensive coverage of entire proteomes of simple organisms.9–10 Imaging Mass Spectrometry (IMS) is a technology that makes regiospecific molecular measurements directly from biological specimens.11–15 This method of imaging capitalizes on all the advantages of modern mass spectrometers, including high sensitivity, high throughput, and molecular specificity, to produce images that visually represent tissue biology on the basis of specific molecules (e.g. peptides, proteins, lipids, drugs and metabolites). The capabilities of mass spectrometry are unique in the imaging world, providing unique insights into biological systems. The distinguishing principle of imaging mass spectrometry from other mass spectrometric techniques is that the preparation of the sample and the acquisition of the MS data must be performed in a manner that preserves the spatial integrity of the sample within the limits of the spatial resolution of the measurement. Therefore, IMS of a biological sample, such as a tissue section, requires that the mass spectral data be registered to specific spatial locations in order to correlate the molecular information to specific cells or groups of cells commonly visualized by microscopy. Images are reconstructed by plotting the intensities of a given ion on a coordinate system that represents the relative position of the mass spectral acquisition from the biological sample. The resulting images create a visual representation of the sample based on the specific molecular information measured from the sample itself. IMS has a number of advantages relative to other imaging techniques currently used for biological and clinical studies. First, MS can be used to detect analytes without the need for labeling or otherwise structurally modifying the native compound. This distinction is important for many reasons, but primarily this avoids potential problems if the tagging reagent affects or changes the physical, chemical, or biological function of the molecules of interest or if the reagent has multiple molecular affinities. Second, MS has the capability of monitoring thousands of molecules in a single experiment. From a systems biology perspective, the advantage of the concurrent measurement of whole pathways or components in multiple pathways is crucial to understanding the function of intact cells. Among the several mass spectrometry ionization techniques that can be used to directly analyze tissues, MALDI has led the way in the development of biological and clinical applications for IMS.16–17 This report describes the essential considerations for performing MALDI IMS experiments on tissue, reviews some of the recent applications to the analysis of clinical specimens, highlights specific contributions of MALDI IMS to our understanding of biology and medicine, and discusses specific advantages and limitations of the technology. This review is not intended to be comprehensive with respect to all aspects of imaging mass spectrometry; rather it focuses on the themes that are essential to the analysis of biological and clinical tissue samples using MALDI IMS. There are excellent reviews that extensively cover both the ionization techniques used in IMS as well as the various mass analyzers that have been adapted for use in IMS and the reader is referred to these for further information.12,18–21