Purchase this article with an account.
V Miller, A Healy, A Wallace, DB Archer, WJ Curry; Neuroretinal Proteomics: Characterisation of the mode of protein extraction . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3631.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose: A wide range of growth factors, cytokines and neurotrophins has been implicated in the survival of photoreceptors. It has been suggested that photoreceptors and other retinal neurones release diffusible survival-promoting factors. Selective analysis of the retinal proteome has the potential to identify novel growth factors that could play key roles in the treatment of retinal degenerative disease. This study aims to examine differential and sequential protein solvation protocols employing 2D-PAGE and electrospray ionisation quadrupole mass spectrometry (ESI-MS). Methods: Porcine retinas were dissected, homogenised in liquid nitrogen and extracted in a range of media (12.5% w/v); 40 mM Tris, MES, CAPS, MOPS, HEPES, ammonium bicarbonate, 0.5 M acetic acid and acid ethanol (24 hr, 4 C), centrifuged, the supernatant decanted, lyophilised and reconstituted in 2% CHAPS. Subsequently, 1 mg of solubilised protein was resolved by 2D-PAGE (1st dimension pH 3-10; 2nd dimension 12-14% polyacrylamide), visualised using silver stain and latterly with Coomassie Blue. Protein spots were excised from the gels and prepared for automated peptide analysis by ESI-MS. Results: Distinct protein arrays were observed following each extraction protocol, demonstrating differential extraction in which separate protein populations were solubilised. Additionally analysis of the application of sequential tissue extraction revealed that this mode of extraction was viable for the characterisation of different protein products from a single tissue specimen. The distinct protein spots arrayed have been subjected to ESI-MS. Conclusion: This study has demonstrated the qualitative variation in proteins recovered using chemically distinct extraction protocols. These methodologies offer an effective mechanism facilitating optimised sample preparation and hence the detection of unique proteins resolved on 2D gels. This will enable the characterisation of the neuroretinal proteome by selective analysis of specific in vivo derived retinal cell populations in health and disease.
This PDF is available to Subscribers Only