Two-dimensional gel electrophoresis is presently the highest resolution method available for analysis of complex mixtures of proteins from cells and biological fluids. The method can be used to follow a host of biological events, e.g. development. However, it is presently difficult to rapidly identify the spots on the gel with a high degree of certainty and to elucidate all-important post-translational modifications and processing events. We are therefore expending considerable resources on developing methods whereby matrix-assisted laser desorption mass spectrometry can be used to identify proteins and to elucidate modifications. Strategies are being developed to remove proteins (after blotting to special media) for mass spectrometric analysis at the picomole level. Strategies are also being developed to obtain mass spectra of the mixtures generated from chemical or enzymatic digestion of proteins directly on blotting media in order to obtain peptide maps of these proteins. These maps will be useful for defining posttranslational modifications of proteins. Initial tests of the new techniques are being carried out on the various phosphorylated forms of the protein synapsin isolated from bovine brain. Proteins used in control studies included casein, bovine serum albumin, phosphorylase b and myoglobin. A paper describing the results appeared in PROTEIN SCIENCE 3 (1994) 677. We are continuing to improve our procedures for extracting peptides from the membranes after digestion of the blotted proteins.
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