An identification of kinases which phosphorylates Ebola virus VP30 protein
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Stephan Becker
PROVIDER: PXD016409 | JPOST Repository | Mon Feb 03 00:00:00 GMT 2020
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 1Baumeister_YT1.raw | Raw | |||
| 1Baumeister_YT2.raw | Raw | |||
| 1Baumeister_YT3.raw | Raw | |||
| 1Baumeister_YT4.raw | Raw | |||
| 1Baumeister_YT5.raw | Raw |
Items per page: 5 1 - 5 of 20 |
Similar Datasets
Project description:Bone marrow-derived dendritic cells were incubated with 50 micro g/mL of H. polygyrus bakery non-polar metabolites in RPMI 1640 containing 10% FBS, 1% of 100X penicillin/streptomycin, 1% of 100 mM sodium pyruvate, and 20 ng/mL GM-CSF at 37°C, 5% CO2 for 4 or 20 h. Supernatants and cell-free media controls were collected and incubated with ice-cold HPLC-grade methanol for 30 min on ice, centrifuged at 10,000 x g for 10 min at 4°C and stored at -80°C until analysis. The profiling of nonpolar metabolites was performed by LC-MS/MS analysis of the deproteinated conditioned media by injecting 3 mL of sample onto a Dionex UHPLC system equipped with an Agilent Eclipse C18 (2.1 x 15 mm, 1.8 mm) column incubated at 45oC. Metabolites were resolved with a 30 min linear running 0-80 % using the buffers system 0.05 % formic acid and 0.05 % formic acid in acetonitrile at a flowrate of 300 mL/min. The column effluent was introduced by electrospray ionization onto a ThermoScientific Velos LTQ Orbitrap Analyzer using a spray voltage of 3.6 kV, a source heater temperature of 350oC, and a sheath gas flow of 40 L/min. Survey scans were performed using the Orbitrap mass spectrometer and the 10 most intense ions were selected for fragmentation using a 30-40 V stepped collision induced dissociation energy. Fragmentation products were analyzed in the linear ion trap mass spectrometer. Fragmentation was used to perform XCMS online database (https://xcmsonline.scripps.edu) search to identify possible metabolites.
2022-07-01 | ST002230 | MetabolomicsWorkbench
Project description:Cervicovaginal lavage (CVL)supernatants were heat inactivated for 30 minutes at 56°C before further processing. Total protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific, Rockford, IL, USA). Sample protein content and volume were normalised with 25mM ammonium bicarbonate (ABC). Soluble proteins were precipitated using an equal volume of ice cold 30% (w/v) TCA in acetone and incubated at -20⁰C for 2 hours. Samples were centrifuged at 12,000g for 10 minutes (4⁰C) to pellet proteins. Pellets were washed three times with ice cold acetone and allowed to air dry. Further sample processing was performed as previously described (Armstrong et al, 2014) with minor modifications. Briefly, protein pellets were resuspended in 25 mM ABC, 0.05%(w/v) rapigest (Waters), reduced and alkylated. Digestion was performed with proteomic-grade trypsin (Sigma-Aldrich, St. Louis, MO, USA) at a protein:trypsin ratio of 50:1. Rapigest was precipitated by addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the nanoACQUITY-nLC system (Waters MS technologies) coupled to an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) mass spectrometer equipped with the manufacturer’s nanospray ion source. The gradient of the analytic column (nanoACQUITY UPLCTM BEH130 C18 15cm x 75µm, 1.7µm capillary column) consisted of 3-40% acetonitrile in 0.1% formic acid for 90 min then a ramp of 40-85% acetonitrile in 0.1% formic acid for 5min.
2016-03-15 | PXD003176 | Pride
Project description:For the analysis of chromosome 11-centric human proteome, we analyzed the human brain hippocampal proteome. The samples from brain tissues were extensively off-gel fractionated by 12 fractions and analyzed by a high resolution mass spectrometer, Orbitrap XL ETD with collision induced dissociation, after LC separation using C18 column.
2016-10-10 | PXD000395 | Pride
Project description:Washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da Oxidation (M) and Carbamidomethyl (C) specified as variable modifications Enzyme specificity was trypsin, 1 missed cleavage was possible
2014-04-28 | PXD000505 | Pride
Project description:Bacteria were washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da; Oxidation (M) and Carbamidomethyl (C) specified as variable modifications, Enzyme specificity was trypsin, 1 missed cleavage was possible
2014-04-28 | PXD000504 | Pride
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
Project description:Flooding is a serious problem for soybean cultivation because it markedly reduces growth. We found that treatment with ABA enhances survival ratio of soybean under flooding stress. To investigate the role of ABA in soybean under flooding stress, proteomic changes were analyzed. Total protein and nuclear protein fractions from root tips of soybean seedlings that treated with flooding or flooding with ABA were analyzed. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin and lysyl endopeptidase. Resulting peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Using an Ultimate 3,000 nanoLC system (Dionex), peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and their separation and spraying were done using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3, Nikkyo Technos) with a spray voltage of 1.5 kV. The elution was done with a linear acetonitrile gradient 8-30% in 120 min for gel free proteomics in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over a mass range of 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy. The top 10 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 sec to prevent repetitive selection of peptides.Identification of proteins were performed by Mascot search engine (version 2.3.0.2, Matrix Science) using soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0)and a common contaminants database (262 sequences). Parameters used in Mascot searches were follows: Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charge was set at +2, +3, and +4. Automatic decoy database search was performed in the search. Mascot results were filtered with Mascot percolator to improve accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were less than 1.0%. The Mascot results were used for differential analysis using SIEVE software (version 2.0, Thermo Fisher Scientific).
2013-12-12 | PXD000160 | Pride
Project description:Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. Two-day-old soybeans, seedlings flooded for 2 days and hypocotyls of seedlings grown for 3 days after flooding were analyzed. The radicles of soybean seedlings were separated to root tips (RT), roots without tip (RwoT) and hypocotyls (Hypo). Protein was extracted from these organs by TCA/Acetone method. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin. Resulting peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Using an Ultimate 3,000 nanoLC system (Dionex), peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and their separation and spraying were done using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3, Nikkyo Technos) with a spray voltage of 1.5 kV. The elution was done with a linear acetonitrile gradient 8-30% in 120 min for gel free proteomics in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over a mass range of 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy. The top 6 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 sec to prevent repetitive selection of peptides.Identification of proteins were performed by Mascot search engine (version 2.3.0.2, Matrix Science) using soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0)and a common contaminants database (262 sequences). Parameters used in Mascot searches were follows: Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charge was set at +2, +3, and +4. Automatic decoy database search was performed in the search. Mascot results were filtered with Mascot percolator to improve accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were less than 1.0%. The Mascot results were used for differential analysis using SIEVE software (version 1.3, Thermo Fisher Scientific)
2013-12-10 | PXD000201 | Pride
Project description:Human primary macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 6 hrs or left untreated. Cells were collected and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the supernatant was collected and the protein content was measured with Bio-Rad DC™ protein assay (Bio-Rad). The proteins were reduced, alkylated, and enzymatically digested in-solution with trypsin. The digestion was stopped by adding FA (final c = 1 %). The samples were centrifuged 9168 x g for 10 min and desalted with Sep-Pak Vac RP C18 cartridges (Waters, MA, USA), following fractionation by strong cation exchange chromatography (SCX). The peptides were separated on a 200 x 4.6 mm, 5 μm, 200 Å PolySULFOETHYL A™ column (PolyLC, USA). The fractions were vacuum centrifuged and desalted as before, following phosphopeptide-enrichment with PHOS-Select™ Iron Affinity Gel (Sigma Aldrich, MO, USA). LC-MS/MS was performed with a Q Exactive hybrid quadrupole-orbitrap tandem mass spectrometer coupled to an EASY-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher Scientific). A 100 μm x 3 cm trap column and a 75 μm x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 μm; Michrom Bioresources). The mobile phases were 2% acetonitrile, 0.2% formic acid (A) and 95% acetonitrile, 0.2% formic acid (B). LC gradient elution condition was 2% B (0 min), 20% B (70 min), 40% B (100 min), and then 100% B (105-110 min), with a flow rate of 300 nl/min. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 300 to m/z 2000 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 120 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation (HCD) with an isolation window of 2 and normalized collision energy of 30. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 250 ms, and the lowest mass fixed at m/z 100. Dynamic exclusion duration was 30 s.
2016-08-08 | PXD001620 | Pride
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.
2022-12-07 | PXD023141 | Pride