W.L. Liao, I.V. Turko

Senescence of retinal pigment epithelial cells is a crucial event in the pathogenesis of age-related macular degeneration. In the current report human ARPE-19 cells subjected to recurring oxidative stress were used as an in vitro model of premature senescence and we focused on quantitative proteomic analysis of partially degraded proteins. Our quantitative approach is based on the use of light (non-deuterated) and heavy (deuterated) acrylamide to alkylate cysteine residues in proteins. Heavy acrylamide has 3 deuterium atoms instead of 3 hydrogen atoms and is 3 mass units heavier than light acrylamide. From the chemical point of view, there is insignificant difference between light and heavy forms of acrylamide. This warrants equal efficiency of alkylation in separate samples and, after mixing, the co-migration of the same protein from separate samples on two-dimensional polyacrylamide gel electrophoresis. The spots of interest are then in-gel digested with trypsin and identified using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). A relative abundance of a protein in the original samples is determined as the ratio of signal intensities for the isotopically heavy and light forms of a cysteine-containing labeled peptide in MALDI-MS spectra. In the current study, we found accumulation of large fragments of several typical cytosolic proteins in the prematurely senescent ARPE-19 cells in comparison to the control cells.

This study is the first demonstration that large fragments of cytosolic proteins can be accumulated in prematurely senescent ARPE-19 cells. These data suggest that protein degradation processes are impaired in these cells and point to a new type of "waste" material in post-mitotic cells that may contribute to the senescent phenotype.

Keywords (Optional): 
retinal pigment epithelial
waste accumulation
quantitative proteomics