Scientific Report 2007

Molecular and Experimental Medicine

Division of Rheumatology Research

Protein Oxidation, Oxidative Stress, and Disease

J.S. Friedman, F.M. Martin, A.C. Takeda, J. Yi

Protein oxidative damage, in particular, protein carbonylation, is increased in inflammatory disorders, neurodegenerative processes, and aging. This type of damage is also a prominent feature of loss of superoxide dismutase 2 (SOD2), an endogenous antioxidant protein, in mouse blood cells. SOD2 deficiency in murine blood cells results in an anemia similar to the human disorder sideroblastic anemia. Using clinical samples, we found that this type of protein oxidation is also characteristic of bone marrow cells from patients with sideroblastic anemia. The samples were provided by our collaborators J. Nieva, J. Andrey, and A. Saven, Scripps Clinic, La Jolla, California; J.C. Barton, Southern Iron Disorders Center, Birmingham, Alabama; and J. Prchal, University of Utah, Salt Lake City, Utah.

To better understand the role of protein carbonylation in disease processes, we developed 2 novel methods for enriching and identifying oxidized proteins. The first method involves the use of multiple fluorophores (e.g., Cy-2, Cy-3, and Cy-5) that can form derivatives of carbonylated proteins via a hydrazide moiety. Individual samples are labeled with distinct fluorophores and then are combined for comparative 2-dimensional gel analysis (Fig. 1). This method is similar to the comparative proteomic method termed difference gel electrophoresis, or DIGE, and thus we coined the term oxo-DIGE. The second method involves the use of a biotin "hook" to obtain oxidized proteins from more complex protein mixtures. Using these techniques, we can enrich, identify, and quantitatively compare oxidized proteins in experimental samples—and allow for whole proteome comparisons of differential oxidation.

Fig. 1. A multiplex oxo-DIGE gel. In this experiment, 50 μg of each sample (Cy-2, internal control; Cy-3, hydrazide Sod2+/+; and Cy-5, hydrazide Sod2–/–) was mixed after fluorophore labeling, precipitated, washed, resuspended for isoelectric focusing, and separated by size. Images were obtained at 100-μm resolution with excitation and emission filter sets specific for each dye. Both pseudocolor and gray-scale images are presented. The Cy-3 and Cy-5 channels identify protein carbonyls; the Cy-2 channel labels all proteins in a pooled control sample to serve as an internal reference for comparison and matching of multiple gels.

The National Center for Research Resources has just awarded us a grant to set up a DIGE facility in the Core Proteomics laboratory of the Department of Molecular and Experimental Medicine. In the next year, we will use the facility and the methods described in new collaborative research with J. Waalen and E. Beutler, Department of Molecular and Experimental Medicine, to study anemia of aging and to investigate basic questions about protein oxidation: What is the hierarchy of protein oxidation? Are specific proteins carbonylated first that serve as a buffer against oxidative injury? Does protein oxidation occur at random in susceptible proteins, or do specific residues (e.g., near metal-binding sites) become oxidized first? Carbonylated proteins are subject to degradation in the proteasome—does degradation of "carbonyl sensor" proteins activate cellular responses to oxidative damage? We think that patterns of protein oxidation probably will be found that correspond to normal cellular responses, whereas other patterns will be found that represent the signature of specific disease states.

Publications

Martin, F.M., Bydlon, G., Friedman, J.S. SOD2-deficiency sideroblastic anemia and red blood cell oxidative stress. Antioxid. Redox. Signal. 8:1217, 2006.