Clinical Ocular Proteomics: Protein Manifestation in Vitreous Humor of Diabetic Retinopathy and Other Retinal Eye Diseases

Abstract

Abstract: : Purpose: Our long term goal is to establish a methodology to use protein information for aiding diagnosis and prognosis of retinal eye diseases. In this work we conducted the proteomic analysis of vitreous humor samples from diabetic retinopathy and other retinal eye diseases. Methods: Vitreous samples collected during surgery were analyzed from patients with diabetic retinopathy (DR), cystoid macular edema (CME) or proliferative diabetic retinopathy (PDR) with vitreous hemorrhage, patients with proliferative vitreoretinopathy (PVR), together with patients with macular hole (MH) and epiretinal membrane (ERM) for control. Plasma samples were also collected from diabetic patients and from normal subjects. The vitreous sample containing 100 µg of proteins was dissolved in lysis buffer and subjected to two–dimensional (2–D) gel electrophoresis. After Coomassie blue staining, the gels were scanned and the images were analyzed by Progenesis Workstation (Nonlinear Dynamics). In the analysis all vitreous proteins were normalized to the total density of the 2–D gel. Protein spots were excised and in–gel digested by trypsin, and the resulting mixtures of peptides were subjected to peptide mass fingerprinting for identification using Voyager Elite (Applied Biosystems) matrix–assisted laser desorption/ionization mass spectrometer (MALDI–TOF MS). Results: The control vitreous and plasma share many common proteins in general. However, both haptoglobin and fibrinogen which are abundant in plasma were barely observed in the control vitreous. We observed some classes of proteins are manifested more abundantly in the vitreous from retinopathy patients compared to control. For example, we observed a tendency in the current set of samples that the fibrinogen level was high in CME, and low in PDR. Conclusions: Since the patient vitreous could have been contaminated with plasma due to the breakdown of the blood–retinal barrier (BRB) and also to the injuries during the surgery, it is rather difficult to attribute the changes of protein levels to a single factor. Nonetheless, the results suggest that 1) vitreous proteins do not seem to represent the set of plasma proteins ultrafilterated evenly through BRB, and 2) it seems that the breakdown of BRB in retinopathies does not always bring out the entire set of plasma proteins into vitreous. Further studies including the statistical analysis of the quantitative proteomics results are needed to establish clinical ocular proteomics of human vitreous.

Keywords: proteomics • clinical research methodology • vitreous