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J. S. Crabb, X. Yuan, G. Dvoriantchikova, D. Ivanov, V. I. Shestopalov, J. W. Crabb; Quantitative Proteomic Analysis of Glaucomatous Rat Retinal Ganglion Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):873.
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© ARVO (1962-2015); The Authors (2016-present)
We seek to quantify proteome changes in retinal ganglion cells (RGCs) associated with elevated intraocular pressure for insights into the molecular mechanisms of glaucoma pathology.
Experimental glaucoma was laser-induced unilaterally in rats. Retinas were isolated after elevated intraocular pressure was demonstrated for 4-6 weeks and RGCs were rapidly isolated by panning. Proteins were extracted from RGCs in SDS, digested with trypsin, peptides were labeled with iTRAQ tags, glaucomatous and control digests combined in equal amounts and fractionated by strong cation exchange chromatography. Proteins were identified by LC MS/MS using a Mascot search engine and the Swiss-Protein sequence database. Relative protein quantification from iTRAQ labeling utilized code written in the statistical program R. Bioinformatic analysis was performed with GeneGo Metacore and Ingenuity Pathways Analysis 5.0.
Relative protein quantitation has been obtained from RGCs pooled from 22 glaucomatous and 22 contra lateral control rat eyes. The LC MS/MS analyses yielded quantification of 273 proteins from two or more unique peptides per protein. Proteins with ratios ≥ 2 SD above the mean included aconitase hydratase, endoplasmic reticulum protein ERp29 and ploy(U)-binding-splicing factor PUF60. Proteins with ratios ≥ 2 SD below the mean included scaffold attachment factor B1, prothymosin alpha, and calnexin. Pathway analysis suggest alterations in several networks including those involving cell death, immune response, RNA post-tanslational modification, gene expression and cellular assembly and organization.
These preliminary results demonstrate the feasiblity of quantitative proteomic analysis of rapidly isolated RGCs. More proteins were detected in glaucomatous RGCs as decreased rather than increased relative to control RGCs, suggesting suppressed biosynthetic activity and downregulation of select functional networks in challenged neurons. Further studies with multiple analyses are required to identify disease mechanisms.
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