Analysis of Differentially Expressed Proteins of Glaucomatous Rat Optic Nerve Head via Proteomics

Abstract

Abstract: : Purpose: Glaucoma is characterized by degeneration of optic nerve and gradual cupping of optic nerve head (ONH). Although the mechanisms of optic nerve damage (mechanical vs ischemic) in glaucoma have not been determined, the ONH may be the initial site of damage. This study's purpose was to identify differentially expressed protein(s) in the ONH of glaucomatous rats. Methods: A laser–induced model of glaucoma was created by cauterization of the episcleral veins and trabecular meshwork previously filled with indocyanine green in the left (OS) eye of Brown Norway rats. The right (OD) eye served as a control. The rise in intraocular pressure was monitored by Tonopen. Fourteen days after induction of chronic ocular hypertension, the ONHs were carefully dissected and pooled from 4 individual animals before performing 2D gel electrophoresis using 200 µg of total protein. Isoelectric focusing was carried out in glass tubes using 2.0% pH 3.5–10 ampholines and 2D gel electrophoresis was performed for 4 hr at 12.5 mA/gel on acrylamide slab gel (0.75 mm thick). The glaucomatous and normal 2D gels were compared. Protein spots showing differences were digested in situ with trypsin and identified by mass spectrometry (MALDI–TOF/MS on an Applied Biosystems Voyager–DE STR and capillary HPLC–ESI/MS/MS on a Thermo Finnigan LCQ), with database searching accomplished by Mascot (Matrix Science). Results: By mass spectrometry, we identified 5 protein spots, which were altered in glaucomatous ONH as compared to the controls. The proteins identified were transferrin, glutamate dehydrogenase–mitochondrial precursor, α–enolase, carbonic anhydrase, and α–crystallin–B chain. Conclusions: Since most of the identified proteins are involved in stress and oxidative damage, these identified proteins point toward the involvement of protein oxidation in glaucomatous ONH damage. These studies also establish that enough protein can be obtained from four pooled rat ONH to permit proteomic analysis. These studies are the first to examine the protein(s) profile in glaucomatous optic nerve heads as compared with the controls via proteomics.

Keywords: proteomics • oxidation/oxidative or free radical damage • pathobiology