Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis

Abstract

Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.