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Am J Physiol Heart Circ Physiol 288: H371-H381, 2005. First published September 2, 2004; doi:10.1152/ajpheart.01030.2003
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Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging

Jaroslaw Kanski,1 Antje Behring,1 Jill Pelling,2 and Christian Schöneich1

1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence; and 2Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, Kansas

Submitted 3 November 2003 ; accepted in final form 31 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 {alpha}-enolase, {alpha}-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 {beta}-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 age-dependent 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, H2O2, 3-nitrotyrosine (3-NT), N-{epsilon}-methyl-lysine, 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 metabolites and/or oxidation products of nitrogen monoxide (NO) such as nitrogen dioxide (·NO2) and peroxynitrite (ONOO–). A hallmark of these species is the conversion of tyrosine to 3-NT (8). This nitration of tyrosine can compromise the functional and/or structural integrity of target proteins (7). In fact, significant tyrosine nitration was detected in the mitochondria of diabetic hearts (65, 64), and a peroxynitrite-dependent inactivation of mitochondrial complex I proteins was established (40). Moreover, the mechanisms and biological relevance of mitochondrial protein nitration were recently investigated (19).

Therefore, in this report we have initiated studies toward the proteomic identification of cardiac proteins that undergo an age-dependent protein tyrosine nitration. We have selected Fisher 344/Brown Norway (BN) F1 rats as a well-defined model of aging (20). These rats show an average life span of ~34 mo and are more resistant to oxidative stress than Fisher 344 rats (average life span, 26–28 mo; Ref. 20).

In general, the identification of protein targets for nitration is important for a large number of different tissues. For example, 3-NT levels correlate with survival rate for certain types of cancer (33), and age represents an important risk factor for cancer (15).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
General. All chemicals were obtained from Sigma (St. Louis, MO) unless stated otherwise. Two-dimensional gel electrophoresis (2-DE) supplies were purchased from Amersham Pharmacia (Piscataway, NJ). The anti-nitrotyrosine monoclonal antibodies were a generous gift of Dr. Beckman (Linus Pauling Institute; Corvallis, OR).

Sample preparation. The research protocol outlined in this report was approved by the University of Kansas Animal Care Facility. Young (5 mo old) and old (26 mo old) Fisher 344/BN F1 rats were housed under a 12:12-h light-dark cycle and were provided with water and food ad libitum. The animals were killed by decapitation, and the hearts were rapidly removed and immediately frozen at –80°C. Small aliquots of specimen were then ground, exposed to the lysis solution [that contained 8 M urea, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM Tris·HCl, 0.5 mM PMSF, 10 µg/ml each of leupeptin and aprotinin, and 20 mM DTT], homogenized with an Ultra-Turrax T8 homogenizer (Fisher; Pittsburgh, PA), and briefly sonicated with a Fisher 550 sonic dismembrator. After 1-h incubation at room temperature with occasional shaking, the samples were spun down in an Eppendorf centrifuge for 15 min at 13,000 g. The middle layer, which contained the proteins, was carefully withdrawn, and a portion of it was saved for determination of protein concentration using Coomassie Plus assay reagent (Pierce; Rockford, IL).

For 2-DE, appropriate amounts of sample were diluted into the rehydration buffer (that contained 8 M urea, 2% CHAPS, 18 mM DTT, 2% of carrier ampholyte, and traces of bromophenol blue; Ref. 39). This solution was used to rehydrate the isoelectric focusing (IEF) strips overnight at room temperature.

For one-dimensional gel electrophoresis (1-DE), a fraction of the samples was diluted to a concentration of 0.25 mg protein/ml with 2x Tris-glycine SDS sample buffer (Invitrogen; Carlsbad, CA). After the addition of DTT (final concentration, 0.1 M), the samples were boiled for 5 min, cooled to room temperature, and then applied (5 µg of protein/lane) to a 4–20% acrylamide gel (1.5 mm thick; Invitrogen) for SDS-PAGE.

Isolation of mitochondria. Mitochondrial-enriched fractions were prepared according to well-established methods (64). Briefly, heart tissue was homogenized on ice into an isolation buffer that contained 0.25 M sucrose, 10 mM phosphate, and 0.1 mM EDTA, pH 7.2, supplemented with 0.5 mM PMSF and protease inhibitor cocktail. The homogenate was then centrifuged at 800 g for 10 min. The supernatant was saved and centrifuged at 10,000 g for 20 min. A pellet containing the mitochondria was saved, washed several times with PBS, and used directly for 2-DE and immunoprecipitation experiments.

2-D gel electrophoresis. Isoelectric focusing was performed on a Multiphor II apparatus using specific voltage ramps (see figure legends) on an EPS 3501 XL power supply (Amersham Pharmacia). The temperature was maintained at 19°C by an RTE-111 cooling apparatus (Neslab; Portsmouth, NH). After completion of the focusing, the IEF strips were stored immediately at –80°C. Second-dimension SDS-PAGE was carried out on the same Multiphor II apparatus using Excel Gel 12–14% gels (Amersham Pharmacia). The strips were first equilibrated for 15 min in an aqueous solution that contained 50 mM Tris·HCl, 6 M urea, 30% (wt/wt) glycerol, 2% (wt/wt) SDS, and 65 mM DTT, pH 8.8, were then placed for an additional 15 min in a solution that contained 50 mM Tris, 6 M urea, 30% (wt/wt) glycerol, 2% SDS, and 135 mM iodoacetamide, pH 8.8. Mark 12 molecular mass standards (Invitrogen) and Precision Plus Protein standards (Bio-Rad; Hercules, CA) were processed along the IEF strips. The second-dimension SDS-PAGE was processed at 1,000 V and 20 mA for 30 min, and after removal of the IEF strip, the electrophoretic conditions were changed to 1,000 V and 40 mA for 165 min. During the process, the water circulation was maintained at 13°C.

1-DE and Western blot analyses. The samples were separated on precast 4–20% acrylamide gels using Tris-glycine running buffer (Invitrogen). Protein separation was conducted for 45 min at 100 V and 12°C, and 1-D gels were then submitted for transfer to a 0.45-µm polyvinylidene difluoride (PVDF) membrane (Millipore; Billerica, MA) for 2 h at 412 mA and 4°C before Western blot analysis.

To specifically reduce 3-NT for control experiments, the membrane was exposed to a solution of 10 mM sodium dithionite (Na2S2O4) in 50 mM pyridine-acetate buffer (pH 5.0) for 2 h at room temperature immediately after the transfer (38). After reduction, the membrane was extensively washed with water and then received three changes with 20 mM Tris·HCl, 150 mM NaCl, and 0.05% Tween 20, pH 7.5 (TBST).

The membranes were incubated overnight at 4°C in 5% dry milk-TBST. After blocking was completed, membranes were rinsed with copious amounts of TBST and exposed to 1A6 mouse anti-3-NT primary antibody solution (1:4,000 dilution in 1% BSA-TBST) for 1 h at room temperature. After incubation, the membranes were washed with TBST and subjected to anti-mouse IgG Fc-peroxide conjugate secondary antibody at 1:10,000 dilution in TBST (Pierce) for 1 h at room temperature. Spots were visualized using an ECL Plus detection kit (Amersham Pharmacia) according to the manufacturer's instructions, and images were captured on Kodak X-ray film using a Kodak developer/fixer kit.

Protein visualization. After completion of electrophoresis, the gels were submitted to either colloidal Coomassie blue or silver staining according to well-established protocols (27, 56). Visualized gels were processed immediately or stored in the dry state in Saran wrap until further processing.

Electrophoretic transfer and Western blot analysis of 2-D gels. The gels were separated from their plastic support using the film remover provided by Amersham Pharmacia. Electrophoretic transfer was then carried out on a Nova Blot (Amersham Pharmacia) apparatus using a 0.45-µm PVDF membrane (Millipore). The transfer was completed at 0.25 mA within 40 min using buffer that contained 40 mM glycine, 48 mM Tris·HCl, and 20% methanol, pH 8.5. The membrane was then submitted to the same Western blot analysis as described (see 1-DE and Western blot analyses).

In-gel digestion. Coomassie blue-stained gel spots of interest were excised from the gels, ground to small pieces, and extensively washed with two changes of a 1:1 (vol/vol) mixture of 200 mM NH4HCO3 and acetonitrile (MeCN) for 45 min at 37°C. Gel pieces were then shrunk in 50 µl of MeCN for 10 min. After the removal of residual solvent, the pieces were rehydrated in 40 mM NH4HCO3, pH 7.8, that contained 0.5 µg of modified trypsin (Promega; Madison, WI). Exhaustive digestion was carried out overnight at 37°C. Formic acid was added to yield a final content of 0.1% (vol/vol), and aliquots were removed and analyzed by mass spectrometry.

Nanoelectrospray ionization-tandem mass spectrometry. In-gel tryptic digests (2 µl) were submitted to nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS) analysis on a Thermo Electron LCQ Duo (San Jose, CA) equipped with a nanospray source (Thermo Electron). Separation of tryptic peptides was achieved online before MS/MS analysis on a BioBasic C-18 nanoflow column (300 , 10 cm x 75 µm, 15-µm tip size; New Objective; Woburn, MA) with the following chromatographic conditions: mobile phase A, 0.1% formic acid in water; mobile phase B, 0.1% formic acid in MeCN; flow rate, 0.5 µl/min (after 1:20 split) delivered by a MicroTech Scientific Ultra Plus II pump. The gradient profile used to increase mobile phase B linearly to the indicated fractions began with a 0- to 5-min gradient held at 10% of phase B that was increased to 60% of phase B within 40 min and continued at 60% for an additional 50 min. After each run, the column was allowed to reequilibrate to the initial conditions for 30 min. Instrumental conditions used for mass spectrometric analysis included the following: number of microscans, 3; length of microscans, 200 ms; capillary temperature, 150°C; spray voltage, 1.8 kV; capillary voltage, 35 V; and tube lens offset, –14 V. The mass spectrometer was tuned using the static nanospray setup with a 5 µM solution of angiotensin I (mol wt, 1,296.5) infused by a picotip emitter (New Objective). Data acquisition was performed in the data-dependent fashion, i.e., an MS scan followed by three MS/MS scans of the three most intense peaks with the normalized collision energy for MS/MS set at 35% and the isolation width of 2.0 m/z. A minimal signal of 2 x 106 was established for MS/MS acquisition. Additionally, the dynamic exclusion option was enabled and set with the following parameters: repeat count, 3; repeat duration, 5 min; exclusion list size, 25; exclusion duration, 5; and exclusion mass width, 3.

Protein identification was achieved with Thermo Electron BioWorks 3.1 software with the most current nonredundant National Center for Biotechnology Information protein database downloaded from ftp.ncbi.nlm.nih.gov/blast/db. Additionally, MS/MS spectra of interest have been manually examined for the presence of 3-NT-containing peptides. For confirmation, some peptides of interest that were previously assigned to the proteins by BioWorks software were subjected to a BLAST search (www.ncbi.nlm.nih.gov/BLAST). In essence, both BioWorks and BLAST have yielded the same identification for the chosen peptides.

In vitro nitration of BSA. Commercially available BSA (Sigma) was nitrated with peroxynitrite to obtain a positive control for the Western blot analysis. Protein portions (1 mg/ml) were nitrated with 250 and 500 µM of peroxynitrite in 200 mM sodium bicarbonate (pH 7.8). The synthesis of peroxynitrite and the determination of its concentration have been described elsewhere (67). The nitrated standards (10 µg of protein) were then submitted to 1-DE and Western blot analysis as described (see 1-DE and Western blot analyses).

Immunoprecipitation with anti-3-NT-antibody. Heart tissue or mitochondrial fraction (6 mg of protein) was suspended into isolation buffer that contained 50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA, and 0.1% Nonidet P-40, pH 7.4. The suspension was then briefly sonicated and centrifuged at 13,000 g for 10 min. The supernatant was collected, adjusted to a concentration of 6 mg/ml, and subjected to immunoprecipitation with an anti-3-NT monoclonal antibody conjugated to protein A agarose beads (40 µl; Cayman; Ann Arbor, MI) for 2 h at room temperature with constant slow shaking. The beads were then collected by centrifugation and were exhaustively washed (five times) with the isolation buffer. Finally, samples were resuspended into Laemmli sample buffer and boiled for 5 min to dissociate the antigen-antibody complex. The solution (100 µl) was then loaded onto the 1-mm-thick, 4–20% SDS-PAGE gel (Invitrogen) and subjected to electrophoretic separation. The gels were visualized by colloidal Coomassie staining, and spots of interest were subjected to tryptic digestion and NSI-MS/MS analysis.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Protein nitration in heart tissue was compared between Fisher 344/BN F1 rats of different ages. Figure 1A shows a representative comparison (total of three separate gels were processed using three different young and old animals) of ~20 µg of soluble cardiac proteins from young (Y, 5 mo old) and old (O, 26 mo old) Fisher 344/BN F1 rats resolved on small, 7-cm, pH 3–10 IEF strips followed by SDS-PAGE and silver staining. For comparison, Fig. 1B shows respective Western blots with the anti-3-NT antibody (a representative example of three independent, reproducible runs). Although silver staining indicated similar levels of protein expression in both samples, the Western blot clearly demonstrated an age-dependent increase in 3-NT-containing proteins (Fig. 1B).



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  		Fig. 1. A: two-dimensional gel electrophoresis (2-DE) analysis of young (Y, right) and old (O, left) Fisher 344/Brown Norway (BN) F1 rat cardiac proteins (silver stain; 20 µg of protein each). Rat heart was homogenized, and the cardiac proteins were extracted into lysis solution that contained 8 M urea, 40 mM Tris·HCl, 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM DTT, 0.5 mM PMSF, and 10 µg/ml each leupeptin and apopritin. Samples were focused on 7-cm, pH 3-10 NL isoelectric focusing (IEF) strips and separated on Excel Gel 12–14% second-dimension gels. Isoelectrofocusing was achieved by the following voltage ramps: 0–200 V in 1 min and 200–3,500 V within 1.5 h, which was held at 3,500 V for 2 h. B: Western blot analysis of cardiac proteins from young (right) and old (left) Fisher 344/BN F1 rats. Proteins from Excel Gel were transferred onto a polyvinylidene difluoride (PVDF) membrane (for 40 min at 250 mA) and analyzed for 3-nitrotyrosine (3-NT) with the 1A6 monoclonal anti-3-NT antibody. Detection was achieved by enhanced chemiluminescence (ECL). Both of these experiments are representative of three independent reproducible experiments.

 
Furthermore, to compare directly the levels of nitration between the young and old cardiac tissue, we performed Western blot analyses on two additional young and old animals together with a nitrated BSA standard using 1-DE (Fig. 2). The gel was stained with colloidal Coomassie blue to show a similar amount of proteins in each sample (Fig. 2A; compare O1 and O2 with Y1 and Y2). In contrast, Western blot analysis clearly showed an age-dependent increase of nitrated proteins (Fig. 2B; compare O1 and O2 with Y1 and Y2) which supports the results obtained by 2-DE (see Fig. 1). The first two lines show BSA, which was exposed to peroxynitrite in vitro. The presence of additional higher and lower molecular mass bands in the Western blot corresponding to nitrated BSA standards in Fig. 2 can be attributed to protein cross-linking and/or oxidative fragmentation resulting from the exposure to peroxynitrite.



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  		Fig. 2. A: SDS-PAGE separation and Coomassie blue detection of cardiac proteins from gel for young and old Fisher 344/BN F1 rats. Proteins from heart homogenate of two young (Y1 and Y2) and two old (O1 and O2) animals as well as BSA, which was nitrated with 0.25 and 0.5 mM of peroxynitrite, were loaded onto a 4–20%, 1.5-mm-thick gel (10 µg of protein/lane). B: Western blot analysis of proteins from A with the 1A6 monoclonal anti-3-NT antibody. Gel from A was transferred as described in the text.

 
To eliminate any possible false-positive results associated with nonspecific binding of the anti-3-NT antibody, control experiments were performed in which the PVDF membrane was reduced with sodium dithionite before Western blot analysis (38). Dithionite selectively reduces 3-NT to 3-amino-tyrosine (3-AT), which is not recognized by the anti-3-NT antibody. Figure 3A shows a Western blot analysis of cardiac proteins from an old-rat specimen together with a nitrated BSA standard. Figure 3B shows the same membrane, except that it was exposed to dithionite reduction before incubation with anti-3-NT antibody. To avoid any potential artifacts due to improper electrophoretic transfer, only one gel was used for this particular experiment, and the PVDF membrane was cut into two pieces for the reduction experiment. During enhanced chemiluminescence signal acquisition, the signals from the membranes were transferred simultaneously to one piece of X-ray film.



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  		Fig. 3. Western blot analysis of cardiac proteins obtained from two old (O1 and O2) Fisher 344/BN F1 rats and of nitrated BSA (0.25 and 0.5 mM peroxynitrite). A: no dithionite reduction before incubation with antibody. B: with dithionite reduction before incubation with anti-3-NT antibody. Dithionite reduction was carried out with 10 mM sodium dithionite (Na2S2O4) in 50 mM pyridine-acetate buffer, pH 5.0, for 2 h before incubation with the antibody.

 
In addition, the presence of 3-NT-containing peptides in the nitrated BSA standard was confirmed by NSI-MS/MS analysis. Nitration was specifically detected at positions Y286, Y423, Y357, and Y520. Figure 4 shows a representative tandem mass spectrum of the tryptic peptide Y286ICDNQDTISSK297 that contains the 3-NT modification at position Y286. Together, the data presented in Figs. 3 and 4 confirm the specificity of the 1A6 antibody toward the 3-NT modification (71).



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  		Fig. 4. Tandem mass spectrometry (MS/MS) spectrum of the tryptic peptide Y286ICDNQDTISSK297 (mol mass, 1,490.62 amu) from BSA, containing 3-NT at position Y286 (indicated as Y*). Respective b and y'' ions are indicated.

 
A more detailed analysis of nitrated cardiac proteins was performed on 18-cm, pH 3-10 NL IEF strips with subsequent SDS-PAGE and Coomassie blue staining (Fig. 5A) and Western blot analysis with the anti-3-NT antibody (Fig. 5B). The Western blot analysis was performed repeatedly (at least three times) on a separate gel after complete transfer of cardiac proteins onto the PVDF membrane. Contrary to results obtained from proteins stained with Coomassie blue (Fig. 5A), the 3-NT immunoreactivity was mainly concentrated in the basic region of the gels, which supports the notion of selective nitration of proteins. To correlate the Coomassie blue-stained gels with the Western blot, the gel and the blot were superimposed according to molecular mass and isoelectric point (pI): a grid corresponding to the exact dimensions of the gels was used to position the gels and the Western blots. It is evident that some highly expressed proteins contain no or minimal 3-NT, whereas some proteins with low expression yielded large amounts of 3-NT-associated signal. For example, some areas, especially in the basic region around apparent mol mass 55–66 kDa and pI 7–8, show an intense anti-3-NT-antibody immunoresponse accompanied by minimal Coomassie staining on the gel. The reason for these differential yields of 3-NT may be 1) the selectivity of the nitrating species, 2) an increased turnover of some nitrated proteins (24), and/or 3) the relative abundance of the target proteins that may, in part, control nitration yields (23, 29). We performed a similar separation on 18-cm IEF strips using only the mitochondrial fraction of heart (see Fig. 5, C and D for protein staining and Western blot analysis, respectively). Here we observed a similar anti-3-NT immunoreactivity pattern as with the crude heart homogenate (Fig. 5B); however, the resolution of the Western blot analysis is significantly better (Fig. 5D).



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  		Fig. 5. A set of examples representative for the results of three to five independent experiments on protein nitration in whole rat heart and heart mitochondria. A: 2-DE separation and colloidal Coomassie blue staining of cardiac proteins (1,000 µg) from 26-mo-old Fisher 344/BN F1 rats. Rat heart was homogenized, and cardiac proteins were extracted into lysis solution for 1 h at room temperature. Samples were focused on 18-cm, pH 3-10 NL IEF strips and separated on Excel Gel 12–14% second-dimension gels. B: Western blot analysis of cardiac proteins of 26-mo-old Fisher 344/BN F1 rat with the anti-3-NT antibody. Proteins (100 µg) from Excel Gel (A) were transferred onto a PVDF membrane and assayed for 3-NT with the 1A6 monoclonal antibody. Detection was achieved by ECL. C: 2-DE separation and colloidal Coomassie blue staining of mitochondrial cardiac proteins (1,000 µg) from 26-mo-old Fisher 344/BN F1 rats. D: Western blot analysis of mitochondrial cardiac proteins (100 µg) of 26-mo-old Fisher 344/BN F1 rats with anti-3-NT antibody.

 
The gel spots of interest were cut out and submitted to in-gel tryptic digestion, and the tryptic peptides were analyzed via NSI-MS/MS to identify the target proteins. Table 1 summarizes the proteins putatively identified in the 3-NT immunoreactive bands that were analyzed with the BioWorks 3.1 software, which uses the SEQUEST search algorithm (70). All peptides identified by SEQUEST were also manually examined, and the following criteria were chosen for accepting peptides as valid: Xcorr > 1.9, 2.2, 3.75 for single-, double-, and triple-charged peptides, respectively; DelCN > 1; and Sp > 500 (16). Two proteins were present in more than one gel spot: for spots 9 and 10, mass spectrometric analysis identified GAPDH (accession no., GenInfo no., 120707), and for spots 12 and 13, NSI-MS/MS analysis characterized the voltage-dependent anion channel (accession no. 6755963). The presence of one protein in more than one gel spot can be explained by different types of posttranslational modifications such as phosphorylation, acylation, etc., which can shift the apparent pI toward the acidic site. In addition, 2-DE often suffers from incomplete focusing in the basic region of the gels (39).


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  		Table 1. Putative nitrated proteins identified by 2-DE and Western blot analysis in heart homogenate and mitochondria

 
We have attempted to identify the sites of protein nitration by screening for potential 3-NT-containing peptides. MS/MS analysis located 3-NT to position Y105 in the sequence Q102FSY(NO2)THIVAGASAFGK117 derived from the mitochondrial electron transport flavoprotein (ETF; accession no., GenInfo no., 2506389). A representative spectrum is shown in Fig. 6, which indicates characteristic fragment ions and their origins from the peptide sequence. We have detected numerous additional peptides that correspond to tyrosine-containing sequences of the proteins listed in Table 1, which were +45 amu higher in molecular mass relative to the native sequence. However, the MS/MS analysis of these peptides yielded ambiguous data; therefore, we did not present these results in this report. There are several possible reasons for our difficulties in obtaining satisfactory MS/MS data for 3-NT-containing peptides from tissue, as follows: 1) poor recovery of the more hydrophobic 3-NT-containing peptides from gel spots and/or the C-18 column; 2) generally low levels of protein nitration in vivo, especially in naturally aged, nonpathological tissues; and 3) insufficient amounts of sample for MS/MS sequencing. These limitations can be partially overcome by a more directed approach, i.e., one that involves the isolation of relatively large amounts (e.g., 50 µg) of a target protein.



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  		Fig. 6. MS/MS spectrum of the sequence Q102FSYTHIVAGASAFGK117 (mol mass, 1,728.8 amu) derived from electron-transfer flavoprotein (accession no. P13803) of a 26-mo-old Fisher 344/BN F1 rat, which contains 3-NT at Y105 (indicated as Y*). The b and y'' ion series are annotated.

 
To confirm 3-NT modification of the proteins putatively identified in Table 1, we performed immunoprecipitation experiments with the anti-3-NT antibody for heart homogenate and mitochondria (Fig. 7). Proteins of interest were submitted to tryptic digestion and NSI-MS/MS analysis (Table 2). A subset of proteins identified by 2-DE and Western blotting was also identified by immunoprecipitation and subsequent 1-DE. These proteins are listed in Table 2 (top). A different set of proteins was only identified through immunoprecipitation with subsequent 1-DE. These proteins are listed in Table 2 (bottom). We successfully confirmed by immunoprecipitation the nitration of desmin, GAPDH, F1-ATPase, and manganese-superoxide dismutase (accession nos., GenInfo no., 11968118, 120707, 114523, and 56691, respectively) that were also identified by Western blot analysis (see Table 1). Six other proteins were found to immunoprecipitate from both heart homogenate and the mitochondrial fraction (Table 2). Sixteen additional proteins were identified in either homogenate or the mitochondrial fraction. The absence of some of these proteins in the 2-DE experiment can be rationalized in part by the limitations of the 2-DE technique. For example, some proteins (accession nos., GenInfo no., 11024680, 27679580, 32189355, and 20302061) are characterized by very basic pI values (9.3, 9.2, 9.8, and 10.03, respectively); this often compromises resolution and identification via 2-DE.



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  		Fig. 7. SDS-PAGE separation and colloidal Coomassie blue detection of immunoprecipitated proteins from heart homogenate (lane 1) and mitochondria (lane 2); 6 mg of starting protein were used for both. Immunoprecipitation was achieved with an anti-3-NT monoclonal antibody conjugated to protein A agarose. Proteins identified are denoted with numbers that refer to the respective numbers in Tables 1 and 2. No attempt was made to identify proteins with apparent molecular masses of 100 kDa and above.

 

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  		Table 2. Identification of putatively nitrated proteins by both 2-DE and Western blot and immunoprecipitation followed by 1-DE or by immunoprecipitation and 1-DE alone

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The objective of the present study was to identify cardiac proteins in Fisher 344/BN F1 rats, which show an age-dependent increase in tyrosine nitration. We have chosen a widely established proteomic approach, i.e., 2-DE for protein separation (3) followed by sensitive protein identification by means of NSI-MS/MS (32). Two-dimensional gels are robust and fairly reproducible, which makes them suitable for Western blot analysis and essentially provides another dimension to protein separation. In addition, we performed an immunoprecipitation experiment to confirm and/or complement the 2-DE results.

Earlier we demonstrated a fairly selective, age-dependent increase of protein nitration of rat skeletal muscle proteins (32) that are constantly exposed to NO and superoxide (50) during muscle contraction and relaxation. Likewise, cardiac proteins are exposed to periodic fluxes of endogenous NO, which controls cardiac muscle function (5, 69) and superoxide (50). This creates conditions for formation of peroxynitrite through the diffusion-controlled reaction of NO with superoxide (41). Therefore, the nitration of protein tyrosine residues even in young animals is not surprising. The important point is that nitrated proteins accumulate at a faster rate in old compared with young tissue. This may indicate a higher yield of nitrating species in old heart muscle or a reduced rate of protein turnover (or both). It is known that nitrated proteins are subject to proteasomal degradation (24) but also that proteasome activity declines with increasing age (13).

Unlike some other reactive oxygen species (e.g., hydroxyl radicals, singlet oxygen), peroxynitrite has a considerable lifetime facilitating diffusion and selective reaction with specific target proteins (7, 8). Consequently, the nitration of cardiac proteins is not only a function of the relative abundance. For example, Fig. 5A shows several highly abundant proteins identified by Coomassie blue staining that are not, however, recognized by the anti-3-NT antibody (see Fig. 5B), which indicates the selective nature of protein nitration. On the other hand, certain spots on the Western blot show little response on the silver- or Coomassie blue-stained gels, which indicates insufficient yields of these proteins for MS analysis under the selected conditions.

NSI-MS/MS analysis identified several proteins in 3-NT-immunoreactive, 2-D gel spots (see Tables 1 and 2). Most of these possibly nitrated proteins are important proteins involved in myocardial energy production, like glycolysis, the tricarboxylic acid (TCA) cycle, or {beta}-oxidation of fatty acids. It is known that the activity of several of these proteins decreases during the aging process (34). The modification of these proteins may in part be associated with an age-dependent decline in heart function such as an inability to increase output when confronted with sudden stress (52) owing to an inadequate energy supply.

As part of the glycolytic machinery, {alpha}-enolase-1, {alpha}-aldolase, and GAPDH were identified as targets for protein nitration. Enolase is a ubiquitous enzyme of which three highly homologous isozymes are expressed (44). Enolase catalyzes the conversion of 2-phosphoglycerate into phosphoenolpyruvate in the glycolysis pathway. Increasing evidence links enolase-dependent pathways to several pathologies and oxidative stress. For example, enolase has been identified as a target for oxidation in bacteria (9, 63) and in Alzheimer's disease victims (10) and as a nitrated protein in aging skeletal muscle (32), which provides a possible rationale for the observed disturbance in energy metabolism in pathologies or aging tissue.

Fructose-biphosphate aldolase catalyzes the hydrolysis of fructose-1,6-bisphosphate into two three-carbon products: dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Evidence exists that this protein suffers nitration as the result of nitric oxide-induced inflammatory processes in the liver as well as exposure of rat retina to excessive light (4, 36).

The last putatively identified glycolytic enzyme in this work is GAPDH. It catalyzes the NAD+-dependent oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate and NADH in the second phase of glucose catabolism, which features the energy-yielding glycolytic reactions that produce ATP and NADH. The GAPDH reaction as well as the aldolase reaction is reversible, and the same enzymes catalyze the reverse reactions during gluconeogenesis. Although there is evidence that GAPDH undergoes nitration in vivo during inflammation (4) and aging in skeletal muscles (32), other proteomic studies of brain cell cultures demonstrate nitrosation of GAPDH (30).

Mitochondrial proteins appear to be especially sensitive to NO-dependent modification (19) under conditions of acute oxidative stress during inflammatory processes or ischemia-reperfusion (2); in these cases, protein 3-NT accumulation was shown to be a dynamic process undergoing enzymatic denitration and superoxide- and L-arginine-dependent renitration (2, 35). The mitochondrial proteins identified in this work include aconitate hydratase (aconitase), which catalyzes the conversion of citrate to cis-aconitate and water. It is one of several mitochondrial enzymes known as non-heme-iron proteins that contain a redox-sensitive iron-sulfur cluster. Aconitate hydratase is highly sensitive toward reactive oxygen species; for example, superoxide exposure leads to enzyme inactivation (48, 49). Our identification of aconitate hydratase as a target for age-dependent nitration is consistent with earlier results showing oxidative damage of this protein during biological aging (35) and loss of protein activity in the presence of peroxynitrite in vitro (11, 26). Furthermore, it was demonstrated that this protein undergoes nitration in hearts of diabetic mice (64).

ETF is a mitochondrial protein that contains a single equivalent of flavin adenine dinucleotide, which serves as a specific electron acceptor for several dehydrogenases. It transfers electrons to the main mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase (51). The 3-NT residue at position 105 is located in the {alpha}-subunit of this protein (belonging to domain I of the protein), which resides in between an {alpha}-helix and a {beta}-sheet. Defects in ETF are responsible for causing the inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired (54). Although not directly involved in binding the flavin adenine dinucleotide cofactor of ETF (51), introduction of 3-NT could destabilize the tertiary structure of domain I of this protein.

ATP synthase produces ATP from ADP and phosphate in the presence of a proton gradient across the membrane (36). It contains several subunits, and the {alpha}-chain represents a regulatory subunit. During enzyme turnover, ATP synthase goes through a sequence of coordinated conformational changes of its major subunits (36). It has been reported that this protein undergoes cysteine-targeted oxidation during renal oxidative stress (17). Other evidence associates ATP synthase deficiency with oxidative stress as in Parkinson's disease (18).

Although aconitase is involved in the early stages of the TCA cycle, malate dehydrogenase is the final enzyme of the TCA pathway oxidizing malate to oxaloacetate. The activity of malate dehydrogenase shows an age-related decline (31), and age-dependent tyrosine nitration may provide a molecular rationale for this observation. Acetyl-CoA acyltransferase is involved in the degradation of fatty acids via {beta}-oxidation, cleaving the {alpha}-{beta} bond of the fatty acid chain, and acetyl-CoA acetyltranferase precursor plays an important role in the ketone body metabolism.

The voltage-dependent anion channel is located in the mitochondria. It supports the effective exchange of metabolites between mitochondria and cytoplasm and is essential for metabolic efficiency and physiological integrity (34, 65). Our detection of age-dependent nitration of this protein is consistent with recent reports demonstrating its nitration in hearts of diabetic mouse models (64). Because diabetes is accompanied by oxidative stress (53), it is not surprising to observe oxidative modification of the voltage-dependent anion channel during diabetes and natural biological aging.

Desmin represents a structural cytosolic protein. It plays an essential role in maintaining the structural and functional integrity of myocytes and is linked to several cardiac diseases including cardiac hypertrophy, ventricular dilatation, and congestive heart failure (68). In addition, desmin seems to be involved in intracellular arrangement and regulation of the mitochondria. Tyrosine nitration of desmin may constitute a potential molecular mechanism by which this protein is involved in these cardiac pathologies.

Some of the identified proteins perform an important role in energy metabolism of heart and offer a potential rationale for biological dysfunction (i.e., heart muscle becomes less able to propel large quantities of blood). The presence of nitrated proteins in young heart tissue can be explained by the inherent involvement of nitric oxide in the modulation of heart function and metabolism (69). It has been recognized that some oxidized and nitrated proteins are subject to accelerated turnover (24, 59), whereas others accumulate (23, 25). Accumulation of specifically modified proteins may especially pose problems for an organism if such proteins are nonfunctional or show adverse effects. Our results show that only a subset of the heart proteome suffers the accumulation of 3-NT despite the fact that on average, proteins are composed of ~4% tyrosine residues relative to other amino acids (60). This suggests that specific structural properties of the respective proteins and/or their cellular locations may be important parameters targeting them for nitration (23).

Elevated levels of peroxynitrite caused by overexpression of inducible nitric oxide synthase in transgenetic mice leads to cardiac enlargement, conduction effects, and heart failure, which constitute a cardiac phenotype that most elderly people experience (28). The results presented here can partly explain the cardiac deficiencies associated with the process of natural aging and also support the notion of importance of oxidative stress in aging (6, 61). The evidence for protein nitration in this work serves as a basis for the targeted isolation of the identified proteins for more detailed functional and biological analysis. Such strategies have, for example, shown the nitration of specific subunits of mitochondrial complexes III and IV (12).


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This work was supported by National Institutes of Health Grants 2P01 AG-12993, CA-072987, and AG-023551, American Heart Association Grant 0051306ZS, and the Center for Bioanalytical Research at the University of Kansas.


   ACKNOWLEDGMENTS
 
Present address for J. Pelling: Dept. of Pathology, Feinberg School of Medicine, Northwestern University, Ward Building, Chicago, IL 60611.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Schöneich, Univ. of Kansas, Dept. of Pharmaceutical Chemistry, 2099 Constant Ave., Lawrence, KS 66047 (E-mail: schoneic{at}ku.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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TOP
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 RESULTS
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 REFERENCES
 

  1. Adachi T, Matsui R, Xu S, Kirber M, Lazar HL, Sharov VS, Schöneich C, and Cohen R. Antioxidant improves smooth muscle sarco/endoplasmic reticulum Ca2+-ATPase function, and lowers tyrosine nitration in hypercholesterolemia and improves nitric oxide-induced relaxation. Circ Res 90: 1114–1121, 2002.[Abstract/Free Full Text]
  2. Aulak KS, Koeck T, Crabb JW, and Stuehr DJ. Dynamics of protein nitration in cells and mitochondria. Am J Physiol Heart Circ Physiol 286: H30–H38, 2004.[Abstract/Free Full Text]
  3. Aulak KS, Koeck T, Crabb JW, and Stuehr DJ. Proteomic method for identification of tyrosine-nitrated proteins. Methods Mol Biol 279: 151–166, 2004.[Medline]
  4. Aulak KS, Miyagi M, Yan L, West KA, Massillon D, Crabb JW, and Stuehr DJ. Proteomic method identifies proteins nitrated in vivo during inflammatory challenge. Proc Natl Acad Sci USA 98: 12056–12061, 2001.[Abstract/Free Full Text]
  5. Balligand J, Kelly RA, Marsden PA, Smith TW, and Michel T. Control of cardiac muscle cell function by endogenous nitric oxide signaling system. Proc Natl Acad Sci USA 90: 347–351, 1993.[Abstract/Free Full Text]
  6. Beal MF. Oxidatively modified proteins in aging and disease. Free Radic Biol Med 32: 797–803, 2002.[CrossRef][ISI][Medline]
  7. Beckman JS. Oxidative damage and tyrosine nitration from peroxynitrite. Chem Res Toxicol 9: 836–844, 1996.[CrossRef][ISI][Medline]
  8. Beckman JS and Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol Cell Physiol 271: C1424–C1427, 1996.[Abstract/Free Full Text]
  9. Cabiscol E, Piulats E, Echave P, Herrero P, and Ros J. Oxidative stress promotes specific protein damage in Saccharomyces cerisiae. J Biol Chem 275: 27393–27398, 2000.[Abstract/Free Full Text]
  10. Castegna A, Aksenov M, Thongbookerd V, Klein JB, Pierce WM, Booze R, Markesbery WR, and Butterfield DA. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part II: dihydropyrimidinase-related protein 2, {alpha}-enolase and heat shock cognate 71. J Neurochem 82: 1524–1532, 2002.[CrossRef][ISI][Medline]
  11. Castro L, Rodrigues M, and Radi R. Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide. J Biol Chem 269: 29409–29415, 1994.[Abstract/Free Full Text]
  12. Choksi KB, Boylston WH, Rabek JP, Widger WR, and Papaconstantinou J. Oxidatively damaged proteins of heart mitochondrial electron transport complexes. Biochim Biophys Acta 1688: 95–101, 2004.[Medline]
  13. Conconi M and Friguet B. Proteasome inactivation upon aging and on oxidation-effects of HSP 90. Mol Biol Rep 24: 45–50, 1997.[CrossRef][ISI][Medline]
  14. Corman B, Duries M, Poitevin P, Heudes D, Bruneval P, Tedgui A, and Levy BI. Aminoguanidine prevents age-related arterial stiffening cardiac hyperthorphy. Proc Natl Acad Sci USA 95: 1301–1306, 1998.[Abstract/Free Full Text]
  15. DePinho RA. The age of cancer. Nature 408: 248–254, 2000.[CrossRef][Medline]
  16. Ducret A, Van Ooxtveen I, Eng JK, Yates JR III, and Aebersold R. High throughput protein characterization by automated reverse-phase chromatography/electrospray tandem mass spectrometry. Protein Sci 7: 706–719, 1998.[Abstract]
  17. Eaton P, Jones ME, McGregor E, Dunn MJ, Leeds N, Byers HL, Leun KY, Ward MA, Pratt JR, and Shattock MJ. Reversible cysteine-targeted oxidation of proteins during renal oxidative stress. J Am Soc Nephrol 8: S290–S296, 2003.
  18. Ebadi M, Govitrapong P, Sharma S, Muralikrishnan D, Shavali S, Pell L, Schafer R, Albano C, and Eken J. Ubiquinone (coenzyme q10) and mitochondria in oxidative stress of Parkinson disease. Biol Signals Recept 10: 224–253, 2001.[CrossRef][ISI][Medline]
  19. Elfering SI, Haynes VL, Traaseth NJ, Ettl A, and Giulivi C. Aspects, mechanism, and biological relevance of mitochondrial protein nitration sustained by mitochondrial nitric oxide synthase. Am J Physiol Heart Circ Physiol 286: H22–H29, 2004.[Abstract/Free Full Text]
  20. Fernandes G, Venkatraman JT, Turturro A, Attwood VG, and Hart RW. Effect of food restriction on life span and immune functions in long-lived Fisher-344 x Brown Norway F1 rats. J Clin Immunol 17: 85–95, 1997.[CrossRef][ISI][Medline]
  21. Gole MD, Souza JM, Choi I, Hertkorn C, Malcolm S, Foust RF III, Finkel B, Lanken PN, and Ischiropoulos H. Plasma proteins modified by tyrosine nitration in acute respiratory distress syndrome. Am J Physiol Lung Cell Mol Physiol 278: L961–L967, 2000.[Abstract/Free Full Text]
  22. Gredilla R, Lopez-Torres M, and Barja G. Effect of time of restriction the decrease in mitochondrial H2O2 production and oxidative DNA damage in the heart of food-restricted rats. Microsc Res Tech 59: 273–277, 2002.[CrossRef][ISI][Medline]
  23. Greenacre SA and Ischiropoulos H. Tyrosine nitration: localization, quantification, consequences for protein function and signal transduction. Free Radic Res 34: 541–581, 2001.[ISI][Medline]
  24. Grune T, Reinheckel T, and Davies KS. Degradation of oxidized proteins in mammalian cells. FASEB J 11: 526–534, 1997.[Abstract]
  25. Harman D. Aging, a theory based on free radicals and radiation chemistry. J Gerontol A Biol Sci Med Sci 2: 298–300, 1956.
  26. Hauslanden A and Fridovich I. Superoxide and peroxynitite inactivate aconitases, but nitric acid does not. J Biol Chem 269: 29405–29409, 1994.[Abstract/Free Full Text]
  27. Heukenshoven J and Dernick R. Simplified method for silver staining of proteins in polyacrylamide gels and the mechanisms of silver staining. Electrophoresis 6: 103–112, 1985.[CrossRef][ISI]
  28. Imran NM, Gros R, You X, Pirani A, Azad A, Csond T, Schulz T, Butany J, Duncan J, and Steward MH. Cardiomyocyte overexpression of iNOS in mice results in peroxynitrite generation, heart block and sudden death. J Clin Invest 109: 735–743, 2002.[CrossRef][ISI][Medline]
  29. Ischiropoulos H. Biological selectivity and functional aspects of protein tyrosine nitration. Biochem Biophys Res Commun 305: 776–783, 2003.[CrossRef][ISI][Medline]
  30. Jaffrey SR, Erdjument-Bromage H, Ferris CD, Tempst P, and Snyder SH. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat Cell Biol 3: 193–197, 2001.[CrossRef][ISI][Medline]
  31. Ji LL, Dillon D, and Wu E. Myocardial aging: antioxidant enzyme systems and related biochemical properties. Am J Physiol Regul Integr Comp Physiol 261: R386–R392, 1991.[Abstract/Free Full Text]
  32. Kanski J, Alterman M, and Schöneich C. Proteomic identification of age-dependent protein nitration in rat skeletal muscle. Free Radic Biol Med 35: 1229–1239, 2003.[CrossRef][ISI][Medline]
  33. Kato H, Miyazaki T, Yoshikawa M, Nakajima M, Fukai Y, Tajima K, Masuda N, Tsutsumi S, Tsukada K, Nakajima T, and Kuwano H. Nitrotyrosine in esophageal squamous cell carcinoma and relevance to p53 expression. Cancer Lett 153: 121–127, 2000.[CrossRef][ISI][Medline]
  34. Kmita H, Budzinska M, and Stobienia O. Modulation of the voltage dependent anion channel by cytoplasmic proteins. Acta Biochim Pol 50: 415–424, 2003.[ISI][Medline]
  35. Koeck T, Fu X, Hazen SL, Crabb JW, Stuehr DJ, and Aulak KS. Rapid and selective oxygen-regulated protein tyrosine denitration and nitration in mitochondria. J Biol Chem 279: 27257–27262, 2004.[Abstract/Free Full Text]
  36. Leyva JA, Bianchet MA, and Amzel LM. Understanding ATP synthesis: structure and mechanism of the F1-ATPase (Review). Mol Membr Biol 20: 27–33, 2003.[CrossRef][ISI][Medline]
  37. Liang-Yun Y, Levine R, and Sohal RS. Oxidative damage during aging targets mitochondrial aconitase. Proc Natl Acad Sci USA 94: 11168–11172, 1997.[Abstract/Free Full Text]
  38. Miyagi M, Skaguchi H, Darrow RM, Yan L, West KA, Aulak K, Stuehr DJ, Hollyfield JG, Organisciak DT, and Crabb JW. Evidence that light modulates protein nitration in rat retina. Mol Cell Proteomics 4: 293–302, 2002.
  39. Molloy MP. Two-dimensional electrophoresis of membrane proteins using immobilized pH gradients. Anal Biochem 280: 1–10, 2000.[CrossRef][ISI][Medline]
  40. Murray J, Taylor SW, Zhang B, Gosh SS, and Capaldi RA. Oxidative damage to mitochondrial complex I due to peroxynitrite; identification of reactive tyrosines by mass spectrometry. J Biol Chem 278: 37223–37230, 2003.[Abstract/Free Full Text]
  41. Nauser T and Koppenol WH. The rate constant of the reaction of superoxide with nitrogen monoxide: approaching the diffusion limit. J Phys Chem A 106: 4084–4086, 2002.[CrossRef]
  42. Nohl H, Breuninger V, and Hegner D. Influence of radical formation on energy-linked respiration. Eur J Biochem 90: 385–390, 1978.[ISI][Medline]
  43. Nohl H and Hegner D. Evidence for existence of catalase in the matrix space of rat-heart mitochondria. FEBS Lett 89: 155–169, 1978.
  44. Ochi H, Horiuchi I, Araki N, Toda T, Araki T, Sato K, Murai H, Osoegawa M, Yamada T, Okamura K, Ogino T, Mizumoto K, Yamashita H, Saya H, and Kira J. Proteomic analysis of human brain identifies {alpha}-enolase as a novel autoantigen in Hashimoto's encephalopathy. FEBS Lett 528: 197–202, 2002.[CrossRef][ISI][Medline]
  45. Oxenham H and Sharpe N. Cardiovascular aging and heart failure. Eur J Heart Fail 5: 427–434, 2003.[CrossRef][ISI][Medline]
  46. Pamplona R, Portero-Otin M, Bellmun MJ, Gredilla R, and Barja G. Aging increases N{epsilon}-(carboxymethyl)lysine and caloric restriction decreases N{epsilon}-(carboxyethyl)lysine and N{epsilon}-(melondialdehyde)lysine in rat heart mitochondrial proteins. Free Radic Res 26: 47–54, 2002.
  47. Pamplona R, Portero-Otin M, Requena J, Gredilla R, and Barja G. Oxidative, glycoxidative, and lipoxidative damage to rat heart mitochondrial proteins is lower after 4 mo of caloric restriction than in age-matched controls. Mech Ageing Dev 123: 1437–1436, 2002.[CrossRef][ISI][Medline]
  48. Patel M and Li QY. Age dependence of seizure-induced oxidative stress. Neuroscience 118: 431–437, 2003.[CrossRef][ISI][Medline]
  49. Powell CS and Jackson RM. Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD. Am J Physiol Lung Cell Mol Physiol 285: L189–L198, 2003.[Abstract/Free Full Text]
  50. Reid MB, Khawli FA, and Moody MR. Reactive oxygen in skeletal muscle. III. Contractility of unfatigued muscle. J Appl Physiol 75: 1081–1087, 1993.[Abstract/Free Full Text]
  51. Roberts DL, Frank EF, and Jung-Ja PK. Three-dimensional structure of human electron transfer flavoprotein to 2.1- resolution. Proc Natl Acad Sci USA 93: 14355–14360, 1996.[Abstract/Free Full Text]
  52. Rockstein M and Sussman M. Biology of Aging. Belmond, CA: Wadsworth, 1979, p. 73–87.
  53. Rosen P, Nawroth PP, King G, Moller W, Tritschler HJ, and Packer L. The role of oxidative stress in the onset and progression of diabetes and its complications: a summary of a Congress Series sponsored by UNESCO-MCBN, the American Diabetes Association, and the German Diabetes Society. Diabetes Metab Res Rev 17: 189–212, 2001.[CrossRef][ISI][Medline]
  54. Salazar D, Zhang L, deGala GD, and Frerman FE. Expression and characterization of two pathogenic mutations in human electron transfer flavoprotein. J Biol Chem 272: 26425–26433, 1997.[Abstract/Free Full Text]
  55. Sawada M and Carlson JC. Changes in superoxide radical and lipid peroxide formation in the brain, heart, and liver during the lifetime of the rat. Mech Ageing Dev 41: 125–137, 1987.[CrossRef][ISI][Medline]
  56. Shevchenko A, Wilm M, Vorm O, and Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68: 850–858, 1996.[Medline]
  57. Sohal RS. Role of oxidative stress and protein oxidation in the aging process. Free Radic Biol Med 33: 37–44, 2002.[CrossRef][ISI][Medline]
  58. Sohal RS, Arnold LA, and Sohal BH. Age-related changes in antioxidant enzymes and prooxidant generation in tissues of the rat with special reference to parameters in two insect species. Free Radic Biol Med 9: 495–500, 1990.[CrossRef][ISI][Medline]
  59. Souza JM, Choi JM, Chen Q, Weisse M, Daikhin E, Yudkoff MO, Ara J, Horwitz J, and Ischiropoulos H. Proteolytic degradation of tyrosine nitrated proteins. Arch Biochem Biophys 380: 360–366, 2000.[CrossRef][ISI][Medline]
  60. Souza JM, Daikhin E, Yudkoff M, Raman CS, and Ischiropoulos H. Factors determining the selectivity of protein tyrosine nitration. Arch Biochem Biophys 371: 169–178, 1999.[CrossRef][ISI][Medline]
  61. Stadtman ER. Protein oxidation and aging. Science 257: 1220–1224, 1992.[Abstract/Free Full Text]
  62. Szweda LI and Lucas DT. Cardiac reperfusion injury: aging, lipid peroxidation, and mitochondrial dysfunction. Proc Natl Acad Sci USA 95: 510–514, 1998.[Abstract/Free Full Text]
  63. Tamarit J, Cabiscol E, and Ros J. Identification of the major oxidatively damaged proteins in Escherichia coli cells exposed to oxidative stress. J Biol Chem 273: 3027–3032, 1998.[Abstract/Free Full Text]
  64. Turko IV, Li L, Aulak KS, Stuehr DJ, Chang JY, and Murad F. Protein tyrosine nitration in the mitochondria from diabetic mouse heart. Implications to dysfunctional mitochondria in diabetes. J Biol Chem 278: 33972–33977, 2003.[Abstract/Free Full Text]
  65. Turko IV and Murad F. Quantitative protein profiling in heart mitochondria from diabetic rats. J Biol Chem 278: 35844–35849, 2003.[Abstract/Free Full Text]
  66. Vadseth C, Souza JM, Thompon L, Seagraves A, Nagaswami C, Scheiner T, Torbet J, Vilaire G, Bennett JS, Murciano JC, Muzynkantov V, Penn MS, Hazen SL, Weisel JW, and Ischiropoulos H. Pro-thrombotic state induced by post-translational modification of fibrinogen by reactive nitrogen species. J Biol Chem 279: 8820–8826, 2004.[Abstract/Free Full Text]
  67. Viner RI, Ferrington DA, Williams TD, Bigelow DJ, and Schöneich C. Protein modification during biological aging: selective tyrosine nitration of the SERCA2 isoform of the sarcoplasmic reticulum Ca-ATPase in skeletal muscle. Biochem J 340: 657–669, 1999.
  68. Wang X, Osinska H, Gerdes AM, and Robbins J. Desmin filaments and cardiac disease: establishing causality. J Card Fail 8: S287–S292, 2002.[CrossRef][ISI][Medline]
  69. Xu KY, Huso DL, Dawson TM, Bredt LC, and Becker LC. Nitric oxide synthase in cardiac sarcoplasmic reticulum. Proc Natl Acad Sci USA 96: 657–662, 1999.[Abstract/Free Full Text]
  70. Yates JR III, Eng JK, McCormack AL, and Schleitz D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal Chem 67: 1426–1436, 1995.[Medline]
  71. Ye YZ, Strong M, Huang ZQ, and Beckman JS. Antibodies that recognize nitrotyrosine. Methods Enzymol 269: 201–209, 1996.[ISI][Medline]



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