The recent completion of the genome sequences for several organisms has opened opportunities for development of new technologies for functional analysis. The structural complexity of proteins, the primary functional components of cells, has hindered development of high-throughput analytical methods comparable to those for nucleic acids. In an effort to bridge the gap between gene sequence and protein function, our laboratory developed a new technology which uses MALDI mass spectrometry to characterize proteins resolved by thin-layer 2-D gel electrophoresis. Proteins are desorbed directly from ultrathin polyacrylamide gels which dry to thicknesses of 10 microns or less. Spectra obtained by this method exhibit very high sensitivity (low fmol), resolution, and mass accuracy. This method works well with IEF, native, and SDS gels, and has been applied to 2-D and 3-D analysis of complex protein mixtures with identification of component proteins by their pI values, intact masses, and CNBr fragment masses. It allows the mass of each spot on a gel to be determined as well as the masses of cleavage fragments generated in situ. The proteins are then identified by searching their fragment masses against a genome database. This technology is also very efficient at identifying post-translationally modified proteins. Protein landmarks may be identified to compare 2-D gels from different tissues or laboratories, and unknown proteins may be fingerprinted. Applications include: 1) identification of proteins whose post-translational modifications change in response to environmental factors (glucose levels, hormones, etc.), 2) identification of proteins affected by deletion of specific genes or over expression of specific proteins, and 3) identification of proteins which change during development. This proposal details efforts to expand our initial feasibility studies and extend them to eukaryotes. The model system chosen for the next stage of this work is yeast, an experimentally versatile organism for which a few hundred major proteins have been identified by much less efficient mass spectrometric mapping methods.
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