Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging

Abstract

Proteomic techniques were used to identify cardiac proteins from whole heart homogenate and heart mitochondria of Fisher 344/Brown Norway F1 rats, which suffer protein nitration as a consequence of biological aging. Soluble proteins from young (5 mo old) and old (26 mo old) animals were separated by one-and two-dimensional gel electrophoresis. One-and two-dimensional Western blots with an anti-nitrotyrosine antibody show an age-related increase in the immunoresponse of a few specific proteins, which were identified by nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS). Complementary proteins were immunoprecipitated with an immobilized anti-nitrotyrosine antibody followed by NSI-MS/MS analysis. A total of 48 proteins were putatively identified. Among the identified proteins were ␣-enolase, ␣-aldolase, desmin, aconitate hydratase, methylmalonate semialdehyde dehydrogenase, 3-ketoacyl-CoA thiolase, acetyl-CoA acetyltransferase, GAPDH, malate dehydrogenase, creatine kinase, electron-transfer flavoprotein, manganese-superoxide dismutase, F1-ATPase, and the voltage-dependent anion channel. Some contaminating blood proteins including transferrin and fibrinogen ␤-chain precursor showed increased levels of nitration as well. MS/MS analysis located nitration at Y105 of the electron-transfer flavoprotein. Among the identified proteins, there are important enzymes responsible for energy production and metabolism as well as proteins involved in the structural integrity of the cells. Our results are consistent with agedependent increased oxidative stress and with free radical-dependent damage of proteins. Possibly the oxidative modifications of the identified proteins contribute to the age-dependent degeneration and functional decline of heart proteins.heart; mitochondria THERE IS INCREASING EVIDENCE for an age-dependent decline of cardiac performance (45). Several studies show this performance decline to be associated with oxidative stress (42, 43, 57, 58), i.e., elevated levels of reactive oxygen species. For example, various biomarkers of oxidative stress such as oxo-2-deoxyguanosine, H 2 O 2 , 3-nitrotyrosine (3-NT), N-⑀-methyllysine, malondialaldehyde, and advanced glycation end products (14,22,47,48,55,62) increase with the age in cardiac tissue. Moreover, dietary restriction significantly reduces the age-dependent accumulation of these oxidative markers in heart, which indicates the importance of oxidative stress in cardiac aging (46).A causal role of reactive oxygen species in the age-dependent decline of cardiac dysfunction can, however, only be defined if biological targets of these species are identified and the physiological impact of biomolecular modification is characterized. A first step to establish molecular mechanisms of age-dependent cardiac dysfunction is the proteomic identification of oxidatively modified proteins. Various studies have indicated the formation of reactive nitrogen species in acute myocardiac disorders such as heart failure (1, 28, 31). These reactive nitrogen species are met...

Age-related muscle dysfunction

“…The function of these proteins include energy production (TPI, β enolase, aconitase, carbonic anhydrase III), and calcium homeostasis (SR Ca-ATPase) Interestingly, mitochondrial aconitase is one of the major intracellular targets of nitric oxide, and the decrease in aconitase activity has been attributed to the direct reactions of nitric oxide with the iron-sulfur cluster (Patel et al, 2003). This protein shows oxidative damage during aging in cardiac tissue (Kanski et al, 2005a). In addition, previous studies demonstrated oxidative modifications of carbonic andydrase III in vivo with a concomitant decrease in catalytic activities in liver tissue (Cabiscol and Levine, 1995).…”

A Method for Selective Enrichment and Analysis of Nitrotyrosine-Containing Peptides in Complex Proteome Samples

“…For example, low levels of nitrated 78 kDa glucose-regulated protein precursor, aspartate aminotransferase, and creatine kinase were found in our previously endogenous nitration study of mouse brain, 6 while acetyl-CoA acetyl transferase, ATP synthase, electron-transfer flavoprotein alpha-subunit, and malate dehydrogenase were found endogenously nitrated in muscle. 23 Approximately 35% of the identified spectra (422 different peptides) contain derivatized nitrotyrosine. A majority (>60%) of the identified non-nitrated peptides are from different proteins and were carried along during the enrichment process (listed as Supporting Information).…”

Mass spectrometry in aging research

“…Similar methodology was applied by Kanski et al to monitor the age-dependent accumulation of 3-nitrotyrosine in rat skeletal muscle (Kanski, Alterman, & Schöneich, 2003) and heart (Kanski et al, 2004) and by Turku et al (2003) for the identification of 3-nitrotyrosine-containing proteins in the mitochondria of diabetic mice. Elfering et al (2003) characterized the relative distribution of nitrated proteins in rat liver mitochondria and quantified half-lives of nitrated proteins.…”

“…The positive identification of a nitrated protein actually requires the enrichment and characterization of 3-nitrotyrosine-containing peptides by MS n analysis. Such data are available for the cardiac mitochondrial electron transfer flavoprotein isolated from aged rats (Kanski et al, 2004). Such experiments may benefit from specific enrichment of 3-nitrotyrosine-containing peptides.…”