Karlfried Groebe

Frank Krause

Birgit Kunstmann

Hermann Unterluggauer

Nicole H Reifschneider

Christian Scheckhuber

Charturvedala Sastri

Werner Stegmann

Wojciech Wozny

Gerhard P Schwall

Slobodan Poznanović

Norbert A Dencher

Pidder Jansen-Dürr

Heinz D Osiewacz

Prof. Dr André Schrattenholz
email: andre.schrattenholz@proteosys.com

Hermann Unterluggauer #

Slobodan Poznanovi

Pidder Jansen-Dürr #

S Goto

H Nakamoto

R Takahashi

Cso

Differential proteomic profiling of mitochondrial from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes

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Introduction

The free radical theory of ageing [START_REF] Harman | Ageing: a theory based on free radical and radiation chemistry[END_REF] implicates molecular damage caused by reactive oxygen species (ROS) as a major cause of ageing processes in most if not all species. The mitochondrial theory of ageing linking mutations in mitochondrial DNA to the ageing process (Miquel et al., 1983; Miquel, 1991) has subsequently found widespread support (for review see: [START_REF] Kowald | The mitochondrial theory of ageing[END_REF]Wallace, 2001;[START_REF] Jacobs | The mitochondrial theory of ageing: dead or alive?[END_REF].

Age-related loss of mitochondrial functions has been demonstrated in various systems over the last several decades (Osiewacz and Hermanns, 1992;[START_REF] Borghouts | Respiration, copper availability and SOD activity in P. anserina strains with different lifespan[END_REF]. In the filamentous fungus Podospora anserina, an extensively studied ageing model, mitochondrial DNA reorganisation is a hallmark of ageing of all wild-type strains [START_REF] Esser | Mitochondrial DNA and Senescence in Podospora anserina[END_REF]Stahl et al., 1978;[START_REF] Belcour | Does Senescence in Podospora anserina Result from Instability of the Mitochondrial Genome?[END_REF][START_REF] Averbeck | Life-span and Senescence in Podospora anserina: Effect of Mitochondrial Genes and Functions[END_REF]. Stabilization of mtDNA leads to increased lifespan. Some strains even appear to have acquired immortality [START_REF] Koll | Ethidium bromide rejuvenation of senescent cultures of Podospora anserina: Loss of senescence-specific DNA and recovery of normal mitochondrial DNA[END_REF] In the yeast Saccharomyces cerevisiae, the level of ROS production increases in naturally aged yeast mother cells [START_REF] Laun | Aged mother cells of Mitochondrial dysfunction, glutamate dysregulation and impaired protein synthesis[END_REF]. In Caenorhabditis elegans agerelated mtDNA deletions were reported previously (Melov et al., 1995;Melov et al., 1995) and a systematic knock-down of mitochondrial genes via RNAi resulted in the generation of long-lived worm mutants (Lee et al., 2003). Using a mouse model, expressing a mutant mtDNA polymerase, it was shown recently that increased levels of point mutations and deletions of mtDNA can indeed cause a phenotype of reduced lifespan and premature onset of ageing-related phenotypes (Trifunovic et al., 2005;[START_REF] Kujoth | Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian ageing[END_REF]. Whereas it is unlikely that increased oxidative stress results from mitochondrial dysfunction in these mouse models, it is currently unknown if the ageing phenotype is due to a disturbed energy metabolism or other causes, such as increased apoptosis [START_REF] Kujoth | Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian ageing[END_REF].

Sporadic loss of mitochondrial function with ageing has also been observed in various rodent and mammalian tissues, and loss of function was correlated to the occurrence of mtDNA mutations, at least in some cases. In numerous nonreproductive tissues of many species, mitochondrial genes (like nuclear genes) accumulate mutations as the animals age (Vijg, 2000). The accumulation of mtDNA mutations can be explained either by a decreased degradation rate of damaged mitochondria or by a replication advantage of smaller mtDNA molecules (reviewed by [START_REF] Kowald | On the relevance of mitochondrial fusions for the accumulation of mitochondrial deletion mutants: a modelling study[END_REF].

The crucial role of mitochondria on intermediary metabolism and regulation is thus Mitochondrial samples from these models were used to establish respective signatures of age-related differential proteins, which were subsequently analyzed for redundant posttranslational isoforms. It has recently been shown that there is a crucial relationship between resolution and differential quantification of proteins requiring isotopic labeling with a detection method providing sufficient dynamic range to enable statistical treatment of differences (Schrattenholz and Groebe, 2007). For relatively complex samples with redundant isoforms, high resolution 2D-PAGE has turned out to be a good compromise.

Despite the fact that the biological systems used in this study are from evolutionary far distant species, the focus on mitochondrial proteins resulted in some striking common mechanistic aspects. We find certain clusters of ROS-related modifications which occur in homologous mitochondrial (or mitochondria-associated) proteins across species.

On this background, systematic molecular profiling of age-related models, using Proteins and pathways identified as being affected by age-and ROS -dependent posttranslational modifications in our study are discussed with a focus on two representative cases, ATP synthase [START_REF] Arrell | Proteomic analysis of pharmacological preconditioning: novel protein targets converge to mitochondrial metabolism pathways[END_REF]; [START_REF] Krause | Active oligomeric ATP synthases in mammalian mitochondria[END_REF]) and SAMdependent O-methyl transferase [START_REF] Averbeck | Life-span and Senescence in Podospora anserina: Effect of Mitochondrial Genes and Functions[END_REF].

Materials and Methods

Isolation of crude mitochondrial fractions from rat brain

Left brains of 5-, 17-, and 31-month-old male rats (F344/DuCrj, purchased from Charles River Japan) were obtained from the animal facility at Toho University (Japan). The ad libitum fed rats had a mean life span of 29 months (Takahashi and Goto, 1987). Brain tissue was frozen in liquid nitrogen immediately upon dissection and stored at -80 °C for 1 -5 years until isolation of mitochondria. Long-term storage of bovine heart mitochondria under these conditions for at least 16 months did not affect the yield of OXPHOS-supercomplexes [START_REF] Krause | Active oligomeric ATP synthases in mammalian mitochondria[END_REF]. Rat brain mitochondria were isolated as gentle as possible (crude mitochondrial fraction) as described by [START_REF] Krause | Active oligomeric ATP synthases in mammalian mitochondria[END_REF]. A half brain was homogenized by a 2 ml tight-fit glass-teflon homogenizer (clearance 45-65 µm) with nine strokes (1100 rpm) after addition of 4 volumes of homogenization buffer (350 mM sucrose, 5 mM Hepes-NaOH, 1 mM EDTA, and 0.5 mM protease-inhibitor Pefabloc SC, pH 7.4) to 1 volume of tissue and the homogenate was centrifuged at 1300g (4 °C, 3 min). The pellet was extracted twice with each 2 and 1 ml homogenization buffer, respectively, and 1500g centrifugation. Thereafter, the combined supernatants were centrifuged with 17,000g (10 min, 4 °C). The crude mitochondrial pellet was washed once and resuspended in 320 mM sucrose, 0.5 mM Pefabloc SC. Finally, all isolated mitochondria were frozen as aliquots (5 -7 mg/ml protein) in liquid nitrogen and stored at -80 °C.

Isolation of mitochondrial proteins from P. anserina

Mitochondria were isolated from juvenile and senescent P. anserina cultures, respectively, according to a previously published protocol [START_REF] Gredilla | Mitochondrial free radical generation and lifespan control in the fungal ageing model Podospora anserina[END_REF]) with the following modifications. Crude mitochondria were isolated by differential centrifugation for 35 minutes at 15.000 g and 4°C. The mitochondrial pellet was resuspended in 1 ml of mitochondria isolation buffer (10 mM Tris, 1mM EDTA, 0.33 M sucrose, pH7.5) and layered on a 20-50 % discontinuous sucrose gradient. After centrifugation for 1 hour at 100.000 g in a swing-out bucket rotor (TH641) the mitochondria were banding between the 50 and the 36 % sucrose step.

Approximately 30 ml mitochondrial isolation buffer without BSA were added to the collected mitochondria fraction and centrifuged for 15 min at 15.000 g at 4°C. Special care was taken to ensure standardized processing times in individual preparations of mitochondria. Following this strategy three pairs of mitochondria (protein content: ~1 mg) from juvenile and senescent wild-type strain were isolated (analytical scale). In addition, one pair of mitochondria from juvenile and senescent cultures was prepared at a preparative scale of approximately 4 mg total mitochondrial protein. The differential pooling scheme for proteomic analyses is shown together with results (Figure 2).

HUVEC cell culture:

Endothelial cells were isolated from human umbilical veins as described by [START_REF] Jaffe | Culture of human endothelial cells derived from umbilical veins: Identification by morphologic and immunologic criteria[END_REF]) and cultured in Endothelial Cell Basal Medium (Cambrex BioScience, Verviers) supplemented with EGM Singlequots (Cambrex BioScience, Verviers), containing 0.1% hEGF, 0.1% hydrocortisone, 0.1% GA-1000, 0.4% BBE and 2% FBS. The cells were subcultured by trypsinization with trypsin-EDTA (Gibco Life Technologies, Vienna, Austria), seeded on cell culture dishes coated with 0.2% gelatine and grown in an atmosphere of 5% CO 2 at 37°C. Cells were passaged at a ratio of 1:5 in regular intervals. At later passages, the splitting ratio was reduced to 1:3 and 1:2, respectively. Cells were passaged such that the monolayers never exceeded 70-80% confluency. PDL were estimated using the following equation: n = (log10 F -log10 I)/301 (where n is the population doublings, F; number of cells at the end of one passage, and I; number of cells that were seeded at the beginning of one passage). After roughly 65 population doublings, the cells reached growth arrest, corresponding to 90-95% SA-ß-Galactosidase positive cells. samples labelled with different iodine isotopes. For each pair of samples, reverse replicate gels were prepared, i.e., the labels ( 125 I and 131 I) used on sample 1 and sample 2 were inverted. Depending on the respective biological system, a total of two to five individual samples per age group entered the study, so the statistical evaluation was based on four to ten data points in each age group.

Depending on the number of age groups in each biological system, different pooling schemes were applied as shown in Figure 2. In the case of 2 groups (juvenile vs. senescent; Figure 2a), young subjects were individually compared to the pool of all old subjects, and old subjects were individually compared to the pool of all young subjects. In the case of three or more age groups (Figure 2b), each subject from each age group was individually compared to a common pool of all subjects.

Abundance ratios of one group vs. another are obtained by dividing the abundance ratios of the respective groups vs. the common pool. The former layout has the advantage that the measured differences in protein abundance are larger (than in comparisons involving a common pool) and that it provides a direct comparison between the conditions of interest (whereas in comparisons via a common pool each comparison between two age groups depends on two individual measurements). As its major disadvantage, the former pooling scheme can only be applied to two samples.

Gel image analysis was performed using the Pic/Greg software package by the Fraunhofer Gesellschaft in Sankt Augustin (http://www.fit.fraunhofer.de/projekte/greg/index_en.xml) as described previously [START_REF] Hunzinger | Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species[END_REF]). Spot quantification and statistical identification of differential spots was performed as described (Schrattenholz and Groebe 2007). In brief, protein spots were included if spot volume was at least 1.e10 (in units used by Greg) and background intensity was at least four times less than spot intensity. All spots were checked manually using the Pic program and accepted or rejected depending on their actual appearance. Individual protein spot abundance ratios for the different conditions were statistically analyzed by t-tests and only those spots meeting significance criteria were subjected to MALDI-TOF peptide mass fingerprinting, as published before (Vogt et al. 2005). To this end, selected spots were excised from the gel by a picking robot (ProPick, Genomic Solutions Ltd, Huntingdon, UK) and proteins in gel pieces were trypsin digested using a ProGestrobot (Genomic Solutions Ltd, Huntington, UK). A ProMS-robot (Genomic Solutions Ltd, Huntingdon, UK) was used to apply samples for MALDI-TOF mass spectrometry onto an anchor target (Bruker, Bremen, Germany). Mass spectra of peptide ions were obtained using an Ultraflex MALDI time-of-flight (TOF) mass spectrometer (Bruker, Bremen, Germany) in reflector mode within a mass range from m/z 800 to 4000. The MS spectra were calibrated and annotated automatically. The resulting peptide mass fingerprints were searched against the non redundant NCBI Protein Sequence Database using Mascot Server software v. 1.8. (Matrix Science, London, UK).

Results

Mitochondria were isolated by differential centrifugation from cell pellets obtained by collecting cultured human endothelial cells, P.anserina cultures and by homogenization of freshly prepared rat brains. The experimental procedures for mitochondrial isolation except that for P. anserina mitochondria were purified by sucrose density centrifugation whereas mitochondria from rats and cell cultures were isolated by differential centrifugation. The subsequent proteome analysis was the same for the three species, providing both mitochondria of comparable quality and statistically significant results. In the case of HUVEC, additional samples were collected after exposure to mild oxidative stress, known to induce a phenotype of premature senescence (Unterluggauer et al., 2003). As shown in Figure 1, by representative false colour images of radioactive spot intensities on high resolution 2D gels (pH range 4-9) of mitochondrial preparations of the three species investigated (rat brain, upper panel, P. anserina in the middle and human cells in the lower part), there are overall quite distinct patterns of protein expression and a variety of age-related differential spots. Despite the differences of patterns, replicates were highly reproducible and identified proteins were homologous in some cases or relating to similar pathway in other cases. The experimental strategy for obtaining statistically significant results about differential protein abundances using ProteoTope inverse replicate gels and identifying them by tracer-controlled preparative gels was performed as described previously [START_REF] Poznanovic | Differential radioactive proteomic analysis of microdissected renal cell carcinoma tissue by 54 cm isoelectric focusing in serial immobilized pH gradient gels[END_REF] Several hundreds of such gels have been differentially quantified in inverse duplicates for each of the following conditions and according to the pooling scheme shown in Figure 2: Thus, excluding contributions from variations of individual protein patterns, mitochondrial preparations of juvenile and senescent P. anserina cultures were compared (Table S1). Then mitochondrial brain proteins of young rats (5 months) were compared against those of middle-aged (17 months; Table S2) and old rats (31 months, Table S3). Also a comparison of the middle-aged group against the old group was included (Table S4). In a further set of experiments human cells were compared in the same way, results from the comparison of young and senescent HUVEC cells are shown in Table S5 and corresponding results from a model of chemically accelerated ageing (young and tBHP-treated HUVEC cells as described in Unterluggauer et al. 2003) are compiled in Table S6.

In Table1, we have summarized examples of age-related proteins with redundant isoforms, i.e. MALDI-TOF based identifications of the same amino acid backbone (and GeneBank accession #), but with slight differences in spectra and 2D gel positions. We listed all redundant isoforms found for SAM-dependent O-methyltransferase (SAM-OMT, PaMTH1), ATP-synthase F1 complex and reticulocalbin.

These differences on the background of a single amino acid sequence always indicate posttranslational modifications, and they can be expected to be age-related because of our particular experimental paradigms.

Proceeding from identifying such multiple redundant isoforms, we see that differential abundances of related spot pairs are quite distinct: As shown in Figure 3 and Table 1, there are subunit isoforms of ATP synthase with relatively large differential amplitudes (e.g. spots 1192 and 1555 from P. anserina), as well as others with relatively subtle differences. This is due to the fact, that underlying posttranslational modifications are associated with distinct domains of ATP synthase subunits; whereas other parts of the protein remain unaffected. As will be shown below, there are indications from detailed inspection of mass spectra that ROS-related oxidative chemical reactions are responsible on the molecular level.

In both age-dependently differential isoforms of SAM-OMT shown in Figure 4 and Table 1, we found N-formylkynurenine-modifications (by typical characteristic mass increments of 4, 16 and 32 at respective peptides) [START_REF] Bienvenut | Matrix-assisted laser desorption/ionizationtandem mass spectrometry with high resolution and sensitivity for identification and characterization of proteins[END_REF] Taken together, our data show that some common targets for protein modification as a result of ageing can be identified in the three model systems applied in the current study. Moreover, at least one particular oxidative modification, the dioxygenation of aromatic tryptophane residues to N-formyl-kynurenine occurs in an age-dependent manner in one of the models investigated. This relatively stable type of carbonylation by ROS will serve as a marker of age-related events in the ongoing study. Moreover, the identification of of PaMTH1 in the sucrose density purified mitochondrial fraction was surprising because the link of such a protein to mitochondria is new. In the initial identification this protein was found to increase in quantity during the ageing of P. anserina in total protein extracts.

Finally, we found ATP synthase affected by age-dependent processes across all species investigated. This is in line with recent reports about specific protein oxidation and protein expression in aged rat brain, conditions of neuronal stress or under caloric restriction, where ATP synthase F1 β and α subunits were consistently among markers found (Poon et Last but not least, our results from mitochondria preparations with HUVEC add to the increasing evidence that mitochondrial proteins interact intimately with the endoplasmic reticulum, Golgi and other organelles [START_REF] Dolman | Stable Golgi-mitochondria complexes and formation of Golgi Ca(2+) gradients in pancreatic acinar cells[END_REF]McMahon et al. 2006), and that this localisational dynamics is moving mitochondrial proteins (or their posttranslational derivatives!) to a variety of cytosolic and membrane compartments including even the outer plasma membrane. We hope that our data can contribute to providing a novel and more comprehensive understanding of the molecular consequences of energy metabolism on ageing, longevity and cellular stress management. It is noteworthy that the best-studied example of this class of proteins is the F 1 F 0 ATP synthase [START_REF] Yonally | The F(1)F(0) ATP synthase and mitochondrial respiratory chain complexes are present on the plasma membrane of an osteosarcoma cell line: An immunocytochemical study[END_REF]), which we were able to profile in considerable detail in this study. Dual isotope labelling allowed differential quantification of proteins from two samples in one experiment: In the upper part the differential display of the pattern of one particular 31 month old rat against the pattern of the pool of all rats is shown. In the middle, mitochondria from senescent P. anserina were compared against a pooled preparation from juvenile cells and in the bottom part, mitochondrial material from senescent HUVEC against pooled material from juvenile HUVEC.

In each case, the load was 10 µg of mitochondrial proteins labelled with I-125 and I-131, respectively. The radioactive quantities of differential spots in inverse replicates of such experiments provided absolute pattern control and served as basis for statistical analysis of age-dependent molecular changes. These experiments were meant for differential and quantitative pattern analysis and statistics only, the bulk of material was subsequently used for mass spectrometry-based identification of agedependent mitochondrial proteins from tracer-controlled and silver-stained preparative gels as described previously [START_REF] Hunzinger | Comparative profiling of the mammalian mitochondrial proteome: multiple aconitase-2 isoforms including N-formylkynurenine modifications as part of a protein biomarker signature for reactive oxidative species[END_REF]). In the paradigm for comparing two conditions only, individual samples (e.g. Ju1-Ju4 or Se1-Se4) of one condition were always compared to the pool of the other condition, and vice versa, as shown in Figure 2a. Thus, individual freak contributions could reliably be eliminated. In the case of three or more conditions of a model (e.g. 5, 17 and 31 months old rats), a common pool of all of these conditions was differentially compared to individual samples (Figure 2b). The differences between conditions are calculated as the total of the differences of the individual conditions with respect to the common pool (Figure 2b). The latter methodology could also be applied if there are only two conditions; it would, however, lead to smaller absolute differences in spot volumes and hence larger relative errors. Both approaches give similar results.

The statistical robustness originated from the application of these schemes to four independent paradigms using models from three species (P. anserina, R. norvegicus, Homo sapiens). 1) include isoforms with highly significant differences in concentration, as well as others with not much of a change. The larger the difference, the clearer the false colur red-blue difference; the pI range of IPG's where the respective spots were found is indicated. Taken together, the underlying biophysical basis is posttranslational modifications, which are expected to yield direct information of hot spots of ROS-related damage. The structural information relating certain domains which are affected and potentially interacting proteins could translate to functional implications and a novel understanding of age-related cascades of dysfunction.

Figure 4: Putative SAM-dependent O-methyltransferase (PaMTH1) from P. anserina, which is homologous to catecholamine-O-methyltransferase (COMT) in higher organisms, is differentially oxidized and carbonylated in an age-dependent manner.

In Figure 4A, the dual false colour images from inverse replicates of a differential and quantitative comparison of senescent and juvenile cells in each one 2D gel are shown (each 10 µg), the differences between the two conditions are consistent and statistically significant (for details see Table S1). In Figure 4B, a similar frame of the corresponding tracer-controlled and silver stained preparative 2D gel is shown (250 µg loaded). The trace amounts of I-125 labelled samples comigrating with the silver stained spots 6 and 7, again helped to control patterns in complex 2D-gel images, as described previously.

In Figure 4C

  ; Schulte et al., 1988; Borghouts et al., 1997; Stumpferl et al., 2004). More recently, the impact of mitochondrial respiration on lifespan has been elaborated. A switch from a standard cyanide dependent respiration to an cyanide resistant, alternative pathway leads to a reduced generation of ROS and an increased lifespan (Schulte et al., 1988; Borghouts et al., 2001; Stumpferl et al., 2004; Gredilla et al., 2006; Dufour et al., 2000).

  well established. Age-related mitochondrial dysfunction affects calcium signalling and downstream processes like apoptosis (Chan 2006). Irreversible lesions of mitochondrial genes or proteins have been shown to result in functional decline (Brookes et al. 2004; Brookes et al. 2002). Neuronal cells are especially vulnerable to mitochondrial damage due to excessive ATP demands of active synaptic regions. The crucial balance of ATP-and calcium homeostasis, explains the importance of mitochondria in many neurodegenerative diseases (Schrattenholz and Soskic 2006). Also, the critical role of ROS during ageing is broadly accepted (Singh 2006; Sanz et al. 2006; Kang and Hamasaki 2003; Osiewacz 2002). The gradual accumulation of irreversible oxidative modifications results in very specific patterns of redundant protein isoforms (Dencher et al. 2006), prompting this work as a systematic attempt to differentially quantitative conserved molecular patterns of age-related changes of mitochondrial proteins. In the present study, we employed high resolution 2D-PAGE (Hunzinger et al. 2006), and three different ageing models: the filamentous fungus P. anserina with a clear mitochondrial etiology of ageing (Scheckhuber et al. 2007; Osiewacz 2002;Osiewacz and Scheckhuber 2006), rats at three different ages, and different stages of a HUVEC (human umbilical vein endothelial cells) in vitro senescence model, applying both replicative senescence and premature (stress-induced) senescence.

  proteomic and genomic platforms has gained considerable steam (Schieke et al. 2006; Weinreb et al. 2007; Vo and Palsson 2006), showing that proteomic signatures are directly correlated to activity-, cellular stress-and energy-dependent protein markers (Ding et al. 2006b; Johnson et al. 2006; Forner et al. 2006).

  Tertiary butylhydroperoxide (tBHP) treatment (stress induced senescence) = chemical ageing: Young cells were treated on five consecutive days with 50µM tBHP (Fluka) for one hour each. After treatment, cells were washed twice with PBS and cultivation continued in standard EGM until the next tBHP-treatment. Following the last stress, the cells were cultivated for 24 hours in EGM and harvested for preparation of crude mitochondrial extracts. Isolation of Mitochondria from HUVEC Preparation of crude mitochondrial extracts was done according to the general guidelines (Rickwood and Hayes 1984) by differential centrifugation. All buffers and devices for extraction were ice-cooled. HUVEC of different replicative age were grown on 145mm culture dishes, rinsed twice with ice-cold PBS, scraped off the dishes and resuspended in 5ml homogenisation buffer [10mM HEPES pH 7.4; 1.1mM EDTA; 0.5%BSA and the antioxidant mixture SCAVEGR [(available at www.brainBitsLLC.com; see (Brewer et al. 2006)] and collected by centrifugation at 500g for 2min. The resulting pellet was again resuspended in Homogenisation buffer and homogenized in an iced -glass Teflon potter at 500rpm. After immediate addition of Sucrose (0.25 molar) the suspension was centrifuged two times for 10min with 1500g and the resulting mitochondria-enriched supernatants collected. In a final step this crude mitochondria were pelleted by centrifugation at 13700g for 10min, washed twice in resuspension buffer [10mM HEPES pH 7.4; 0.25M Sucrose; 1.1mM EDTA plus protease inhibitor cocktail (Complete Mini EDTA free tablets from Roche)] and snap frozen in liquid nitrogen. Protein concentration of samples was determined by Bradford assay. High resolution IEF/SDS-PAGE: 2D-PAGE was performed by 54 cm daisy chain serial IPG-IEF as described (Hunzinger et al. 2006). Briefly, shock-frozen samples were thawed at 25°C and dissolved in 8M Urea, 4% CHAPS, 0.1M Tris pH 7.4. The volume was adjusted to 20 µL if necessary, followed by incubation of the sample at room temperature for 30 min with shaking at 1000 rpm in a Thermomixer comfort (Eppendorf). The samples were centrifuged for 5 min at 12000 rcf and 25°C, and the soluble extracts were collected by removing the supernatant. Experimental design, quantification and protein identification: The experimental strategy for obtaining statistically significant results about differential protein abundances using ProteoTope inverse replicate gels was performed as described previously (Poznanovic et al. 2005;Schrattenholz and Groebe 2007;Neubauer et al. 2006). In brief, protein iodination reactions with either 125 I or 131 I were conducted with identical chemical iodine concentrations. Less than 1 mg of protein from each radiolabelled sample were mixed and separated by 2-D PAGE high resolution daisy chains covering a pH range of 4-9. A high sensitivity radio imaging technique was applied to discriminate between 125 I and 131 I signals in one 2-D PAGE gel and to generate a quantitative multicolour differential display of proteins from separate

  ;Schrattenholz and Groebe 2007;Neubauer et al. 2006).

  ; Simat and Steinhart 1998). The specific oxidized sites are at tryptophanes W 35, 37 und 56. and abundances of this oxidative modification are different between juvenile and senescent P. anserina cultures: W 35 and 37 are more oxidized in senescent strains (~50% N-formylkynurenine in senescent vs. ~25% N-formylkynurenine in juvenile cells).

  Alterations in mitochondrial structure or function have been implicated in a variety of human diseases, ageing and longevity for quite some time. Since the initial reports of age-related mtDNA reorganization in P. anserina in the late 1970's and early 1980's(Stahl et al., 1978; Cummings et al., 1979) [START_REF] Kück | Plasmid-like DNA is part of mitochondrial DNA in Podospora anserina[END_REF] Osiewacz and Esser, 1984) and complete sequencing of the human mitochondrial genome in 1981, enormous progress has been made in understanding the the relationship between mitochondrial energy metabolism and ageing (Gillardon 2006;Schrattenholz and Soskic 2006;Marin-Garcia et al. 2006;Lesnefsky and Hoppel 2006;Ruiz-Romero et al. 2006). So far 800-1200 mt-proteins have been described (Reinders et al. 2006; Alonso et al. 2005;Bailey et al. 2005) and organized in corresponding public databases (Prokisch et al. 2006;Basu et al. 2006). This also includes mtDNA information, with ROS-related mutations and subsequent consequences on the protein level (Rottenberg 2006; Capri et al. 2006). Obviously mitochondrial genes and even more so, proteins can have different biological roles at different ages, and the constant generation of ROS accumulates to age-dependent decline of function. Recent data from invertebrate (Dufour et al., 2000; Gredilla et al., 2006; Borghouts et al., 2002) and animal models for ageing (Trifunovic et al., 2005) as well as from human cell cultures {Stoeckl et al., 2006} tend to suggest that, besides mitochondrial ROS production, other consequences of mitochondrial dysfunction, e.g. a reduction of ATP levels (Stoeckl et al., 2006) (Zwerschke et al., 2003; Wang et al., 2003; Stoeckl et al., 2007 in press), may contribute to cellular and organismic ageing. Nevertheless, the available data still suggest a key role for ROS-induced damage in ageing. To address a potential role of ROS-induced protein damage in an unbiased way, we used a proteomics approach in the current study. Concerning the chemical nature of oxidative modifications, we focussed on the differential analysis of ROS-related posttranslational modifications, like N-formyl-kynurenine (Hunzinger et al. 2006) in redundant protein spots, like the ones demonstrated for putative SAM-dependent O-methyltransferase (PaMTH1), which was age-dependently carbonylated at distinct W-residues in P. anserina is homologous to catecholamine O-methyltransferase (COMT) in higher organisms (Averbeck et al. 2000) which in mammals is part of dopamine metabolism and has been shown to play key role in a variety of age-related human neurological disorders. By finding a COMT-homologue as being associated with mitochondria and modified by ROS-and age-related molecular changes, we support recent reports about impairments in mitochondrial functions contribute to the pathogenesis of Morbus Parkinson (Eriksen et al. 2005; Ding et al. 2006a; Jin et al. 2005). For the first time, we have indications linking dopamine-related metabolism and mitochondrial processes and it would be interesting to compare our proteomic results to agedependent mtDNA deletions causing functional impairment in aged human substantia nigra neurons and Parkinson disease (Bender et al. 2006; Kraytsberg et al. 2006).

  al. 2006c; Poon et al. 2006a; Sultana et al. 2006; Poon et al. 2005; Poon et al. 2006b; Kim et al. 2006). Our findings establish ATP synthase firmly as one of the prime mitochondrial targets of age-related molecular changes. Since the protein was uniformly affected in all models investigated in this study, we assume here a conserved mechanism of ageing represented by a "hot spot" of accumulating ROS-related damage. This finding suggests that functional inactivation of ATPase by posttranslational modification may directly contribute to the loss of intracellular ATP levels found in senescent human cells (Wang et al., 2003; Zwerschke et al., 2003; Stoeckl et al., 2006; 2007)(and possibly in aged tissue as well). Some of the differential proteins found in the HUVEC model and others in rat and P. anserina point to downstream consequences of ROS-related damage of ATP synthase on dopamine-and proteasome-related pathways. Moreover we found indications that the intrinsic mitochondrial pathway of apoptosis regulated via the mitochondrial transition pore (Deniaud et al. 2006;Schrattenholz and Soskic 2006;Soskic et al. 2007) is involved. The mitochondrial transition in turn regulates mitochondrial autophagy observed in ageing (Cavallini et al. 2007;Donati et al. 2006;Terman and Brunk 2006). ROS-effects from oxidative phosphorylation and ATP synthase upon reductive defence mechanisms and apoptosis (Piec et al. 2005;Soti and Csermely 2006;Deocaris et al. 2006) are in line with our finding of agedependence of cyclophilin D, a part of the mitochondrial permeability transition pore and support the free radical theory of ageing (Poon et al. 2006c;Poon et al. 2006a;Sultana et al. 2006;Poon et al. 2005;Poon et al. 2006b;Kim et al. 2006).

Figure 1 :

 1 Figure 1: Representative images of high resolution 2D gels of mitochondrial

Figure 2 :

 2 Figure 2: Schematic cartoon showing the design of the pooling paradigms for

Figure 3 :

 3 Figure 3: Dual false colour images of all redundant protein isoforms and subunits

  , details of the MALDI-TOF identification are given; both spots 6 and 7 come to the same protein identity. The more detailed MS-based investigation revealed an age-dependent difference of N-formylkynurenine modifications of tryptophane residues W-35 and W-37 of the protein. accumulating during senescence of Podospora anserina cultures. Curr Genet 37:200-208 4. Bailey S.M., Landar A., Darley-Usmar V. (2005) Mitochondrial proteomics in free radical research. Free Radic Biol Med 38:175-188 5. Barros, M. H., Bandy, B., Tahara, E. B. and Kowaltowski, A. J. (2004) Higher respiratory activity decreases mitochondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J Biol Chem 279, 49883-49888. 6. Basu S., Bremer E., Zhou C., Bogenhagen D.F. (2006) MiGenes: a searchable interspecies database of mitochondrial proteins curated using gene ontology annotation. Bioinformatics 22:485-492 14. Brewer J., Benghuzzi H., Tucci M. (2006) Effects of thymoquinone, lycopene, and selenomethione in the presence of estrogen on the viability of SiHa cells in vitro. Biomed Sci Instrum 42, 37-41 15. Brookes P.S., Pinner A., Ramachandran A., Coward L., Barnes S., Kim H., Darley-Usmar V.M. (2002) High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2, 969-977 16. Brookes P.S., Yoon Y., Robotham J.L., Anders M.W., Sheu S.S. (2004) Calcium, ATP, and ROS: a mitochondrial love-hate triangle. Am J Physiol Cell Physiol 287, C817-C833 17. Capri M., Salvioli S., Sevini F., Valensin S., Celani L., Monti D., Pawelec G., De B.G., Gonos E.S., Franceschi C. (2006) The genetics of human longevity. Ann N Y Acad Sci 1067, 252-263 18. Cavallini G., Donati A., Taddei M., Bergamini E. (2007) Evidence for Selective Mitochondrial Autophagy and Failure in Ageing. Autophagy 3, 26-27 19. Chan D.C. (2006) Mitochondria: dynamic organelles in disease, ageing, and development. Cell 125, 1241-1252 20. Cummings, D.J., Belcour, L., and Grandchamp, C. (1979) Mitochondrial DNA from Podospora anserina and the occurrence of multimeric circular DNA in senescent cultures. pp. 268-77. In: Engberg. J., et al., eds. Specific Eukaryotic. Genes. Copenhagen, Munksgaard. , 1979. W3. AL36. 13th. 1978. Saccharomyces cerevisiae show markers of oxidative stress and apoptosis. Mol Microbiol 39, 1166-1173 51. Lesnefsky E.J., Hoppel C.L. (2006) Oxidative phosphorylation and ageing. Ageing Res Rev 5, 402-433 52. Marin-Garcia J., Pi Y., Goldenthal M.J. (2006) Mitochondrial-nuclear Cross-talk in the Ageing and Failing Heart. Cardiovasc Drugs Ther. 20, 477-91 53. Marley K., Mooney D.T., Clark-Scannell G., Tong T.T., Watson J., Hagen T.M., Stevens J.F., Maier C.S. (2005) Mass tagging approach for mitochondrial thiol proteins. J Proteome Res 4, 1403-1412 54. McMahon K.A., Zhu M., Kwon S.W., Liu P., Zhao Y., Anderson R.G. (2006) Detergent-free caveolae proteome suggests an interaction with ER and mitochondria. Proteomics 6, 143-152 55. Miquel, J. (1991). An integrated theory of ageing as the result of mitochondrial-DNA mutation in differentiated cells. Arch Gerontol Geriatr 12, 99-117 56. Miquel, J., Binnard, R., and Fleming, J. E. (1983). Role of metabolic rate and DNA-repair in Drosophila ageing: implications for the mitochondrial mutation theory of ageing. Exp Gerontol 18, 167-171 57. Neubauer H., Clare S.E., Kurek R., Fehm T., Wallwiener D., Sotlar K., Nordheim A., Wozny W., Schwall G.P., Poznanovic S., Sastri C., Hunzinger C., Stegmann W., Schrattenholz A., Cahill M.A. (2006) Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection. Electrophoresis 27, 1840-1852 58. Osiewacz H.D. (2002) Ageing in fungi: role of mitochondria in Podospora anserina. Mech Ageing Dev 123, 755-764 59. Osiewacz H.D., Scheckhuber C.Q. (2006) Impact of ROS on ageing of two fungal model systems: Saccharomyces cerevisiae and Podospora anserina. Free Radic Res 40, 1350-1358 60. Osiewacz, H.D. (2002). Mitochondrial functions and ageing. Gene 286, 65-71 61. Osiewacz, H.D. and Esser, K. (1984). The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene. Curr. Genet. 8, 299-305 62. Osiewacz, H.D. and Hermanns, J. (1992). The role of mitochondrial DNA rearrangements in ageing and human diseases [see comments]. Ageing (Milano) 4, 273-286 63. Piec I., Listrat A., Alliot J., Chambon C., Taylor R.G., Bechet D. (2005) Differential proteome analysis of ageing in rat skeletal muscle. FASEB J 19, 1143-1145 64. Poon H.F., Calabrese V., Calvani M., Butterfield D.A. (2006a) Proteomics analyses of specific protein oxidation and protein expression in aged rat brain and its modulation by L-acetylcarnitine: insights into the mechanisms of action of this proposed therapeutic agent for CNS disorders associated with oxidative stress. Antioxid Redox Signal 8, 381-394 65. Poon H.F., Shepherd H.M., Reed T.T., Calabrese V., Stella A.M., Pennisi G., Cai J., Pierce W.M., Klein J.B., Butterfield D.A. (2006b) Proteomics analysis provides insight into caloric restriction mediated oxidation and expression of brain proteins associated with age-related impaired cellular processes: 71. Reinders J., Zahedi R.P., Pfanner N., Meisinger C., Sickmann A. (2006) Toward the complete yeast mitochondrial proteome: multidimensional separation techniques for mitochondrial proteomics. J Proteome Res 5, 1543-1554 72. Rickwood D., Hayes A. (1984) An evaluation of methods used to prepare yeast mitochondria for transcriptional studies. Prep Biochem 14, 163-171 73. Rottenberg H. (2006) Longevity and the evolution of the mitochondrial DNAcoded proteins in mammals. Mech Ageing Dev 127, 748-760 74. Ruiz-Romero C., Lopez-Armada M.J., Blanco F.J. (2006) Mitochondrial proteomic characterization of human normal articular chondrocytes. Osteoarthritis Cartilage 14, 507-518 75. Sanz A., Pamplona R., Barja G. (2006) Is the mitochondrial free radical theory of ageing intact? Antioxid Redox Signal 8, 582-599 76. Scheckhuber C.Q., Erjavec N., Tinazli A., Hamann A, Nystrom T., Osiewacz H.D. (2007) Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models. Nat Cell Biol 9, 99-105 77. Schieke S.M., Phillips D., McCoy J.P., Jr., Aponte A.M., Shen R.F., Balaban R.S., Finkel T. (2006) The mammalian target of rapamycin (mTOR) pathway regulates mitochondrial oxygen consumption and oxidative capacity. J Biol Chem 281, 27643-27652 78. Schrattenholz A., Groebe K. (2007) What does it need to be a biomarker? Relationships between resolution, differential quantification and statistical validation of protein surrogate biomarkers. Electrophoresis, May 22, Epub ahead of print 79. Schrattenholz A., Soskic V. (2006) NMDA receptors are not alone: dynamic regulation of NMDA receptor structure and function by neuregulins and transient cholesterol-rich membrane domains leads to disease-specific nuances of glutamate-signalling. Curr Top Med Chem 6, 663-686 80. Schulte, E., Kück, U., and Esser, K. (1988). Extrachromosomal mutants from Podospora anserina: Permanent vegetative growth in spite of multiple recombination events in the mitochondrial genome. Mol. Gen. Genet. 211, 342-349 81. Simat T.J., Steinhart H. (1998) Oxidation of Free Tryptophan and Tryptophan Residues in Peptides and Proteins. J Agric Food Chem 46, 490-498 82. Singh K.K. (2006) Mitochondrial damage checkpoint, ageing, and cancer. Ann N Y Acad Sci 1067, 182-190 83. Soskic V., Klemm M., Proikas-Cezanne T., Schwall GP., Poznanovic S., Stegmann W., Groebe K., Zengerling H., Schoepf R., Burnet M., Schrattenholz A. (2007) A connection between the mitochondrial permeability transition pore, autophagy and cerebral amyloidogenesis. submitted 84. Soti C., Csermely P. (2007) Ageing cellular networks: Chaperones as major participants. Exp Gerontol. 42, 113-139 84. Stahl, U., Lemke, P.A., Tudzynski, P., Kück, U. and Esser, K. (1978) Evidence for plasmid like DNA in a filamentous fungus, the ascomycete Podospora anserina. Mol. Gen. Genet. 162, 341-343 85. Stockl, P., Hutter, E., Zwerschke, W., and Jansen-Durr, P. (2006). Sustained inhibition of oxidative phosphorylation impairs cell proliferation and induces premature senescence in human fibroblasts. Exp Gerontol 41, 674-682 86. Stumpferl, S.W., Stephan, O. and Osiewacz, H.D. (2004). Impact of a disruption of a pathway delivering copper to mitochondria on Podospora anserina metabolism and life span. Eukaryotic Cell 3, 200-211 87. Sultana R., Boyd-Kimball D., Poon H.F., Cai J., Pierce W.M., Klein J.B., Merchant M., Markesbery W.R., Butterfield D.A. (2006) Redox proteomics identification of oxidized proteins in Alzheimer' s disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD. Neurobiol Ageing 27, 1564-1576 88. Takahashi R. and Goto S. (1987) Influence of dietary restriction on accumulation of heat-labile enzyme molecules in the liver and brain of mice, Arch. Biochem. Biophys. 257 200-206 89. Terman A., Brunk U.T. (2006) Oxidative stress, accumulation of biological ' garbage' , and ageing. Antioxid Redox Signal 8, 197-204 90. Trifunovic, A., Hansson, A., Wredenberg, A., Rovio, A. T., Dufour, E., Khvorostov, I., Spelbrink, J. N., Wibom, R., Jacobs, H. T., and Larsson, N. G. (2005). Somatic mtDNA mutations cause ageing phenotypes without affecting reactive oxygen species production. Proc Natl Acad Sci U S A 102, 17993-17998 91. Trifunovic, A., Wredenberg, A., Falkenberg, M., Spelbrink, J. N., Rovio, A. T., Bruder, C. E., Bohlooly, Y. M., Gidlof, S., Oldfors, A., Wibom, R., et al. (2004). Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417-423 92. Unterluggauer H., Hampel B., Zwerschke W., Jansen-Durr P. (2003) Senescence-associated cell death of human endothelial cells: the role of oxidative stress. Exp Gerontol 38, 1149-1160 93. Vijg, J. (2000). Somatic mutations and ageing: a re-evaluation. Mutat Res 447, 117-135 94. Vo T.D., Palsson B. (2007) Building the power house: Recent advances in mitochondrial through proteomics and systems biology. Am J Physiol Cell Physiol 292, C164-177 95. Vogt J.A., Hunzinger C., Schroer K., Holzer K., Bauer A., Schrattenholz A., Cahill M.A., Schillo S., Schwall G., Stegmann W., Albuszies G. (2005) Determination of fractional synthesis rates of mouse hepatic proteins via metabolic 13C-labeling, MALDI-TOF MS and analysis of relative isotopologue abundances using average masses. Anal Chem 77, 2034-2042 96. Wallace, D. C. (2001). A mitochondrial paradigm for degenerative diseases and ageing. Novartis Found Symp 235, 247-263; discussion 263-246 97. Wang, W., Yang, X., Lopez De Silanes, I., Carling, D., and Gorospe, M. (2003). Increased AMP: ATP ratio and AMP-activated kinase activity during cellular senescence linked to reduced HuR function. J Biol Chem 278, 27016-27023 98. Weinreb O., Drigues N., Sagi Y., Reznick A.Z., Amit T., Youdim M.B. (2007) The Application of Proteomics and Genomics to the Study of Age-Related Neurodegeneration and Neuroprotection. Antioxid Redox Signal 9, 169-179

Table 1 :

 1 Redundant posttranslational age-related isoforms from the different models in three species:

	Species	Description	Theoretical .	Experiment	Spot ID
			MW	pI	MW	pI
	Podospora	ATP synthase β chain,	60792 5.5	58000	4.6-4.7	904, 907, 917, 920,
	anserina	mitochondrial precursor			14800	4.87	2249
		ATP synthase subunit 4,	26458	9.2	25000	5.6	1192
		mitochondrial precursor	55802
		ATP synthase oligomycin	23573 9.6	24000	8.5	1555
		sensitivity conferral protein-
		like protein
		ATP synthase γ chain,	33019 8.9	32000	6.8	1337
		mitochondrial precursor
	Rattus	ATP synthase, mitochondrial	56318 5.0 53000, 33000	4.9, 4.8	1436, 1470, 1685, 1694,
	norvegicus	F1 complex, β subunit			59000, 43000	5.1, 5.1	1715, 2476,
	Homo	ATPase 5A1 protein	49119	9.2	57500, 56500	6.8, 7.0	760, 762, 1225, 1255
	sapiens	ATP synthase, H+	61456	9.2	56.500, 55000	6.85, 6.6
		transporting, mitochondrial
		F1 complex, α subunit,
		precursor
	Podospora	Putative SAM-dependent O-	27774 5.4	27000	5.2, 5.35	1079, 1092
	anserina	methyltransferase
		(PaMTH1)
	Homo	Reticulocalbin 1, precursor	38866		47500	4.6-4.8	xxx
	sapiens	Reticulocalbin 3	37470
		Reticulocalbin precursor	37401

Table S1 Juvenile versus senescent Podospora anserina (mitochondrial preparation) All accession numbers are from Podospora anserina if not stated otherwise Identification Quantification

 S1

	.1943841) Enoyl-CoA hydratase,	ps|29563	152 20387 6.2 28666 6.567 63.2	36.8 6.3754	0.0003
	putative Elongation factor 1-beta	ps|26538	67	25342 4.3 37000	4.3	36.5	63.5 8.0793 <0.0001
	Protein SC_J_chrm5.seq:55882..56 625 3-hydroxyacyl-CoA dehydrogenase, NAD binding domain (SC_A_chrm2.seq:1878728. Methylmalonate-(SC_C_chrm7.seq:1806496. [Aspergillus terreus .1807056) NIH2624]. semialdehyde Glycine cleavage system H (SC_C_chrm5.seq:162845.. dehydrogenase, protein 163892) mitochondrial precursor (SC_A_chrm2.seq:2279444. ATP synthase oligomycin (SC_B_chrm1.seq:2405475. .2280030) sensitivity conferral protein-.2407298) Acyl-CoA dehydrogenase ATP synthase subunit 4, like protein mitochondrial precursor (SC_B_chrm3.seq:2193212. (SC_D_chrm6.seq:109461.. (SC_E_chrm1.seq:1244142. .2194235) 111164) Flavohemoglobin .1245072) ATP synthase beta chain, Cytochrome c oxidase mitochondrial precursor (SC_A_chrm5.seq:647906.. polypeptide IV, (SC_B_chrm6.seq:781183.. 649171) Hypothetical protein (SC_E_chrm1.seq:962729.. 192859) [Neurospora crassa OR74A] mitochondrial precursor 783469) (SC_A_chrm7.seq:191738.. Vacuolar-ATPase	Accession Number ps|27328 ps|22964 ps|20766 ps|23086 ps|24700 ps|28197 ps|21763 ps|26217 ps|27916 ps|28599 gi|85106801 ps|21700	PMF score 71 151 35136 9.2 31000 Mass pI exp. mass 22181 9.6 16500 151 63908 8.3 63333 81 18719 4.8 18000 4.25 63.2 exp. pI Juv (%) 5.2 72.0 7.8 71.1 5.9 66.7 194 23573 9.6 22666 8.5 35.2 152 61265 8.2 50500 5.2 66.6 162 26458 9.2 24000 5.6 63.0 113 46652 5.3 50500 75 60792 5.5 15000 4.8 35.1 5.2 107 20630 6.2 19666 4.967 62.8 108 47942 7.9 50500 5.2 73 37859 9.1 22000 6.9 32.8	Sen (%) 28.0 14.433 <0.0001 StdDev (%) p-value 28.9 5.5467 <0.0001 33.3 8.2418 <0.0001 36.8 8.1601 <0.0001 64.8 7.0871 0.0002 37.0 7.9239 <0.0001 33.4 7.4067 <0.0001 64.9 6.8754 <0.0001 37.2 7.1631 <0.0001 67.2 7.7993 0.0002
	.1879998) Isocitrate dehydrogenase subunit 1, mitochondrial precursor (SC_B_chrm1.seq:1727818. .1729170) Acetyl-CoA acetyltransferase (SC_A_chrm6.seq:563339.. 564758) Ubiquinol-cytochrome-c reductase complex core protein 2, mitochondrial Hypothetical protein Ubiquinol-cytochrome-c 963867) reductase complex core ATP synthase gamma chain, MG09367.4 protein 2, mitochondrial mitochondrial precursor grisea 70-15] Malate dehydrogenase, precursor. (SC_C_chrm1.seq:218135.. (SC_B_chrm6.seq:411876.. 219388) mitochondrial precursor 413451) SC_D_chrm4.seq:604601..6 [Chaetomium globosum Isocitrate dehydrogenase 05847 CBS 148.51] Malate dehydrogenase, subunit 1, mitochondrial Hypothetical protein precursor CHGG_09308 [Chaetomium mitochondrial precursor (SC_B_chrm1.seq:1727818. globosum CBS 148.51] [Chaetomium globosum .1729170) CBS 148.51]. Acetyl-CoA SC_A_chrm2.seq:4588577.. (SC_B_chrm3.seq:251337.. acetyltransferase 4589920 251966) (SC_A_chrm6.seq:563339.. Aspartate aminotransferase, precursor. (SC_B_chrm6.seq:411876.. 413451) Acetyl-CoA acetyltransferase (SC_A_chrm6.seq:563339.. 564758) Isocitrate dehydrogenase subunit 1, mitochondrial precursor (SC_B_chrm1.seq:1727818. .1729170) ATP synthase beta chain, mitochondrial precursor (SC_B_chrm6.seq:781183.. 783469) Malate dehydrogenase, mitochondrial precursor (SC_B_chrm3.seq:251337.. 251966) Hypothetical protein [Neurospora crassa OR74A] Hypothetical protein MG09367.4 [Magnaporthe grisea 70-15] NADH-quinone oxidoreductase (SC_B_chrm1.seq:1943172. ATP synthase beta chain, 564758) mitochondrial precursor (SC_C_chrm3.seq:293121.. mitochondrial precursor Mitochondrial import 294666) (SC_B_chrm6.seq:781183.. receptor subunit TOM20 Vacuolar protease A 783469) Electron transfer flavoprotein (MOM19 protein) precursor (SC_A_chrm6.seq:652966.. (SC_F_chrm5.seq:579358.. subunit alpha 653662) 580684) (SC_A_chrm6.seq:473832.. 474884) Glycine cleavage system H protein (SC_A_chrm2.seq:2279444. .2280030) Glycine cleavage system H protein (SC_A_chrm2.seq:2279444. .2280030) Peptidylprolyl isomerase (SC_A_chrm2.seq:1458028. .1458541) ATP synthase beta chain, mitochondrial precursor (SC_B_chrm6.seq:781183.. 783469) Putative peroxiredoxin (SC_F_chrm5.seq:280296.. 280916) Cytochrome c subunit Vb SC_A_chrm2.seq:4588577.. 4589920 (SC_A_chrm7.seq:191738.. Nucleoside diphosphate 192859) ATP synthase beta chain, kinase (SC_A_chrm2.seq:3204186. mitochondrial precursor .3204993) (SC_B_chrm6.seq:781183.. Putative mitochondrial 783469) Heat shock protein SSC1, cyclophilin 1 (SC_B_chrm1.seq:1416610. mitochondrial precursor .1417548) (SC_A_chrm6.seq:904733.. Putative SAM-dependent O-906927) Vacuolar-ATPase methyltranferase Putative SAM-dependent O-(SC_E_chrm1.seq:962729.. methyltranferase. 963867) Ubiuqinol-cytochrome c (SC_A_chrm2.seq:2823633. .2824514) reductase complex core Manganese superoxide dismutase (SC_A_chrm5.seq:663911.. 664819) protein I Putative SAM-dependent O-(SC_B_chrm1.seq:1974671. methyltranferase .1976261) Heat shock protein SSC1, 906927) .2824514) (SC_A_chrm6.seq:904733.. (SC_A_chrm2.seq:2823633. mitochondrial precursor methyltranferase. Putative SAM-dependent O-	ps|20585 ps|27514 ps|27828 gi|39960255 ps|27828 ps|20945 gi|88181085 ps|25644 ps|20585 ps|24151 gi|88177826 ps|27514 ps|23766 ps|25020 ps|27514 ps|20585 ps|27916 ps|24151 gi|32407475 gi|39960255 ps|20639 ps|27916 ps|27547 ps|26947 ps|27491 ps|23086 ps|23086 ps|22843 ps|27916 ps|26871 ps|28599 ps|23766 ps|23368 ps|27916 ps|20492 ps|27612 gi|9968599 ps|21700 ps|23254 ps|20652 ps|26221 gi|9968599 ps|27612 ps|23254	211 43057 147 45657 8.9 44333 7.067 9 44333 7.067 71.1 110 46451 7.8 44333 7.067 131 35878 8.3 35000 7.55 66.3 202 46451 7.8 44666 6.967 61.5 119 33019 8.9 31000 6.85 32.4 120 35865 8.6 35000 7.55 184 42440 9.4 39666 6.4 32.0 170 43057 9 44666 6.967 117 33545 9 39666 6.4 115 22580 5.8 35000 7.55 96 45657 8.9 44666 6.967 67 42347 4.7 67500 4.6 202 43289 8.3 41333 6.933 29.5 197 45657 8.9 44333 7.167 70.4 135 43057 9 44333 7.167 122 60792 5.5 58333 4.6 67.8 124 22580 5.8 35000 7.5 67.2 110 36027 9.1 35000 7.5 110 35878 8.3 35000 7.5 126 21865 5.4 24333 5.4 66.8 249 60792 5.5 58000 4.633 66.2 81 20350 4.8 18000 4.8 61.4 113 43599 5 46666 4.5 28.9 213 36424 5.8 34000 5.1 65.8 98 18719 4.8 18000 4.4 65.7 84 18719 4.8 17500 4.4 65.2 121 15688 9.3 16666 5.367 64.9 282 60792 5.5 57666 4.7 64.2 125 20204 7.8 16666 5.367 63.9 111 20630 6.2 18333 4.967 59.8 72 42347 4.7 70000 4.6 28.9 153 17062 7.8 15666 7.167 28.6 310 60792 5.5 57333 4.7 59.4 122 26116 8.6 18333 5.6 23.5 106 72862 5.9 33333 4.9 40.9 140 27774 5.2 26666 5.333 18.4 85 37859 9.1 22000 6.6 37.7 90 28975 7 26666 5.333 251 48004 4.9 52666 5.067 37.2 155 25479 6.5 22666 5.5 63.6 131 27774 5.2 26666 5.2 17.6 74 72862 5.9 52666 5.067 102 28975 7 26666 5.2	28.9 33.7 10.942 8.04 <0.0001 38.5 9.166 0.0054 0.0020 67.6 6.7909 <0.0001 68.0 4.4705 <0.0001 70.5 19.133 0.0123 29.6 6.0058 <0.0001 32.2 7.945 <0.0001 32.8 7.2148 <0.0001 33.2 8.8648 <0.0001 33.8 6.0773 <0.0001 38.6 6.209 <0.0001 71.1 7.4027 <0.0001 34.2 7.0367 <0.0001 34.3 7.1411 <0.0001 34.8 7.15 <0.0001 35.1 6.2908 <0.0001 35.8 6.8307 <0.0001 71.1 7.0036 <0.0001 40.2 6.8626 <0.0001 71.4 6.0651 <0.0001 40.6 9.493 0.0013 76.5 6.5778 <0.0001 59.1 16.392 0.0427 81.6 8.41 <0.0001 62.3 6.6932 0.0006 62.8 10.405 0.0005 36.1 5.4021 <0.0001 36.4 7.1141 <0.0001 82.4 7.084 <0.0001

Table S2 Rat brain 5 months versus 17 months (mitochondrial preparation) All accession numbers are from Rattus norvegicus if not stated otherwise Identification Quantification

 S2

	Protein	Accession	PMF	Mass	pI	exp.	exp.	5Mo	17Mo	StdDev	p-value
		Number	score			mass	pI	(%)	(%)	(%)
	ATP synthase, H+	gi|54792127	275 56318	5	53000 4.9	63.2	36.8 6.3252 0.0006
	transporting, mitochondrial
	F1 complex, beta subunit
	Tubb5 protein	gi|38014544	117 24787 4.7 36000 4.95	37.2	62.8 2.5173 <0.0001
	Tubb2 protein [Mus	gi|13097483	87 34688 4.5 36000 4.95
	musculus]
	Tubulin beta chain 15 -rat	gi|92930	77 51322 4.5 36000 4.95
	Calretinin [Mus musculus]	gi|393387	116 22406 4.6 29000 4.8	40.1	59.9 5.8426 0.0020
	Calbindin 2	gi|16758892	100 31739 4.7 29000 4.8
	Neurofilament, light	gi|13929098	164 61475 4.3 61000 5.2	57.3	42.7 6.6337 0.0168
	polypeptide
	Tubulin alpha	gi|223556	130 52335 4.7 61000 5.2
	Gamma-actin [Mus	gi|809561	139 42055 5.6 50000 5.5	59.8	40.2 8.3588 0.0125
	musculus]
	Actin beta -rat	gi|71620	138 42786 5.2 50000 5.5
	COP9 signalosome subunit	gi|6753490	109 47141 5.6 48000 5.5	39.2	60.8 7.7569 0.0056
	4 [Mus musculus]
	Guanine nucleotide-binding	gi|204444	77 37040 5.3 38000 5.1	56.3	43.7 3.882 0.0026
	protein alpha subunit
	GTP-binding protein alpha o gi|8394152	75 41814 5.2 38000 5.1
	Tubulin beta chain 15 -rat	gi|92930	99 51322 4.5 39500 5.3	59.7	40.3 5.1443 0.0011
	Crystallin, mu	gi|56789444	98 34064 5.2 39500 5.3
	Tubulin alpha	gi|223556	71 52335 4.7 39500 5.3
	Eno1 protein	gi|50926833	83 48161 6.5 56000 6.1	57.4	42.6 1.6752 <0.0001
	Malate dehydrogenase,	gi|42476181	185 37077 8.9 36000 7.6	43.1	56.9 5.2035 0.0072
	mitochondrial
	Similar to hemoglobin alpha	gi|34870607	77 15806 8.5 15000 7.3	41.6	58.4 6.5707 0.0084
	chain

Table S3 Rat brain 5 months versus 31 months (mitochondrial preparation) All accession numbers are from Rattus norvegicus if not stated otherwise Identification Quantification

 S3

	Actin beta -rat	gi|71620	186 42786 5.2 51000 5.2
	COP9 signalosome subunit	gi|6753490	109 47141 5.6 48000 5.5	30.0	70.0 7.2891	0.0001
	4 [Mus musculus]
	Gamma-actin [Mus	gi|809561	80 42055 5.6 48000 5.3	65.4	34.6 5.5958	0.0001
	musculus]
	Actin beta -rat	gi|71620	79 42786 5.2 48000 5.3
	Isocitrate dehydrogenase 3	gi|16758446	171 41005 6.9 45000 5.5	57.9	42.1 8.619	0.0352
	(NAD+) alpha
	Protein ATP synthase, H+	Accession gi|54792127	PMF 80 56318 Mass	pI 5	exp. 43000 5.1 exp.	5Mo 58.2	31Mo 41.8 7.2202 StdDev	p-value 0.0145
	transporting, mitochondrial	Number	score		Mass	pI	(%)	(%)	(%)
	Ubiquilin 1 F1 complex, beta subunit Tubulin alpha Guanine nucleotide-binding Rab GDP dissociation protein alpha subunit GTP-binding protein alpha o gi|8394152 gi|48675852 gi|223556 gi|204444 gi|1707888 inhibitor alpha (Rab GDI Acidic ribosomal protein P0 gi|450370 alpha) (GDI-1) Tubulin beta chain 15 -rat gi|92930 Similar to Beta-soluble NSF gi|34859344 Unknown (protein for gi|50926985 attachment protein (SNAP-beta) (N-ethylmaleimide-MGC:94172) Type II cAMP-dependent gi|206671 sensitive factor attachment protein, beta) protein kinase regulatory Similar to heme-binding gi|34858543 subunit ATP synthase, H+ gi|54792127 protein Ubiquitin-conjugating gi|58477429 transporting, mitochondrial enzyme E2N (homologous F1 complex, beta subunit Tubb2 protein [Mus gi|13097483 to yeast UBC13) Cytochrome c oxidase gi|55971 musculus] Tubulin beta chain 15 -rat gi|92930 subunit Va preprotein Tubb5 protein gi|38014544 Cytochrome c oxidase, gi|21707954 Tubb2 protein [Mus gi|13097483 subunit Va [Mus musculus] Eno1 protein gi|50926833 musculus] tubulin beta chain 15 -rat gi|92930 Tyrosine 3/tryptophan 5 -gi|6981710 mitochondrial Malate dehydrogenase, gi|42476181	84 61975 4.6 74000 4.7 90 52335 4.7 63000 5 77 37040 5.3 38000 5.1 73 52275 4.8 63000 5 75 41814 5.2 38000 5.1 73 34725 6.2 40000 5.6 71 51322 4.5 63000 120 40357 6.1 37000 5.3 5 89 48314 4.5 61500 4.7 75 47202 4.6 61500 4.7 97 21465 5.1 25000 5.2 275 56318 5 53000 4.9 105 17290 6.5 16000 5.7 103 34688 4.5 38000 4.8 89 16828 6.5 15000 5.2 92 51322 4.5 38000 4.8 117 24787 4.7 36000 4.95 89 16800 6.5 15000 5.2 87 34688 4.5 36000 4.95 83 48161 6.5 56000 6.1 82 28725 4.5 30000 4.8 77 51322 4.5 36000 4.95 185 37077 8.9 36000 7.6	63.1 57.7 65.7 60.0 59.5 64.4 57.1 62.8 61.9 58.8 59.9 55.9 57.8 64.9 55.8	36.9 6.5456 42.3 8.556 34.3 5.1981 <0.0001 0.0110 0.0378 40.0 6.9207 0.0046 40.5 9.7324 0.0286 35.6 6.3856 0.0004 42.9 7.26 0.0279 37.2 7.8943 0.0025 38.1 6.8741 0.0017 41.2 10.578 0.0499 40.1 7.2557 0.0061 44.1 5.4041 0.0175 42.2 6.3626 0.0103 35.1 3.7409 <0.0001 44.2 5.6511 0.0236
	monooxygenase activation
	protein, eta polypeptide
	Hsc70-ps1	gi|56385	86 71593 5.2 80000	5	64.0	36.0 5.1318	0.0001
	Heat shock protein 8 [Mus	gi|55250073	86 71536 5.2 80000	5
	musculus]
	Internexin, alpha	gi|9506811	381 56613 4.9 73000 5.2	67.1	32.9 15.393	0.0161
	Tubulin alpha	gi|223556	140 52335 4.7 63000 5.1	60.4	39.6 6.248	0.0022
	Tubulin alpha	gi|223556	162 52335 4.7 63000 5.1	57.9	42.1 6.7756	0.0132
	Tubulin alpha	gi|223556	156 52335 4.7 63000 5.1	57.9	42.1 7.5551	0.0208
	Tubulin alpha	gi|223556	159 52335 4.7 64000 5.1	59.2	40.8 7.151	0.0082
	Tubulin beta chain 15 -rat	gi|92930	106 51322 4.5 64000 5.1
	Tubulin alpha	gi|223556	142 52335 4.7 64000 5.1	59.1	40.9 5.5095	0.0023
	Tubulin beta chain 15 -rat	gi|92930	84 51322 4.5 64000 5.1
	Neurofilament, light	gi|13929098	164 61475 4.3 61000 5.2	63.3	36.7 6.9854	0.0010
	polypeptide
	Tubulin alpha	gi|223556	130 52335 4.7 61000 5.2
	Tubulin alpha	gi|223556	155 52335 4.7 60000 5.1	61.3	38.7 4.5432	0.0002
	Enolase 2, gamma	gi|37805239	143 48350 4.8 51000	5	59.9	40.1 9.9273	0.0257
	Gamma-actin [Mus	gi|809561	139 42055 5.6 50000 5.5	61.0	39.0 7.4007	0.0039
	musculus]
	Actin beta -rat	gi|71620	138 42786 5.2 50000 5.5
	Gamma-actin [Mus	gi|809561	157 42055 5.6 49500 5.4	68.3	31.7 5.564 <0.0001
	musculus]
	Actin beta -rat	gi|71620	155 42786 5.2 49500 5.4
	Gamma-actin [Mus	gi|809561	187 42055 5.6 51000 5.2	60.4	39.6 11.566	0.0382
	musculus]

Table S4 Rat brain 17 months versus 31 months(mitochondrial preparation) All accession numbers are from Rattus norvegicus if not stated otherwise Identification Quantification

 S4

	Isocitrate dehydrogenase 3	gi|16758446	171 41005 6.9 45000 5.5	57.6	42.4 8.1177 0.0339
	(NAD+) alpha
	ATP synthase, H+	gi|54792127	80 56318	5	43000 5.1	60.0	40.0 8.1932 0.0105
	transporting, mitochondrial
	F1 complex, beta subunit
	GTP-binding protein alpha o gi|8394152	99 41814 5.2 42000	5	57.6	42.4 6.0174 0.0093
	ATP synthase, H+	gi|54792127	73 56318	5	42000	5
	transporting, mitochondrial
	Protein F1 complex, beta subunit	Accession	PMF	Mass	pI	exp.	exp.	17Mo	31Mo	StdDev	p-value
	Guanine nucleotide-binding	Number gi|204444	score 77 37040 5.3 38000 5.1 Mass pI	(%) 59.9	(%) 40.1 5.7678 0.0019 (%)
	Tubulin alpha protein alpha subunit Rab GDP dissociation inhibitor alpha (Rab GDI GTP-binding protein alpha o gi|8394152 gi|223556 gi|1707888 Acidic ribosomal protein P0 gi|450370	90 52335 4.7 63000 73 52275 4.8 63000 75 41814 5.2 38000 5.1 5 5 73 34725 6.2 40000 5.6	58.9 56.3	41.1 9.0029 0.0272 43.7 7.246 0.0447
	alpha) (GDI-1) Tubulin beta chain 15 -rat	gi|92930	99 51322 4.5 39500 5.3	43.2	56.8 5.3885 0.0089
	Tubulin beta chain 15 -rat Crystallin, mu	gi|92930 gi|56789444	71 51322 4.5 63000 98 34064 5.2 39500 5.3 5
	Unknown (protein for Tubulin alpha	gi|50926985 gi|223556	89 48314 4.5 61500 4.7 71 52335 4.7 39500 5.3	63.6	36.4 5.551 0.0002
	MGC:94172) Similar to Beta-soluble NSF	gi|34859344	120 40357 6.1 37000 5.3	60.5	39.5 7.8568 0.0070
	Type II cAMP-dependent attachment protein (SNAP-	gi|206671	75 47202 4.6 61500 4.7
	protein kinase regulatory beta) (N-ethylmaleimide-
	subunit sensitive factor attachment
	Tubb2 protein [Mus protein, beta)	gi|13097483	103 34688 4.5 38000 4.8	58.1	41.9 5.872 0.0060
	musculus] Gamma-actin [Mus	gi|809561	73 42055 5.6 34000 5.5	55.0	45.0 2.7393 0.0013
	Tubulin beta chain 15 -rat musculus]	gi|92930	92 51322 4.5 38000 4.8
	Tubb5 protein Actin beta -rat	gi|38014544 gi|71620	117 24787 4.7 36000 4.95 72 42786 5.2 34000 5.5	68.1	31.9 4.5623 <0.0001
	Tubb2 protein [Mus Ubiquitin-conjugating	gi|13097483 gi|58477429	87 34688 4.5 36000 4.95 105 17290 6.5 16000 5.7	59.7	40.3 5.0217 0.0009
	musculus] enzyme E2N (homologous
	Tubulin beta chain 15 -rat to yeast UBC13)	gi|92930	77 51322 4.5 36000 4.95
	Tyrosine 3/tryptophan 5 -Cytochrome c oxidase	gi|6981710 gi|55971	82 28725 4.5 30000 4.8 89 16828 6.5 15000 5.2	62.3 62.3	37.7 4.6227 0.0001 37.7 6.2369 0.0008
	monooxygenase activation subunit Va preprotein
	protein, eta polypeptide Cytochrome c oxidase,	gi|21707954	89 16800 6.5 15000 5.2
	Calretinin [Mus musculus] subunit Va [Mus musculus]	gi|393387	116 22406 4.6 29000 4.8	61.0	39.0 5.6596 0.0009
	Calbindin 2 Malate dehydrogenase,	gi|16758892 gi|42476181	100 31739 4.7 29000 4.8 185 37077 8.9 36000 7.6	62.7	37.3 5.0763 0.0002
	Vacuolar adenosine mitochondrial	gi|1184661	106 57788 5.5 69000 5.6	54.9	45.1 5.612 0.0418
	triphosphatase subunit B Similar to hemoglobin alpha	gi|34870607	77 15806 8.5 15000 7.3	62.7	37.3 4.3678 <0.0001
	Glucose regulated protein, chain	gi|38382858	87 58004 6.1 69000 5.6
	58 kDa
	Tubulin alpha	gi|223556	140 52335 4.7 63000 5.1	58.4	41.6 3.9629 0.0005
	Tubulin alpha	gi|223556	162 52335 4.7 63000 5.1	55.7	44.3 5.1568 0.0170
	Tubulin alpha	gi|223556	156 52335 4.7 63000 5.1	56.5	43.5 6.9693 0.0341
	Tubulin alpha	gi|223556	159 52335 4.7 64000 5.1	59.1	40.9 5.2161 0.0017
	Tubulin beta chain 15 -rat	gi|92930	106 51322 4.5 64000 5.1
	Tubulin alpha	gi|223556	142 52335 4.7 64000 5.1	55.8	44.2 6.6918 0.0444
	Tubulin beta chain 15 -rat	gi|92930	84 51322 4.5 64000 5.1
	Neurofilament, light	gi|13929098	164 61475 4.3 61000 5.2	56.4	43.6 3.2626 0.0009
	polypeptide
	Tubulin alpha	gi|223556	130 52335 4.7 61000 5.2
	Tubulin alpha	gi|223556	155 52335 4.7 60000 5.1	57.6	42.4 5.1025 0.0040
	Gamma-actin [Mus	gi|809561	157 42055 5.6 49500 5.4	67.4	32.6 6.1202 <0.0001
	musculus]
	Actin beta -rat	gi|71620	155 42786 5.2 49500 5.4
	Gamma-actin [Mus	gi|809561	187 42055 5.6 51000 5.2	59.8	40.2 8.5599 0.0143
	musculus]
	Actin beta -rat	gi|71620	186 42786 5.2 51000 5.2
	COP9 signalosome subunit	gi|6753490	109 47141 5.6 48000 5.5	39.8	60.2 4.1461 0.0002
	4 [Mus musculus]
	Gamma-actin [Mus	gi|809561	80 42055 5.6 48000 5.3	62.4	37.6 6.2746 0.0008
	musculus]
	Actin beta -rat	gi|71620	79 42786 5.2 48000 5.3

Table S5 Juvenile versus senescent HUVEC (mitochondrial preparation) All accession numbers are from Homo sapiens if not stated otherwise Identification Quantification

 S5

	Tropomyosin 4 Chaperonin Chain A, Cys302ser mutant PREDICTED: similar to N-(E.C.3.4.22.1) Annexin A2	gi|4507651 gi|41399285 gi|33357547 gi|114678095 gi|18645167	137 28504 4.4 31000 387 61548 5.5 99250 272 54394 5.8 82000 192 32179 5.4 42000 193 39260 0 18000	4.6 5.2 5.9 5.3 3.4	59.4 52.5 45.6	40.6 5.0676 <0.0001 47.5 4.4749 0.0198 54.4 4.9095 0.0006
	Tropomyosin 4 Heat shock protein 60 of human mitochondrial ethylmaleimide-sensitive Cathepsin B Chain A, Annexin A2: Does	gi|12803959 gi|77702086 gi|741376 gi|56966699	99 28907 4.4 31000 377 61706 5.7 99250 117 17143 5.5 30500 193 39346 0 18000	4.6 5.2 5.1 3.4
	Protein Tubulin, beta 6 Tubulin, beta polypeptide Tubulin, beta, 2 Tubulin, beta polypeptide TUBB protein Tubulin, beta 2 disulfide isomerase-related 5 Calumenin precursor Calumenin Crocalbin-like protein Reticulocalbin 3, EF-hand calcium binding domain Reticulocabin precursor RCN3 protein Reticulocalbin 3, EF-hand calcium binding domain RCN3 protein Reticulocabin precursor Reticulocalbin 3, EF-hand calcium binding domain RCN3 protein Reticulocabin precursor Reticulocalbin 1, precursor Reticulocalbin 1 precursor Proliferation-inducing gene 20 protein Reticulocalbin 1 precursor Reticulocalbin 1, precursor Reticulocalbin 3, EF-hand calcium binding domain Reticulocalbin 1, precursor Reticulocalbin 1 precursor Vimentin Proliferation-inducing gene 20 protein Laminin-binding protein Ribosomal protein SA Chain A, 14-3-3 Protein Epsilon (Human) Complexed To Peptide Tyrosine 3/tryptophan 5 -monooxygenase activation protein, epsilon polypeptide 14-3-3 protein epsilon isoform transcript variant 1 TPM4-ALK fusion oncoprotein type 2 Unknown HIRA interacting protein 5 isoform 1 PREDICTED: similar to myosin:SUBUNIT=regulatory light chain isoform 2 [Pan troglodytes] Human Complement Regulatory Protein Cd59 (Extracellular Region, Residues 1 -70) (Nmr, Restrained Minimized Average Structure) Human Complement Regulatory Protein Cd59 (Extracellular Region, Residues 1 -70) (Nmr, 10 Structures) Cd59 Complexed With Glcnac-Beta-1,4-(Fuc-Alpha-1,6)-Glcnac-Beta-1 (Nmr, 10 Structures) Transmembrane protein 4 Human Complement Regulatory Protein Cd59 (Extracellular Region, Residues 1 -70) (Nmr, 10 Structures) Human Complement Regulatory Protein Cd59 (Extracellular Region, Residues 1 -70) (Nmr, Restrained Minimized Average Structure) Human Complement Regulatory Protein Cd59 (Extracellular Region, Residues 1 -70) (Nmr, 10 Structures) CD59 antigen p18-20 Smooth muscle and non-muscle myosin alkali light chain, isoform 1 Myosin, light polypeptide 6, alkali, smooth muscle and non-muscle, isoform 2 MYL6 protein MYL6 protein Myosin, light chain 6, alkali, smooth muscle and non-muscle isoform 1 Motor protein Transmembrane protein IMMT Heat shock 70kDa protein 9B (mortalin-2) Chaperonin Heat shock protein 60 Chaperonin Chaperonin Chaperonin Heat shock protein 60 Protein disulfide isomerase-associated 3 precursor T-complex protein 1 isoform a T-complex polypeptide 1 T-complex protein 1 isoform b Vimentin Vimentin variant VIM Unnamed protein product G-rich RNA sequence binding factor 1 G-rich sequence factor-1 unnamed protein product G-rich RNA sequence binding factor 1 G-rich sequence factor 1 (GRSF-1) Chain H, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ Chain A, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ Chain H, Cys302ser mutant of human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mg2+ Chain A, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ Chain A, Cys302ser mutant of human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mg2+ Chain H, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ Chain A, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ aldehyde dehydrogenase factor attachment protein, Refined Crystal Structure Of It Induce Membrane complexed with NAD+ and alpha isoform 7 [Pan Aggregation By A New Human Procathepsin B At Mg2+ Chain H, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ troglodytes] Multimeric State Of The 2.5 Angstrom Resolution Chain A, Human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ Chain A, Cys302ser mutant of human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ Chain H, human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+ N-ethylmaleimide-sensitive Peroxiredoxin 3 Protein factor attachment protein, alpha N-ethylmaleimide-sensitive factor attachment protein, alpha HSPC124 Inorganic pyrophosphatase 2 isoform 1 precursor Pyrophosphatase HSPC124 Inorganic pyrophosphatase 2 isoform 1 precursor Peroxiredoxin 3 isoform b Mitochondrial ribosomal Peroxiredoxin 3, isoform a protein S22 VDAC2 precursor Peroxiredoxin 3 isoform a Voltage-dependent anion channel 2 precursor variant SPRY domain containing 4 Voltage-dependent anion Mitochondrial ribosomal channel VDAC2 -human Peroxisomal enoyl-protein L50 Mitochondrial ribosomal coenzyme A hydratase-like protein protein L50 PREDICTED: glutaminase C isoform 3 [Pan troglodytes] Enoyl Coenzyme A hydratase 1, peroxisomal Putative protein STRF8 Putative protein STRF8 Solute carrier family 25 (mitochondrial carrier\; phosphate carrier), member 24 Solute carrier family 25 member 24, isoform 1 Calcium-binding transporter gi|6841066 Accession Number gi|14210536 gi|57209813 gi|23958133 gi|57209813 gi|16198437 gi|4507729 gi|1710248 gi|4502551 gi|2809324 gi|8515718 gi|28626510 gi|9963785 gi|15488585 gi|28626510 gi|15488585 gi|9963785 gi|28626510 gi|15488585 gi|9963785 gi|14603330 gi|4506455 gi|41350407 gi|4506455 gi|14603330 gi|28626510 gi|14603330 gi|4506455 gi|62414289 gi|41350407 gi|34234 gi|31419811 gi|67464424 gi|5803225 gi|62131678 gi|10441386 gi|62822279 gi|50593025 gi|114672411 gi|515118 gi|515119 gi|640301 gi|7657176 gi|515119 gi|515118 gi|515119 gi|10835165 gi|16924329 gi|62531190 gi|113812151 gi|113812151 gi|17986258 gi|516764 gi|1160963 gi|48145703 gi|12653415 gi|31542947 gi|77702086 gi|41399285 gi|31542947 gi|31542947 gi|77702086 gi|21361657 gi|57863257 gi|36796 gi|57863259 gi|37852 gi|62896523 gi|47115317 gi|21748975 gi|53759145 gi|517196 gi|21748975 gi|53759145 gi|55977848 gi|6137684 gi|6137677 gi|33357604 gi|6137677 gi|33357547 gi|6137684 gi|6137677 gi|2982152 gi|6137684 gi|6137677 gi|33357547 gi|6137684 gi|47933379 gi|14250063 gi|54696006 gi|6841470 gi|29171702 gi|5931600 gi|6841470 gi|29171702 gi|32483377 gi|9910244 gi|14250063 gi|48146045 gi|62896877 gi|55664661 gi|46409324 gi|346412 gi|21265096 gi|70995211 gi|21618939 gi|114582297 gi|16924265 gi|41152530 gi|41152530 gi|55959506 gi|46249805 ACTB protein gi|15277503 Actin, beta gi|14250401 Actin, gamma 1 gi|54696574 Follistatin-like 1, precursor gi|12652619 Follistatin-like 1 precursor gi|5901956 Follistatin-like 1 [Bos taurus] gi|62988316 ACTB protein gi|15277503 Actin, beta gi|14250401 Actin, gamma 1 gi|54696574 Similar to heme oxygenase-2 isoform 2 [Pan troglodytes] gi|114660701 HMOX2 gi|48145637 Annexin A2 gi|18645167 Potassium channel tetramerisation domain containing 12 gi|15489330 ANXA2 protein gi|73909156 Annexin A2 gi|18645167 Protein phosphatase 1, catalytic subunit, alpha [Canis familiaris] gi|50978728 Protein phosphatase 1, catalytic subunit, alpha isoform 1 gi|4506003 Protein phosphatase 1, catalytic subunit, alpha isoform 3 gi|56790945 PPA2 protein gi|34980942 Glutaminase isoform C gi|6002671 Endoplasmic reticulum gi|75517652 Peroxisomal enoyl-coenzyme A hydratase-like protein gi|70995211 Peroxisomal enoyl-coenzyme A hydratase-like protein gi|15080016 Enoyl Coenzyme A hydratase 1, peroxisomal gi|16924265 PREDICTED: similar to prohibitin isoform 4 [Macaca mulatta] gi|109114264 PREDICTED: similar to prohibitin isoform 3 [Macaca mulatta] gi|109114262 Prohibitin gi|46360168 P2ECSL gi|11275665 Unnamed protein product gi|28317 Chloride intracellular channel 2 gi|66346733 PREDICTED: similar to Chloride intracellular channel 2 isoform 2 [Pan troglodytes] gi|114690746 PHB gi|49456373 DCI protein gi|16307101 Chain A, Crystal Structure Of Human Mitochondrial Delta3-Delta2-Enoyl-Coa Isomerase gi|60593479 Dodecenoyl-Coenzyme A delta isomerase (3,2 trans-enoyl-Coenzyme A isomerase) gi|12653937 PREDICTED: apolipoprotein A-I binding protein isoform 2 [Pan troglodytes] gi|114560275 APOA1BP protein gi|71681685 Apolipoprotein A-I binding protein precursor gi|91984773 Chain B, Cathepsin B gi|999909 Glutaminase C gi|5690372 protein 29, isoform 1 Succinate dehydrogenase gi|4759080 precursor Dihydropteridine reductase gi|118600 complex, subunit A, (HDHPR) (Quinoid flavoprotein precursor Succinate dehydrogenase gi|62087562 dihydropteridine reductase) Chain , Dihydropteridine gi|640447 complex, subunit A, Reductase (Dhpr) flavoprotein precursor (E.C.1.6.99.7) Complexed variant Glutaminase isoform C gi|6002671 With Nadh ATP synthase, H+ gi|24660110 Unnamed protein product gi|28071074 X-Ray Crystal Structure Of gi|3402185 transporting, mitochondrial The Human Galectin-3 F1 complex, alpha subunit, Carbohydrate Recognition precursor ATP synthase, H+ gi|4757810 Domain (Crd) At 2.1 Angstrom Resolution transporting, mitochondrial Chain A, Crystal Structure gi|66360056 F1 complex, alpha subunit Of The Human Galectin-3 precursor ATP5A1 protein gi|34782901 Crd In Complex With A 3' -Keratin, type I cytoskeletal 9 gi|81175178 Derivative Of N-Acetyllactosamine (Cytokeratin-9) (CK-9) Hydroxyacyl-Coenzyme A gi|83715985 (Keratin-9) (K9) keratin 1 gi|17318569 dehydrogenase, type II ATP5A1 protein gi|34782901 isoform 2 Chain A, Latent Form Of gi|10835819 Hydroxyacyl-Coenzyme A gi|4758504 dehydrogenase, type II Plasminogen Activator isoform 1 Inhibitor-1 (Pai-1) Chain A, Human gi|4699714 Endoplasmic reticulum-gi|20336350 associated amyloid beta Plasminogen Activator peptide-binding protein Inhibitor Type-1 In Complex RAB5C, member RAS gi|41393545 With A Pentapeptide Plasminogen activator gi|755747 oncogene family isoform b RAB5C, member RAS gi|41393545 inhibitor ACTB protein gi|15277503 oncogene family isoform b ALDOA gi|49456715 Rab5c-like protein, similar to gi|508285 Flotillin gi|6563242 Canis familiaris Rab5c Annexin A2 (Annexin II) gi|113950 protein, PIR Accession Number S38625 (Lipocortin II) (Calpactin I Superoxide dismutase [Mn], gi|38503334 heavy chain) (Chromobindin-mitochondrial 8) (p36) (Protein I) protein IV) (PAP-IV) Superoxide Dismutase (Placental anticoagulant Chain A, Human Manganese gi|2780818	PMF score 203 51242 4.5 60333 4.933 Mass pI exp. Mass exp. pI 159 48976 4.4 60333 4.933 150 51225 4.6 60333 4.933 181 47736 4.4 63000 4.9 152 30340 4.5 63000 4.9 146 49875 4.5 63000 4.9 167 46170 4.7 51333 5 100 37084 4.2 48000 4.5 89 37404 4.2 48000 4.5 79 34968 4.2 48000 4.5 164 37470 4.5 46500 4.6 148 37401 4.5 46500 4.6 112 37470 4.5 46500 4.6 146 37470 4.5 47333 4.6 131 37470 4.5 47333 4.6 130 37401 4.5 47333 4.6 238 37470 4.5 47000 4.633 189 37470 4.5 47000 4.633 172 37401 4.5 47000 4.633 231 38866 4.6 47000 4.667 178 38866 4.6 47000 4.667 128 19549 4.2 47000 4.667 172 38866 4.6 47333 4.7 154 38866 4.6 47333 4.7 118 37470 4.5 47333 4.7 92 38866 4.6 46000 4.7 108 38866 4.6 48000 4.75 103 53619 4.8 48000 4.75 87 19549 4.2 48000 4.75 148 32128 4.6 45500 4.8 147 33188 4.5 45500 4.8 120 26740 4.9 34666 4.6 117 29155 4.4 34666 4.6 104 27018 4.8 34666 4.6 98 27513 4.5 31000 4.6 100 22142 4 26000 4.3 99 25907 4.3 26000 4.3 79 17091 4.2 19000 4.5 75 8081 6.9 19000 4.1 107 9851 6.9 18000 4.1 105 10726 5 18000 4.1 100 21702 4.5 19000 4.8 105 9851 6.9 18000 4.1 112 8081 6.9 17333 4.5 96 9851 6.9 17333 4.5 92 14168 6.5 17333 4.5 231 17450 4.3 16000 4.433 217 17481 4.2 16000 4.433 120 15809 4.6 16000 4.433 118 15809 4.6 16000 4.5 117 16919 4.3 16000 4.5 262 80191 5.8 158000 5.733 258 84856 6.4 158000 5.733 246 83628 6.4 158000 5.733 116 73682 6.2 111666 5.4 324 61016 5.5 99250 5.2 418 61706 5.7 95000 5.225 414 61548 5.5 95000 5.225 316 61016 5.5 95000 5.225 113 61016 5.5 93000 5.45 113 61174 5.7 93000 5.45 96 57986 6.3 93000 5.45 166 61899 6 106000 5.75 166 61949 6.3 106000 5.75 143 44920 7.8 106000 5.75 415 53830 4.8 85333 5.1 397 53828 4.8 85333 5.1 290 53724 4.8 85333 5.1 231 48428 5.4 91000 5.2 231 49544 5.4 91000 5.2 214 49563 5.5 91000 5.2 164 48428 5.4 79250 5.2 164 49544 5.4 79250 5.2 150 51732 5.8 79250 5.2 167 55475 5.8 82500 5.5 167 53881 5.8 82500 5.5 166 55811 5.8 82500 5.5 327 53881 5.8 77500 5.75 326 54394 5.8 77500 5.75 276 55475 5.8 77500 5.75 273 53881 5.8 82000 5.9 75 37801 6.3 30500 5.1 193 55475 5.8 82000 5.9 302 53881 5.8 77500 5.75 301 54394 5.8 77500 5.75 189 33211 5 42000 5.3 60 27705 7.6 25000 5.6 167 34628 5 42000 5.3 156 36519 5.7 40500 5.8 155 37896 7.5 40500 5.8 73 32604 7.2 40500 5.8 151 36519 5.7 40000 5.933 137 37896 7.5 40000 5.933 180 26708 7.5 27000 5.85 109 41786 0 20000 3.1 178 28768 7.6 27000 5.85 112 31810 7.2 16500 3.7 112 31923 7.9 16500 3.7 166 28670 7.9 27000 5.85 172 23084 7 22000 5.2 111 33168 7.7 16500 3.7 153 18845 8.1 16500 5.875 76 36856 8.1 16250 3.25 81 18347 8.1 16500 5.875 169 67897 8.1 49500 4.7 76 36798 8.4 16250 3.25 234 55475 5.8 77500 5.75 125 42802 5.6 71000 5.35 175 42802 5.6 61333 5 289 54029 6.2 63666 5.733 277 54013 6.2 63666 5.733 240 46676 5.1 63666 5.733 133 41257 5.6 72000 5.1 118 42041 5.6 72000 5.1 117 42828 5.2 72000 5.1 204 38504 5.2 52333 5.2 184 34963 5.2 52333 5.2 170 34834 5.3 52333 5.2 73 41257 5.6 58000 5.9 72 42041 5.6 58000 5.9 71 42828 5.2 58000 5.9 147 32816 5.7 40000 5.4 144 35992 5.1 40000 5.4 124 38552 7.9 37000 5.4 78 36564 5.3 43500 5.3 78 40503 8.4 43500 5.3 63 38552 7.9 43500 5.3 195 37504 6.3 45666 5.833 192 37488 6.2 45666 5.833 162 41085 6.6 45666 5.833 145 60239 6.5 49500 4.7 127 29152 7.5 13500 3.3 101 35875 8.6 40000 5.933 163 35793 8.1 37333 5.933 150 36856 8.1 37333 5.933 146 35735 8.4 37333 5.933 219 28047 5.5 32250 5.3 216 29074 5.4 32250 5.3 204 29979 5.5 32250 5.3 106 24298 5.7 32000 5.55 70 60200 4.9 32000 5.55 163 28338 5.3 33500 5.4 144 24360 5.8 33500 5.4 98 29991 5.5 33500 5.4 127 26965 6.8 32000 5.933 125 28571 6.3 32000 5.933 118 32779 8.7 32000 5.933 144 20418 7 31500 5.3 126 21126 7 31500 5.3 124 31654 7.8 31500 5.3 127 22401 5 30500 5.1 143 67909 8 49500 4.7 122 75831 7.4 45333 4.133 68 26495 0 13500 3.3 122 76317 7.3 45333 4.133 68 26481 0 13500 3.3 73 60239 0 45333 4.133 285 61456 0 38000 4.533 88 14065 9.7 28000 7.6 86 15750 9.9 28000 7.6 282 60068 0 38000 4.533 84 16518 9.4 28000 7.6 223 49119 9.5 38000 4.533 113 62800 5 42000 4.85 101 26676 7.3 16666 4.967 110 66558 0 42000 4.85 82 49119 9.5 42000 4.85 132 42742 0 31666 4.433 99 27614 7.9 16666 4.967 91 21099 8.1 16666 4.967 101 42800 7.2 31666 4.433 63 24176 0 12000 3.9 83 43389 7.2 31666 4.433 101 24176 0 11500 4.1 105 41257 5.6 22250 3.975 180 40840 8.4 28333 5.267 99 24261 0 11500 4.1 62 28216 8.6 28333 5.267 193 39288 0 18000 3.4 121 22549 7.3 10750 3.425 121 22530 7.4 10750 3.425	Juv (%) 55.6 57.2 64.4 39.1 33.8 34.1 35.1 32.1 35.7 34.7 36.4 56.8 52.9 52.8 65.7 23.4 21.3 63.9 16.3 66.4 56.6 53.6 53.5 52.6 58.5 56.8 58.3 60.2 61.3 40.0 34.2 36.7 39.6 44.6 30.6 40.1 46.6 55.6 44.9 44.0 52.0 38.7 33.0 59.8 59.8 40.0 62.9 46.5 55.7 41.9 45.8 43.9 62.7 41.0 31.6 43.2 40.0 29.1 36.3 40.5 22.1 34.8 40.2 41.4 39.8 45.6 58.4 40.9 38.8 40.8 48.2 40.6 36.7	Sen (%) 44.4 7.4303 StdDev (%) 42.8 8.8069 35.6 8.6217 <0.0001 p-value 0.0024 0.0012 60.9 6.6477 <0.0001 66.2 8.6449 <0.0001 65.9 7.7465 <0.0001 64.9 7.1699 <0.0001 67.9 5.6503 <0.0001 64.3 5.9005 <0.0001 65.3 8.6589 <0.0001 63.6 8.6692 <0.0001 43.2 7.4033 0.0004 47.1 4.2196 0.0046 47.2 4.8803 0.0177 34.3 7.8525 <0.0001 76.6 8.8621 <0.0001 78.7 10.016 <0.0001 36.1 6.0685 <0.0001 83.7 14.212 <0.0001 33.6 7.037 <0.0001 43.4 6.4376 0.0001 46.4 6.9544 0.0284 46.5 5.9509 0.0132 47.4 5.6461 0.0459 41.5 8.4936 0.0002 43.2 12.932 0.0248 41.7 9.3182 0.0006 39.8 6.7373 <0.0001 38.7 7.3964 <0.0001 60.0 8.5166 <0.0001 65.8 8.458 <0.0001 63.3 9.6807 <0.0001 60.4 8.0352 <0.0001 55.4 4.8576 <0.0001 69.4 10.659 <0.0001 59.9 7.3374 <0.0001 53.4 6.7102 44.4 9.0574 0.0175 0.0300 55.1 8.5338 0.0212 56.0 5.3599 <0.0001 48.0 3.7915 0.0285 61.3 11.703 0.0007 67.0 19.735 0.0020 40.2 6.4415 <0.0001 40.2 11.523 0.0009 60.0 5.7997 <0.0001 37.1 12.049 <0.0001 53.5 6.2261 0.0191 44.3 8.716 0.0074 58.1 6.3795 <0.0001 54.2 5.7594 0.0032 56.1 6.9257 0.0007 37.3 10.087 <0.0001 59.0 13.563 0.0119 68.4 11.699 <0.0001 56.8 9.8958 0.0049 60.0 11.013 0.0005 70.9 13.388 <0.0001 63.7 9.5303 <0.0001 59.5 8.839 <0.0001 77.9 14.079 <0.0001 65.2 16.511 0.0011 59.8 11.233 58.6 9.3998 0.0012 0.0018 60.2 14.312 0.0076 54.4 7.0474 0.0177 41.6 11.957 0.0084 59.1 9.8391 0.0011 61.2 9.8909 0.0002 59.2 10.477 0.0017 51.8 3.5515 0.0484 59.4 15.065 0.0168 63.3 13.34 0.0006

Table S6 Juvenile versus tBHP-treated HUVEC (mitochondrial preparation) All accession numbers are from Homo sapiens if not stated otherwise Identification Quantification

 S6

	Glutathione peroxidase	gi|14717805	105 22818 6.5 29000	5.8
	Enolase 1	gi|4503571	121 48202 7.5 25000	3.3	39.2	60.8 0.7271	0.0304
	Enolase 1 variant	gi|62897945	121 48230 7.5 25000	3.3
	Enolase 1 variant	gi|62896593	121 48174 7.5 25000	3.3
	Citrate synthase precursor,	gi|38327627	107 45206 7.2 22750 3.65	35.8	64.2 1.2985	0.0411
	isoform b
	CS protein	gi|48257138	107 46118	7	22750 3.65
	Citrate synthase	gi|3288815	106 52559 8.2 22750 3.65
	Protein ACTB protein	Accession gi|15277503	PMF 65 41257 Mass	pI 0	exp. 20750 3.475 exp.	Poolt	PoolY	StdDev	p-value
	T255e, E376g Mutant Of Chain A, Structure Of	Number gi|2392312	score 70 44678	0	Mass 22500 3.35 pI	BHP (%) 41.6	(%) 58.4 0.3814 (%)	0.0203
	Tubulin, beta 6 Human Medium Chain Acyl-	gi|14210536	203 51242 4.5 60333 4.933	39.0	61.0 0.6173	0.0253
	Tubulin, beta polypeptide Coa Dehydrogenase	gi|57209813	159 48976 4.4 60333 4.933
	Tubulin, beta, 2 PREDICTED: acyl-Tubulin, beta polypeptide Coenzyme A TUBB protein dehydrogenase, C-4 to C-12 Tubulin, beta 2 straight chain [Pan Laminin-binding protein troglodytes] Acyl-Coenzyme A	gi|23958133 gi|114557331 gi|57209813 gi|16198437 gi|4507729 gi|34234 gi|4557231	68 51915 150 51225 4.6 60333 4.933 0 22500 3.35 181 47736 4.4 63000 4.9 152 30340 4.5 63000 4.9 146 49875 4.5 63000 4.9 148 32128 4.6 45500 4.8 68 47975 0 22500 3.35	33.0 40.5	67.0 1.5181 59.5 0.6006	0.0402 0.0284
	Ribosomal protein SA dehydrogenase, C-4 to C-12	gi|31419811	147 33188 4.5 45500	4.8
	Corneal endothelium straight chain	gi|20069113	80 25551 4.1 39000	4.2	31.3	68.7 1.9331	0.0465
	specific protein 1 Chain A, Human	gi|83755034	89 43347 7.8 21000	3.8	38.0	62.0 0.695	0.0260
	Ovary-specific acidic protein gi|12584947 Mitochondrial Acetoacetyl-	77 28734 4.2 39000	4.2
	Tropomyosin 4 Coa Thiolase	gi|4507651	137 28504 4.4 31000	4.6	34.7	65.3 0.3927	0.0116
	Tropomyosin 4 Mitochondrial ribosomal	gi|12803959 gi|9910244	99 28907 4.4 31000 109 41786 0 20000	4.6 3.1	33.8	66.2 0.5715	0.0159
	TPM4-ALK fusion oncoprotein type 2 protein S22 Torsin A	gi|10441386 gi|4557541	98 27513 4.5 31000 154 38847 7 17750 3.075 4.6	38.7	61.3 1.2518	0.0498
	Unknown Mitochondrial malate	gi|62822279 gi|89574129	100 22142 259 33365	4 0	26000 20600 5.04 4.3	37.7 37.4	62.3 0.3273 62.6 0.6478	0.0119 0.0231
	HIRA interacting protein 5 dehydrogenase 2, NAD	gi|50593025	99 25907 4.3 26000	4.3
	isoform 1 Unknown	gi|41472053	223 34624	0	20600 5.04
	Motor protein mitochondrial malate	gi|516764 gi|21735621	262 80191 5.8 158000 5.733 219 36897 0 20600 5.04	37.5	62.5 1.1014	0.0397
	Transmembrane protein dehydrogenase precursor	gi|1160963	258 84856 6.4 158000 5.733
	IMMT Porin 31HM [human,	gi|48145703 gi|238427	246 83628 6.4 158000 5.733 235 30977 0 15250 3.925	34.2	65.8 0.1883	0.0054
	Heat shock protein 60 skeletal muscle membranes,	gi|77702086	418 61706 5.7 95000 5.225	36.0	64.0 0.341	0.0109
	Chaperonin Peptide, 282 aa] Chaperonin Voltage-dependent anion Alpha-tubulin channel 1 PREDICTED: similar to 2,4-dienoyl CoA reductase 1 precursor Tubulin alpha-6 chain (Alpha-tubulin 6) (Alpha-Fracture callus 1 homolog	gi|41399285 gi|31542947 gi|4507879 gi|37492 gi|73996530 gi|4503301 gi|6912382	414 61548 5.5 95000 5.225 235 31108 0 15250 3.925 316 61016 5.5 95000 5.225 211 52251 4.8 74500 5.1 203 46042 5 74500 110 36930 0 15250 3.925 5.1 131 11579 7.5 15000 6.6	33.2 37.9	66.8 0.8983 62.1 0.2034	0.0241 0.0076
	tubulin isotype M-alpha-6)
	isoform 1 [Canis familiaris]
	PREDICTED: tubulin alpha	gi|114644943	202 46028	5	74500	5.1
	6 isoform 2 [Pan troglodytes]
	Unnamed protein product	gi|21748975	231 48428 5.4 91000	5.2	32.2	67.8 1.4282	0.0362
	G-rich RNA sequence	gi|53759145	231 49544 5.4 91000	5.2
	binding factor 1
	G-rich sequence factor-1	gi|517196	214 49563 5.5 91000	5.2
	Acyl-Coenzyme A	gi|15559225	179 48490	7	52000 5.733	39.0	61.0 0.6147	0.0251
	dehydrogenase,
	short/branched chain,
	precursor
	Hypothetical protein	gi|30268183	164 47975 7.2 52000 5.733
	Acyl-Coenzyme A	gi|4501859	159 47455	7	52000 5.733
	dehydrogenase,
	short/branched chain
	precursor
	Glutathione peroxidase 1,	gi|49522058	105 22960 6.5 29000	5.8	40.5	59.5 0.3182	0.0151
	isoform 1

This work was supported by EC FP6 Contract Nr. LSHM-CT-2004-512020;

(http://www.mimage.uni-frankfurt.de).

Mutant Q143n

Superoxide dismutase [