December 2002
Volume 43, Issue 13
Free
ARVO Annual Meeting Abstract  |   December 2002
Neuroretinal Proteomics: Characterisation of the mode of protein extraction
Author Affiliations & Notes
  • V Miller
    Centre of Ophthalmology & Vision Science
    Queens University Belfast United Kingdom
  • A Healy
    Centre for Peptide & Protein Engineering
    Queens University Belfast United Kingdom
  • A Wallace
    Centre for Peptide & Protein Engineering
    Queens University Belfast United Kingdom
  • DB Archer
    Centre of Ophthalmology & Vision Science
    Queens University Belfast United Kingdom
  • WJ Curry
    Centre of Ophthalmology & Vision Science
    Queens University Belfast United Kingdom
  • Footnotes
    Commercial Relationships   V. Miller, None; A. Healy, None; A. Wallace, None; D.B. Archer, None; W.J. Curry, None.
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 3631. doi:
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      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.

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Abstract

Abstract: : 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.

Keywords: 526 protein purification and characterization • 554 retina • 489 neuroprotection 
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