Identifying Novel Proteins in Nervous Tissue Using Microsequencing Techniques

Identifying signaling molecules that participate in pathways leading from receptor activation to changes in gene expression in cells is the fundamental focus of many research endeavors. For example, neurotrophins interacting with their trk receptors mediate a number of cellular effects, including cell differentiation, neurite outgrowth, and neuronal survival (1 –4 ). This results from specific protein-protein interactions in different signaling pathways. The primary aim of many research efforts is to elucidate these signaling pathways by identifying the proteins that participate in them. The most common method for isolating protein-protein interactions is by coimmunoprecipitation of protein complexes from cell lysates, where an appropriate antibody is available. Isolated proteins are separated using polyacrylamide gel electrophoresis and then transferred from the gel to a membrane support and identified using immunoblotting. These techniques have been well documented (see refs. 5 –8 ). The major drawback of this technique is its inability to find novel proteins. Currently, the best technique available for identifying novel proteins is by chemical or enzymatic fragmentation of isolated proteins in a gel matrix following one- or two-dimensional electrophoresis or after electrotransfer onto a suitable membrane (9 ,10 ). In most cases, reverse-phase high-performance liquid chromatography (RP-HPCL) is used to separate the cleavage fragments.