Dataset Information

TFRE (Transcription factor response elements)

ABSTRACT: TFRE pulldowns, and trypsin digestion Tumor and paired NAT breast tissues were washed twice with ice-cold phosphate-buffered saline (PBS) and minced and lysed in lysis buffer (8M Urea, 100mM Tris Hydrochloride, pH=8.0) containing protease and phosphatase Inhibitors (Thermo Scientific) followed by 1 min sonication (3s on and 3s off, amplitude 25%). Lysates were then centrifuged at 16,000 g for 10 min at 4℃ to collect the supernatants. Protein concentration was determined by Bradford protein assay. One hundred µg of protein samples were reduced at 56°C for 30 min with 10 mM dithiothreitol (DTT) and alkylated with 10 mM iodoacetamide (IAA) at room temperature in the dark for an additional 30 min. Samples were then digested using the FASP method (Wisniewski et al., 2009) with trypsin at 1:50 (w/w) ratios. Tryptic peptides were then dried using a SpeedVac concentrator (Thermo Scientific). Nuclear extracts were incubated with biotinylated DNA at 4°C for 2 h. The complexes were precipitated on M-280 streptavidin-coupled dynabeads, washed with NETN (100 mM NaCl, 20 mM Tris-HCl, 0.5 mM EDTA, and 0.5% [vol/vol] NP-40) buffer twice and water once, respectively. Proteins on the Dynabeads were then digested with trypsin at 1:50 (w/w) ratios in 50 mM ammonium bicarbonate at 37 °C for 6 h. Peptide mixtures were dissolved in 0.1% formic acid before they were subjected to LC-MS/MS analysis (Ge et al., 2018; Gong et al., 2020).LC-MS/MS Analysis for Proteomic Analysis Liquid chromatography For proteome profiling, phosphoproteome, and TFRE experiments, LC-MS/MS analysis was performed using three mass spectrometry platforms: an Easy-nLC 1000 nanoflow LC system (Thermo Fisher Scientific) connected to (1) Orbitrap Fusion, (2) Orbitrap Fusion Lumos, (3) and a Q Exactive HF mass spectrometer (Thermo Fisher Scientific, Rockford, IL, USA) coupled to an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) (Ge et al., 2018). The peptide mixture was then resuspended in Solvent A (0.1% formic acid in water) for LC-MS/MS analysis. The obtained peptides were separated on a home-made 150 μm × 12 cm silica microcolumn (particle size, 1.9 μm; pore size, 120 Å; SunChrom, USA) at a flow rate of 600 nl/min. The gradient was set 5–35% mobile phase B (0.1% formic acid in acetonitrile) for 75 min. The MS analysis for QE HF was performed with one full scan (300–1400 m/z, R = 60,000 at 200 m/z) at automatic gain control target of 3e6 ions, followed by up to 20 data-dependent MS/MS scans. The AGC was set as 5e4 ions with max injection time 40 ms. A 1.6 m/z isolation window was used. The normalized collision energy was 27% and the resolution was 15,000 at 200 m/z. 16 For detection with Fusion or Fusion Lumos mass spectrometry, a precursor scan was carried out in the Orbitrap by scanning m/z 300−1400 with a resolution of 120,000 at 200 m/z. The most intense ions selected under top-speed mode were isolated in Quadrupole with a 1.6 m/z window and fragmented by higher energy collisional dissociation (HCD) with normalized collision energy of 35%. The ACG was setting as 5e5 ions with a max injection time of 50 ms for full scans and 5e3 with 35 ms for MS/MS scans. Dynamic exclusion time was set to 18s to avoid repeated sequencing of identical peptides

TISSUE(S): ['breast', 'cell Line']

DISEASE(S): ['triple-receptor Negative Breast Cancer']

SUBMITTER: Weimin Zhu

PROVIDER: OEX00014791 | NODE | 2023-07-26

REPOSITORIES: NODE

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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

Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:Cells were treated with DMSO (biological triplicate) or degrader at indicated dose and time (Meta Treatment Table) and cells were harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets and homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 1 - 4 mg/mL. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg of protein for each sample were reduced and alkylated as previously described1. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 M Urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein) for 6 hours at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 11 or 16-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. Each of the sample channels were combined in a 1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid and acidified (pH 2 - 3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific) or an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a/ES803a.rev2 75 μm inner diameter microcapillary column (Thermo Fisher Scientific) or a 100 µm inner diameter microcapillary column packed with ~ 50 cm of Accucore C18 resin (2.6 µM, 100 Å, Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6 - 27% acetonitrile in 1.0% formic acid with a flow rate of 300 or 350 nL/min. Each analysis used an MS3-based TMT method as described previously. The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.

Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other

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TFRE (Transcription factor response elements)

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