2 Dimensional Gel Electrophoresis and Computer Analysis of Proteins Synthesized by Clonal Cell Lines

An improved method of 2-dimensional gel electrophoresis was developed which produces high resolution, reproducible images suitable for computer analysis. In the images that are presented, more than 800 proteins were resolved without significant overlap, and many more proteins can be detected after longer exposures. To establish the usefulness of such methods for detailed quantitative comparisons of cultured cells, extensive controls were carried out to test the reproducibility of the electrophoretic procedures, the sample preparation procedures and the cell culture conditions. A computerized scanning system was developed which can automatically detect and integrate the densities of the spots on a 2-dimensional fluorogram or autoradiogram. The corresponding proteins from 2 or more samples can then be matched and their intensities compared. Several types of graphic output were used to display the number and magnitude of the differences between the compared samples. These methods were used to study the patterns of protein synthesis in the nerve cell line B103 and the glial cell line B9 [rat neoplastic cell lines]. Both exponentially dividing and stationary cultures were analyzed and the relative rates of synthesis of .apprx. 300 proteins were compared by computer. For each cell line, no major qualitative differences were found between dividing and stationary phase cells although numerous quantitative differences of up to 15-fold were detected. The proteins that were increased or decreased in rate of synthesis as B103 cells became confluent were in general not the same proteins that were increased or decreased in rate of synthesis as B9 cells reached confluence, indicating that most of the changes do not reflect growth control responses common to all cells. When the 2 cell lines were analyzed in the same state of growth and compared by computer, qualitative differences were found in .apprx. 5% of the proteins analyzed, and at least 40% of the shared proteins were involved in quantitative differences of 2-fold or more. The rates of synthesis of the shared proteins were more divergent between the 2 cell lines than between dividing and stationary phase cells of either line. These cell lines can therefore be distinguished, regardless of growth state, by their cell-specific proteins and by their characteristic rates of synthesis of many of the shared proteins