Visualizing the proteome: mapping protein changes in disease states with mass spectrometry imaging
Version of Record online: 18 FEB 2013
© 2013 International Society for Neurochemistry
Journal of Neurochemistry
Volume 124, Issue 5, pages 581–583, March 2013
How to Cite
Lanni, E. J., Rubakhin, S. S. and Sweedler, J. V. (2013), Visualizing the proteome: mapping protein changes in disease states with mass spectrometry imaging. Journal of Neurochemistry, 124: 581–583. doi: 10.1111/jnc.12037
- Issue online: 18 FEB 2013
- Version of Record online: 18 FEB 2013
- Manuscript Accepted: 25 SEP 2012
- Manuscript Received: 17 SEP 2012
amyotrophic lateral sclerosis
matrix-assisted laser desorption/ionization
mass spectrometry imaging
In this issue, Hanrieder et al. (2012) demonstrate an elegant application of mass spectrometry imaging to probe the chemical changes in the spinal cord proteome related to a neurodegenerative disease. Before discussing their results, some background on the technology is appropriate.
As biological organisms, human beings can be thought of as a large collection of molecules that interact to generate body movement, behavior, consciousness, memory, and other higher and lower order functions. These complex functions depend not only on the diverse chemical composition of organs, tissues, and structures, but also on their spatial arrangements and temporal dynamics. The comprehensive, chemical-level modeling of a biological organism should capture this complexity, a key aspect of which is a chemical inventory encompassing monatomic ions to massive biopolymers, along with their metabolism and spatial distributions on scales ranging from the entire organism down to organelles within single cells. Clearly, no chemical imaging technique is yet sufficient for this task, although we can easily imagine the progress in our understanding of biology that could be made with such a tool.
Molecular imaging approaches used in biology, such as fluorescence microscopy, autoradiography, and positron emission tomography, normally target one or a few chemical species of interest at once, pre-selected for relevance to a hypothesis. These approaches have yielded a vast amount of important information about biological systems, but researchers also face a number of challenges in their pursuit of comprehensive biochemical visualization. Certainly not every biomolecule in a cell, or even a significant fraction of them, can be labeled with a fluorescent affinity tag and simultaneously imaged, regardless of fluorophore availability and technology advancements. Moreover, affinity tags are not always selective; minor changes in biomolecule structure (e.g., protein post-translational modification) may go undiscerned, and similar species (e.g., membrane phospholipids) are difficult to specifically label.
In contrast with the techniques mentioned above, mass spectrometry (MS) is capable of distinguishing hundreds of compounds in a single sample. By ionizing the compounds collected from a chemically complex sample, sorting these ions by their mass and charge, and detecting them to generate a representative mass spectrum, MS is chemically specific, multiplexed, and remarkably versatile. Moreover, tandem MS approaches (such as MS/MS) permit fragmentation of molecular analytes to yield increased chemical information, even allowing the identification of unknown molecules. Importantly, the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) in the late 1980s—‘soft’ ionization sources that successfully desorb and ionize large intact biomolecules such as proteins—opened a new era for bioanalytical MS with greatly relaxed size limits for characterizing molecules (Karas and Hillenkamp 1988; Tanaka et al. 1988; Fenn et al. 1989). As a result, MALDI MS and ESI MS now routinely allow label-free, non-targeted, and highly multiplexed detection of analytes ranging from monatomic ions to intact proteins and other biopolymers. Therefore, it is not surprising that both approaches have become critical tools for the chemical analysis of complex biological mixtures.
While MALDI-MS is traditionally used to analyze cell homogenates or analyte extracts, it can also be performed directly on tissue sections, including those of brain and spinal cord. This capability enables the profiling of tissue sections or regions of interest by collecting a complete mass spectrum at an ordered array of points. Detected compounds can then be selected post hoc and displayed as a chemical image by filtering the mass spectra and representing signal intensity as pixel brightness or false color (see Fig. 1). This technology, called mass spectrometry imaging (MSI) or imaging mass spectrometry, utilizes specific approaches for sample preparation, data acquisition and processing, and the generation of chemical images. As tens to hundreds of distinct compounds are detected simultaneously in an MSI experiment, this corresponds to a similar number of coregistered chemical images that can be created. Current MSI-enabled commercial instrumentation offers lateral resolutions for imaging large biomolecule distributions down to a few tens of microns, which is suitable for most tissue-level studies. However, cutting-edge research instrumentation now exceeds this by nearly an order of magnitude (Guenther et al. 2011).
The mammalian brain and spinal cord are both useful model tissues for MSI method development as they include distinct and well-known anatomical features across centimeter to micrometer length scales. These features often can be characterized by distinct profiles of relatively well-characterized biomolecules such as metabolites (including lipids), peptides, and proteins. Lipids are particularly amenable to detection using MSI, and recent method optimization has allowed the detection of many dozens at once in rat brain (Angel et al. 2012). This approach has also shown promising results in uncovering changes in the lipid profiles of injured rat spinal cord (Girod et al. 2010). The capability of MSI to survey rat spinal cord, detect and visualize the localization of dozens of peptides by MALDI MSI, and small molecules via SIMS imaging, has been demonstrated (Monroe et al. 2008). The distribution of proteins has also been determined in spinal cord using MSI; multiple proteins were located in this structure using a bottom-up approach where trypsin digestion was performed on-tissue prior to MSI (Tucker et al. 2011). Protocols for MSI are often optimized for a specific class of biomolecules per experiment. However, the technology has the ultimate capacity to detect a wide variety of analytes, present in tissues at different abundances, resulting in broad coverage of the metabolome, peptidome, and proteome of a specimen.
In this issue, Bergquist's group (Hanrieder et al. 2012) reports an exciting application of MSI to the examination of chemical changes occurring in the human spinal cord proteome during a poorly understood neurodegenerative disease—amyotrophic lateral sclerosis (ALS), a.k.a. Lou Gehrig's disease. In their exciting work, MSI methods were optimized for the high-sensitivity detection of proteins (with a trade-off of lateral resolution, which was less important here), revealing the distributions of many ions; ultimately, 18 identified proteins were visualized in disease-affected tissues and control specimens. Despite small sample sizes, statistically significant disease-related changes were observed in the abundance of two proteins. The MSI results demonstrated that these two proteins are localized within the cell body-containing central gray matter of the spinal cord, and the abundance of both is reduced in ALS-afflicted tissue. Analyte identification by separate MS/MS experiments allowed one of these compounds to be defined as truncated ubiquitin (Ubc-T), while the other protein remains unidentified, presenting an interesting target for future investigations. Importantly, western blotting and immunological tissue staining with available anti-Ubc antibodies were not able to help discern Ubc-T from its non-truncated endogenous counterpart; thus, the chemical specificity of MSI was critical in detecting this subtle difference between disease-affected tissues and controls. Nevertheless, these other techniques were useful in validating the qualitative and quantitative MS data. The results of this study allowed the authors to evaluate two established hypotheses to explain ALS pathophysiology, one based on cathepsin B RNA down-regulation, and another based on non-specific enzymatic cleavage, and to conclude that the former is more likely.
The work presented by Hanrieder et al. (2012) contributes new information related to ALS. It also demonstrates that MSI is a potent tool with unique capabilities to bring us closer to comprehensive, label-free chemical imaging of complex biological samples. The availability of a non-targeted molecular imaging tool allows us to significantly broaden the scope of imaging experiments; rather than pursue a defined hypothesis with readouts based on a few labeled analytes, the experimenter can approach research topics by asking open-ended questions: ‘What's there?’ or ‘What changes?’ The answers to these questions should aid a broad range of future efforts. The chemical specificity of MS also allows the discernment of subtle, yet significant, condition-related molecular changes, as shown successfully by Bergquist and colleagues (Hanrieder et al. 2012). Current MSI systems are able to generate exciting data. However, MSI is not yet able to handle the exceptional chemical complexity of tissues, their broad range of analyte concentrations, and mass spectral complications (e.g., chemical interference, adduct formation, ion suppression, etc.). Anticipated advances in the MSI instrumentation and sample preparation methodology will continue to enhance the applicability of MSI in fundamental and applied neurochemistry.
The authors have no conflicts of interest to declare.
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