We propose to develop a system, based on matrix-assisted laser desorption (MALDI) mass spectrometry, which is capable of producing images of tissues or other relevant biological samples in discrete m/z values in order to determine the spatial arrangement o specific molecules in these samples. Ion images would be produced by a repetitive exposure of the sample by the laser beam where adjacent spots are irradiated. The result would be an ordered array of mass spectra, keyed to specific locations in the sample, which would be the database for the production of individual or summed ion images. From one raster of the sample, specific ion image, at any chosen m/z value, could be produced to give the spatial arrangement of molecules of interest. Proteins and peptides will be identified using post-source decay techniques and proteolytic digestion with endo- and exo-peptidases followed by post-source decay analysis. The overall goal is to develop and apply a technique that would permit the localization of specific molecules in tissue and other biological samples. The proposal is targeted toward analysis of peptides and proteins in mammalian tissue sections. The specific goals are 1) Instrument development involving modifications to an existing commercial MALDI (TOF) mass spectrometer, including changes to the laser optics, target movement system, and instrument control hardware and software. This would permit us to achieve approximately 1-5 micrometer resolution and at sensitivities in the range of attomoles/(10 micrometers-squared). Image resolution and sensitivity would be variable in order to optimize the analysis for a particular sample. (2) Development of methods for sample preparation and target surface modifications to achieve hig sensitivity and high image resolution, with molecular sensitivity. Tissues wil be measured both directly and indirectly; the latter as blotted images. A number of different active target surfaces would be used for blotting techniques. (3) Applications to specific research areas involving questions about certain spatial distributions of molecules within specific tissues. Thes include: i) processing of peptides and proteins in rat pituitary and pancreas, ii) binding of proteins to antibodies, iii) location of some specific forms of cytochrome P450 in mammalian tissue, iv) location of isoforms of selenoprotein in human kidney tissue, and v) identification and location of neuropeptides in brain tissue from a parkinsonian animal model.
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