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M. J. Rico, A. V. Rapkiewicz, V. Espina, W. Brunken, E. Shrier, L. S. Liotta, D. Lazzaro; Comparison of Vitreous Proteome in Cadavaric Subjects and Human Subjects Requiring Vitrectomy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3418.
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To create and compare a catalog of proteins expressed in the vitreous of cadaveric subjects and human subjects in both normal and pathologic states.
Samples were obtained from cadavers and therapeutic vitrectomies performed on human subjects. Vitreous samples were digested by trypsin and then analyzed by liquid chromatography nanospray tandem mass spectrometry using a Thermo Electron LTQ. Separations will be performed using 100 µm i.d. × 10 cm long fused silica capillary columns packed in-house with 5 µm C18 resin. The mass spectrometer will be operated in a data dependent MS/MS mode in which each full MS scan will be followed by five MS/MS scans with dynamic exclusion. Tandem mass spectra will be searched against human database with SEQUEST using tryptic cleavage constraints. For a peptide to be considered legitimately identified, it will have to achieve cross correlation scores of 1.5 for [M+H]1+, 2.0 for [M+2H]2+, 2.5 for [M+3H]3+, and a maximum probability of randomized identification of 0.01. At least one iteration is performed for each specimen.
Albumin and immunoglobulin account for more than 80% of whole vitreous proteins. We have recently determined that such carrier proteins may sequester and amplify low abundance proteins emanating from diseased tissues. We have developed a method to elute and identify them by mass spectrometry. In this proof-of-concept study, we applied this technique to vitreous samples obtained at autopsy in a research hospital and to therapeutic vitrectomy samples for human subjects. Over 400 proteins were identified from these samples.
Novel vitreous proteins not previously reported were identified in multiple samples studied. Some proteins known to be involved in angiogenesis were identified in cadaveric specimens and human subjects. By better defining the post-mortem vitreous proteome in comparison to pre-mortem vitreous proteome, we hope to generate a complete vitreous proteome which may elucidate links to pre-mortem disease.
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