Dataset Information

TMT10plex_cTEC_mTEC_beta5tKO-cTEC

ABSTRACT: 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.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD013132 | 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:The brains of neonatal mice (Atg5+/+ in triplicate, Atg5-/- in quadruplicate, and Atg5-/-;NSE-Atg5 in triplicate) were lysed in 500 µL of 6 M guanidine-HCl containing 100 mM Tris-HCl, pH8.0, and 2 mM DTT. The lysates were dissolved by heating and sonication, followed by centrifugation at 20,000 g for 15 min at 4 ºC. Proteins (100 µg each) were reduced in 5 mM DTT at room temperature for 30 min, alkylated in 27.5 mM iodoacetamide at room temperature for 30 min in the dark, and subjected to methanol/chloroform precipitation. After solubilization with 25 µL of 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate, the proteins were digested with 1 µg of trypsin/Lys-C mix (Promega) for 16 h at 37 ºC. The peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay. Approximately 25 µg of peptides for each sample was labeled with 0.2 mg of TMT-10plex reagents (Thermo Fisher Scientific) for 1 h at 25 ºC. After the reaction was quenched with hydroxylamine, all the TMT-labeled samples were pooled, acidified with trifluoroacetic acid (TFA), and fractionated using the Pierce high pH reversed-phase peptide fractionation kit. Eight fractions were collected using 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, 25%, and 50% acetonitrile (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 X 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear gradient of 4–20% ACN for 0–180 min and 20–32% ACN for 180–220 min, followed by an increase to 80% ACN for 220–230 min. 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 3e6, and a mass range of 375–1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000, an AGC target of 1e5, an isolation window of 0.4 m/z, a maximum injection time of 100 ms, and a normalized collision energy of 32. Dynamic exclusion was set to 30 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer version 2.2 (Thermo Fisher Scientific) with Mascot search engine version 2.5 (Matrix Science) for identification and TMT quantification. The search parameters were as follows: (a) trypsin as an enzyme with up to two missed cleavages; (b) precursor mass tolerance of 10 ppm; (c) fragment mass tolerance of 0.02 Da; (d) TMT of lysine and peptide N-terminus and carbamidomethylation of cysteine as fixed modifications; and (e) oxidation of methionine as a variable modification. Peptides were filtered at a false-discovery rate of 1% using the percolator node.

			Action	DRS
	TEC_TMT10.xlsx	Xlsx
	TEC_TMT10_bRP1.raw	Raw
	TEC_TMT10_bRP10.raw	Raw
	TEC_TMT10_bRP2.raw	Raw
	TEC_TMT10_bRP3.raw	Raw