Towards the Establishment of the Vesicular Proteome of Porcine Retina

Abstract

Abstract: : Purpose: Cell:cell communication is essential for maintaining the integrity of the neural retina. Extracellular factors involved in this process, some of which are likely to have neuroprotective activity, are exported via secretory vesicles. The purpose of this investigation was to purify a vesicle-enriched fraction from porcine retina and identify the proteins within the vesicular proteome. Methods: Subcellular fractions were obtained by differential ultracentrifugation. The quality of the vesicular preparations was assessed by several techniques; electron microscopy was used to analyse the ultrastructural integrity and to view potential organelle contamination. Vesicular proteins were identified by Western blot analysis. Potential mitochondrial and lysosomal contamination was assessed by PCR and Western blot analysis of specific protein markers, respectively. 2-D gel electrophoresis was performed on the total protein extracted from the vesicle preparation and mass spectrometry (MS/MS) was performed on selected spots from the 2-D gel. Results: A vesicle-enriched retinal preparation was generated by modification of previously established subcellular fractionation protocols using ultracentrifugation. Western blot analysis of the vesicle preparation showed positive staining for the known vesicle markers and was negative for Cathepsin D (lysosomal marker). Electron microscopy confirmed that the protocol generated a heterogeneous vesicle preparation devoid of larger organelles such as lysosomes and mitochondria. PCR analysis, using primers directed against the porcine mitochondrial genome confirmed that mitochondria were absent from the vesicle preparation. The application of a Tris/chaps protein extraction protocol and subsequent 2D electrophoresis employing pH 3-10 IEF strips and 12-14% gels revealed the protein profile of the vesicle preparation. Parallel silver and Coomassie Blue staining in conjunction with densitometry and MS/MS, respectively, were employed to investigate the profiles and proteins present in enriched porcine retinal vesicular preparations. Conclusions: A protocol for the isolation of vesicles from porcine retina has been established. This facilitates the generation of a discrete subcellular protein database, the "vesicular proteome".

Keywords: cell-cell communication • protein purification and characterization • animal model