Abstract
We have applied proteomic analysis to the degeneration of photoreceptors. In the rd1 mouse, a recessive mutation in the PDE6B gene leads to rapid loss of rods through apoptosis. By 5 wk postnatal, virtually all rod photoreceptors have degenerated, leaving one row of cones that degenerates secondarily. In order to assess comparative protein expression, proteins extracted from whole retina were resolved on a two-dimensional gel and identified by mass spectrometry combined with database screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry coupled to peptide mass fingerprinting was sufficient to identify most of the proteins, the remaining being identified with additional sequence information obtained by nano-electrospray ionization tandem mass spectrometry or liquid chromatography tandem mass spectrometry. The study revealed 212 spots, grouped into 109 different proteins. Differential analysis showed loss of proteins involved in the rod-specific phototransduction cascade, as well as induction of proteins from the crystallin family, in response to retinal degeneration. Identification of such pathways may contribute to new therapeutic approaches.
Highlights
We have applied proteomic analysis to the degeneration of photoreceptors
The rd1 mouse carries a recessive mutation in the gene coding for the beta subunit of cGMP-phosphodiesterase (PDE6B) selectively expressed by rod photoreceptors (PR)1 and is a widely used model of inherited retinal degeneration [1]
In order to display retinal proteins, we separated retinal extracts by two-dimensional (2D) gel electrophoresis and identified stained protein spots by mass spectrometry followed by database searching
Summary
1. 2D gel electrophoresis separation of neural retina extracts after Coomassie staining. The strips were incubated 30 min at 20 °C in electrophoresis buffer: 50 mM Tris-HCl, pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 65 mM DTT and followed by 30 min in the same buffer containing 26 mM iodacetamide. Western Blotting—For Western blotting, 40 g of protein from 2and 5-wk-old C57BL/6 and C3H/He neural retina extracts were loaded on a 10% SDS-PAGE transferred onto nitrocellulose membrane (Optitran; Schleicher & Schuell, Dassel, Germany). 2. 2D gel electrophoresis separation of photoreceptors extracted from wild-type mouse retina isolated by vibratome sectioning. Dilution of 1/5,000 in blocking solution washed four times in PBS 0.1% Tween-20, incubated 1 h at room temperature with goat anti-rabbit coupled to peroxydase (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1/15,000 in blocking solution.
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