MALDI imaging of lipid biochemistry in tissues by mass spectrometry.

Abstract

As a result of recent advances, remarkable images revealing the distribution of complex lipids in tissues are now generated by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Lipids are amphipathic biomolecules with hydrophobic structural characteristics made by either an initial anion thioester condensation reaction (fatty acid synthase) or by carbocation condensation of branched chain pyrophosphate intermediates (isoprene pathway).1 Lipids play essential roles in cellular function including the self-assembly of phospholipids to form the constitutive outer and inner membrane bilayer of every living cell. Specific components of these membrane phospholipids include species that contain esterified arachidonate that can be enzymatically released to a free acid and transformed to potent signaling molecules (prostaglandins, leukotrienes) with myriad biological effects. The lipid cholesterol is an essential component of bilayer membranes that has a complicated, yet highly regulated biosynthesis. Elevation of cholesterol levels (predominantly as cholesteryl esters) in blood has been implicated in heart disease and is commonly monitored in consideration of human health. Some lipid molecules play the central role in biochemical energy storage in the form of triacylglycerol molecules stored in lipid bodies within most all cells. Mass spectrometry has historically been a tool of choice in biochemical studies of lipids. The sensitivity and specificity of mass spectral data are useful to sort out the complexity of lipid structures to begin to follow biological changes. While techniques such as fluorescence confocal microscopy or ability to engineer proteins that can be expressed in cells with fluorescent tags have become the mainstream of modern biochemical research, such techniques are not amenable to most lipids due to the relatively small size of the lipid molecules and the dynamic nature of their structure in the cell. Recent developments in MALDI IMS have merged specificity of lipid identification with two-dimensional molecular mapping to enable biochemical studies of lipids across regions of a biological tissue. Several significant reasons for the success of MALDI IMS applied to lipid imaging have emerged. The first is the high abundance of various lipids in biological tissues because these hydrophobic molecules constitute the external and internal defining membranes of each cell. These membranes are almost exclusively bilayers composed of phospholipids, sphingolipids, and cholesterol that are closely packed in high local concentrations to render the membrane only semipermeable to water. A second reason is that many lipids, e.g. phospholipids, are already ionized as either phosphate anions or nitrogen centered cations and generate abundant positive or negative ions during the MALDI process. An equally important factor in the success of MALDI IMS of lipids is that the molecular weight of these biomolecules is generally below 1,000 Da, which is an optimal mass range for the most sensitive operation of modern mass spectrometers. Additionally this low molecular weight facilitates diffusion of lipids into a matrix crystal driven by the high concentration of the lipid within the microstructure of the tissue. Because of these fundamental factors coupled with the exciting potential of MALDI IMS, lipid molecules have been frequently used as substrates for the advancement of IMS methodology and instrumentation. Research groups that utilize secondary ion mass spectrometry (SIMS) imaging have embraced lipid biochemistry by moving from inorganic to biological applications, development of larger particle size beams and demonstrations of sub-micron lateral resolution.2,3 Similar development and implementation of instrumentation for MALDI IMS has leveraged lipid diversity, abundance and contrast in rodent brain samples to achieve advancements in technology.4-8 The development of different matrices useful for MALDI IMS,9-16 different methods of matrix application17-23 and different matrix modifiers24,25 have been employed in MALDI IMS experiments to establish the value and parameters of these method modifications for lipid analysis. Advances in biology have been a direct result of our ability to observe biochemical events at the micron and submicron regimes within a tissue. Having a sensitive technique that reveals molecular structure information about specific lipids in a tissue with 10-50 μm resolution and provides information relative to concentration of that lipid, has already provided insight into lipid biochemistry at the tissue level. Since lipids are products of complex, intertwined enzymatic processes, MALDI IMS data reveals the integrated solution to complex reaction pathways that define the living cell in terms of lipid biochemistry. It has become apparent to a host of scientists converging into the use of MALDI IMS from fields as diverse as neuroscience, chemistry, and instrument development that there is a richness and complexity of lipid biochemistry suggested by the exquisite, molecule specific MALDI images created in the course of developing this technology. Many reviews have focused on the technological developments of MALDI IMS of lipids with respect to the issues mentioned above.2,26,27 This review focuses on the lipid biochemistry revealed by MALDI IMS.