We used the comparative proteomic technique iTRAQ coupled with offline 2DLC-MS/MS to analyze a rare specimen of the poorly understood, potentially blinding ophthalmic condition Macular Telangiectasia type 2 (MacTel type 2). We refined the technique using an internal standard consisting of pooled samples for each iTRAQ experiment to allow for multiple comparisons between different regions of the retina and different tissue donors. A total of 594 nonredundant proteins were identified in the retina and 168 in the vitreous, of which approximately half were found in significantly different abundance in the various comparisons made. The most prominent differences were found within the glycolytic pathway, where 8 proteins were reduced in the diseased macula compared with peripheral retina of the same eye, and 10 were also reduced in comparison with the macula of a control eye. Furthermore, Müller cell-associated proteins, including GFAP, VIME, and GLNA, were also reduced in the diseased macula, consistent with a link between the glycolytic pathway and Müller cells. These changes were validated by Western blotting and immunohistochemical studies. Proteomic analysis of the vitreous revealed an increase of proteins that were reduced in the retina. This supports proteomic analysis of the more easily available vitreous, which may reveal retina-specific protein changes associated with disease. Furthermore, our study has highlighted changes in the glycolytic pathway as a possible component of MacTel type 2 pathobiology.