Dataset Information

TMT-based quantitative proteomic analysis of cardiac proto-myofibroblasts after Bcat1 knockdown

ABSTRACT: Proto-myofibroblasts were treated with siRNA, washed with ice-cold HEPES-saline (20 mM HEPES-NaOH [pH 7.5], 137 mM NaCl), detached by Accutase (Nacalai Tesque), and lysed in guanidine-TCEP buffer (8 M guanidine-HCl, 100 mM HEPES-NaOH [pH 7.5], 10 mM TCEP, 40 mM CCA). After heating and sonication, 200 Î¼g of protein was purified by methanol-chloroform precipitation and resuspended in 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. Proteins were further sonicated, heated, and digested with trypsin/Lys-C mix (Promega) for 16 hours at 37Â°C. Peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 130 Î¼g of peptides was labeled with TMTpro 16-plex reagent (Thermo Fisher Scientific) for 1 hour at 25Â°C. After quenching with hydroxylamine, labeled samples were pooled. One-tenth of the pooled sample was then acidified with trifluoroacetic acid (TFA) and fractionated by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific). The 48 fractions were consolidated into 16 fractions, evaporated in a SpeedVac concentrator, and disolved in 3% acetonitrile (ACN) and 0.1% TFA. LC-MS/MS analysis of the resulting peptides (500 ng each) was performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 Î¼m inner diameter Ã— 150 mm C18 reversed-phase column (Nikkyo Technos) using a linear gradient of 4%â€“20% ACN for 0â€“115 minutes and 20%â€“32% ACN for 115â€“160 minutes, followed by an increase to 80% ACN for 10 minutes and a final 10-minute hold at 80% ACN. The mass spectrometer was operated in data-dependent acquisition mode with a top-10 MS/MS method. MS1 spectra were acquired at a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375â€“1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000 with an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 seconds. Raw data were analyzed against the Mus musculus SwissProt database using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine for protein identification and TMT quantification. Search parameters included trypsin with up to 2 missed cleavages, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. TMTpro labeling of lysine residues and peptide N-termini and carbamidomethylation of cysteine were set as fixed modifications, and methionine oxidation was set as a variable modification. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the percolator node and protein FDR validator node, respectively. Normalization was performed so that the total abundance across all peptides was equal for each TMT channel.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Michio Nakaya

PROVIDER: PXD075667 | JPOST Repository | Wed Jul 08 00:00:00 BST 2026

REPOSITORIES: jPOST

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			Action	DRS
	208_Nakaya_siBcat1_TMTpro16_Total_Vanquish16_1_QEp.raw	Raw
	210_Nakaya_siBcat1_TMTpro16_Total_Vanquish16_2_QEp.raw	Raw
	212_Nakaya_siBcat1_TMTpro16_Total_Vanquish16_3_QEp.raw	Raw
	214_Nakaya_siBcat1_TMTpro16_Total_Vanquish16_4_QEp.raw	Raw
	216_Nakaya_siBcat1_TMTpro16_Total_Vanquish16_5_QEp.raw	Raw

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Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.

Project description:Proto-myofibroblasts were treated with siRNA, washed with ice-cold HEPES-saline (20 mM HEPES-NaOH [pH 7.5], 137 mM NaCl), detached by Accutase (Nacalai Tesque), and lysed in guanidine-TCEP buffer (8 M guanidine-HCl, 100 mM HEPES-NaOH [pH 7.5], 10 mM TCEP, 40 mM CCA). After heating and sonication, 200 Î¼g of protein was purified by methanol-chloroform precipitation and resuspended in 0.1% RapiGest SF (Waters) in 50 mM triethylammonium bicarbonate. Proteins were further sonicated, heated, and digested with trypsin/Lys-C mix (Promega) for 16 hours at 37Â°C. Peptide concentrations were determined using the Pierce quantitative colorimetric peptide assay (Thermo Fisher Scientific). Approximately 130 Î¼g of peptides was labeled with TMTpro 16-plex reagent (Thermo Fisher Scientific) for 1 hour at 25Â°C. After quenching with hydroxylamine, labeled samples were pooled. Nine-tenths of the pooled sample were subjected to phosphopeptide enrichment using the High-Select Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific). The enriched phosphopeptides were subsequently fractionated into 16 fractions by offline high-pH reversed-phase chromatography on a Vanquish DUO UHPLC system (Thermo Fisher Scientific), evaporated in a SpeedVac concentrator, and dissolved in 3% acetonitrile (ACN) and 0.1% TFA. LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC coupled to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). Peptides were separated on a 75 Î¼m inner diameter Ã— 150 mm C18 reversed-phase column (Nikkyo Technos) using a linear gradient of 4%â€“20% ACN for 0â€“115 minutes and 20%â€“32% ACN for 115â€“160 minutes, followed by an increase to 80% ACN for 10 minutes and a final 10-minute hold at 80% ACN. The mass spectrometer was operated in data-dependent acquisition mode with a top-10 MS/MS method. MS1 spectra were acquired at a resolution of 70,000, an automatic gain control (AGC) target of 3e6, and a mass range of 375â€“1400 m/z. HCD MS/MS spectra were acquired at a resolution of 35,000 with an AGC target of 1e5, an isolation window of 0.7 m/z, a maximum injection time of 150 ms, and a normalized collision energy of 33. Dynamic exclusion was set to 20 seconds. Raw data were analyzed against the Mus musculus SwissProt database using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) with the Sequest HT search engine for protein identification and TMT quantification. Search parameters included trypsin with up to 2 missed cleavages, a precursor mass tolerance of 10 ppm, and a fragment mass tolerance of 0.02 Da. TMTpro labeling of lysine residues and peptide N-termini and carbamidomethylation of cysteine were set as fixed modifications, and methionine oxidation and serine/threonine/tyrosine phosphorylation were set as a variable modifications. Peptides were filtered at a false discovery rate (FDR) of 1% using the percolator node. Normalization was performed so that the total abundance across all peptides was equal for each TMT channel.

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:Approximately 1 x 10^8 GFP-HA-KERP2 expressing Entamoeba histolytica cells were cross-linked in 0.5% formaldehyde (in PBS) for 10 minutes at room temperature. Cross-linking was quenched with 125 mM glycine for 5 minutes. Cells were washed once with PBS and subjected to nuclear fractionation using nuclei isolation buffer (10 mM Hepes-KOH pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 2% NP-40, 1 uM E-64, 0.5 mM PMSF, and 1x cOmplete protease inhibitor cocktail). Nuclear pellets were resuspended in sonication buffer (50 mM Tris-HCl pH 8.0, 140 mM NaCl2, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 5 mM DTT, 1 uM E-64, 0.5 mM PMSF, 1x cOmplete) and sonicated using a Covaris instrument. Sonicated samples were centrifuged at 16000 x g for 20 minutes. A 200 uL aliquot of supernatant was transferred to a 1.5 mL Protein LoBind tube. Remaining sample was stored as input control. For immunoprecipitation, 1 mL of ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, 0.5% BSA, 1x cOmplete) containing 50 uL of GFP(ab290)-antibody conjugated Dynabeads was added and rotated at 4 C for 12 hours. Beads were washed five times with low-salt wash buffer (20 mM Tris-HCl pH 8.0, 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl), followed by two washes with high-salt wash buffer (same as above, but 500 mM NaCl). DNA-protein complexes were eluted with 200 uL of ChIP elution buffer (10 mM Tris-HCl pH 8.0, 1% SDS, 5 mM EDTA, 300 mM NaCl) at room temperature for 20 minutes. Input samples were thawed and diluted with 190 uL of elution buffer. All samples were incubated at 65 C for 8 hours to reverse cross-links, followed by RNase A treatment (1 uL) at 37 C for 1 hour and Proteinase K digestion (5 uL) at 55 C for 2 hours. DNA was purified by phenol/chloroform extraction with 1 uL glycogen as carrier, followed by ethanol precipitation overnight at -30 C. DNA pellets were washed with 80% ethanol, air-dried, and resuspended in 20 uL TE (ChIP) or 40 uL TE (input). DNA concentrations were quantified using a Qubit dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific). Purified DNA was submitted to Rhelixa for ChIP-seq.

Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.

Dataset Information

TMT-based quantitative proteomic analysis of cardiac proto-myofibroblasts after Bcat1 knockdown

Dataset's files

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