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X.–J. Yang, G. Tezel; Proteomic Analysis of Retinal Ganglion Cells: Toward Retinal Ganglion Cell Protein Mapping . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3771.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Experimental investigation of the proteome (PROTEins expressed by the genOME) is essential to the interpretation of data arising from genomic studies. The ultimate function of the gene resides in the protein, which can be modulated by post–translational modifications as also evident in retinal ganglion cells (RGCs). We therefore performed proteomic analysis to build a database of proteins expressed in RGCs under normal conditions to be used in future studies to identify proteome changes under pathologic conditions including glaucoma. Methods: Immunomagnetic selection of Thy–1.1–positive RGCs was performed in a two–step process. Protein expression was determined by Sypro Ruby staining after 2D PAGE of RGC protein lysates. Protein spots were excised and digested with trypsin. Peptide masses were identified by MALDI–TOF mass spectrometry. The Mascot search engine was used to analyze the peptide masses and proteins were identified using bioinformatics. Results: Western blot analysis of RGC protein lysates first confirmed the expression of Thy–1.1 and neurofilament protein, but the absence of other retinal cell markers, including GFAP, an astrocyte marker; syntaxin, an amacrine cell marker; vimentin and glutamine synthetase, Müller cell markers; mGluR6, a bipolar cell marker, and rhodopsin, a photoreceptor marker. Proteomic study utilizing these lysates revealed more than 300 spots on each gel. Through the peptide mass fingerprinting, 44 proteins were identified during the initial period of the study. These included a wide variety of proteins with predicted protein locations within the entire spectra of cellular compartments, including nuclear, mitochondrial, endoplasmic, and membrane proteins, which are involved in the cell structure/cytoskeleton, energy metabolism, signal transduction, and stress response. The probability–based Mowse scores of the identified proteins were >70 (p<0.05), and the sequence coverage ranged between 16% and 83%. Peptide mass fingerprinting using the Profound search engine confirmed the protein identification (Z scores >1.65, p<0.05), and 2D Western blot analysis confirmed the identified proteins. Conclusions: The generated RGC–specific proteome maps can be used to analyze the qualitative and quantitative differences in protein expression under pathologic conditions. Large–scale identification of RGC proteome alterations can reveal cellular pathways associated with glaucomatous neurodegeneration, thereby providing novel targets for neuroprotective interventions in glaucoma.
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