Proteomic Analysis of the Vitreous following Experimental Retinal Detachment in Rabbits.

Purpose. The pathogenesis of rhegmatogenous retinal detachment (RRD) remains incompletely understood, with no clinically effective treatment for potentially severe complications such as photoreceptor cell death and proliferative vitreoretinopathy. Here we investigate the protein profile of the vitreous following experimental retinal detachment using a comparative proteomic based approach. Materials and Methods. Retinal detachment was created in the right eyes of six New Zealand red pigmented rabbits. Sham surgery was undertaken in five other rabbits that were used as controls. After seven days the eyes were enucleated and the vitreous was removed. The vitreous samples were evaluated with two-dimensional polyacrylamide gel electrophoresis and the differentially expressed proteins were identified with tandem mass spectrometry. Results. Ten protein spots were found to be at least twofold differentially expressed when comparing the vitreous samples of the sham and retinal detachment surgery groups. Protein spots that were upregulated in the vitreous following retinal detachment were identified as albumin fragments, and those downregulated were found to be peroxiredoxin 2, collagen-Iα1 fragment, and α-1-antiproteinase F. Conclusions. Proteomic investigation of the rabbit vitreous has identified a set of proteins that help further our understanding of the pathogenesis of rhegmatogenous retinal detachment and its complications.

Analytical platforms in vitreoretinal proteomics.

Current proteomic technologies can effectively be used to study the proteins of the vitreous body and retina in health and disease. The use of appropriate samples, analytical platform and bioinformatic method are essential factors to consider when undertaking such studies. Certain proteins may hinder the detection and evaluation of more relevant proteins associated with pathological processes if not carefully considered, particularly in the sample preparation and data analysis stages. The utilization of more than one quantification technique and database search program to expand the level of proteome coverage and analysis will help to generate more robust and worthwhile results. This review discusses important aspects of sample processing and the use of label and label-free quantitative proteomics strategies applied to the vitreous and retina.

Protein changes in the retina following experimental retinal detachment in rabbits.

PURPOSE: Retinal detachment leads to the widespread cellular remodeling of the retina. The purpose of this study was to identify protein changes that accompany these cellular alterations by comparing the proteomic profiles of sham and experimentally detached rabbit retina. Elucidation of the proteins most dramatically affected by retinal detachment would add further understanding to the pathophysiology of this condition, and potentially identify therapeutic targets useful in preventing the deleterious effects of detachment, including photoreceptor cell death and the activation of non-neuronal microglial and Müller cells. METHODS: Retinal detachments were induced in the right eyes of six New Zealand Red pigmented rabbits. Sham surgery was performed in the right eyes of six other rabbits that were used as controls. At seven days, the eyes were enucleated and the retinal tissue was harvested. The individual retinal samples were subjected to high resolution two-dimensional polyacrylamide gel electrophoresis. Differentially expressed protein spots were processed for identification by liquid chromatography-tandem mass spectrometry. Further investigation was undertaken with western blotting, and immunocytochemical studies on a further set of four sham and four detached retinas. RESULTS: Eighteen protein spots were found to be at least twofold differentially expressed between the sham and detached retinas. These protein spots were identified as: vimentin; tubulin ß-2C; fragments of α-enolase; fructose-bisphosphate aldolase A; ATP synthase subunit ß; mitochondrial creatine kinase; N-terminal fragments of albumin; prohibitin; and transducin-ß(1). CONCLUSIONS: The differentially expressed proteins determined in this study may play an important role in the cellular responses of the retina after its detachment, subsequent ability to recover following surgical reattachment, as well as in serious complications such as subretinal fibrosis and proliferative vitreoretinopathy.

Proteomics of uveal melanomas suggests HSP-27 as a possible surrogate marker of chromosome 3 loss.

PURPOSE: To compare the proteomic profiles of primary uveal melanomas, with and without loss of chromosome 3. METHODS: Frozen specimens from three uveal melanomas with disomy 3 and from four tumors with monosomy 3, according to fluorescence in situ hybridization (FISH) analysis, were subjected to high-resolution, two-dimensional (2-D) gel electrophoresis. The protein expression profiles of the two uveal melanoma cytogenetic groups were compared: Proteins that differed significantly were excised and analyzed by tandem mass spectrometry. Differentially expressed proteins were further analyzed with Western blot analysis. An independent cohort of 41 formalin-fixed, paraffin-embedded (FFPE) uveal melanomas, whose chromosome 3 status had been determined by multiplex ligation-dependent probe amplification (MLPA), was examined for the appropriate antigens by immunohistochemistry. RESULTS: Four protein spots were 1.5-fold (Student's t-test, P < 0.05) differentially expressed in the two uveal melanoma types: two spots were overexpressed in the disomy 3 group compared with the monosomy 3 group, whereas two spots were underexpressed. Identification of the four spots yielded nine proteins. Western blot analysis confirmed the results for heat shock protein (HSP)-27, vimentin, and pyruvate dehydrogenase beta (PDHB), with a statistical significance for the first two proteins. HSP-27 was significantly downregulated, whereas vimentin was upregulated in the monosomy 3 tumors (Student's t-test, P = 0.003 and P = 0.005, respectively). Immunohistochemistry confirmed low-to-negative HSP-27 protein expression in monosomy 3 uveal melanomas (Student's t-test; P = 0.011). CONCLUSIONS: Low-to-negative HSP-27 protein expression in uveal melanoma correlates strongly with monosomy 3. Further validation is necessary to determine whether immunohistochemical assessment of HSP-27 expression correlates with metastatic mortality.

Ocular proteomics with emphasis on two-dimensional gel electrophoresis and mass spectrometry.

The intention of this review is to provide an overview of current methodologies employed in the rapidly developing field of ocular proteomics with emphasis on sample preparation, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Appropriate sample preparation for the diverse range of cells and tissues of the eye is essential to ensure reliable results. Current methods of protein staining for 2D-PAGE, protein labelling for two-dimensional difference gel electrophoresis, gel-based expression analysis and protein identification by MS are summarised. The uses of gel-free MS-based strategies (MuDPIT, iTRAQ, ICAT and SILAC) are also discussed. Proteomic technologies promise to shed new light onto ocular disease processes that could lead to the discovery of strong novel biomarkers and therapeutic targets useful in many ophthalmic conditions.