Dataset Information

LFQ_K5D1-cTEC_K5D1-beta5tKO-cTEC

ABSTRACT: Cortical TECs from K5D1 and K5D1-β5tKO mice were lysed, reduced, alkylated, and subjected to methanol/chloroform precipitation. Proteins were digested with trypsin/Lys-C mix. The digests were desalted using GL-Tip SDB, and the eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% TFA. LC-MS/MS analysis of the resultant peptides (400 ng each) was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer. The mass spectrometer was operated in a data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 1×106 and a mass range from 350 to 1,500 m/z. HCD MS/MS spectra were acquired at a resolution of 17,500, an AGC target of 5×104, an isolation window of 2.0 m/z, a maximum injection time of 60 msec and a normalized collision energy of 27. Dynamic exclusion was set to 10 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 label-free precursor ion quantification. Peptides and proteins were filtered at a FDR of 1% using the percolator node and the protein FDR validator node, respectively. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD013133 | JPOST Repository | Tue Oct 08 00:00:00 BST 2019

REPOSITORIES: jPOST

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Publications

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 Y

Cell reports 20191101 9

The 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 mic ...[more]

PMID: 31775054

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Project description:In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized over 1.3-fold by ARF, while BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. ICAT-labeled Cys peptides were analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Peptide eluents were monitored in an MS survey scan followed by ten data-dependent MS/MS scans on an LTQ-Orbitrap ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LTQ was used to acquire MS/MS spectra (2 m/z isolation width, 35% collision energy, 5,000 AGC target, 200 ms maximum ion time). The Orbitrap was used to collect MS scans (300-1600 m/z, 1,000,000 AGC target, 500 ms maximum ion time, resolution 30,000). All data were converted from .raw files to the .dta format using ExtractMS version 2.0 (Thermo Finnigan, San Jose, CA). The acquired MS/MS spectra were searched against a concatenated target-decoy human RefSeq (release 37 - September 2009 - 38108 target proteins) database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm (version 3.11, SAGE-N) searching parameters included partially tryptic restriction, parent ion mass tolerance (+/- 20 ppm), dynamic modifications of oxidized Met (+15.9949 Da), differential ICAT-modified Cys (+9.0302 Da) and static ICAT modification of Cys (+227.1270 Da). The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS), and then dynamically by increasing XCorr and deltaCn values to reduce protein false discovery rate to less than 1%, according to the target-decoy strategy.

Project description:TEC 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.

Project description:Tissue lysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012)) Briefly, greater than 300 mg of frozen tumor mass was homogenized and sonicated in urea lysis buffer (20 mM HEPES pH 8.0, 9 M urea, 2.5 mM sodium pyrophosphate, 1.0 mM beta-glycerophosphate, 1% N-octyl glycoside, 2 mM sodium orthovanadate). Total protein was measured using the BCA Protein Assay Kit (Thermo Scientific/Pierce) and 25 mg of total protein was used for phospho-proteomic analysis. Phospho-tyrosine peptide enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012); Rubbi, L., et al. Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling. Sci Signal 4, ra18 (2011); Graham, N.A., et al. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death. Molecular systems biology 8, 589 (2012)) Phospho-peptides were identified using the Proteome Discoverer software (version 1.3.0.339, Thermo Fisher Scientific). MS/MS fragmentation spectra were searched using SEQUEST against the Uniprot human reference proteome database with canonical and isoform sequences (downloaded January 2012 from uniprot.org). Search parameters included carbamidomethyl cysteine (*C) as a static modification. Dynamic modifications included phosphorylated tyrosine, serine, or threonine (pY, pS, pT, respectively) and oxidized methionine (*M). The Percolator node of Protein Discoverer was used to calculate false-discovery rate (FDR) thresholds and the FDR for the datasets was adjusted to 1% (version 1.17, Thermo Scientific). The Percolator algorithm uses a target-decoy database search strategy and discriminates true and false identifications with a support vector machine. The PhosphoRS 2.0 node was used to more accurately localize the phosphate on the peptide44. Only phospho-peptides with at least one phospho-tyrosine assignment with a reported probability above 20% were considered. MS2 spectra for all reported phosphopeptides are available under the PRIDE accession numbers

			Action	DRS
	WT_cTEC_1st.raw	Raw
	WT_cTEC_2nd.raw	Raw
	beta5tKO_cTEC_1st.raw	Raw
	beta5tKO_cTEC_2nd.raw	Raw
	cTEC_LFQ.xlsx	Xlsx