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Tim U. Krohne, Sebastian Aretz, Boris V. Stanzel, Martina Schnölzer, Frank G. Holz, Jürgen Kopitz; Ultrasensitive Mass Spectrometric Analysis Of The Human Vitreous Proteome. Invest. Ophthalmol. Vis. Sci. 2011;52(14):900.
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© ARVO (1962-2015); The Authors (2016-present)
Proteins in the vitreous are in part derived from retinal cells, and proteomic analysis of vitreous samples may thus provide insights in both physiological and pathological retinal processes. However, many proteins are released into the vitreous in very low abundance and therefore difficult to detect by conventional methods. We applied ultrasensitive high-throughput proteomic screening technologies to characterize the complete human vitreous proteome.
Human vitreous samples were obtained by vitrectomy from patients with epiretinal gliosis. Proteins were prefractionated by liquid phase electrophoresis, and the resulting factions were further separated by SDS gel electrophoresis. Lanes on the SDS-gel were sliced into 25 pieces, and proteins in each individual piece were subjected to in-gel digestion by trypsin. The resulting peptides were extracted and analyzed by nano-HPLC ESI-MS/MS on a nanoACQUITY UPLC system (Waters, Milford, MA) coupled to a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA). The obtained mass peptide fingerprints were searched against the MSIPI database using the Mascot search engine (Matrix Science, London, UK). Bioinformatics tools were used to annotate known biological functions to the detected proteins.
We analyzed the vitreous proteomes of 3 individual patients and identified a total of 2037 different proteins. In addition to structural and binding proteins, 307 proteins with known enzymatic activity (excluding proteases), 94 proteases, 38 protease inhibitors, 38 members of the complement and coagulation cascades, 20 peptide hormones, 13 growth factors, and 12 cytokines were detected. No known function could be assigned to 54 proteins, and 24 proteins were hypothetical. Many identified proteins are secreted by retinal cells, such as PEDF, retinoschisin, and complement factor H.
Human vitreous contains a highly complex mixture of proteins that is amenable to comprehensive assessment by ultrasensitive proteomic screening technologies. The presented catalogue of human vitreous proteins will enhance our understanding of physiological processes in the eye and provides the groundwork for future studies of vitreous proteome changes associated with retinal diseases.
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