Proteome survey of proliferating and differentiating rat RPE-J cells

Karen A. West, Lin Yan, Masaru Miyagi, John S. Crabb, Alan D. Marmorstein, Lihua Marmorstein, John W. Crabb

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

The suitability of the rat derived SV-40T immortalized RPE-J cell line for identifying proteome changes associated with RPE differentiation was evaluated by surveying changes in protein expression levels. Rat RPE-J cells were induced to undergo differentiation in culture by growth at the nonpermissive temperature of 40°C in the presence of retinoic acid. Total proteins were extracted from cells grown under proliferating or differentiating conditions and separated by 1D and 2D gel electrophoresis. Gel spots were excised, 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 differentiating RPE-J cell cultures exhibited detectable levels of cellular retinaldehyde-binding protein, RPE65, 11-cis-retinol dehydrogenase or lecithin retinol acyl transferase, suggesting that RPE-J cells are not appropriate for visual cycle studies. About 18% of the 61 identified proteins appear to change expression levels with the cell growth conditions. Seven proteins appeared to be up-regulated and four proteins down-regulated when the cells were changed from proliferating to differentiating culture conditions. The majority of the apparent changes in protein expression levels were associated with stress response genes. Significant changes in the apparent mass and charge properties of proteins were also observed and for select proteins, the modifications appeared to be correlated with cell growth conditions. The results demonstrate that proteome differences in RPE-J cells associated with growth conditions can be identified and support the suitability of RPE-J cells for more targeted and/or more global proteome analysis of RPE differentiation.

Original languageEnglish (US)
Pages (from-to)479-491
Number of pages13
JournalExperimental Eye Research
Volume73
Issue number4
DOIs
StatePublished - Jan 1 2001

    Fingerprint

Keywords

  • 2D gel electrophoresis
  • Mass spectrometry
  • Proteomics
  • Retinal pigment epithelium

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

West, K. A., Yan, L., Miyagi, M., Crabb, J. S., Marmorstein, A. D., Marmorstein, L., & Crabb, J. W. (2001). Proteome survey of proliferating and differentiating rat RPE-J cells. Experimental Eye Research, 73(4), 479-491. https://doi.org/10.1006/exer.2001.1058