) The recent completion of the genome sequences for several organisms has opened opportunities for development of new technologies for analysis of cells. 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 has 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. The spectra we obtain exhibit very high sensitivity (low fmol), resolution, and mass accuracy. This method works well with isoelectric focusing (IEF), native, and SDS gels, and has been applied to 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 intact mass of each spot on a gel to be determined as well as the masses of cleavage fragments generated in situ. The ultimate in parallel sample processing is achieved because protein cleavage is performed on an entire gel simultaneously. 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 identification of: proteins whose post-translational modifications alter in response to environmental factors, proteins affected by deletion or mutation of specific genes, and proteins that change during carcinogenesis or normal development. Our goal is to develop this technology allowing changes in living cells to be routinely examined at the molecular level (proteins) with very high throughput, sensitivity, and accuracy. We expect our technology to radically shift the experimental paradigm for cell biologists.
