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Molecular & Cellular Proteomics 1:293-303, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Evidence That Light Modulates Protein Nitration in Rat Retina ^*

Masaru Miyagi^,, Hirokazu Sakaguchi, Ruth M. Darrow^¶, Lin Yan, Karen A. West, Kulwant S. Aulak^||, Dennis J. Stuehr^||, Joe G. Hollyfield, Daniel T. Organisciak^¶ and John W. Crabb^,||,**

Cole Eye Institute
^|| Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195
^¶ Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio 45435

As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. In the course of the research, Western methodology for detecting nitrotyrosine-containing proteins has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific nitrotyrosine immunoreactivity to be distinguished. A liquid chromatography MS/MS detection strategy was used that selects all possible nitrotyrosine peptides for MS/MS based on knowing the protein identity. Quantitative liquid chromatography MS/MS analyses with tetranitromethane-modified albumin demonstrated the approach capable of identifying sites of tyrosine nitration with detection limits of 4–33 fmol. Using two-dimensional gel electrophoresis, Western detection, and mass spectrometric analyses, several different nitrotyrosine-immunoreactive proteins were identified in light-exposed rat retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. These results suggest that light modulates retinal protein nitration in vivo and that nitration may participate in the biochemical sequela leading to light-induced photoreceptor cell death. Furthermore, the identification of nitrotyrosine-containing proteins from rats maintained in the dark, under non-pathological conditions, provides the first evidence of a possible role for protein nitration in normal retinal physiology.

^** To whom correspondence may be addressed: Cole Eye Inst. and Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-0425; Fax: 216-445-3670; E-mail: crabbj{at}ccf.org

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