Portal de Periódicos da CAPES

Global Protein Oxidation Profiling Suggests Efficient Mitochondrial Proteome Homeostasis During Aging

2016; Elsevier BV; Volume: 15; Issue: 5 Linguagem: Inglês

10.1074/mcp.m115.055616

1535-9484

Carina Ramallo Guevara, Oliver Philipp, Andréa Hamann, Alexandra Werner, Heinz D. Osiewacz, Sascha Rexroth, Matthias Rögner, Ansgar Poetsch,

Cholinesterase and Neurodegenerative Diseases

The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age. The free radical theory of aging is based on the idea that reactive oxygen species (ROS) may lead to the accumulation of age-related protein oxidation. Because themajority of cellular ROS is generated at the respiratory electron transport chain, this study focuses on the mitochondrial proteome of the aging model Podospora anserina as target for ROS-induced damage. To ensure the detection of even low abundant modified peptides, separation by long gradient nLC-ESI-MS/MS and an appropriate statistical workflow for iTRAQ quantification was developed. Artificial protein oxidation was minimized by establishing gel-free sample preparation in the presence of reducing and iron-chelating agents. This first large scale, oxidative modification-centric study for P. anserina allowed the comprehensive quantification of 22 different oxidative amino acid modifications, and notably the quantitative comparison of oxidized and nonoxidized protein species. In total 2341 proteins were quantified. For 746 both protein species (unmodified and oxidatively modified) were detected and the modification sites determined. The data revealed that methionine residues are preferably oxidized. Further prominent identified modifications in decreasing order of occurrence were carbonylation as well as formation of N-formylkynurenine and pyrrolidinone. Interestingly, for the majority of proteins a positive correlation of changes in protein amount and oxidative damage were noticed, and a general decrease in protein amounts at late age. However, it was discovered that few proteins changed in oxidative damage in accordance with former reports. Our data suggest that P. anserina is efficiently capable to counteract ROS-induced protein damage during aging as long as protein de novo synthesis is functioning, ultimately leading to an overall constant relationship between damaged and undamaged protein species. These findings contradict a massive increase in protein oxidation during aging and rather suggest a protein damage homeostasis mechanism even at late age. Reactive oxygen species (ROS) 1The abbreviations used are:ROSreactive oxygen speciesFAformic acidFASPFilter-aided sample preparationFDRfalse discovery rateFwhmfull width at half maximumHCDhigher-energy collisional dissociationIAAIodoacetamideiTRAQisobaric tags for relative and absolute quantificationP. anserinaPodospora anserinaPSMpeptide spectrum matchPTMposttranslational modificationSRMselected reaction monitoringTEABtriethylammonium bicarbonate. are highly reactive intermediates leading to oxidative damage of virtually all biomolecules (1Halliwell B. Gutteridge J. The antioxidants of human extracellular fluids.Arch. Biochem. Biophys. 1990; 280: 1-8Crossref PubMed Scopus (1201) Google Scholar, 2D'Autreaux B. Toledano M.B. ROS as signalling molecules: mechanisms that generate specificity in ROS homeostasis.Nat. Rev. Mol. Cell Biol. 2007; 8: 813-824Crossref PubMed Scopus (2481) Google Scholar). Mitochondria are known as the main source of endogenous ROS, mainly generated as by-products of oxidative phosphorylation (OXPHOS) at complexes I and III of the respiratory electron transport chain (3Lanciano P. Khalfaoui-Hassani B. Selamoglu N. Ghelli A. Rugolo M. Daldal F. Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.Biochim. Biophys. Acta. 2013; 1827: 1332-1339Crossref PubMed Scopus (54) Google Scholar, 4Dröse S. Brandt U. Molecular mechanisms of superoxide production by the mitochondrial respiratory chain.Adv. Exp. Med. Biol. 2012; 748: 145-169Crossref PubMed Scopus (390) Google Scholar). Consequently, mitochondria are inevitably the most prominent target of ROS-induced damage (5Calabrese V. Scapagnini G. Giuffrida Stella A.M. Bates T.E. Clark J.B. Mitochondrial involvement in brain function and dysfunction: relevance to aging, neurodegenerative disorders and longevity.Neurochem. Res. 2001; 26: 739-764Crossref PubMed Scopus (230) Google Scholar). The accumulation of oxidatively damaged macromolecules, particularly proteins, has critical consequences, for example on mitochondrial structure and activity of the respiratory chain (6Lenaz G. Bovina C. D'Aurelio M. Fato R. Formiggini G. Genova M.L. Giuliano G. Pich M.M. Paolucci U.G.O. Castelli G.P. Role of mitochondria in oxidative stress and aging.Ann. N.Y. Acad. Sci. 2002; 959: 199-213Crossref PubMed Scopus (331) Google Scholar, 7Cui Z.J. Han Z.Q. Li Z.Y. Modulating protein activity and cellular function by methionine residue oxidation.Amino Acids. 2011; 43: 505-517Crossref PubMed Scopus (41) Google Scholar, 8Wang C.H. Wu S.B. Wu Y.T. Wei Y.H. Oxidative stress response elicited by mitochondrial dysfunction: implication in the pathophysiology of aging.Exp. Biol. Med. (Maywood). 2013; 238: 450-460Crossref PubMed Scopus (230) Google Scholar). Accordingly, ROS are involved in several diseases (9Jenner P. Oxidative stress in Parkinson's disease.Ann. Neurol. 2003; 53 (discussion S36–38): S26-36Crossref PubMed Scopus (1701) Google Scholar, 10Lin M.T. Beal M.F. Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases.Nature. 2006; 443: 787-795Crossref PubMed Scopus (4599) Google Scholar), and the free radical theory of aging postulates that the cumulative ROS-induced damage plays a causative role in aging (11Harman D. Aging: a theory based on free radical and radiation chemistry.J. Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6497) Google Scholar, 12Harman D. The biologic clock: the mitochondria?.J. Am. Geriatr. Soc. 1972; 20: 145-147Crossref PubMed Scopus (1531) Google Scholar). From the chemical and analytical point of view, oxidative damage of proteins is complex and leads to a variety of products, with the accumulation of irreversible oxidative protein modifications contributing to the development of disease (13Levine R.L. Carbonyl modified proteins in cellular regulation, aging, and disease.Free Radic. Biol. Med. 2002; 32: 790-796Crossref PubMed Scopus (562) Google Scholar, 14Cui H. Kong Y. Zhang H. Oxidative stress, mitochondrial dysfunction, and aging.J. Signal Transduct. 2012; 2012: 646354Crossref PubMed Google Scholar) and to aging (12Harman D. The biologic clock: the mitochondria?.J. Am. Geriatr. Soc. 1972; 20: 145-147Crossref PubMed Scopus (1531) Google Scholar, 15Harman D. Free radical theory of aging.Mutat. Res. 1992; 275: 257-266Crossref PubMed Scopus (643) Google Scholar). reactive oxygen species formic acid Filter-aided sample preparation false discovery rate full width at half maximum higher-energy collisional dissociation Iodoacetamide isobaric tags for relative and absolute quantification Podospora anserina peptide spectrum match posttranslational modification selected reaction monitoring triethylammonium bicarbonate. Although ROS inflicted damage has frequently been reported for higher organisms and humans (16Beal M.F. Aging, energy, and oxidative stress in neurodegenerative diseases.Ann. Neurol. 1995; 38: 357-366Crossref PubMed Scopus (1261) Google Scholar, 17Groebe K. Krause F. Kunstmann B. Unterluggauer H. Reifschneider N.H. Scheckhuber C.Q. Sastri C. Stegmann W. Wozny W. Schwall G.P. Poznanović S. Dencher N.A. Jansen-Dürr P. Osiewacz H.D. Schrattenholz A. Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes.Exp. Gerontol. 2007; 42: 887-898Crossref PubMed Scopus (77) Google Scholar), their long lifespan and laborious molecular manipulation has drawn attention toward alternative model systems such as Podospora anserina. This fungus is a well-established model system in aging research because of its senescence syndrome and a short lifespan of ∼25 days (18Osiewacz H.D. Aging in fungi: role of mitochondria in Podospora anserina.Mech. Ageing Dev. 2002; 123: 755-764Crossref PubMed Scopus (59) Google Scholar, 19Osiewacz H.D. Mitochondrial functions and aging.Gene. 2002; 286: 65-71Crossref PubMed Scopus (58) Google Scholar, 20Lorin S. Dufour E. Sainsard-Chanet A. Mitochondrial metabolism and aging in the filamentous fungus Podospora anserina.Biochim. Biophys. Acta. 2006; 1757: 604-610Crossref PubMed Scopus (63) Google Scholar, 21Osiewacz H.D. Hamann A. Zintel S. Assessing organismal aging in the filamentous fungus Podospora anserina.Methods Mol. Biol. 2013; 965: 439-462Crossref PubMed Scopus (42) Google Scholar). Importantly, for P. anserina, it has been shown that in mitochondria the generation of ROS at the electron transport chain increases with age: For instance, the age-related accumulation of endogenous hydrogen peroxide is indicated by increased secretion of this ROS from old individuals. Deletion of a gene encoding for a mitochondrial fission factor leads to a strong increase in the healthy lifespan and goes along with a delay in hydrogen peroxide release in comparison to the wild type (22Scheckhuber C.Q. Erjavec N. Tinazli A. Hamann A. Nyström T. Osiewacz H.D. Reducing mitochondrial fission results in increased life span and fitness of two fungal ageing models.Nat. Cell Biol. 2007; 9: 99-105Crossref PubMed Scopus (267) Google Scholar). The mutation of a nuclear gene, Grisea, coding for a transcription factor that is involved in the control of high affinity copper uptake leads to a switch from a copper-dependent standard respiration to an iron-dependent alternative respiration. This switch, because of a loss of respiratory complex III, a major generator of superoxide anion, leads to a decreased generation of this ROS (23Gredilla R. Grief J. Osiewacz H.D. Mitochondrial free radical generation and lifespan control in the fungal aging model Podospora anserina.Exp. Gerontol. 2006; 41: 439-447Crossref PubMed Scopus (65) Google Scholar). Although carbonylation of proteins visualized by the commonly used Western blot technique did not identify a prominent age-related change of carbonylated proteins in P. anserina (24Luce K. Osiewacz H.D. Increasing organismal healthspan by enhancing mitochondrial protein quality control.Nat. Cell Biol. 2009; 11: 852-858Crossref PubMed Scopus (113) Google Scholar), a strong decrease of carbonylated proteins was found in a strain in which a gene was overexpressed that encodes a mitochondrial methyltransferase which protects against ROS generation. The healthy lifespan of the corresponding transgenic strain was increased by 115% compared with that of the wild type (25Kunstmann B. Osiewacz H.D. Over-expression of an S-adenosylmethionine-dependent methyltransferase leads to an extended lifespan of Podospora anserina without impairments in vital functions.Aging Cell. 2008; 7: 651-662Crossref PubMed Scopus (46) Google Scholar). So far, a proteomic view to understand the effects of ROS-induced protein damage, such as carbonylation, in molecular detail during aging is missing. Because of its high resolution power of (modified) proteins, 2D-electrophoresis has successfully been applied for the identification and quantification of oxidative protein modifications on the proteome level (26Bakala H. Ladouce R. Baraibar M.A. Friguet B. Differential expression and glycative damage affect specific mitochondrial proteins with aging in rat liver.Biochim. Biophys. Acta. 2013; 1832: 2057-2067Crossref PubMed Scopus (28) Google Scholar). Carbonylated proteins were detected with fluorophore-labeling, differential ProteoTope radioactive quantification (17Groebe K. Krause F. Kunstmann B. Unterluggauer H. Reifschneider N.H. Scheckhuber C.Q. Sastri C. Stegmann W. Wozny W. Schwall G.P. Poznanović S. Dencher N.A. Jansen-Dürr P. Osiewacz H.D. Schrattenholz A. Differential proteomic profiling of mitochondria from Podospora anserina, rat and human reveals distinct patterns of age-related oxidative changes.Exp. Gerontol. 2007; 42: 887-898Crossref PubMed Scopus (77) Google Scholar, 27Chaudhuri A.R. de Waal E.M. Pierce A. Van Remmen H. Ward W.F. Richardson A. Detection of protein carbonyls in aging liver tissue: a fluorescence-based proteomic approach.Mech. Ageing Dev. 2006; 127: 849-861Crossref PubMed Scopus (130) Google Scholar), and the immunochemical detection technique (25Kunstmann B. Osiewacz H.D. Over-expression of an S-adenosylmethionine-dependent methyltransferase leads to an extended lifespan of Podospora anserina without impairments in vital functions.Aging Cell. 2008; 7: 651-662Crossref PubMed Scopus (46) Google Scholar, 28Aksenov M.Y. Aksenova M.V. Butterfield D.A. Geddes J.W. Markesbery W.R. Protein oxidation in the brain in Alzheimer's disease.Neuroscience. 2001; 103: 373-383Crossref PubMed Scopus (437) Google Scholar, 29Kunstmann B. Osiewacz H.D. The S-adenosylmethionine dependent O-methyltransferase PaMTH1: a longevity assurance factor protecting Podospora anserina against oxidative stress.Aging. 2009; 1: 328-334Crossref PubMed Google Scholar, 30Surco-Laos F. Cabello J. Gómez-Orte E. González-Manzano S. González-Paramás A.M. Santos-Buelga C. Dueñas M. Effects of O-methylated metabolites of quercetin on oxidative stress, thermotolerance, lifespan and bioavailability on Caenorhabditis elegans.Food Funct. 2011; 2: 445-456Crossref PubMed Scopus (54) Google Scholar). Despite their popularity, antibody and 2D gel-based methods for identification and quantification of oxidative protein damage have well-known limitations, such as under-representation of certain protein categories, limited dynamic range and comigration of proteins (31Sheehan D. McDonagh B. Bárcena J.A. Redox proteomics.Expert Rev. Proteomics. 2010; 7: 1-4Crossref PubMed Scopus (38) Google Scholar, 32Törnvall U. Pinpointing oxidative modifications in proteins—recent advances in analytical methods.Anal. Methods. 2010; 2: 1638-1650Crossref Scopus (27) Google Scholar, 33Fedorova M. Bollineni R.C. Hoffmann R. Protein carbonylation as a major hallmark of oxidative damage: update of analytical strategies: protein carbonylation.Mass Spectrom. Rev. 2014; 33: 79-97Crossref PubMed Scopus (311) Google Scholar). In particular, these detection techniques allow only the analysis of one specific protein modification at a time. Furthermore, observed oxidative protein modifications by gel electrophoresis have to be interpreted with caution, because proteins can undergo artificial oxidation in polyacrylamide gels (34Froelich J.M. Reid G.E. The origin and control of ex vivo oxidative peptide modifications prior to mass spectrometry analysis.Proteomics. 2008; 8: 1334-1345Crossref PubMed Scopus (35) Google Scholar, 35Perdivara I. Deterding L.J. Przybylski M. Tomer K.B. Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: chemical artifact or post-translational modification?.J. Am. Soc. Mass Spectrom. 2010; 21: 1114-1117Crossref PubMed Scopus (78) Google Scholar). Because oxidation results in a specific mass shift, it can be precisely pinpointed with tandem mass spectrometry of intact proteins or their proteolytic digest (36Møller I.M. Rogowska-Wrzesinska A. Rao R.S.P. Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective.J. Proteomics. 2011; 74: 2228-2242Crossref PubMed Scopus (186) Google Scholar). Hence, gel-free mass spectrometry analyses with previous enrichment step of low-abundant oxidized peptides have been developed (37Meany D.L. Xie H. Thompson L.V. Arriaga E.A. Griffin T.J. Identification of carbonylated proteins from enriched rat skeletal muscle mitochondria using affinity chromatography-stable isotope labeling and tandem mass spectrometry.Proteomics. 2007; 7: 1150-1163Crossref PubMed Scopus (108) Google Scholar). However, they are limited to only a few selected protein oxidations (38Mann M. Jensen O.N. Proteomic analysis of post-translational modifications.Nat. Biotechnol. 2003; 21: 255-261Crossref PubMed Scopus (1629) Google Scholar, 39Baraibar M.A. Ladouce R. Friguet B. Proteomic quantification and identification of carbonylated proteins upon oxidative stress and during cellular aging.J. Proteomics. 2013; 92: 63-70Crossref PubMed Scopus (87) Google Scholar). In order to perform a large-scale, unbiased temporal analysis of prominent ROS-induced protein oxidations, we developed a gel-free quantitative proteomic workflow using chemical labeling of peptides with iTRAQ reagents (40Ross P.L. 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 (3680) Google Scholar, 41Evans C. Noirel J. Ow S.Y. Salim M. Pereira-Medrano A.G. Couto N. Pandhal J. Smith D. Pham T.K. Karunakaran E. Zou X. Biggs C.A. Wright P.C. An insight into iTRAQ: where do we stand now?.Anal. Bioanal. Chem. 2012; 404: 1011-1027Crossref PubMed Scopus (242) Google Scholar). This enables parallel quantification of protein species from mitochondria at different age stages; also, a beforehand data analysis with a novel statistical framework allows an interpretation and comparison of temporal trends of both oxidized and nonoxidized protein species to verify proteome homeostasis during increasing ROS exposure with age. The cultivation of the wild-type strain s and the isolation of crude mitochondrial fractions from P. anserina were performed as previously described (42Rexroth S. Poetsch A. Rögner M. Hamann A. Werner A. Osiewacz H.D. Schäfer E.R. Seelert H. Dencher N.A. Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.Biochim. Biophys. Acta. 2012; 1817: 381-387Crossref PubMed Scopus (37) Google Scholar). Cultures of six individuals serving as biological replicates were harvested at four different age stages (6 days, 9 days, 13 days and 16 days) resulting in a total of 24 mitochondrial samples. The mitochondrial samples were processed according to the in-filter protein digestion (FASP II) procedure described by (43Wiśniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5042) Google Scholar) with minor modifications. FASP combines the advantages of in-gel and in-solution digestion for mass spectrometry-based proteomics and enables the solubilization of crude mitochondria with SDS and detergent removal prior to LC-MS analysis. Because we focus on the mitochondrial proteome a solubilization step is inevitable for high protein coverage. Fifty micrograms of crude mitochondria were denatured and solubilized in SDT-lysis buffer by sonication for 3 min and subsequent shaking at RT for 30 min. Next, samples were mixed with 8 m urea and prepared using the FASP II protocol. All solutions for FASP II included additionally 1 mm DTT and 1 mm EDTA to avoid artificial oxidation during sample processing. In addition, triethylammonium bicarbonate (TEAB) was used as a tertiary amine buffer instead of ammonium bicarbonate. After centrifugation the resulting peptides were acidified with 50% (v/v) ACN in 0.5% (v/v) TFA and dried using a SpeedVac. Digested and carbamidomethylated samples were labeled with iTRAQ 4plex tags according to the manufacturer's protocol. Deviant from manufacturer's protocol, peptide samples were labeled with half the amount of iTRAQ reagent after tryptic digestion for multiplexing all age stages from one of each biological replicate. Briefly, each iTRAQ reagent was dissolved in 70 μl ethanol and the aliquots of iTRAQ 114, 115, 116, and 117 were combined with the peptide mixtures of mitochondria from four different age stages (6, 9, 13 and 16 days), respectively. Consequently, for each experiment 35 μl of reagent solution was used for 50 μg of sample, which was resuspended in 25 μl iTRAQ dissolution buffer (0.5 m TEAB). After 1.5 h of incubation at RT, labeling reaction was stopped with 10 mm glycine. The differently-labeled samples of all 4 age stages from one individual were pooled and dried again in a SpeedVac. Prior to nLC-MS analysis all samples were desalted by solid-phase extraction using Spec C18AR pipette tips according to the manufacturer's protocol. To address any potential label bias, labels for all age stages of one biological replicate were swapped, too. The assignment of label tags to samples is summarized in supplemental Table S1. All desalted samples were resuspended in 2% ACN in 0.1% FA (1 μg/μl) by sonication for 10 min prior to one-dimensional nLC-ESI-MS/MS analysis. Measurements were performed on a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA) coupled to a nanoACQUITY gradient UPLC pump system combined with an autosampler (all Waters, Milford, MA). The nanoACQUITY UPLC system was equipped with a reversed phase UPLC HSS T3 column (1.8 μm, 75 μm x 250 mm, Waters) and a PicoTip Emitter (Silica TipTM, 10 μm i.d, New Objective, Woburn, MA) as a nanospray source. Four microliters of the sample were loaded directly onto the analytical column using the nanoACQUITY autosampler with 99% buffer A (0.1% FA) and 1% buffer B (0.1% FA in ACN) for 60 min. Peptides were eluted by a multiple step gradient of buffer A and B at a flow rate of 300 nL/min in 270 min. Subsequently the concentration of buffer B was increased to 3% within 20 min, followed by a linear gradient to 30% buffer B in 225 min (60–80 min: 3% buffer B; 80–200 min: 13% buffer B; 200–260 min: 20% buffer B; 260–305 min: 30% buffer B). To elute all peptides from the column, the concentration of buffer B was raised to 99% in 20 min and kept constant for 5 min before the column was re-equilibrated at 1% buffer B within 40 min. The column oven was set to 45 °C and the heated desolvation capillary was set to 275 °C. Data-dependent acquisition on the LTQ Orbitrap Velos was operated via instrument method files of Xcalibur (Rev. 2.1.0) in positive ion mode (91Olsen J.V. de Godoy L.M. Li G. Macek B. Mortensen P. Pesch R. Makarov A. Lange O. Horning S. Mann M. Parts per million mass accuracy on an Orbitrap mass spectrometer via lock mass injection into a C-trap.Mol Cell Proteomics. 2005; 4: 2010-2021Abstract Full Text Full Text PDF PubMed Scopus (1241) Google Scholar) at a spray voltage of 1.3–1.8 kV. The full MS scan was performed in the Orbitrap in a range of 400–1400 m/z at a resolution of 60.000 using polysiloxane (m/z 445.120024) for internal lock mass calibration (Olsen et al., 2005). The ten most intense precursors per cycle were isolated and fragmented consecutively with CID and HCD. For peptide identification, CID was performed with relative collision energy of 35%, an isolation width of 1.5 Th and an activation time of 10 ms before analysis at normal scan rate and mass range in the linear ion trap. For reporter ion quantification, HCD fragmentation spectra were acquired with normalized collision energy of 65%, an isolation width of 1.2 Th and an activation time of 0.1 ms at a resolution of 7500. Dynamic exclusion was enabled with a repeat count of four and a 90 s exclusion duration window. Unassigned charge states, singly and more than triply charged ions were rejected from MS/MS. For each biological replicate three technical replicates were performed with following MS-settings in Xcalibur: 1. Top10, 2. Bottom10, and 3. Top10 with exclusion list which contained both the retention times and the m/z of previously identified peptides to be excluded for MS/MS. Furthermore, Top10 method meant that each of the ten most intense peaks in a full scan were fragmented in the order of highest to lowest intensity, whereas fragmentation order was from lowest to highest in the Bottom 10 method. MS data search was performed against a P. anserina protein database (version 6.32) from http://podospora.igmors.u-psud.fr/, containing 10612 sequences (44Espagne E. Lespinet O. Malagnac F. Da Silva C. Jaillon O. Porcel B.M. Couloux A. Aury J.-M. Ségurens B. Poulain J. The genome sequence of the model ascomycete fungus Podospora anserina.Genome Biol. 2008; 9: R77Crossref PubMed Scopus (240) Google Scholar), using the SEQUEST algorithm embedded in ProteomeDiscoverer 1.3.0.339 (Thermo Electron © 2008–2011). All acquired raw-files from three technical replicates of one biological replicate were combined in one data search, for which following search parameters were applied: (1) fully tryptic as enzyme specificity, (2) a maximum of two missed cleavages, (3) precursor ion mass tolerance of 5 ppm, (4) fragment ions mass tolerance of 1 Da, (5) Carbamidomethylation of Cys and (6) iTRAQ4plex(N-term) as fixed as well as (7) iTRAQ4plex(K,Y) as variable modifications. The S/N threshold of Peak Filters in the Orbitrap was set to 3. Of note, database searchesfor suspected protein modifications are limited in the ProteomeDiscoverer 1.3.0.339 to four. Therefore, database searches had to be sequentially conducted considering different oxidative amino acid modifications as fixed modification to allow detection of all possible posttranslational modifications (PTMs). All searched oxidative modifications are specified in Table I. Protein quantification is performed with the Reporter Ions Quantifier tool embedded in the ProteomeDiscoverer software. Settings were kept at the default values: (1) integration window tolerance of 20 ppm, (2) integration method of most confident centroid, (3) mass analyzer is the Orbitrap with (4) MS2 order, and (5) HCD as activation type. Reporter based quantification is normalized by the protein ratio median. The factor normalizes all peptide ratios by the median protein ratio. Additionally, to minimize unwanted quantification of co-isolated peptides the allowed relative isolation interference was set to < 20% of precursor signal intensity. For the determination of the false discovery rate (FDR) a decoy database search was performed with the percolator validation in Proteome Discoverer. The q-value is the minimal FDR at which the identification is considered correct and was set to 1%. The q-values are estimated using the distribution of scores from the decoy database search (45Käll L. Storey J.D. MacCoss M.J. Noble W.S. Posterior error probabilities and false discovery rates: two sides of the same coin.J. Proteome Res. 2008; 7: 40-44Crossref PubMed Scopus (210) Google Scholar). For data analysis the mass spec format-(msf)-files were filtered with peptide confidence "high" and one peptide per protein with peptide rank 1. Protein or peptide grouping were disabled to achieve the highest number of protein identifications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://www.proteomexchange.org) via the PRIDE partner repository (46Vizcaíno J.A. Deutsch E.W. Wang R. Csordas A. Reisinger F. Ríos D. Dianes J.A. Sun Z. Farrah T. Bandeira N. Binz P.-A. Xenarios I. Eisenacher M. Mayer G. Gatto L. Campos A. Chalkley R.J. Kraus H.-J. Albar J.P. Martinez-Bartolomé S. Apweiler R. Omenn G.S. Martens L. Jones A.R. Hermjakob H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination.Nat. Biotechnol. 2014; 32: 223-226Crossref PubMed Scopus (2070) Google Scholar) with the dataset identifier PXD001023.Table IAll examined single amino acid modifications induced by oxidative damage. Molecular formula and monoisotopic mass shift of the difference between the native amino acid and the oxidized product are given. Based on protein modifications for mass spectrometry (www.unimod.org)Amino acidProductMolecular formulaMonoisotopic mass shiftArgGlutamic semialdehyde−5H −1C −3N +1O−43.05343Arg+14 DaaPrecise structure of these products is unknown.−2H +1O+13.97927Asp3-hydroxyaspartic acid+1O+15.99492CysSulfinic acid+2O+31.98983CysS-nitrosylationaPrecise structure of these products is unknown.H(-1) N O+28.990164His2-oxohistidine+1O+15.99492His4-hydroxynonenalaPrecise structure of these products is unknown.H(16) C(9) O(2)+156.115030Leu+14 Daa−2H +1O+13.97927Leu3-hydroxyleucine+1O+15.99492MetMethionine sulfoxide+1O+15.99492MetMethionine sulfone+2