Proteome survey of proliferating and differentiating rat RPE-J cells

The suitability of the rat derived SV-40T immortalized RPE-J cell line for identifying proteome changes associated with RPE differentiation was evalua ted by surveying changes in protein expression levels. Rat RPE-J cells were induced to undergo differentiation in culture by growth at the nonpermissi ve temperature of 40 degreesC in the presence of retinoic acid. Total prote ins were extracted from cells grown under proliferating or differentiating conditions and separated by 1D and 2D gel electrophoresis. Gel spots were e xcised, digested in situ with trypsin, and analysed by mass spectrometry to identify proteins. Computer assisted image analysis was used to align gel patterns and quantify spot intensities. Neither proliferating nor different iating RPE-J cell cultures exhibited detectable levels of cellular retinald ehyde-binding protein, RPE65, 11-cis-retinol dehydrogenase or lecithin reti nol acyl transferase, suggesting that RPE-J cells are not appropriate for v isual cycle studies. About 18% of the 61 identified proteins appear to chan ge expression levels with the cell growth conditions. Seven proteins appear ed to be up-regulated and four proteins down-regulated when the cells were changed from proliferating to differentiating culture conditions. The major ity of the apparent changes in protein expression levels were associated wi th stress response genes. Significant changes in the apparent mass and char ge properties of proteins were also observed and for select proteins, the m odifications appeared to be correlated with cell growth conditions. The res ults demonstrate that proteome differences in RPE-J cells associated with g rowth conditions can be identified and support the suitability of RPE-J cel ls for more targeted and/or more global proteome analysis of RPE differenti ation. (C) 2001 Academic Press.