Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized Ϸ16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.

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Like many effective therapeutics, interleukin-12 (IL-12) therapy often causes side effects. Tumor targeted delivery may improve the efficacy and decrease the toxicity of systemic IL-12 treatments. In this study, a novel targeting approach was investigated. A secreted alkaline phosphatase (SEAP) reporter gene-based screening process was used to identify a mini-peptide which can be produced in vivo to target gene products to tumors. The coding region for the best peptide was inserted into an IL-12 gene to determine the antitumor efficacy. Affinity chromatography, mass spectrometry analysis, and binding studies were used to identify a receptor for this peptide. We discovered that the linear peptide VNTANST increased the tumor accumulation of the reporter gene products in five independent tumor models including one human xenogeneic model. The product from VNTANST-IL-12 fusion gene therapy increased accumulation of IL-12 in the tumor environment, and in three tumor models, VNTANST-IL-12 gene therapy inhibited distal tumor growth. In a spontaneous lung metastasis model, inhibition of metastatic tumor growth was improved compared to wild-type IL-12 gene therapy, and in a squamous cell carcinoma model, toxic liver lesions were reduced. The receptor for VNTANST was identified as vimentin. These results show the promise of using VNTANST to improve IL-12 treatments.

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The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following ingel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome. Molecular & Cellular Proteomics 2:37-49, 2003.The RPE 1 is a simple cuboidal epithelium that separates the photoreceptor cells of the retina from their principal blood supply in the choroid (1). In all vertebrates, the RPE forms an integral part of the blood-retinal barrier and is responsible for vectorial transport of nutrients to rod and cone photoreceptors and removal of waste products to the blood. In addition, the RPE phagocytizes shed photoreceptor outer segments, absorbs scattered light, and functions in the retinoid visual cycle and regeneration of bleached visual pigment (2, 3). To facilitate studies of retina and RPE in health and disease (4), we have initiated the development of a human RPE protein database. EXPERIMENTAL PROCEDURESIn Vivo RPE Samples-Forty-two normal human eyes were used in this study and were obtained from the Cleveland Eye Bank within 3-12 h postmortem. Following bisection of the globe behind the limbus, the anterior segment and vitreous were discarded, and the retina was removed. RPE cells were gently brushed from the eye cup using an artist's 7-mm angular paint brush (Langnickel L7160) and Ca 2ϩ -and Mg 2ϩ -free phosphate-buffered saline containing 5 mM EDTA and 1 mM phenylmethylsulfonyl fluoride (two to three times with 500 l). The RPE cells were then washed two to three times by centrifugation in phosphate-buffered saline. For select preparations, red blood cells were removed by centrifugation in a Percoll gradient (1.0 -1.1 g/ml, Amersham Biosciences); the pigmented RPE cells float near the top of the gradient and were collected with a Pasteur pipette and then washed two times in phosphatebuffered saline. Whole cell lysates were prepared by homogenizing RPE cell pellets in isoelectric focusing (IEF) solvent B (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% Triton X-100, 2% carrier ampholyte, 1% dithiothreitol), the solution was clarified by centrifugation, and protein was quantified by a modified Bradford assay (5). About 95 g of soluble whole cell RPE protein was recovered per eye (n ϭ 11 eyes).Subcellular Fractionation-Subcellular RPE fractions were prepared according to Saari et al. (6). Briefly, freshly isolated RPE cells were suspended in 1-4 ml of 25 mM Tris acetate, pH 7, 0.25 M sucrose, 1 mM dithiothreitol, homogenized with 25-125 manual passes of a glass homogenizer, and clari...