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X. Yang, C. Luo, J. Cai, G. Tezel; Proteomic Identification of Phosphorylated Proteins in a Chronic Pressure–Induced Rat Model of Glaucoma . Invest. Ophthalmol. Vis. Sci. 2006;47(13):198.
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Recent studies have provided evidence that phosphorylation regulates the functional activity of many proteins involved in RGC signaling in glaucoma. As an initial proteomic approach to identify signaling molecules involved in glaucomatous neurodegeneration, differential protein phosphorylation was determined in the retina following experimental elevation of IOP.
IOP elevation was induced in rats by hypertonic saline injections into episcleral veins. Phosphorylated retinal proteins were detected by Pro–Q Diamond staining of 2D–gels followed by Sypro Ruby staining for total protein profiling. Phosphorylation–related shift of protein spots from their initial position on 2D–gels was determined before and after phosphatase treatment. For large–scale quantitation, differential display analysis was performed to compare the intensity of protein spots matched on 2D–gel images obtained from ocular hypertensive and control eyes. The phosphoproteins revealed by the specific staining were identified through peptide mass fingerprinting and peptide sequencing using tandem mass spectrometry. 2D–western blot analysis and immunohistochemistry using phosphorylation site–specific antibodies were also performed for further validation of the proteomic findings.
Comparison of Pro–Q Diamond–stained and Sypro Ruby–stained 2D–gels revealed that the number and intensity of protein spots exhibiting phosphorylation were greater in ocular hypertensive retinas compared with the controls (p<0.01). Among over hundred spots exhibiting an increased or new phosphorylation, the proteins identified with significant matches (p<0.05; sequence coverage 18–84%; and sequence matches for multiple peptides) included those involved in cell signaling (collapsin response mediator proteins, 14–3–3 family proteins, dC stretch–binding protein, ubiquinol–cytochrome c reductase core protein, and annexin) and stress response (hsp60, hsp70, and matricin). 2D–western blots confirmed the selected proteins, and immunolabeling of retina sections with specific antibodies demonstrated their cellular localization in ocular hypertensive eyes, which included RGCs.
These in vivo findings reveal some signaling molecules that function after phosphorylation during glaucomatous neurodegeneration. Ongoing proteomic studies should fully identify the signaling cascades promoting RGC death in glaucoma, thereby providing novel treatment targets for neuroprotective interventions.
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