May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Porcine Neural Retinal Proteomics
Author Affiliations & Notes
  • W.J. Curry
    Ophthalmology, Queens University, Belfast, United Kingdom
  • A. Healy
    Biochemistry, Queens University, Belfast, United Kingdom
  • A. Wallace
    Biochemistry, Queens Univ of Belfast, Belfast, United Kingdom
  • V. Miller
    Ophthalmology, Queens Univ of Belfast, Belfast, United Kingdom
  • Footnotes
    Commercial Relationships  W.J. Curry, None; A. Healy, None; A. Wallace, None; V. Miller, None.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 4562. doi:
  • Views
  • Share
  • Tools
    • Alerts
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      W.J. Curry, A. Healy, A. Wallace, V. Miller; Porcine Neural Retinal Proteomics . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4562.

      Download citation file:

      © ARVO (1962-2015); The Authors (2016-present)

  • Supplements

Abstract: : 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.

Keywords: protein purification and characterization • retina • animal model 

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.