Mitochondrial Dysregulation of Osteoarthritic Human Articular Chondrocytes Analyzed by Proteomics

Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-α receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis. Mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including osteoarthritis (OA). Mitochondrial proteins are attractive targets for the study of metabolism of the chondrocyte, the unique cell type present in mature cartilage, and its role in tissue degradation. Using a proteomics approach based on two-dimensional DIGE and MALDI-TOF/TOF mass spectrometric identification of mitochondria- enriched protein fractions from human articular chondrocytes, we analyzed mitochondrial protein changes that are characteristic of OA chondrocytes. A total of 73 protein forms were unambiguously identified as significantly altered in OA; 23 of them have been previously described as mitochondrial. An extensive statistical and cluster analysis of the data revealed a mitochondrial protein profile characteristic for OA. This pattern includes alterations in energy production, maintenance of mitochondrial membrane integrity, and free radical detoxification. Real time PCR, Western blot, and immunohistofluorescence assays confirmed a significant decrease of the major mitochondrial antioxidant protein manganese-superoxide dismutase (SOD2) in the superficial layer of OA cartilage. As possible outputs for this antioxidant deficiency, we found an increase of intracellular reactive oxygen species generation in OA chondrocytes and also verified an OA-dependent increase in the mitochondrial tumor necrosis factor-α receptor-associated protein 1 (TRAP1), a chaperone with a reported reactive oxygen species antagonist role. Our results describe the differences between the mitochondrial protein profiles of normal and OA chondrocytes, demonstrating that mitochondrial dysregulation occurs in cartilage cells during OA and highlighting redox imbalance as a key factor in OA pathogenesis. The mitochondrion is one of the most complex and important organelles found in eukaryotic cells and carries out a wide variety of biochemical processes. Mitochondria are critical subcellular organelles responsible for energy production through the coupling of respiration to the generation of ATP. Mitochondria consist of four components: an outer membrane, an intermembrane space, an inner membrane, and a matrix. These components all function in concert to convert pyruvate and fatty acids to acetyl CoA, which is metabolized by the citric acid cycle to produce NADH. High energy electrons from NADH are then passed to oxygen by means of the respiratory chain in the inner membrane, producing ATP by a chemiosmotic process. Transcription and translation take place in mitochondria, which also actively import proteins and metabolites from the cytosol, influence programmed cell death, and respond to cellular signals such as oxidative stress (1Green D.R. Reed J.C. Mitochondria and apoptosis.Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). In addition to their central role in energy metabolism, mitochondria are involved in many cellular processes; mitochondrial dysfunctions have been associated with apoptosis, aging, and a number of pathological conditions, including Parkinson disease, diabetes mellitus, Alzheimer disease, and OA 1The abbreviations used are: OA, osteoarthritis; 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; BVA, biological variation analysis; Cy, cyanine; DCF, dichlorofluorescein; DIA, differential in-gel analysis; MRC, mitochondrial respiratory chain; PMF, peptide mass fingerprinting; ROS, reactive oxygen species; SOD2, manganese-superoxide dismutase; TNFα, tumor necrosis factor-α; TRAP1, TNFα receptor-associated protein 1; N, normal; PCA, principal component analysis; HC, hierarchical clustering; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBGD, porphobilinogen deaminase; FACS, fluorescence-activated cell sorting; IMMT, inner membrane protein mitofilin; SOD, superoxide dismutase. 1The abbreviations used are: OA, osteoarthritis; 2-D, two-dimensional; 2-DE, two-dimensional gel electrophoresis; BVA, biological variation analysis; Cy, cyanine; DCF, dichlorofluorescein; DIA, differential in-gel analysis; MRC, mitochondrial respiratory chain; PMF, peptide mass fingerprinting; ROS, reactive oxygen species; SOD2, manganese-superoxide dismutase; TNFα, tumor necrosis factor-α; TRAP1, TNFα receptor-associated protein 1; N, normal; PCA, principal component analysis; HC, hierarchical clustering; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBGD, porphobilinogen deaminase; FACS, fluorescence-activated cell sorting; IMMT, inner membrane protein mitofilin; SOD, superoxide dismutase. (1Green D.R. Reed J.C. Mitochondria and apoptosis.Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar, 2Finkel T. Holbrook N.J. Oxidants, oxidative stress and the biology of ageing.Nature. 2000; 408: 239-247Crossref PubMed Scopus (7120) Google Scholar, 3Blanco F.J. Lopez-Armada M.J. Maneiro E. Mitochondrial dysfunction in osteoarthritis.Mitochondrion. 2004; 4: 715-728Crossref PubMed Scopus (132) Google Scholar). OA, the most common age-related cartilage and joint pathology (4Heinegard D. Bayliss M. Lorenzo P. Biochemistry and metabolism of normal and osteoarthritic cartilage.in: Brandt K.D. Doherty M. Lohmander L.S. Osteoarthritis. Oxford University Press, New York1998: 74-84Google Scholar), is a slowly progressive degenerative disease characterized by degradation of the matrix and cell death, which result in a gradual loss of articular cartilage integrity (5Pritzker K. Pathology of osteoarthritis.in: Brandt K.D. Doherty M. Lohmander L.S. Osteoarthritis. Oxford University Press, New York1998: 50-61Google Scholar, 6Kim H.A. Blanco F.J. Cell death and apoptosis in osteoarthritic cartilage.Curr. Drug Targets. 2007; 8: 333-345Crossref PubMed Scopus (171) Google Scholar). The only cell type present in mature cartilage is the chondrocyte, which is responsible for repairing cartilage tissue damaged by OA. Recently the role of mitochondrial dysfunction in OA has been the subject of renewed interest. Some studies have shown that mitochondrial dysfunction mediates several pathways implicated in cartilage degradation (7Blanco F. Loépez-Armada M. Rego I. Mitochondria and chondrocytes: role in osteoarthritis.in: Buckwalter J.A. Lotz M. Stoltz J.-F. Osteoarthritis, Inflammation and Degradation: A Continuum. IOS Press, Amsterdam2007: 192-205Google Scholar). These include oxidative stress, inadequacy of chondrocyte biosynthetic and growth responses, up-regulated chondrocyte cytokine-induced inflammation and matrix catabolism, pathologic cartilage matrix calcification, and increased chondrocyte death (necrosis or apoptosis). For example, mitochondrial respiratory chain (MRC) activity in OA chondrocytes showed decreases in complexes I, II, and III compared with normal chondrocytes that caused a reduction in mitochondrial membrane potential (Δψm) and in ATP synthesis. On the other hand, as a compensatory mechanism, the number of mitochondria is increased in OA chondrocytes as demonstrated by a significant increase in mitochondrial mass and in citrate synthase activity (8Maneiro E. Martin M.A. de Andres M.C. Lopez-Armada M.J. Fernandez-Sueiro J.L. del Hoyo P. Galdo F. Arenas J. Blanco F.J. Mitochondrial respiratory activity is altered in osteoarthritic human articular chondrocytes.Arthritis Rheum. 2003; 48: 700-708Crossref PubMed Scopus (178) Google Scholar). Other reports implicate decreased mitochondrial bioenergy reserve as a pathogenic factor in degenerative cartilage disease (9Johnson K. Jung A. Murphy A. Andreyev A. Dykens J. Terkeltaub R. Mitochondrial oxidative phosphorylation is a downstream regulator of nitric oxide effects on chondrocyte matrix synthesis and mineralization.Arthritis Rheum. 2000; 43: 1560-1570Crossref PubMed Scopus (150) Google Scholar, 10Tomita M. Sato E.F. Nishikawa M. Yamano Y. Inoue M. Nitric oxide regulates mitochondrial respiration and functions of articular chondrocytes.Arthritis Rheum. 2001; 44: 96-104Crossref PubMed Scopus (65) Google Scholar, 11Carlo Jr., M.D. Loeser R.F. Increased oxidative stress with aging reduces chondrocyte survival: correlation with intracellular glutathione levels.Arthritis Rheum. 2003; 48: 3419-3430Crossref PubMed Scopus (207) Google Scholar). Taken together, these findings suggest that mitochondrial proteins would be an attractive target for study of the metabolism of chondrocytes and the role they play in cartilage degradation. Most studies analyzing mitochondrial proteins in chondrocytes evaluated single proteins without addressing the mitochondrial proteome. The introduction of proteomics has enabled the simultaneous analysis of changes in multiple proteins. Currently many proteomics studies use two-dimensional gel electrophoresis (2-DE) to separate proteins, and this technology remains one of the key methodologies in proteomics studies. 2-DE gel-based approaches typically resolve hundreds to thousands of intact proteins according to their charge and molecular mass and compare the presence and intensity of protein spots among gel images to allow both qualitative and quantitative analysis. Using this proteomics approach, we have recently described the proteome of normal and OA human chondrocytes as well as their differential protein profile (12Ruiz-Romero C. Lopez-Armada M.J. Blanco F.J. Proteomic characterization of human normal articular chondrocytes: a novel tool for the study of osteoarthritis and other rheumatic diseases.Proteomics. 2005; 5: 3048-3059Crossref PubMed Scopus (99) Google Scholar, 13Ruiz-Romero C. Carreira V. Rego I. Remeseiro S. Lopez-Armada M.J. Blanco F.J. Proteomic analysis of human osteoarthritic chondrocytes reveals protein changes in stress and glycolysis.Proteomics. 2008; 8: 495-507Crossref PubMed Scopus (91) Google Scholar). Nevertheless traditional 2-D gel-based strategies have, until recently, lacked the ability of directly quantifying changes in abundance in the same fashion as stable isotope strategies using liquid chromatography coupled with tandem mass spectrometry (14Gygi S.P. Rist B. Gerber S.A. Turecek F. Gelb M.H. Aebersold R. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags.Nat. Biotechnol. 1999; 17: 994-999Crossref PubMed Scopus (4293) Google Scholar, 15Ross P.L. Huang Y.N. Marchese J.N. Williamson B. Parker K. Hattan S. Khainovski N. Pillai S. Dey S. Daniels S. Purkayastha S. Juhasz P. Martin S. Bartlet-Jones M. He F. Jacobson A. Pappin D.J. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.Mol. Cell. Proteomics. 2004; 3: 1154-1169Abstract Full Text Full Text PDF PubMed Scopus (3616) Google Scholar). Therefore, issues such as gel-to-gel variation and normalization of spot intensities across gel sets highly affect both accuracy and sensitivity of quantification. DIGE technology (16Unlu M. Morgan M.E. Minden J.S. Difference gel electrophoresis: a single gel method for detecting changes in protein extracts.Electrophoresis. 1997; 18: 2071-2077Crossref PubMed Scopus (1810) Google Scholar) adds an essential quantitative advantage to 2-D gel-based strategies and allows the detection of slight changes in protein abundance with statistical confidence (17Tonge R. Shaw J. Middleton B. Rowlinson R. Rayner S. Young J. Pognan F. Hawkins E. Currie I. Davison M. Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology.Proteomics. 2001; 1: 377-396Crossref PubMed Scopus (796) Google Scholar). In this approach, samples are labeled with different fluorescent dyes (Cy2, Cy3, and Cy5), not only increasing sensitivity and dynamic range but also allowing sample multiplexing so that two different samples can be run in the same gel together with an internal standard. The use of a pooled sample internal standard permits direct quantitative evaluation of changes and reduces intergel variation and false positives (18Lilley K.S. Friedman D.B. All about DIGE: quantification technology for differential-display 2D-gel proteomics.Expert Rev. Proteomics. 2004; 1: 401-409Crossref PubMed Scopus (243) Google Scholar, 19Marouga R. David S. Hawkins E. The development of the DIGE system: 2D fluorescence difference gel analysis technology.Anal. Bioanal. Chem. 2005; 382: 669-678Crossref PubMed Scopus (505) Google Scholar), resulting in highly reproducible data with biological significance. Moreover one of the advantages of 2-D DIGE versus non-gel-based quantitative proteomics techniques is that it detects not only changes in protein quantity but also posttranslational modifications of the protein (20Kolkman A. Dirksen E.H. Slijper M. Heck A.J. Double standards in quantitative proteomics: direct comparative assessment of difference in gel electrophoresis and metabolic stable isotope labeling.Mol. Cell. Proteomics. 2005; 4: 255-266Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Because gel-based techniques have a bias toward abundant proteins, proteins in lower quantity are not often detected in the 2-DE analysis of total cellular proteins because of the complexity of these samples. The use of prefractionation methods by subcellular isolation or selective enrichment of a specific group of proteins provides an effective approach to eliminate this drawback. We previously optimized the methodology for isolating mitochondria from human articular chondrocytes and reported their mitochondrial 2-DE reference map (21Ruiz-Romero C. Lopez-Armada M.J. Blanco F.J. Mitochondrial proteomic characterization of human normal articular chondrocytes.Osteoarthritis Cartilage. 2006; 14: 507-518Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In this work, we took advantage of 2-D DIGE technology to study mitochondria-enriched protein fractions and analyze the differential mitochondrial protein profile of osteoarthritic human articular chondrocytes to identify new mitochondrial proteins related with OA pathogenesis. Culture media and FCS were from Invitrogen. Culture flasks and plates were purchased from Costar (Cambridge, MA). DIGE materials (IPG buffer and strips and Cy dyes) were from GE Healthcare. Unless indicated, all other chemicals and enzymes were obtained from Sigma-Aldrich. Monoclonal antibodies against human tumor necrosis factor-α (TNFα) receptor-associated protein 1 (TRAP1) and manganese-superoxide dismutase (SOD2) were from BD Biosciences. The corresponding horseradish peroxidase- or phycoerythrin-conjugated secondary antibodies were from Santa Cruz Biotechnologies (Santa Cruz, CA) and DAKO Diagnostics (Glostrup, Denmark), respectively. MitoTracker Green was purchased from Invitrogen. Macroscopically normal human knee cartilage from adult donors having no history of joint disease was provided by the Tissue Bank and the Autopsy Service at Complejo Hospitalario Universitario A Corunña. Osteoarthritic cartilage was obtained from patients undergoing joint surgery. The study was approved by the institutional Ethics Committee. Once cartilage surfaces were rinsed with saline, scalpels were used to cut parallel vertical sections 5 mm apart from the cartilage surface to the subchondral bone. These cartilage strips were dissected from the bone, and the tissue was incubated with trypsin at 37 °C for 10 min. After removing the trypsin solution, the cartilage slices were treated for 12–16 h with type IV clostridial collagenase in Dulbecco’s modified Eagle’s medium with 5% FCS to release cartilage cells. The isolated chondrocytes were recovered and plated at high density in Dulbecco’s modified Eagle’s medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, 1% glutamine, and 10% FCS. The cells were then seeded onto 162-cm2 flasks for proteomics studies or 12-well culture dishes for total RNA extraction. The seeded cells were incubated at 37 °C in a humidified gas mixture containing 5% CO2 balanced with air. The chondrocytes were used at confluency (2–3 weeks in primary culture) after making them quiescent by incubation in a medium containing 0.5% FCS for 48 h. Cell viability was assessed by trypan blue dye exclusion. Chondrocytes (20–30 × 106 cells) were recovered from culture flasks by trypsinization and collected by centrifugation at 4 °C. After one wash in 130 mm NaCl, 5 mm KCl, 2.5 mm Tris-HCl (pH 7.5), and 0.7 mm Na2HPO4, the cells were transferred to microcentrifuge tubes and resedimented. For mitochondrial isolation, a differential centrifugation procedure (21Ruiz-Romero C. Lopez-Armada M.J. Blanco F.J. Mitochondrial proteomic characterization of human normal articular chondrocytes.Osteoarthritis Cartilage. 2006; 14: 507-518Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) was performed. All centrifugations were carried out at 4 °C. Briefly chondrocytes were resuspended in a swelling buffer containing protease inhibitors and incubated for 20 min on ice. Then 0.4 volumes of an ice-cold 2.5× sucrose buffer was added, and the cells were homogenized by 30 passes through a 25-gauge needle. The resulting homogenates were centrifuged twice for 10 min at 1200 × g to remove nuclei and large cell debris. The supernatants were then centrifuged at 9000 × g for 15 min; each homogenate yielded a mitochondrial pellet and a cytosol-enriched fraction. The mitochondria were thoroughly resuspended in 500 μl of sucrose buffer and centrifuged at 1200 × g to remove further contaminants. The supernatant was finally centrifuged at 9000 × g to obtain the crude mitochondrial pellet. This pellet was solubilized by 1-h incubation with gentle agitation in an isoelectric focusing-compatible urea lysis buffer (21Ruiz-Romero C. Lopez-Armada M.J. Blanco F.J. Mitochondrial proteomic characterization of human normal articular chondrocytes.Osteoarthritis Cartilage. 2006; 14: 507-518Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). For protein quantification, 2–4 μl of each protein extract was diluted to 50 μl with water, and protein was quantified in triplicate by the BCA technique (Pierce). Correct quantification was confirmed by loading 5 μg of each sample on a standard SDS-PAGE gel and subsequent Coomassie staining. The proteomics comparison between OA and normal chondrocyte mitochondrial proteins was performed across six DIGE gels using the same pooled sample internal standard to reduce intergel variation. The 12 individual samples were generated from six normal (N) donors (mean age, 65.8 years; age range, 53–83 years) and six OA donors (mean age, 69.8 years; age range, 51–87 years). Proteins in each sample were fluorescently tagged with a set of matched fluorescent dyes according to the manufacturer’s protocol for minimal labeling. To eliminate any dye-specific labeling artifacts, three samples of each group (N and OA) were labeled with Cy3, and the other three were labeled with Cy5. The pooled sample internal standard was always Cy2-labeled. In every case, 400 pmol of dye was used for 50 μg of protein. Briefly labeling was performed for 30 min on ice in darkness, and the reaction was quenched with 1 μl of 10 mm l-lysine for 10 min under the same conditions. The six pairs of Cy3- and Cy5-labeled samples (each containing 50 μg of protein) were combined and mixed with a 50-μg aliquot of the Cy2-labeled pooled standard. The mixtures containing 150 μg of protein were diluted 1:1 with rehydration buffer (7 m urea, 2 m thiourea, 4% CHAPS, 4% ampholytes (pH 3–11), and 200 mm DTT). The IPG strips (24 cm, pH 3–11 non-linear) were rehydrated overnight with 450 μl of a rehydration buffer as above but with 2% ampholytes, 0.002% bromphenol blue, and 97 mm DeStreak reagent instead of DTT. The labeled samples were then applied to the strips by cup-loading on a manifold-equipped IPGphor II IEF system (GE Healthcare). Isoelectric focusing was carried out for a total of 70 kV-h using the following conditions: 1 h at 120 V, 1 h at 500 V, 1 h at 1000 V, gradient to 4000 V in 1 h, and finally 12 h at 4000 V. Prior to the second dimension run, the strips were equilibrated first for 15 min in equilibration buffer (100 mm Tris-HCl (pH 8.0), 6 m urea, 30% glycerol, and 2% SDS) with 2% DTT and then for another 15 min in the same buffer supplemented with 2.5% iodoacetamide and 0.002% bromphenol blue. The equilibrated strips were transferred onto 12% homogenous polyacrylamide gels (2.6% C) cast in low fluorescence glass plates using an Ettan-DALT six system (GE Healthcare). Electrophoresis was run at 2 watts/gel for about 17 h at 20 °C. The differentially labeled co-resolved proteins within each gel were imaged at a resolution of 100 dots/inch using a Typhoon 9400 laser scanner (GE Healthcare). Cy2-, Cy3-, and Cy5-labeled images of each gel were acquired at excitation/emission values of 488/520, 523/580, and 633/670 nm, respectively. Gels were scanned directly between the glass plates, and the 16-bit image file format images were exported for data analysis. After imaging for Cy dyes, the gels were removed from the plates and subjected to colloidal Coomassie staining. The DeCyder version 6.5 software (GE healthcare) was used for spot detection and determination of quantity, intergel matching, and statistics. The differential in-gel analysis (DIA) module was used for automatic spot detection and abundance measurements for each individual gel by comparing the normalized volume ratio of each spot from a Cy3- or Cy5-labeled sample to the corresponding Cy2 signal from the pooled sample internal standard. The DIA data sets from each individual gel were collectively analyzed using the biological variation analysis (BVA) module, which allows intergel matching and calculation of average abundance for each protein spot among the six gels of our study. Statistical significance was assessed for each change in abundance using Student’s t test and analysis of variance analyses. We considered statistical significance to be at the 95% confidence level when standardized average spot volume ratios exceeded 1.3 in at least four of the six analyzed gels (i.e. 12 of the 18 analyzed images). Calculation of experimental molecular weight and pI for each differential protein spot was carried out using PDQuest 7.3.1 software. Unsupervised principal component analysis (PCA), hierarchical clustering (HC), and k-means clustering analyses were performed using the DeCyder extended data analysis module on the group of spots identified as significantly changed. These multivariate analyses clustered the individual Cy3- and Cy5-labeled samples based on collective comparison of expression patterns from the set of proteins. The groups of protein expression characteristics are represented by each data point in the PCA plots and by each column in the HC expression matrixes. Mapping of proteins identified by mass spectrometry, biological association network analysis, and database search onto existing pathways and cellular networks was carried out using Pathway Studio 5.0 (Ariadne Genomics, Rockville, MD). The gel spots of interest were manually excised from the gels and transferred to microcentrifuge tubes. Samples selected for analysis were in-gel reduced, alkylated, and digested with trypsin according to Sechi and Chait (22Sechi S. Chait B.T. Modification of cysteine residues by alkylation. A tool in peptide mapping and protein identification.Anal. Chem. 1998; 70: 5150-5158Crossref PubMed Scopus (354) Google Scholar). Briefly spots were washed twice with water, shrunk with 100% ACN, and dried in a Savant SpeedVac. Then samples were reduced with DTT and subsequently alkylated with iodoacetamide. Samples were digested with 12.5 ng/μl sequencing grade trypsin (Roche Applied Science) for at least 6 h at 37 °C. After digestion, the supernatant was collected, and 1 μl was spotted onto a MALDI target plate (384-spot Teflon®-coated plates) and allowed to air dry at room temperature. Subsequently 0.5 μl of a 3 mg/ml solution of α-cyano-4-hydroxy-trans-cinnamic acid matrix in 0.1% TFA and 50% ACN was added to the dried peptide digest spots and again allowed to air dry. The samples were analyzed using the MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (Applied Biosystems, Framingham, MA) and 4000 Series Explorer™ software (Applied Biosystems). MALDI-TOF spectra were acquired in reflector positive ion mode using 1000 laser shots per spectrum. Data Explorer version 4.2 (Applied Biosystems) was used for spectra analyses and generating peak picking lists. All mass spectra were internally calibrated using autoproteolytic trypsin fragments and externally calibrated using a standard peptide mixture (Sigma-Aldrich). TOF/TOF fragmentation spectra were acquired by selecting the 10 most abundant ions of each MALDI-TOF peptide mass map (excluding trypsin autolytic peptides and other known background ions) and averaging 2000 laser shots per fragmentation spectrum. The parameters used to analyze the data were a signal to noise threshold of 20, a minimum area of 100, and a resolution higher than 10,000 with a mass accuracy of 20 ppm. The monoisotopic peptide mass fingerprinting data obtained from MS and the amino acid sequence tag obtained from each peptide fragmentation in MS/MS analyses were used to search for protein candidates using Mascot version 1.9 from Matrix Science. Peak intensity was used to select up to 50 peaks per spot for peptide mass fingerprinting and 50 peaks per precursor for MS/MS identification. Tryptic autolytic fragment-, keratin-, and matrix-derived peaks were removed from the data set used for the database search. The searches for peptide mass fingerprints and tandem MS spectra were performed in the Swiss-Prot release 53.0 and TrEMBL release 37.0 databases without taxonomy restriction, containing 269,293 and 4,672,908 sequence entries, respectively, for each software version and database release. Fixed and variable modifications were considered (Cys as S-carbamidomethyl derivate and Met as oxidized methionine, respectively), allowing one trypsin missed cleavage site and a mass tolerance of 50 ppm. For MS/MS identifications, a precursor tolerance of 50 ppm and MS/MS fragment tolerance of 0.3 Da were used. Identifications were accepted as positive when at least five matching peptides and at least 20% of the peptide coverage of the theoretical sequences matched within a mass accuracy of 50 or 25 ppm with internal calibration. In every case probability scores were significant at p < 0.01. Intracellular localization of the identified proteins was predicted from the amino acid sequence using PSORT II program. One-dimensional Western blot tests were performed according to standard procedures. Briefly 50 μg of total cellular or mitochondria-enriched proteins were loaded and resolved on standard 10% polyacrylamide SDS-PAGE gels. Separated proteins were then electroblotted onto PVDF membranes (Immobilon P, Millipore, Bedford, MA). Equivalent loadings were verified by Ponceau Red staining after transference. Membranes were blocked in Tris-buffered saline (pH 7.4) containing 0.1% Tween 20 (TBST) and 5% nonfat dried milk for 60 min at room temperature. The blots were then hybridized overnight at 4 °C with antibodies against TRAP1 (1:500) or SOD2 (1:1000) and the housekeeping controls α-ATPase (1:5000, in tests performed on crude mitochondria extracts) or α-Tubulin (1:5000 in tests performed on whole cell extracts). All antibodies were diluted in TBST with 2% nonfat milk. After thorough washing with TBST, immunoreactive bands were detected by chemiluminescence using corresponding horseradish peroxidase-conjugated secondary antibodies and ECL detection reagents (GE Healthcare) and then digitized using an LAS 3000 image analyzer. Quantitative changes in band intensities were evaluated with ImageQuant 5.2 software (GE Healthcare). The densitometric values of the Western blot bands containing the protein of interest (TRAP or SOD2) were normalized against those of α-ATPase or α-Tubulin obtained from the s