JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10.xlsxhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP9.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP10.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP5.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP4.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP2.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP1.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP6.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP3.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP7.rawhttps://storage.jpostdb.org/JPST000568/files/TEC_TMT10_bRP8.rawprimaryOK200ProteomicsHidetaka KosakoMus Musculus (mouse)https://repository.jpostdb.org/entry/JPST000568Tokushima UniversityjPOSTThe thymic function to produce self-protective and self-tolerant T cells is chiefly mediated by cortical thymic epithelial cells (cTECs) and medullary TECs (mTECs). Recent studies including single-cell transcriptomic analyses have highlighted a rich diversity in functional mTEC subpopulations. Because of their limited cellularity, however, the biochemical characterization of TECs, including the proteomic profiling of cTECs and mTECs, has remained unestablished. Utilizing genetically modified mice that carry enlarged but functional thymuses, here we show a combination of proteomic and transcriptomic profiles for cTECs and mTECs, which identified signature molecules that characterize a developmental and functional contrast between cTECs and mTECs. Our results reveal a highly specific impact of the thymoproteasome on proteasome subunit composition in cTECs and provide an integrated trans-omics platform for further exploration of thymus biology.Trans-omics Impact of Thymoproteasome in Cortical Thymic Epithelial Cells.Ohigashi Izumi I, Tanaka Yu Y, Kondo Kenta K, Fujimori Sayumi S, Kondo Hiroyuki H, Palin Amy C AC, Hoffmann Victoria V, Kozai Mina M, Matsushita Yosuke Y, Uda Shinsuke S, Motosugi Ryo R, Hamazaki Jun J, Kubota Hiroyuki H, Murata Shigeo S, Tanaka Keiji K, Katagiri Toyomasa T, Kosako Hidetaka H, Takahama Yousuke YcTEC, cTEC., mTECbig, 3.4.22.-, d230, Epithelial Cell, Laboratory, Mus domesticus, Thymus, thymus organ, dTAFII250, mini-ICE, CASP-14, Glandular Epithelial, Glandular, EfW1, Ximpact, House Mouse, Thymus Glands, dmTAF[[II]]230, large, Multicatalytic Proteinase, Squamous Epithelial Cells, dmTAF1, House, Taf230, Multicatalytic Endopeptidase Complex, CG9063, Mus musculus domesticus, Mice, Squamous Epithelial, 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<eudicots>, Adenomatous Epithelial Cell, Taf[[II]]250, proteasome, Mus musculus, Adenomatous Epithelial Cells, Squamous Cell, TAF[[II]]230, Caspase-14 subunit p10, Prosome, mice, BcDNAGH03694, Swiss Mouse, expanded, Glandular Epithelial Cells, House Mice, 20S, TAF[II]250, Caspase-14 subunit p19, CG17603, MICE, TAF[[II]], 3L6, Laboratory Mice, domesticus, DmelCG17603, Taf250, rich, enlarged, SR3-5, Cells, Mouse, E430016J11Rik, multicatalytic endopeptidase, Laboratory Mouse, RWDD5, TAF230, proteasome endopeptidase complex, TAF1Methane, fic, src-4, Raw, Laboratory, TFA, NINA C, heavy chain disease, House Mouse, trichloro-, prevention, Long Term, nodus primitivus., 5730420M11Rik, Polypeptides, KL receptor activity, protein polypeptide chains, hydrogen chloride, dorsal marginal zone, Method, SRC 29A, btk29A, B1, SCO5, SEC, SCO1, Gsfsow3, WMS, C79691, SET, Dsrc28C, Divorced, F, C79325, sec, Divorces, proteins, W, T6G21.3, DmelCG4299, Lccp, isolation and purification, Dsrc29A, set, NOR-1, Wasserstoffchlorid, column, ACN, sample, Acn, D1, Bs, SGS, LC-MS2, Stars, preventive therapy, CHN, Trypsin/K, Longterm Effect, Guanidine Monohydroiodine, Procedure, not genetically inherited, CG18355, mice C57BL/6xCBA/CaJ hybrid, ACMICD, DmelCG5125, PBT, TPSG1, Chlorwasserstoff, Tec29A, DRONINAC, pbt, PSCTK4, HLA-DR-associated protein II, DI-2, I-2Dm, Swiss Mouse, pooled, Guanidine Phosphate, MASCOT, Methodological, beta Trypsin, CT2415, I-2PP1, TAF-IBETA, src28C, spirit of wood, liquid chromatography-tandem mass spectroscopy, PRSS31, DMZ, TAF-Ibeta, Mouse, chlorane, STARS, TMT, Methyl Alcohol, krk1, Hydroxylamine Hydrochloride, Effects, False, chlorure d'hydrogene, Pierce, mouse <Mus musculus>, Dm SRC2, CH3OH, thomson, protein-containing complex, 17alpha)-isomer, mass-to-charge ratio, Guanidium, isolation, LC-MSMS, Gene Products, Mus musculus domesticus, NINAC, Technique, CG54125, T6K12_14, Monohydroiodine, NinaC, Acinus, Hydrogen chloride, Longterm, nodus primitivus, beta-Trypsin, 2pp2a, Long-Term, CG10574, acinusL, Study, Injectable, mKIAA0989, Monohydrobromide, Guanidinium Chloride, 2PP2A, acinusS, dSET, dSet, peptidos, Tec29, 2.2, PTHB1, proto-oncogene c-Kit, Guanidine Monohydrate, Guanidine Nitrate, Proteins, Methyl alcohol, DSrc28, chloridohydrogen, backward, buffer, Cell, mKIAA0670, Engine, CD142, polypeptide, Temperatures, native protein, I-2PP2A, KIT ligand receptor activity, C18, chemical analysis, Long Term Effects, Dm I-2, Nitrate, 5alpha, MFS1, S13, Sodium, MIXL, MeOH, CT41718, XKrk1, T6K12.14, Guanidine Monohydrobromide, Guanidine, Col4a-1, Guanidine Sulfate, underdeveloped, ensemble, prophylaxis, Separations, Striated muscle activator of Rho-dependent signaling, Chloride, House Mice, CD117, Longterm Effects, plan specification, Gene Proteins, C-Kit, control, Ssm, xkl-1, liquid chromatography tandem mass spectrometry, Sulfate, hydrochloric acid, Methoxide, reversed, liquid chromatography tandem mass spectroscopy, IPP2A2, Bru, determination, Mus domesticus, Dm NinaC, classic hairy cell leukaemia, Monohydrate, Guanidine Sulfate (2:1), CG5125, CSMF, Hydrogenchlorid, Xkl-1, protein, peptide, Techniques, Gsfsco1, peptido, BANF, protein aggregate, Gsfsco5, prevention and control, Injectables, Effect, MIX, wood alcohol, SOW3, Svc, 3-hydroxy-5-estrane-17-carbonitrile, LCMSMS, peptides, reference sample, TAF-I, 2610036I19Rik, 2610510L13Rik, hypoplasia, Swiss Mice, Phosphate, purification, btk, c-src/fps, preventive measures, reaction, Hydroxylammonium Chloride, IGAAD, fSAP152, Nina C, Methodological Studies, DmelCG10574, TF, Guanidine Sulfate (1:1), TEAB, Sodium Methoxide, house mouse, Sl, GPHYSD2, Long-Term Effects, CT16120, Src29A, phapii, Monohydrochloride, Alcohol, LC-MS/MS, mouse, StF-IT-1, Tr-kit, Th, Search, classic hairy cell leukemia, S-adenosyl-L-methionine:thiol S-methyltransferase activity, HCD, kl1-A, Henson's node, KIT, Carbinol, carbinol, tyrosine-protein kinase Kit, MILD1, Mus musculus, Src2, Xylella fastidiosa str. Temecula1, HCl, Guanidine Monohydrochloride, kit, MASS, CG4299, Methodological Study, Trichloromethane, domesticus, nanospray, Wood, src4, Acetamide, Polypeptide, i2pp2a, src2, DmelCG8049, Procedures, AW549739, SCF receptor activity, FBN, Gene, Methyl, ACINUS, Methylalkohol, C-src2, C-src4, PHAPII, LC-MS-MS, Buffer, method, reduced, scfr, polypeptide chain, Ms1, House, ECTOL1, Injection, DTec29, method used in an experiment, Studies, Wood Alcohol, Del(8)44H, tiny, SCFR, Separated, Mice, cloruro de hidrogeno, Guanidium Chloride, Search Engines, Fdc, AI573420, MS1, ninac, tec29, Swiss, Selb, ipp2a2, CG 5125, Xylella fastidiosa (strain Temecula1 / ATCC 700964), OCTD, wood naphtha, taf-ibeta, stem node, male sterility 1, Long-Term Effect, dsrc29A, Guanidine Hydrochloride, 2-iodo-, thiol methyltransferase activity, Controlled, small, NOR1, Controlling, Nor1, protein complex, igaad, Guanidinium, Peptide, group, Mus muscaris, [HCl], LC/MS/MS, secret agent, natural protein, Mus, Protein, I2PP2A, Hydrochloride, Guanidine Sulfite (1:1), CT42491, CG8049, (3alpha, WMS2, Data Base, Separation, c-KIT, PRSS, MINOR, nodal stem, MALE STERILITY 1 PROTEIN, btk29a, Tripcellim, sample population, Laboratory Mice, Protein Gene Products, Trypure, dSET/TAF-Ibeta, c-kit, 2610030F17Rik, wood spirit, SSKS, TEC, Tec, Peptid, assay, SCH, AA407739, Laboratory Mouse, 3-OHECNimprinted and ancient gene protein, Transitional Epithelial Cell, Transitional, Adenomatous Epithelial Cell, Squamous Cells, Adenomatous Epithelial Cells, Squamous Cell, Epithelial Cell, Glandular Epithelial Cell., Glandular Epithelial Cells, Adenomatous, Adenomatous Epithelial, Glandular Epithelial, Squamous, Glandular, Ximpact, Transitional Epithelial, impact-a, Cell, Squamous Epithelial Cells, Cuboidal Glandular Epithelial Cells, Epithelial Cells, Transitional Epithelial Cells, Cells, IMPACT, imprinted and ancient gene protein homolog, Columnar Glandular Epithelial Cells, E430016J11Rik, Squamous Epithelial Cell, Squamous Epithelial, RWDD5, EpithelialfalseTMT10plex_cTEC_mTEC_beta5tKO-cTECTEC cells (K5D1 cTECs in quadruplicate, K5D1 mTECs in triplicate, and K5D1-β5tKO cTECs in triplicate) were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl, pH 8.0, and 2 mM DTT. The clarified lysates were reduced in 5 mM DTT and alkylated in 27.5 mM iodoacetamide. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate buffer. The proteins were digested with trypsin/Lys-C mix for 16 hr. Approximately 10 µg of peptides for each sample were labeled with 0.2 mg of TMT10-plex reagents for 1 hr at room temperature. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with TFA and fractionated using Pierce high pH reversed-phase peptide fractionation kit. Ten fractions were collected using 5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, 50%, and 80% ACN. Each fraction was evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis of the resultant peptides (1 µg each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a 75 µm inner diameter × 150 mm C18 reversed-phase column. The mass spectrometer was operated in a data-dependent acquisition mode with a top 15 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control (AGC) target of 3×106 and a mass range from 375 to 1,400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1×105, an isolation window of 0.4 m/z, a maximum injection time of 100 msec and a normalized collision energy of 32. Dynamic exclusion was set to 30 sec. Raw data were directly analyzed against the Swiss-Prot database restricted to Mus musculus using Proteome Discoverer version 2.2 with Mascot search engine version 2.5 for identification and TMT quantification. Peptides and proteins were filtered at a false-discovery rate (FDR) of 1 % using the percolator node and the protein FDR validator node, respectively.Tue Oct 08 00:00:00 BST 2019PXD0131321009031775054