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John Heckenlively, A. Karoukis, Mohammad Othman, Thomas Gardner; Auto Anti-retinal Antibodies in Diabetic Retinopathy. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2427.
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Purpose: To identify the retinal proteins targeted by serum antibodies from diabetic retinopathy patients.
Western blots were performed on diabetic retinopathy patients' serum using eye bank normal retinal proteins. Protein A magnetic beads captured the antibody/antigens immune complexes. We then ran the eluted complexes on SDS gels stained with Sypro Ruby stain and excised the bands and sent them for mass spectrometery for protein identification. The immunoglobulins in the serum were bound to the protein A magnetic beads and then incubated with normal retinal extracts. The immune complexes were eluted and ran on SDS gels. The gels were stained with Sypro Ruby stain and the distinct band were excised from the gels and sent for mass spec analysis. Gel slices were processed for mass spec including an overnight digestion with trypsin, the peptides were extracted and the samples were run on Thermo Q exactive mass spectrometer. Data analysis was performed with Proteome Discoverer 1.3 and retinal protein sequences were blasted against the SwissProt_2012_09 database for identification. Scaffold, proteome software, was used for secondary analysis.
Over 280 proteins were identified from the runs. The initial mass spec results identified several retinal proteins from the immunocomplexes with diabetic patients’ sera. These proteins include: type I and II cytoskeletal keratin, fibronectin, Complement C4-B and C3, myosin 9, cytoplasmic I actin, microtubule-associated protein 1, non-erythrocytic spectrin alpha and beta chains and filamin-A. These proteins were differentially pulled down by the diabetic patients’ sera compared to control sera.
This is a new approach to study the role of serum antibodies from diabetic patients. Immune complexes were generated first using protein A magnetic beads. This is a simpler method than the time consuming 2D gels. Various proteins were identified as work is in progress to generate more data. These proteins need to be verified regarding their role in diabetic retinopathy. Further work is planned to investigate the retinal targets of the antibodies by indirect immunolohistology.
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