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J. Biol. Chem., Vol. 280, Issue 25, 24261-24266, June 24, 2005

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Proteomic Analysis of Protein Nitration in Aging Skeletal Muscle and Identification of Nitrotyrosine-containing Sequences in Vivo by Nanoelectrospray Ionization Tandem Mass Spectrometry^*

Jaroslaw Kanski, Sung J. Hong, and Christian Schöneich

From the Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas 66047

The nitration of protein tyrosine residues represents an important post-translational modification during development, oxidative stress, and biological aging. To rationalize any physiological changes with such modifications, the actual protein targets of nitration must be identified by proteomic methods. While several studies have used proteomics to screen for 3-nitrotyrosine-containing proteins in vivo, most of these studies have failed to prove nitration unambiguously through the actual localization of 3-nitrotyrosine to specific sequences by mass spectrometry. In this paper we have applied sequential solution isoelectric focusing and SDS-PAGE for the proteomic characterization of specific 3-nitrotyrosine-containing sequences of nitrated target proteins in vivo using nanoelectrospray ionization-tandem mass spectrometry. Specifically, we analyzed proteins from the skeletal muscle of 34-month-old Fisher 344/Brown Norway F1 hybrid rats, a well accepted animal model for biological aging. We identified the 3-nitrotyrosine-containing sequences of 11 proteins, including cytosolic creatine kinase, tropomyosin 1, glyceraldehyde-3-phosphate dehydrogenase, myosin light chain, aldolase A, pyruvate kinase, glycogen phosphorylase, actinin, -actin, ryanodine receptor 3, and neurogenic locus notch homolog. For creatine kinase and neurogenic locus notch homolog, two 3-nitrotyrosine-containing sequences were identified, i.e. at positions 14 and 20 for creatine kinase and at positions 1175 and 1205 for the neurogenic locus notch homolog. The selectivity of the in vivo nitration of creatine kinase at Tyr¹⁴ and Tyr²⁰ does not correspond to the product selectivity in vitro, where exclusively Tyr⁸² was nitrated when creatine kinase was exposed to peroxynitrite. The latter experiments demonstrate that the in vitro exposure of an isolated protein to peroxynitrite may not always be a good model to mimic protein nitration in vivo.

Received for publication, February 16, 2005 , and in revised form, April 19, 2005.

^* This work was supported in part by National Institutes of Health Grants AG23551, AG12993, and CA072987 and by the Center for Bioanalytical Research (CBAR) at the University of Kansas. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: University of Kansas, Dept. of Pharmaceutical Chemistry, 2095 Constant Ave., Lawrence, KS 66047. Tel.: 785-864-4880; E-mail: schoneic{at}ku.edu.

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