Comparative Proteome Analysis of Light Exposed and Unexposed Photoreceptor Rod Outer Segments

Abstract

Purpose:: To gain insight into changes in photoreceptor rod outer segment (ROS) proteins during photo transduction and as possible mediators of light induced photoreceptor degeneration we have initiated efforts to quantitatively analyze the ROS membrane proteome.

Methods:: Proteolytic ¹⁸O labeling strategy was employed to examine the changes in protein expression in light exposed and unexposed ROS membranes. Cyclic light reared rats (2 month old) were exposed to intense green light (1,200-1,400 lux; 490-580 nm) for 8 hours; unexposed rats were used as controls. ROS were prepared by sucrose density ultracentrifugation. ROS were washed in hypotonic buffer (5 mM Tris-HCl, pH 7.1 containing 15 mM 2-mercaptoethanol), soluble proteins removed by centrifugation, and the resulting pellet was used in this study. Proteins from light exposed and unexposed ROS membranes were digested by trypsin in either H₂¹⁸O or H₂¹⁶O solvent containing 0.1% RapiGest SF (acid cleavable surfactant). After digestion, the two digests were mixed together in equal proportions and the relative abundance of ¹⁸O- and ¹⁶O-labeled peptides from the two samples was determined by mass spectrometry.

Results:: Over 35 different proteins have been identified from initial analyses of these ROS membranes. Among the proteins identified, 15 were increased by at least 3-fold in the light exposed ROS membranes and three proteins were decreased by at least 3-fold. The proteins that increased included sodium/potassium-transporting ATPase, voltage-dependent anion-selective channel protein and heat shock cognate 70 kDa protein 1A/1B. The proteins that decreased were guanine nucleotide-binding protein (transducin) alpha, beta and gamma. The membrane protein, opsin, did not exhibit changes. About 80% of the proteins identified as changing during light exposure were membrane associated proteins.

Conclusions:: The comparative proteomic method utilizing protease-catalyzed ¹⁸O labeling appears to be an effective method for studying changes in the ROS membrane proteome. Information about the ROS proteome will lead to a better understanding of the changes in protein levels that occur during the pathophysiology of light induced photoreceptor degeneration and may be useful in studying other retinal degenerations.

Keywords: proteomics • photoreceptors • apoptosis/cell death