Neuropeptides are a diverse set of complex cell-cell signaling molecules that modulate behavior, learning and memory. Their spatially heterogeneous distributions, large number of post-translational modifications, and wide range of physiologically active concentrations make their characterization challenging. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging is well-suited to characterizing and mapping neuropeptides in the central nervous system. Because matrix application can cause peptide migration within tissue samples, application parameters for MALDI typically represent a compromise between attaining the highest signal quality and preserving native spatial distributions. The stretched sample approach minimizes this tradeoff by fragmenting the tissue section into thousands of spatially isolated islands, each ~40 microns in size. This inhibits analyte migration between the pieces and, at the same time, reduces analyte-salt adduct formation. Here we present methodological improvements that enable the imaging of stretched tissues and reveal neuropeptide distributions in nervous tissue from Aplysia californica. The distributions of known neuropeptides are shown to correspond with previous immunohistochemical results, demonstrating that the stretched imaging method is well-suited for working with easily redistributed molecules and heterogeneous tissues, and reduces adducts from physiological salts.
Neurons often exhibit a complex chemical distribution and topography; therefore, sample preparation protocols that preserve structures ranging from relatively large cell somata to small neurites and growth cones are an important factor in secondary ion mass spectrometry (SIMS) imaging studies. Here, SIMS was used to investigate the subcellular localization of lipids and lipophilic species in neurons from Aplysia californica. Using individual neurons cultured on silicon wafers, we compared and optimized several SIMS sampling approaches. After an initial step to remove the high salt culturing media, formaldehyde, paraformaldehyde and glycerol, and various combinations thereof, were tested for their ability to achieve cell stabilization during and after the removal of extracellular media. These treatments improved the preservation of cellular morphology as visualized with SIMS imaging. For analytes >250 Da, coating the cell surface with a 3.2 nm-thick gold layer increased the ion intensity; multiple analytes previously not observed or observed at low abundance were detected, including intact cholesterol and vitamin E molecular ions. However, once a sample was coated, many of the lower molecular mass (<200 Da) analyte signals were suppressed. The optimum approach depended on the analyte being studied, and these approaches included rinsing with water and cell stabilization with glycerol and 4% paraformaldehyde. The sample preparation methods described here enhance SIMS imaging of processes of individual cultured neurons over a broad mass range with enhanced image contrast.
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been used to create spatial distribution maps from lipids, peptides, and proteins in a variety of biological tissues. MALDI-MSI often involves trade-offs between the extent of analyte extraction and desired spatial resolution, compromises that can adversely affect detectability. For example, increasing the extraction time can lead to unwanted analyte spatial redistribution. With the stretched sample method (SSM), the extraction period can be extended, resulting in reduced analyte redistribution while suppressing detection of cationic salt adducts. The SSM involves thaw-mounting a thin tissue section onto a substrate of small glass beads embedded in Parafilm M and then stretching the membrane to fragment the tissue into thousands of bead-sized pieces. Here, we applied the SSM method to MALDI-MSI using rat spinal cord as a model. We used surface-modified beads coated with trypsin or chymotrypsin in order to facilitate controlled digestion and detection of proteins. The enzymatic reactions were maintained by repeatedly condensing water on the stretched sample surface. As a result, new peptides formed by tryptic or chymotryptic protein digestion were detected and identified using a combination of MALDI-MSI and offline liquid chromatography tandem mass spectrometric analysis. Localization of these peptides indicated the distribution of their proteins of origin, including myelin basic protein, actin beta, and tubulin alpha chain. Additionally, we used uncoated beads to create distribution maps of many endogenous lipids and small peptides. The extension of the SSM using modified beads resulted in the creation of mosaic bead surfaces where adjacent beads were coated with different enzymes or other reactive chemicals, permitting investigation of the distributions of a wider range of analytes in biological samples within a single experiment.
Techniques that map the distribution of compounds in biological tissues can be invaluable in addressing a number of critical questions in biology and medicine. One of the newest methods, mass spectrometric imaging, has enabled investigation of spatial localization for a variety of compounds ranging from atomics to proteins. The ability of mass spectrometry to detect and differentiate a large number of unlabeled compounds makes the approach amenable to the study of complex biological tissues. This chapter focuses on recent advances in the instrumentation and sample preparation protocols that make mass spectrometric imaging of biological samples possible, including strategies for both tissue and single cell imaging using the following mass spectrometric ionization methods: matrix-assisted laser desorption/ionization, secondary ion, electrospray and desorption electrospray.
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