RAPID DETECTION OF CELLULAR PROTEIN DIFFERENCES

描述：（申请人摘要）蛋白质组成和

DESCRIPTION: (Applicant's abstract) Differences in protein composition and modification are the primary determinants of differences in cellular structure, function and behavior. In many cases, these differences are generated by differential gene expression. However, many cellular changes and features are the result of differences in post-translation modification. Understanding the molecular basis for cell differences is a major driving force in modern biology. A great deal of emphasis has been placed on differential gene expression. To complement these efforts, we have taken a protein-based approach for identifying the protein differences that are the basis for cellular differences. Typical eukaryotic cells are composed of more than 5,000 different proteins, many of which are common to all cells of the organism. The distinguishing features of different cell types are created by a minority of proteins. The standard method for displaying the array of cellular proteins is to separate them according to their charge and mass by two-dimensional polyacrylamide gel electrophoresis (2DE), a technique that allows one to visualize approximately 2,000 of the most abundant cellular proteins. 2DE suffers from a reproducibility problem-no two gels are perfectly alike. Therefore, one must resort to computer-based methods to align different gels, which does not perfectly resolve the heterogeneity problem. We have developed a technique that bypasses these problems by running the different protein samples on the same gel. This is accomplished by covalently coupling each protein sample with a different colored fluorescent dye that was designed to have no effect on the relative migration of labeled proteins during electrophoresis. The fluorescently tagged proteins are visualized by a fluorescent-gel imager that can discriminate between the two fluorescent dyes. Common proteins appear as spots composed of both fluorescent dyes; while proteins that differ between the two samples, referred to as "difference proteins", display a bias toward one or the other dye. This technique is called Difference Gel Electrophoresis (DIGE). Currently, DIGE is more sensitive than silver staining and can detect difference proteins down to 0.01% of total protein in a whole cell extract. This proposal describes three specific aims to improve DIGE so that one can visualize nearly all cellular proteins and detect very low abundance differences. Software for automated detection and isolation of difference proteins will also be described. Once a difference protein is detected, mass spectrometer analysis coupled to the genome data base will be used to determine the identity of the genes encoding the difference proteins.