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Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells
Published on: December 12, 2014
Proteomic analysis of the porcine interphotoreceptor matrix
Stefanie M Hauck1, Stephanie Schoeffmann, Cornelia A Deeg
1GSF-National Research Centre for Environment and Health, Institute of Human Genetics, Neuherberg, Germany.
Insights
Researchers identified new proteins in the interphotoreceptor matrix (IPM), crucial for retinal function. This study distinguishes true IPM components from contaminants, revealing potentially neuroprotective proteins like GRP78.
Area of Science:
- Ophthalmology
- Retinal Biology
- Proteomics
Background:
- The interphotoreceptor matrix (IPM) is a vital retinal layer between photoreceptors and pigment epithelium.
- It plays key roles in the visual cycle, including chromophore exchange and retinal adhesion.
- IPM preparations are often contaminated with intracellular proteins, complicating analysis.
Purpose of the Study:
- To identify novel components of the IPM.
- To differentiate between genuinely secreted IPM proteins and contaminants.
- To develop a predictive strategy for identifying in vivo IPM proteins.
Main Methods:
- Extraction of soluble and insoluble porcine IPM using PBS and water.
- Two-dimensional electrophoresis (2-DE) for protein separation.
- Mass spectrometry (MALDI-TOF, CapLC Q-TOF MS) for protein identification.
- Computational prediction methods combined with high-resolution separation.
Main Results:
- Identified 140 protein spots, with a majority not previously described in the IPM.
- Classified identified proteins into nine functional networks.
- Discovered a set of potentially neuroprotective proteins, including GRP78, peroxiredoxin 5, and alpha-B-crystallin.
- Confirmed GRP78 localization in the IPM via immunohistochemistry.
Conclusions:
- The study successfully identified novel IPM proteins and established a method to distinguish true components from contaminants.
- The findings reveal a set of previously unrecognized, potentially neuroprotective proteins within the IPM.
- The validated prediction strategy offers a robust approach for future IPM research.
Abstract:
The interphotoreceptor matrix (IPM) is located between photoreceptors and pigment epithelium in the retina and is involved in fundamental functions of the visual cycle. These include visual pigment chromophore exchange, retinal adhesion, metabolite trafficking, and growth factor presentation. In general, IPM preparations are contaminated with intracellular proteins, as has also been described for other body fluids. This study aimed at identifying new components of the IPM by discriminating between truly secreted proteins and proteins that are part of the IPM for secondary reasons. "Soluble" porcine IPM was extracted from retina and pigment epithelium with PBS by two different procedures, followed by extraction with water alone that released "insoluble" IPM matrix sheets. Samples from all preparations were separated by 2-DE and a total of 140 protein spots were identified by MALDI-TOF and/or CapLC Q-TOF MS. Although identified proteins included several already known in the IPM, the majority had not been previously described in this structure. Gene ontology classifications allocated the identified proteins into nine different functional networks. The IPM preparations also included intracellular proteins from cells adjacent to the IPM, which may have resulted from cell disruption. This underlines the experimental difficulties of a biochemical analysis of the IPM as an intact compartment. We show here a strategy for predicting the probability of identified IPM proteins occurring in vivo by combined high-resolution protein separation methods with computational prediction methods. Thus, a set of potentially neuroprotective proteins could be extracted, including PEA-15, peroxiredoxin 5, alpha-B-crystallin, macrophage migration inhibitory factor, 78 kDa glucose-regulated protein (GRP78), protein disulfide-isomerase, and PEP-19, which have not been previously associated with the IPM. Furthermore, with immunohistochemical staining we could confirm the localization of GRP78 in the IPM on porcine eye sections, thus validating the proposed prediction method.
