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W.J. Curry, A. Healy, A. Wallace, V. Miller; Porcine Neural Retinal Proteomics . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4562.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Proteomics provides an unbiased approach offering an inclusive method of studying normal cell function relative to the changing cellular events that reflect pathological processes. Human tissue-specific proteome databases are being established and it is evident that the porcine proteome will become increasingly important as the potential for xenotransplantation is fulfilled. The aim of this study was to employ 2D electrophoresis to commence studies to determine the normal porcine neural retinal proteome. Methods: Porcine eyes were resected at the abattoir and transported to the laboratory on ice. The retinae (n=10/preparation) were dissected, snap-frozen, ground up in liquid nitrogen and proteins extracted overnight in 40 mM Tris, 4% CHAPS. The protein extracts were dialysed, freeze-dried and reconstituted in 8 M urea, 0.5 M thiourea, 2% CHAPS, 1% DTT and 1% IPG buffer. IPG strips were rehydrated and proteins separated by 2D PAGE using a Multiphor II system (Pharmacia Biotech). A range of IPG strips with various pH gradients were employed in conjunction with 12-14% gels. Coomassie blue stain was employed to visualise the proteome. Prominent protein spots were excised, trypsin-digested and subjected to tandem mass spectrometry (MS/MS). The resulting m/z data of the fragment ions was searched against the NCBInr database using the Mascot search engine. Results: Distinct proteins were identified after separation in the pH ranges 3-10, 4-7 and 4.5-5.5. To date 62 protein spots have been characterised; these include retinal proteins, antioxidants and metabolic enzymes. Several protein populations resolved by IEF exhibited co migration on the second dimension, indicating charge isomers corresponding to posttranslational modifications. Conclusions: The application of broad spectrum and narrow range IEF strips in conjunction with consistent second dimension resolution has demonstrated the complexity of the neural retinal proteome. The present study has commenced the establishment of a porcine neural retinal proteome database. Comparison of the normal porcine retinal proteome with that of models of retinal disease will offer an insight into the pathological processes.
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