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M. Miyagi, V. Palamalai, R.M. Darrow, D.T. Organisciak; Comparative Proteome Analysis of Light Exposed and Unexposed Photoreceptor Rod Outer Segments . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4569.
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© ARVO (1962-2015); The Authors (2016-present)
To gain insight into protein movement during photo transduction and possible mediators of light induced photoreceptor degeneration we have initiated efforts to quantitatively analyze photoreceptor rod outer segments (ROS) proteomes.
ROS were prepared by a sucrose density ultracentrifugation method from rats exposed to intense green light or not for 1 h. The unexposed rats were used as controls. Proteins were extracted from the light exposed and unexposed ROS with 100 mM Tris–HCl (pH 8.0) containing 2% (w/v) SDS and digested in H218O and H216O solvent, respectively, by peptidyl–Lys metalloendopeptidase. Thus, resulting peptides from light exposed and unexposed ROS were labeled with 18O and 16O, respectively. After the digestion, the two digests were mixed together in equal proportions and the relative abundance of each particular peptide from the two samples determined by mass spectrometry. By comparing the peak intensities of the 16O– and 18O–labeled peptide, the relative abundance of each particular peptide was determined, which equals the relative abundance in the original samples of the parent protein from which the peptide was generated.
Over 50 different proteins have been identified from initial analyses of the ROS, including most of the proteins involved in phototransduction. Among the proteins identified two proteins were increased at least 3–folds in the light exposed ROS and five proteins were decreased at least 2–folds. The proteins increased were arrestin and heat shock cognate 71 kDa protein. About 25% of the identified proteins were membrane proteins.
The comparative proteomic method utilizing protease–catalyzed 18O labeling appears to be effective method for studying ROS proteome. This technique allows us to quantify soluble and membrane proteins. Information about the ROS proteome will lead to a better understanding of the normal protein movement during phototransduction and the pathophysiology of light induced photoreceptor degeneration.
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