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Mattia MancusoMass SpectrometryBottom-up proteomicsProteome Discoverer 1.3.0.339Lc-msmsRatCardiac muscleFaspPhosphoproteomeMithocondriahttp://www.ebi.ac.uk/pride/archive/projects/PXD000354Cardiac MuscleNot availablePrideNot availableNot availableNot availableProteomicsinstrument modelCOMPLETERattus Norvegicus (rat)24467267 Ferreira R, Vitorino R, Padrao AI, Espadas G, Mancuso FM, Moreira-Gonçalves D, Castro-Sousa G, Henriques-Coelho T, Oliveira PA, Barros AS, Duarte JA, Sabidó E, Amado F. Lifelong exercise training modulates cardiac mitochondrial phosphoproteome in rats. J Proteome Res. 2014 Jan 28francescomattia.mancuso@gmail.comPrime-xs projectCRG/UPF Proteomics Unit10.6019/PXD000354Moderate physical activity has traditionally been associated with the improvement of cardiac function and, consequently, with the extension of life span. Mitochondria play a key role in the adaptation of heart muscle to exercise-related metabolic demands. In order to disclose the molecular mechanisms underlying the beneficial effect of lifelong physical activity in cardiac function, we performed label-free quantitative mass spectrometry-based proteomics of Sprague-Dawley rat heart mitochondrial proteome and phosphoproteome. Our data revealed that 54 weeks of moderate treadmill exercise modulates the abundance of proteins involved in the generation of precursor metabolites and cellular respiration, suggesting an increase in carbohydrate oxidation-based metabolism. Moreover, from the 1335 phosphopeptides identified in this study, 6 phosphosites were exclusively assigned to heart mitochondria from sedentary rats and 17 to exercised animals, corresponding to 6 and 16 proteins, respectively. Most proteins exhibiting significant alterations in specific phosphorylation sites were involved in metabolism. Analysis of the acquired data led to the identification of several kinases potentially modulated by exercise training, which were selected for further validation. Indeed, higher protein abundance levels of RAF and p38 in mitochondria were confirmed to be modulated by sustained exercise. Our work describes the plasticity of heart mitochondria in response to long exercise programs manifested by the reprogramming of phosphoproteome and provides evidence for the kinases involved in the regulation of metabolic pathways and mitochondrial maintenance.Lifelong exercise training modulates cardiac mitochondrial phosphoproteome in rats.Ferreira Rita R, Vitorino Rui R, Padrão Ana Isabel AI, Espadas Guadalupe G, Mancuso Francesco M FM, Moreira-Gonçalves Daniel D, Castro-Sousa Gonçalo G, Henriques-Coelho Tiago T, Oliveira Paula A PA, Barros António S AS, Duarte José Alberto JA, Sabidó Eduard E, Amado Francisco F617Heart, Rat, extracellular signal-regulated kinase activity, GrpL, pp44mapk, GRPL, Herz, Growth factor receptor-binding protein, CRKII, Laboratory, NURF38, Physical, Metabolic Concepts, adult heart, raf, Mitochondrial Contractions, glucide, p38-alpha, Norway Rats, phosphorylation, p38MAPK, p38a, Sprague-Dawley Rats, carbohydrates, Hog, p38ALPHA, circulatory vessel, Motor, rat, protein polypeptide chains, LeMPK3, Physical Exercises, p44mpk, hnRNP A2/B1, glucido, responsivity, Dp38, Analysis, Metabolism Concept, SAPK2, Acute Exercises, Mpk34C, p38B, p38A, Animalia, oxidative metabolic process, Aerobic Exercises, mapk14a, Motor Activities, Analyses, Draf-1, pp42, heart or heart like organ, catabolism, hrp40, Physical Activity, hnRNP36, proteins, metabolic process resulting in cell growth, W, cardiac structure, Dm p38b, Hrb87Fa, D-raf1, Hrb85CD, hrp36, Grf40, Role Concepts, dMKK3, GRBLG, ESTS:186F5S, DmMPK2, vertebrate heart, dorsal tube, l(1)phl, 6.3.2.-, metabolism resulting in cell growth, csbp, ERK-2, CG4634, saccharides, PMK-2, PMK-1, Spectrum Analysis, not genetically inherited, PMK-3, Hrb87f, oxidative metabolism, CSBP, Role Concept, heart myocardium, Double ring-finger protein, Playthings and Play, Role, secretion, D-MPK2, GRAP-2, Kohlenhydrat, Aerobic Exercise, DmelCG5475, Mek3, MEK3, DMEK3, l(1)pole hole, training, Spectrometry, Heart Muscles, l(1)polehole, carbohidratos, D-raf, c-Crk, rats, Activities, NURF-38, CSBP2, carbohidrato, CSBP1, Csbp2, Afr-1, EG:BACH48C10.3, Csbp1, D-p38 MAPK, Grf40 adapter protein, Peptidomics, Processes, carbohydrate, number, saccharide, DRaf, branchial heart, Exercise Training, precursor, Metabolic Processes, protein-containing complex, AFR1, animals, Afr1, GRB-2-like protein, Exip, draf, PRKM15, Cardiac, Rats, dMPK2, p38 MAPK, PRKM14, Mitochondrion, Gene Products, sapk2a, dNURF, EXIP, D-RAF, D-Raf, Toy, ERK, PRKM1, p38beta, PRKM2, mKIAA0854, Hrp36, JTV1, p38Ka, Playthings, Contraction, p38Kb, Metabolic Concept, Phl, AI848995, BC023857, GRB2L, CG12749, Puppets, curriculum, Draf, HRP36, STK26, p38-2, myelin basic protein kinase activity, Puppet, Dp38b, Dp38a, RING finger protein 19A, SAPK2A, degradation, raf-1, Proteins, Heart Muscle, Phosphorylations, MAP-2 kinase activity, Protein GADS, function, Hearts, Concept, DmMKK3, PDIP38, native protein, p38MAPKK, Laboratory Rats, heart, chemical analysis, Acute Exercise, glucides, metabolism, p38 beta, Mass Spectrum Analyses, Metabolic Phenomenon, Nurf38, multicellular organism metabolic process, Locomotor Activities, P42MAPK, Crk1, Crk3, CSPB1, Exercise Trainings, multicellular animals, l(1)polehole/draf, Gene Proteins, 38 kDa DNA polymerase delta interaction protein, Acute, D-MKK3, CRK1, regulation, Norway, quantitative, ph, Anabolism, Holtzman, biochemical pathways, Metabolic Process, Nurf 38, Activity, determination, sapk2, Motor Activity, Mpk2, Mpk3, Sprague Dawley Rats, metazoans, Crko, Norway Rat, p42mapk, hydrates de carbone, protein, D-p38, Crk-II, p38 alpha, NURF, Cardiac Muscle, DmelCG4634, Cardiac Muscles, p38/NURF-38, Roles, Concepts, Norway rat, mitogen-activated protein kinase activity, Q14, Q16, protein aggregate, Phenomenon, RK, mpk2, Mass Spectrum Analysis, Adapter protein GRID, Kohlenhydrate, DMKK3, MEK3/MKK3, long, Myocardia, free, Laboratory Rat, MP kinase activity, 38, Muscles, Dmp38a, AHA1, Hematopoietic cell-associated adapter protein GrpL, Dmp38b, Holtzman Rat, MBP kinase II activity, DmelCG12749, P11, biotransformation, associated, CG5475, Exercises, Catabolism, Holtzman Rats, MAP kinase 2 activity, Process, Sprague-Dawley, Aerobic, ERK1, ERK2, Erk2, A230093K24Rik, Rattus, Hrb2, D-P38a, GRBX, GrbX, Programs, Spectroscopy, D-p38b, heart muscle, D-p38a, chambered heart, Mus norvegicus, p41mapk, Syn, MAPK, Plaything, muscle of heart, P38, cardium, C14orf3, Physical Exercise, DRaf1, CG12244, saccharidum, HRB87F/hrp36, parent ion, l(1)G0475, Mitochondrial, SH3-SH2-SH3 adapter Mona, GADS, JTV-1, Draf1, raf1, HRB87F, Mona, Toys, Sprague Dawley, p38alpha, Maintenances., p38, Prkm15, Prkm14, Proto-oncogene c-Crk, precursor ion, p41, p40, C110, DmelCG12304, a carbohydrate, glucidos, 186F5S, Proteomes, rafl, Plays, l(1)G0252, norvegicus, respiration, CG7393, Training, Isometric, Gene, Spectrum Analyses, Locomotor, presence, CrkIII, p38Hog, dp38a, dp38b, Gads, polypeptide chain, Metabolism, Mass, N38, Animal, p42-MAPK, Metabolism Phenomena, Raf1, Mass Spectroscopy, Sprague-Dawley rat, brown rat, study, Mkk3, MKK3, reactivity, Rattus norvegicus, teaching, DmelCG2845, DmelCG12244, Grf-40, Locomotor Activity, p42 mitogen-activated protein kinase activity, Crk-III, ATP:protein phosphotransferase (MAPKK-activated) activity, Sprague-Dawley Rat, cardiac pump, GRID, l(1)raf, Play, data, protein complex, DMPK2, Sprague Dawley Rat, anon-sts23, Exercise, total expressed protein, l(1)2Fe, P11/Hrb87F, Muscle, cardiac muscle, Crk-I, DMKK3/lic, DmelCG7393, Metabolic Phenomena, p38delta, Contractions, Metabolism Concepts, count in organism, Raf/phl, Phosphopeptide, natural protein, group 2, Protein, Metazoa, Phenomena, MBP kinase I activity, Isometric Exercises, l(2)k16102, Mass Spectrum, biodegradation, Metabolic, mitogen activated kinase activity, Mxi2, POLD4, Sprague-Dawley rats, Sapk2A, MAPK2, l(1)ph, Trainings, Carbohydrate, 11-29, dp38, Isometric Exercise, Dorfin, Mitochondrial Contraction, Protein Gene Products, single-organism metabolic process, mitochondria, Physical Activities, mxi2, ERT1, BG:DS00797.3, assay, RAF, Raf, response, MAP kinase 1 activity, presence or absence in organism, CG2845Not Available, Unavailable.brown rat, rat, Rat, Mus norvegicus, Rattus norvegicus, Rats, norvegicus, Laboratory, Laboratory Rats, Proteomes., total expressed protein, Norway rat, Norway Rat, Norway, Rattus, rats, Norway Rats, Laboratory Ratbrown rat, Aerobic Exercise, Rat, Aerobic Exercises, Rattus norvegicus, Activity, norvegicus, training, teaching, Laboratory, Physical Activity, Training, Isometric, Physical, Exercise, Aerobic, Norway Rat, Exercise Training, Rattus, Trainings, rats, Norway Rats, Exercise Trainings, Isometric Exercise, Activities, Laboratory Rat., rat, Mus norvegicus, Acute, Physical Activities, Rats, Physical Exercises, curriculum, Laboratory Rats, Acute Exercise, Norway rat, Norway, Isometric Exercises, Exercises, Physical Exercise, Acute ExercisesNot Available, Unavailable.sodium salt, Water, l(4)17, l(4)13, ammonium formate, Gli, 13C-labeled, Size, cadmium salt, ion, Raw, Cid[Mel], Ci[D], Particle, Physical, BOUND WATER, SDH, Basodexan, magnesium formate, Apaf-1, Mitochondrial Contractions, oxidane, zinc salt, CENP-A, phosphorylation, Long Term, WATER, CENP-C, HOH, 5730420M11Rik, SDS, Polypeptides, ref2p, protein polypeptide chains, rat, Drug Tolerance, cobalt(II) formate dihydrate, DmelCG1921, Physical Exercises, Method, B1, 1, Urea, 2, Software Engineering, Faroe Islands, Acute Exercises, INSL3R, C79691, Animalia, (R*, ALY|REF, ABC, SET, Divorced, Aerobic Exercises, BcDNA:RE21270, C79325, hac-1, Paul, Physical Activity, HOIP, phosphatase 2A inhibitor I2PP2A, Salt, Divorces, proteins, procedures, D17Mit170, SURGICAL AND MEDICAL PROCEDURES, HPLC, AI047805, DmelCG4299, Lccp, isolation and purification, BEF, set, decreased, CI, column, ACN, sample, D1, PAUL, Acn, 2-(14)C-labeled, REF, drug tolerance, nickel salt, CiD, LC-MS2, Ce, Ci, cobalt (+2) salt, 6.3.2.-, immune system tolerance, anon-53Fa, Longterm Effect, ref, l(2)SH0173, Procedure, Tl3, Tl2, female, collisionally activated dissociation, CID, ciD, Cid, Software Tools, Computer Applications, H2O, sodium (4:1:1) salt, Rattus norwegicus, Dm1, magnesium salt, Computer Applications Software, Sizes, ci-D, END, CG6829, activation, dark/hac-1/dapaf-1, Aerobic Exercise, LGR8, Lgr8, Software Applications, HLA-DR-associated protein II, instrument, DI-2, CENP-A/Cid, CENP-A/CID, CENPA, Source Software, FT, I-2Dm, formate, expanded, cromium (+3), MASCOT, Methodological, inhibitor of granzyme A-activated DNase, beta Trypsin, rats, glycolate, Activities, I-2PP1, Applications, TAF-IBETA, Self Tolerance, liquid chromatography-tandem mass spectroscopy, ammonium (4:1) salt, TAF-Ibeta, lead (+2) salt, Ci-155, umat, Computer Software Applications, Cleland Reagent, big, F10B6_15, Particle Sizes, CLK, nickel formate dihydrate, lead formate, Effects, Ref(2)P, fractured, aluminum salt, 4-dimercapto-, Exercise Training, Carbamide, R*)-, protein-containing complex, thomson, animals, DUH3, body system, combined T and B cell immunodeficiency, CG7826, F10B6.15, agua, mass-to-charge ratio, period, CenpA, large, Rats, isolation, Filtrations, Ref(2)p, THOC4, LC-MSMS, hydroxyacetic acid, CG7835, Mitochondrion, CG42273, system, Carmol, Siah, Technique, Application, Open Source Softwares, anatomical systems, Acinus, Longterm, interventionDescription, thallium (+1) salt, potassium glycolate, Software Application, Contraction, ALY/REF, beta-Trypsin, Monosodium Salt, rubidium salt, spry, Open Source Software, Argos, 4733401P19Rik, 2pp2a, Sciences, CG4531, Long-Term, methanoic acid, ions, CG10574, Solution, Computer Software Application, acinusL, Study, Injectable, mKIAA0989, Faeroe Islands, 2PP2A, Tools, acinusS, dSET, dSet, CG2125, peptidos, LAN, rat <Rattus norvegicus>, rats <Rattus norvegicus>, 4-(2-hydroxyethyl)-, GPR106, rlt, PTHB1, CS3-3, constant, dark/dapaf-1/hac-1, grupo, cou, 1-(14)C-labeled, female human body, dApaf-1, ALY, Proteins, Flj10111, Phosphorylations, Clelands Reagent, split, glycolic acid, potassium formate, copper (+2) salt, Mischung, buffer, mKIAA0670, Tool, polypeptide, CLK/STY, Spry, APAF1, Lr, native protein, Immune Tolerance, carbonyldiamide, Hac-1, I-2PP2A, ci[D], C18, cupric formate, Long Term Effects, chemical analysis, Dm I-2, hemorrhaged, Acute Exercise, Putative Ariadne-like ubiquitin ligase, aluminum formate, l(3)05959, Cleland's, Intervention, AU020952, torn, lithium formate, DmelCG2125, Col4a-1, ci155, CenH3, underdeveloped, RING finger protein 31, Separations, monolithium salt, SWDS, Dark/Dapaf-1/HAC1, Sry, l(3)05845, Exercise Trainings, Longterm Effects, ME-IV, multicellular animals, Cleland's Reagent, Computer Programs, DmelCG4531, Aos, Acute, Applications Softwares, l(3)rJ472, Bra, liquid chromatography tandem mass spectrometry, CG10360, [OH2], CenpA/CID, Immunological Tolerance, liquid chromatography tandem mass spectroscopy, Hac-1/Dark, IPP2A2, Bru, Activity, determination, DmelCG6829, instrument configuration, metazoans, zinc formate, HEPES Monosodium Salt, lead salt, protein, combined T cell and B cell immunodeficiency, STY, Sty, peptide, Techniques, ammonium tetraformate, peptido, ClvPrd, BANF, BC031509, Min, Norway rat, Apaf1, protein aggregate, sty, Effect, present in fewer numbers in organism, ARK, Injectables, Computer Program, DmelCG42273, CG13329, Svc, HEPES Monosodium, me75, LCMSMS, peptides, reference sample, TAF-I, E927b, 2610036I19Rik, Open, 2610510L13Rik, hypoplasia, CLK|STY, min, arc, Computer Programs and Programming, mAPC, purification, ABC14, arg, copper, ark, SCAN, T1, dSPRY, IGAAD, GREAT, fSAP152, lithium salt, Methodological Studies, Sputolysin, SOLO DANCERS, 1-Piperazineethanesulfonic acid, DmelCG10574, ppm, Cenp-A, Great, dihydridooxygen, MCOPCB7, dApaf1, dihydrogen oxide, Exercises, Long-Term Effects, MTABC3, ammonium (2:1) salt, phapii, D-Apaf-1, DmelCG10360, T5E21.12, PRP, LC-MS/MS, aqua, Dmel_CG7826, Aerobic, AI836084, StF-IT-1, Th, Mitochondrial Protein, fragmented, ur, Source Softwares, CG1921, Programs, Ref2P, Program, carbamide, ref(2)Po2, Mus norvegicus, Cleland, Ionen, Computer Applications Softwares, Immunologic Tolerance, Softwares, nickel (+2) salt, Physical Exercise, Dmel_CG7835, T5E21_12, cenpA, Mnb, MNB, ammonium salt, Mitochondrial, monoammonium salt, Ci/Gli, Ci/GLI, CenH3/CID, cracked, cobaltous formate, hydrogen hydroxide, Sprague Dawley, common, 3-Butanediol, hac1, CG4299, AW124434, Methodological Study, monosodium salt, acqua, organ system, congenital combined immunodeficiency, dapaf-1S, nanospray, H2NC(O)NH2, apaf-1, dapaf-1L, Tolerance, enlarged, Acetamide, copper salt, Polypeptide, Wasser, HEPES, i2pp2a, time, 1728, Gruppe, Procedures, Dapaf-1/HAC-1, AW549739, Reagent, Training, cesium salt, Isometric, ACINUS, Computer, PHAPII, LC-MS-MS, Intervention or Procedure, formic acid, Buffer, Dark/Hac-1/dApaf1, Karbamid, l(4)102ABc, Rattus sp. strain Wistar, Hac1, potassium salt, l(3)rQ526, Dark/Hac-1/dApaf-1, reduced, polypeptide chain, template-activating factor I, subnumerary, Injection, 14C-labeled, p62, Harnstoff, Studies, Low, Del(8)44H, Animal, tiny, Rattus rattiscus, l(3)j5E11, Separated, Sprague-Dawley rat, CenH3[Cid], CenH3[CID], brown rat, Gpr106, iones, ipp2a2, uniform, dapaf-1, DmelCG13329, Interventional, dapaf, RXFPR2, dark, eau, decreased number, calcium formate, X-linked combined immunodeficiency, 10^[-6], DARK, grupos, taf-ibeta, DmAos, great, AL033293, Long-Term Effect, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, cromium (+3) salt, dArk, Dapaf-1, HHT1, 2-iodo-, sodium formate, ref(2)Pn, Controlled, combined immunodeficiency, Rattus norvegicus8, small, Applications Software, Intervention Strategies, Open Source, DYRK1, data, Controlling, Computer Software, nickel formate, protein complex, mFLJ00217, igaad, Exercise, strontium formate, ref(2)p, cenH3, apaf1, strontium salt, HOIL-1-interacting protein, Peptide, group, Contractions, l(3)rJ812, LC/MS/MS, Software Tool, Phosphopeptide, CAD, natural protein, Ci155, water, Metazoa, Protein, I2PP2A, Dyrk1, techniques, Dark, Vacuums, Isometric Exercises, connected anatomical system, Software, Dark/Apaf-I, Data Base, Separation, uree, gil, urea, CC1, Engineering, Sprague-Dawley rats, Rest, Tripcellim, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, monopotassium salt, CGI-97, Trainings, LGR8.1, dApaf-1/DARK/HAC-1, sample population, chromic formate, UREA, Isometric Exercise, Mitochondrial Contraction, Trypure, dSET/TAF-Ibeta, 2610030F17Rik, mitochondria, Physical Activities, Pressures, Ion, calcium salt, Peptid, assay, ORW1, variable, SCH, AA407739, Runnings, groupe, Peptide., Females, dAPAF-1, methodology, CID/CENP-A, Gunn rats0trueRat cardiac mitochondrial proteome and phosphoproteomeFemale Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.2014-02-142013-07-22PXD0003544454464152499175593117388043330432359839062887053271602461971603293581136352758385118794713512251177505855571468353649215891030629159364138858033271591002263888691513558814647969282803836545833398580118259063295712472725639286728139545134081691390363453011409810871355477142450732644355512744327146945597293751461575464784224220896431744712701390114319338293550823074111481194669489693529372512646907275802402165993665812165952941281283296543128033952321818431832920435315257554787575930797253623114367331159556143213818492224261910969762261551531117957334798645586265281736309431796989361604885664162425678253175136303555432046565050632633NCBITaxon:1313456586068860769938942795545351201488779592161294100264203289096763107618543121574577760494144242324113NCBITaxon:17739297931023910117870974990799131122734982171701873674510116569156931036098334133679566330399258839100058987329940224911343053115631201755442193287263178723666987246426872754762422311151043055468911180066125522856213616299767982510216998231174673116761080772446896776744685960039376511822635735784588086363266689NCBITaxon:2697049103129838241368108931997596123197479465228319662776689605960615729513722117110125673712334351052311009040549986387833578840193733496151982157574878229719721519089850764802563184189856239190264946454637463601134506649125314594352638NCBITaxon:6157977872374081263380394526411459536123517861672272432307469112386940979541NCBITaxon:29544243277627912924970361251802848124264281504751029938865990346941734751541554036329979662877323962899548657558462823823522862871736231399463601068842043601043994796858355847954440426071016780781440772NCBITaxon:619184023171993128118361003610502318557919080237499070384766416922591065922607046183260705334542260707NCBITaxon:150288531870292115869103209285124966842528619212638541094343197192875NCBITaxon:103592829126806395602959870448300641802242752235443224308469008150257541298396427119136451699632810412169792726232726243008521238993643680109779135622334747269704958331803082688297603818583342409061147787127441415368163958885813076749402243264081721281612042546876138370812744239901191274420127442651953491137025437341316462615675060285321895177311935018464514505114924121532395648188229709158002571256148015490313847360314231663859628596310151014505204932275969373332648373153NCBITaxon:9615297221097677112111449376083334191107927628333223756126662555592924847974920036009434073352472515151438992374724467267