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ButterMass SpectrometryBottom-up proteomicsSilacRna pull-downhttps://www.ebi.ac.uk/pride/archive/projects/PXD000342Not availablePrideNot availableNot availableProteomicsF ButterPARTIALQuantitative Proteomics Institute of Molecular Biology (IMB)Homo Sapiens (human)Not availablef.butter@imb-mainz.deQuantitative Proteomics Institute of Molecular Biology (IMB)Germany1Not Available, Unavailable.DAT1, rock salt, PKDYS, Spalt, ionic compound, sales, Salt, chlorure de sodium, Msal, B130022O04Rik, Salze, natrii chloridum, sel, Salz, salts, sal, Doublesex-like 2 protein, cloruro sodico, salt, common salt, halite, Solute carrier family 6 member 3, Natriumchlorid, Dietary Sodium, sels, ionic compounds, Kochsalz, Msal-1, table salt, DAT, DA transporter, NaCl, Dat1, Kochsalz., Terra, SalNot Available, Unavailable.l(4)17, Ribonucleic, l(4)13, Gli, cox4, telomere, MeCN, Raw, miRNA-mediated gene silencing, Vinegar, Cid[Mel], Ci[D], aneurysmal cyst of bone, SDH, CENP-A, Social Controls, CENP-C, 5730420M11Rik, dmTAF[[II]]230, SDS, Polypeptides, KL receptor activity, %, E260, Drug Tolerance, CH3-C#N, ionic compounds, B1, High Performance Liquid Chromatography, Post Transcriptional, SCO5, nuclear chromosome, Msal-1, SCO1, 2-amino-3-mercaptopropanoic acid, Analysis, myd, Gsfsow3, Non Polyadenylated, WMS, aneurysmal bone cyst (disease), ABC, SET, C, Divorced, Grain, BcDNA:RE21270, F, TFIID TAF250, Analyses, cel, COXIV, Aneurysmal, M, Salt, Mbp-1, Msal, Divorces, W, heterochromatin assembly involved in chromatin silencing at centromere outer repeat region, Acetic Acid Glacial, sel, Salz, COXA_DROME, HPLC, salts, DmelCG4299, Social, salt, set, Gene Silencings, Dehydrated ethanol, heterochromatic silencing, CI, heterochromatin assembly involved in chromatin silencing, h, column, Cox4, D1, establishment of heterochromatin architecture involved in chromatin silencing at centromere outer repeat region, Chromatography, MeCO2H, Bs, CT, stage, High Speed Liquid, 1.3.3.3, Half Cystine, INSDC_feature:telomere, COX-VA, miRNA biogenesis, drug tolerance, Histological Labeling, SGS, spiritus vini, CiD, Ce, dTAF[[II]]230, Ci, Zinc Cysteinate, immune system tolerance, acetylation, Ct, Liquid Chromatography, TAF200, telomeric sequence, Spectrum Analysis, collisionally activated dissociation, Cid, CID, ciD, Absolute Alcohol, ACMICD, PBT, common salt, Glacial, aneurysmal bone cyst, mKIAA0609, AL024441, ci-D, Methionin, Ribonucleic acids, Hmet, (2R)-2-amino-3-sulfanylpropanoic acid, [OEtH], Post-Transcriptional Gene, fg, Acid, pbt, HLA-DR-associated protein II, Ethylic acid, DI-2, CENP-A/Cid, CENP-A/CID, ionic compound, Coprogen oxidase, CENPA, I-2Dm, Spectrometry, expanded, Shwachman-Diamond type metaphyseal dysplasia, srrt, Shwachman syndrome, MORFL2, Controls, Co Suppression, beta Trypsin, alcohol etilico, Methylcarbinol, human, Post Transcriptional Gene Silencing, I-2PP1, MDC1D, TAF-IBETA, Gene Silencing, Self Tolerance, enr, Taf250, TAF-Ibeta, methyl cyanide, Liquimeth, Ci-155, TAF230, umat, krk1, big, Regulations, F10B6_15, post-transcriptional gene silencing by RNA, telomeric region, lifespan, production of microRNAs, l(1)VE614, 2-amino-4-(methylsulfanyl)butanoic acid, Peptidomics, developmental stage, etanol, number, L-Zystein, Bone Cysts, precursor, protein-containing complex, DUH3, F10B6.15, 2410017O14Rik, Human, CenpA, CG10664, large, IV, Gene Products, Half-Cystine, production of microRNAs involved in gene silencing by microRNA, abacavir, Siah, Man, Terra, dTAF[[II]]250, cell, MDDGB6, ethanol, L Cysteine, ligand, E-920, beta-Trypsin, Mrgx, 2pp2a, 4733401P19Rik, Hcys, Cosuppression, kf, High-Performance Liquid Chromatographies, CG10574, Solution, DmelCG7843, dTAF250, C2H5OH, 2PP2A, 2-amino-4-(methylthio)butanoic acid, CYSTEINE, congenital lipomatosis of pancreas, COX, Kochsalz, dSET, dSet, negative regulation of homeostatic process, MRGX, CG2125, FREE CYSTEINE, peptidos, UPLC, establishment of heterochromatic silencing, (2R)-2-amino-3-mercaptopropanoic acid, LAN, metionina, PTHB1, proto-oncogene c-Kit, RNA, AcOH, 1-(14)C-labeled, Proteins, microRNA-mediated gene silencing, RNS, BG:DS00004.13, Stainings, cosuppression, acetonitrile, L-Isomer, buffer, Cell, Engine, dTAF230, MS, Experiment, Interference, Temperatures, native protein, Immune Tolerance, Natriumchlorid, I-2PP2A, Social Control, ci[D], KIT ligand receptor activity, C18, Dm I-2, chemical analysis, alcool ethylique, TAF[[II]]250/230, MFS1, background, NaCl, regulation of homeostatic process, Mass Spectrum Analyses, Silencing, Met, XKrk1, Post-Transcriptional Gene Silencings, Taf[[II]]250, miRNA processing, DmelCG2125, Col4a-1, CenH3, ci155, ensemble, underdeveloped, storage, Posttranscriptional, ASR2, Asr2, {(1S-cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]cyclopent-2-en-1-yl}methanol, Separations, CG7843, SWDS, transcriptional gene silencing, establishment of chromatin silencing, B130022O04Rik, Shwachman-Bodian-Diamond syndrome, (R)-2-amino-3-mercaptopropanoic acid, Ribonucleic Acid, ethanenitrile, CD117, 2-Amino-3-mercaptopropionic acid, Gene Proteins, Post-Transcriptional, High Pressure, CG14724, C-Kit, MeCOOH, Dietary Sodium, Pancreatic insufficiency and bone marrow dysfunction, Ssm, TIP60 histone acetylase complex, xkl-1, E920, regulation, sequestering, Post-Transcriptional Gene Silencing, CenpA/CID, Ethanoic acid, Immunological Tolerance, IPP2A2, Bru, Grain Alcohol, E 920, NCMe, determination, Mbp1, Essigsaeure, Xkl-1, protein, 1-hydroxyethane, alcohol, Cut, Ethanol, L-Isomer Methionine, halite, Gsfsco1, heterochromatin formation involved in chromatin silencing at centromere outer repeat region, Methionine, peptido, Glacial Acetic Acid, l(1)7Ba, congenital, l(1)7Bb, ARS2, Alkohol, Ars2, heterochromatin assembly involved in chromatin silencing at pericentric region, Cystein, DmelCG10664, protein aggregate, Gsfsco5, Formal Social Controls, RNA Gene Products, ars2, SOW3, Mass Spectrum Analysis, CG13329, Svc, Man (Taxonomy), peptides, TAF-I, cisteina, entire life cycle, Ethyl, hypoplasia, Acetic Acid, ABC14, Aethanol, quelling, Sid393p, [CH2Me(OH)], CG11387, lipomatosis of pancreas, retention, cloruro sodico, IGAAD, High-Performance, DmelCG10574, SOLO DANCERS, ppm, Cenp-A, MCOPCB7, High-Performance Liquid, ethoic acid, Shwachman-Bodian syndrome, Sl, GPHYSD2, Racemethionine, MTABC3, phapii, rock salt, Alcohol, ribose nucleic acid, gyltl1b-b, PRP, Modern, ribonucleic acids, 2-Amino-4-(methylthio)butyric acid, StF-IT-1, AI836084, Tr-kit, Labeling and Staining, Search, Ethyl alcohol, TAFII-250, TAF250/230, Cys, results, Spectroscopy, TAFII250, positive regulation of homeostatic process, VA, MDDGA6, Ribonukleinsaeure, LRRG00119, kl1-A, Immunologic Tolerance, pentosenucleic acids, overlap, KIT, BcDNA:GH10590, COX IV, KIAA0609, RNA Silencing, ETHANOL, aneurysmal bone cysts, cenpA, parent ion, tyrosine-protein kinase Kit, Epistemology, gyltl1b, EtOH, CenH3/CID, Ci/Gli, Ci/GLI, Spalt, 2810019G02Rik, Va, aneurysmal cyst of the bone, life, COX IV-1, mdc1d, Control, anon-WO0118547.166, L-Cysteine, CG4299, kit, MASS, CG17603, natrii chloridum, TAF[[II]], L-Cystein, Labelings, Tolerance, precursor ion, enlarged, SR3-5, Acetamide, heterochromatin formation involved in chromatin silencing at pericentric region, telomeric DNA, Liquid, Polypeptide, DmelCG14724, Co-Suppression, mKIAA0026, i2pp2a, Regulation, Cox4a, serrate, MOPS, d230, E-260, human being, Cysteine Hydrochloride, ASR2D, ASR2B, ASR2C, Histological Labelings, SCF receptor activity, L Isomer, FBN, Gene, CH3-COOH, dTAFII250, ASR2A, Spectrum Analyses, EfW1, presence, froggy, PHAPII, Gyltl1a, Buffer, Aethylalkohol, l(4)102ABc, Methanecarboxylic acid, morpholine propanesulphonic acid, reduced, scfr, Homo sapiens, dmTAF1, Taf230, ECTOL1, Mass, L-Methionine, Histological, Del(8)44H, tiny, Separated, SCFR, CH3CO2H, Pedameth, Mass Spectroscopy, Pro-Mega, TAF250, CenH3[Cid], CenH3[CID], Search Engines, Taf200, Fdc, Coproporphyrinogenase, telomeres, Staining, entire lifespan, Absolute, ipp2a2, chlorure de sodium, DmelCG13329, Acetylations, Taf1p, LARGE, Non-Polyadenylated RNA, OCTD, X-linked combined immunodeficiency, BPFD#36, Abstract, DmelCG11387, 10^[-6], taf-ibeta, sels, great, production of miRNAs, Ultra Performance Liquid Chromatography, False., TAF, 2-iodo-, High-Performance Liquid Chromatography, small, Autoregulation, TAF[[II]]250, 3H-labeled, Formal Social Control, DL-Methionine, protein complex, sales, igaad, posttranscriptional gene silencing by siRNA, l(3)84Ab, cenH3, Labeling, Peptide, group, RNAi, count in organism, CAD, TIP60 histone acetyltransferase complex, HOAc, Ci155, yeast nucleic acid, p230, Protein, I2PP2A, Telomeres, TFIID, table salt, IV-1, ACETONITRILE, Zystein, cyanomethane, WMS2, Data Base, Sal, regulation of telomere length, Ethyl Alcohol, ribonucleic acid, Mass Spectrum, Separation, ars2-B, c-KIT, ars2-A, TAF[[II]]230, PRSS, E 260, 2-amino-3-sulfanylpropanoic acid, heterochromatin formation involved in chromatin silencing, Posttranscriptional Gene Silencing, Non Polyadenylated RNA, tend, Non-Polyadenylated, Tripcellim, L-2-Amino-3-mercaptopropionic acid, TAF[II]250, Salze, acide acetique, establishment of heterochromatin architecture involved in chromatin silencing at pericentric region, CGI-97, introduction, sal, Protein Gene Products, Doublesex-like 2 protein, Trypure, dSET/TAF-Ibeta, hydroxyethane, 2610030F17Rik, c-kit, DmelCG17603, gene silencing by miRNA, Modern Man, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, Peptid, INS No. 260, High Performance Liquid, assay, variable, AA407739, TAF1, CID/CENP-A452falseSILAC dat for two TERRA pull-down experiments using different salt conditions (1 M NaCl and 250 mM NaCl)ABSTRACT: Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)-based proteomics to obtain a high confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pull-down experiment, and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtain good overlap to both studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knock-down of ten proteins significantly affected total cellular levels of TERRA and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/Ars2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knock-down of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation. MS SECTION: Pull-downs were separated on a 4-12% NOVEX gradient SDS gel (Life Technologies) for 1 hour at 170V in 1x MOPS buffer (Life Technologies). Proteins were fixated and stained with the Colloidal Blue staining Kit (Life Technologies). The gel lane was cut into 8 slices, minced and de-stained with a 50 percent Ethanol/50 mM ABC solution. Proteins were reduced in 10 mM DTT for 1 hour at 56 °C and then alkylated with 55 mM iodoacetamide for 1 hours at room temperature. Proteins were digested with trypsin (Promega) at 37 °C overnight. Peptides were extracted from the gel using acetonitrile which was subsequently removed in a concentrator (Eppendorf) and loaded on stage tips for storage. For mass spectrometric analysis, peptides were separated on a C18 column with 75 µm diameter packed with 3 µm Reprosil beads (Dr. Maisch) mounted to a EASY HPLC (Thermo Fisher) and sprayed online into an Orbitrap-XL mass spectrometer (Thermo Fisher). We used a 120 minutes gradient from 2% to 60% acetonitrile in 0.5% acetic acid at a flow of 200 nl/minutes. The mass spectrometer was operated with CID fragmentation for top10 data-dependent MS/MS per full scan. Mass spectrometry raw data was searched using the Andromeda search engine (Cox et al. 2011) integrated into MaxQuant suite 1.2.2.0 (Cox and Mann 2008) using the IPI human database v3.68. Carbamidomethylation at cysteine was set as fixed modification while methionine oxidation and protein N-acetylation were considered as variable modifications. The search was performed with an initial mass tolerance of 7 ppm mass accuracy for the precursor ion and 0.5 Da (CID) for the MS/MS spectra. Search results were processed with MaxQuant and identification up to a false discovery rate of 0.01 were accepted.2013-08-022013-07-15PXD000342NEWT:3555NEWT:71647NEWT:35554NEWT:32046NEWT:544496NEWT:2042546NEWT:32049NEWT:259447NEWT:45351NEWT:445974NEWT:43179NEWT:180454NEWT:5722NEWT:1129NEWT:55153NEWT:309800NEWT:281395NEWT:1211601NEWT:876138NEWT:10036NEWT:498019NEWT:1351NEWT:1438992NEWT:1352NEWT:2649997NEWT:1147161NEWT:224326NEWT:1333499NCBITaxon:79857NEWT:1096976NEWT:95648NEWT:1589NEWT:135622NEWT:383379NEWT:10029NEWT:913645NEWT:317447NEWT:4688NEWT:7955NEWT:7959NEWT:2261NEWT:31156NEWT:4442NEWT:192875NEWT:59729NEWT:2164133NEWT:1316931NEWT:224308NEWT:3347NEWT:511145NEWT:931281NEWT:658457NEWT:77133NEWT:145481NCBITaxon:79824NCBITaxon:4563NEWT:5755NEWT:44689NEWT:5759NEWT:1736231NEWT:1392NEWT:498217NEWT:2242NEWT:11320NEWT:286NEWT:391619NEWT:246196NEWT:287NEWT:246197NEWT:10239NEWT:44685NEWT:161934NEWT:1148NEWT:5508NEWT:5507NEWT:55571NEWT:35500NEWT:1140NEWT:1143NEWT:4896NEWT:1390NEWT:11557NEWT:1094343NEWT:1336795NEWT:644042NEWT:1773NEWT:1182590NEWT:3712NEWT:82380NEWT:935293NEWT:375146NEWT:263NEWT:52283NEWT:284812NEWT:8175NEWT:43330NEWT:1603293NEWT:408169NEWT:47946NEWT:3702NEWT:243277NEWT:7067NEWT:2850NEWT:408172NEWT:408170NEWT:3708NEWT:332648NEWT:536231NEWT:1436733NEWT:460519NEWT:572307NEWT:1432138NEWT:1424507NEWT:1194599NEWT:272844NEWT:9483NEWT:1513458NCBITaxon:40559NEWT:84588NEWT:480NEWT:67767NEWT:46835NEWT:109757NEWT:300852NEWT:1502NEWT:376686NEWT:95486NEWT:508771NEWT:1883446NEWT:253NCBITaxon:2759NEWT:1233435NEWT:93061NEWT:93062NCBITaxon:4932NEWT:644223NEWT:235443NEWT:108458NEWT:272623NEWT:272624NEWT:983964NEWT:118499NEWT:32644NEWT:527796NEWT:499175NEWT:109779NEWT:3745NEWT:1715989NCBITaxon:4751NEWT:476272NEWT:3747NEWT:195051NEWT:3988NEWT:1255228NEWT:649908NEWT:410289NEWT:373153NEWT:352472NEWT:1071661NEWT:360094NEWT:470NEWT:41364NEWT:1313NEWT:39491NCBITaxon:5811NEWT:29491NEWT:2014887NEWT:33952NEWT:571256NEWT:40483NEWT:9031NEWT:1872122NEWT:7091NEWT:108931NEWT:1055524NEWT:9778NEWT:150475NEWT:303NEWT:7111NEWT:347515NEWT:9534NEWT:5180NEWT:256737NEWT:9541NEWT:185579NEWT:941442NEWT:13076NEWT:7108NEWT:40479NEWT:317NEWT:1006581NEWT:885318NEWT:39488NEWT:550NEWT:4081NEWT:273068NEWT:554NEWT:98334NEWT:451516NEWT:36185NEWT:1225786NEWT:7574NEWT:1715256NEWT:575412NEWT:929793NEWT:29204NEWT:28112NEWT:6494NEWT:6491NEWT:507601NEWT:643680NCBITaxon:6157NEWT:6239NEWT:162425NEWT:216257NEWT:102169NEWT:9986NEWT:4054NEWT:1268063NEWT:1263854NEWT:118503NEWT:2059687NEWT:160488NEWT:28104NEWT:407821NCBITaxon:2NEWT:985076NEWT:1215323NEWT:986NEWT:52641NEWT:7159NEWT:28532NEWT:353152NEWT:27448NEWT:1194669NEWT:51329NEWT:243230NEWT:1080772NEWT:519NEWT:8479NEWT:6063NEWT:49240NEWT:104395NEWT:6289NEWT:6287NEWT:300641NEWT:727NEWT:9796NEWT:725NEWT:469008NEWT:256318NEWT:206411NCBITaxon:6191NEWT:596153NEWT:1836NEWT:80863NEWT:105231NEWT:52638NEWT:4097NEWT:884204NEWT:9785NEWT:6279NEWT:1123869NEWT:9544NEWT:7370NEWT:83906NCBITaxon:780NCBITaxon:1502NEWT:6282NEWT:1134506NEWT:38783NEWT:4006NEWT:6426NCBITaxon:5476NEWT:6669NEWT:9935NEWT:89453NEWT:51515NEWT:51511NEWT:8845NEWT:92867NEWT:92866NEWT:5334NEWT:51750NEWT:202950NEWT:382352NEWT:413071NEWT:632957NEWT:9925NEWT:89462NEWT:8839NEWT:4232NEWT:2758385NEWT:11298NEWT:171101NEWT:714NEWT:421932NEWT:312017NEWT:196627NEWT:870435NEWT:5315NEWT:9913NEWT:105841NEWT:337330NEWT:2666255NEWT:2285NEWT:1392998NEWT:380394NEWT:981087NEWT:180066NEWT:226900NEWT:563041NEWT:326423NEWT:452467NEWT:36111NEWT:326424NEWT:1416333NEWT:1678078NEWT:749906NEWT:418985NEWT:749907NEWT:145263NEWT:694569NEWT:27606NEWT:9739NEWT:59202NEWT:9975NEWT:51953NEWT:3197NEWT:860688NEWT:13443NEWT:36329NEWT:62615NCBITaxon:3044782NEWT:72407NEWT:349741NEWT:157295NEWT:2096NEWT:9721NEWT:137221NEWT:9963NEWT:1617910NEWT:565050NEWT:2785785NEWT:411901NEWT:105884NCBITaxon:9615NEWT:58334NEWT:1193501NEWT:58331NEWT:6689NEWT:70448NEWT:698936NEWT:9940NEWT:521001NEWT:192011NEWT:1813887NEWT:224911NCBITaxon:11552NEWT:6945NEWT:184922NEWT:6703NEWT:2104NEWT:2NEWT:157546NEWT:474186NEWT:38942NEWT:307972NEWT:1496NEWT:2102NEWT:4513NEWT:1247NEWT:6938NEWT:376741NCBITaxon:10407NEWT:1736309NEWT:10360NEWT:47664NEWT:317548NEWT:3654NEWT:237561NEWT:5833NEWT:6928NEWT:36745NEWT:272563NEWT:82688NEWT:3885NEWT:3888NEWT:5821NEWT:44139NCBITaxon:4896NEWT:6915NEWT:3649NEWT:101510NEWT:28903NEWT:3880NEWT:272559NEWT:28909NEWT:515849NEWT:3641NEWT:466585NEWT:1000589NEWT:85963NEWT:85962NEWT:143361NEWT:398580NEWT:4565NEWT:1264690NEWT:515619NEWT:34305NCBITaxon:183674NEWT:167387NEWT:84645NEWT:44586NEWT:626528NEWT:139927NEWT:4558NEWT:209285NEWT:5888NEWT:211586NEWT:747078NEWT:1282NEWT:1283NEWT:431241NEWT:4550NEWT:1000561NEWT:2133NEWT:294381NEWT:197NEWT:1390363NEWT:288705NEWT:29176NEWT:4787NEWT:3218NEWT:556182NEWT:1270NEWT:374990NEWT:1432059NEWT:156471NEWT:4784NEWT:360106NEWT:156476NEWT:360104NEWT:10117NEWT:10116NEWT:1280NEWT:10358NEWT:1735272NEWT:83334NEWT:83332NEWT:317513NEWT:580240NEWT:294128NEWT:11676NEWT:100226NEWT:4530NEWT:75058NEWT:13616NEWT:172NEWT:296543NEWT:173NEWT:316435NEWT:42157NEWT:1895NEWT:1034304NEWT:105023NEWT:866628NEWT:215358NEWT:64152NEWT:4924NEWT:749200NEWT:9378NEWT:990346NEWT:145953NEWT:257309NEWT:10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