LFQ_TurboID-STING_anti-biotin_antibody
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus (mouse)
SUBMITTER: Hidetaka Kosako
PROVIDER: PXD018803 | JPOST Repository | Wed Jun 17 00:00:00 BST 2020
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| TurboID_STING_Antibody.pdResult | Other | |||
| TurboID_STING_Antibody.xlsx | Xlsx | |||
| TurboID_STING_Antibody_DMSO_1.raw | Raw | |||
| TurboID_STING_Antibody_DMSO_2.raw | Raw | |||
| TurboID_STING_Antibody_DMSO_3.raw | Raw |
Items per page: 5 1 - 5 of 8 |
Publications
BioID screening of biotinylation sites using the avidin-like protein Tamavidin 2-REV identifies global interactors of stimulator of interferon genes (STING).
Motani Kou K Kosako Hidetaka H
The Journal of biological chemistry 20200617 32
Stimulator of interferon genes (STING) mediates cytosolic DNA-induced innate immune signaling via membrane trafficking. The global identification of proteins that spatiotemporally interact with STING will provide a better understanding of its trafficking mechanisms and of STING signaling pathways. Proximity-dependent biotin identification (BioID) is a powerful technology to identify physiologically relevant protein-protein interactions in living cells. However, biotinylated peptides are rarely d ...[more]
Similar Datasets
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 30 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM HEPES-NaOH (pH 7.5), 10 mM TCEP, and 40 mM CAA. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, and proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with trypsin at 37ºC overnight. The resultant peptide solutions were captured on MagCapture HP Tamavidin 2-REV magnetic beads in the presence of 1 mg/ml Pefabloc SC during 3 h of incubation at 4ºC. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 1 mM biotin at 95ºC twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrato, and redissolved in 0.1% TFA and 3% ACN.
2020-06-17 | PXD018802 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 100 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl (pH 8.0) and 2 mM DTT. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, followed by reduction in 5 mM DTT at room temperature for 30 min and alkylation in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with 20 µg trypsin at 37ºC overnight. The resultant peptide solutions were captured on the anti-biotin antibody-coupled beads in the presence of 1 mg/ml Pefabloc SC during 16 h of incubation at 4ºC. After washing with TBS four times, and with ultrapure water twice, the biotinylated peptides were eluted with 50 µL of 0.1% TFA in 80% acetonitrile three times. The combined eluates were evaporated, and desalted using GL-Tip SDB. The desalted eluates were further evaporated, and redissolved in 0.1% TFA.
2021-01-10 | PXD017044 | JPOST Repository
Project description:The STING-null RAW264.7 cells expressing TurboID-STING were treated with DMSO or 100 μg/ml DMXAA, and 500 µM biotin was added to the medium. After 1-h incubation, the cells were lysed in 6 M guanidine-HCl containing 100 mM Tris-HCl (pH 8.0) and 2 mM DTT. The lysates were dissolved by heating and sonication, and centrifuged at 20,000 g for 15 min at 4ºC. The supernatants were recovered, followed by reduction in 5 mM DTT at room temperature for 30 min and alkylation in 27.5 mM iodoacetamide at room temperature for 30 min in the dark. Proteins were purified by methanol/chloroform precipitation and solubilized by 0.1% RapiGest SF in 50 mM triethylammonium bicarbonate. After repeated sonication and vortex, the proteins were digested with 20 µg trypsin at 37ºC overnight. The resultant peptide solutions were captured on MagCapture Tamavidin 2-REV magnetic beads in the presence of 1 mg/ml Pefabloc SC during 16 h of incubation at 4ºC. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 2 mM biotin at 95ºC twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrato, and redissolved in 0.1% TFA.
2023-04-17 | PXD017042 | JPOST Repository
Project description:To evaluate the optimized workflow, biotinylated peptides enriched by the original and optimized workflows from 300 µg proteins of RAW264.7 cells expressing TurboID-STING were compared by LC-MS/MS analysis. We assessed reproducibility of each workflow using six technical replicates.
Original workflow: A 15-µL slurry of MagCapture HP Tamavidin 2-REV magnetic beads per sample was washed three times with TBS2 (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). The digested peptides containing 0.1% RapiGest SF were diluted 5-fold with TBS2 and incubated with the Tamavidin 2-REV beads in the presence of 1 mg/mL Pefabloc SC for 3 h at 4 °C. After washing five times with TBS2, biotinylated peptides were eluted with 100 μL of 1 mM biotin in TBS2 for 15 min at 95 °C twice. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% acetonitrile (ACN).
Optimized workflow: A 15-µL slurry of the Tamavidin 2-REV beads per sample was washed twice with 10% ACN and then three times with TBS2. The digested peptides in diluted PTS buffer were heated at 95 °C for 10 min to inactivate trypsin, diluted 2-fold with TBS2, and incubated with the ACN-prewashed Tamavidin 2-REV beads for 3 h at 4 °C. During the incubation period, 1 mM biotin solution in TBS3 (50 mM Tris-HCl, pH 7.5, and 500 mM NaCl) was passed through GL-Tip SDB. After washing five times with TBS2, the beads were incubated with 200 μL of TBS3 for 15 min at 37 °C as a mock elution. After removing TBS3, biotinylated peptides were eluted for 15 min at 37 °C twice with 100 µL of the biotin solution that had been passed through GL-Tip SDB. The combined eluates were desalted using GL-Tip SDB, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.
2022-08-25 | PXD035220 | JPOST Repository
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.
2014-02-14 | PXD000354 | Pride
Project description:Chem-seq analysis was conducted to reveal genome-wide localization of a cell penetrating peptide, SVS-1. A549 cells treated with 15 mM of biotin-PEG-GG-SVS-1 for 1 hour were passaged once to remove dead cell debris. After 24 hours incubation from the passage, the treated cells were fixed with formaldehyde to crosslink SVS-1 and chromatin, and total chromatin was subjected to fragmentation by sonication. Chromatin bound by the biotin-PEG-GG-SVS-1 was isolated with avidin-conjugated resin, followed by DNA purification. The input DNAs were also purified and employed as controls for the peak callings. The purified DNAs were subjected to library preparation for deep sequencing by the illumina platform. Sequencing was run by the NextSeq with paired end sequencing (2x76), 100 million clusters /sample.
2018-11-07 | GSE122241 | GEO
Project description:Bacteria was grown at 30C in 3 different conditions, i.e. SYN (syngas and minimal medium- ATCC no 1789), AC (0.3% acetate in minimal medium- ATCC no 1789) and TSB (tryptic soy broth). After harvesting by centrifugation, O. carboxidovorans pellets (1g) were lysed in 100 mM Tris-Cl pH 8.0, 2% Triton X-100, 2.6 mg/ml sodium azide, 8 mM PMSF by sonication on ice (4 pulses of 15 s duration each). For each condition of growth 4 samples (1g pellets) were separately treated (i.e. lysed and processed further). The supernatants were treated with 50% cold TCA, and the precipitated protein washed with acetone. The pellets were resuspended in solubilization buffer (7M urea, 20 mM tris-Cl, pH 8.0, 5 mM EDTA, 5 mM MgCl2, 4% CHAPS), and protein concentration was determined using the Plus One 2-D Quant Kit (Amersham) following the manufacturers instructions. Protein samples from each treatment were stored at -80 C. One hundred micrograms of each protein sample was resuspended in 0.1 M ammonium bicarbonate, 5% HPLC grade ACN, reduced in 5 mM DTT (65 C, 5 min), alkylated in 10 mM iodoacetamide (30 C, 30 min), and then trypsin digested until there was no visible pellet (1:50 w/w 37 C, 16 h). Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA) and eluted using a 0.1% TFA, 95% ACN solution. Desalted peptides were dried in a vacuum centrifuge and resuspended in 20 ?l of 0.1% formic acid. Peptides were separated by strong cation exchange (SCX) liquid chromatography (LC) followed by reverse phase (RP) LC coupled directly in line with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). 2DLC ESI MS/MS was done exactly as described (1). All searches were done using TurboSEQUEST (Bioworks Browser 3.2; Thermo Electron). Mass spectra and tandem mass spectra were searched against all annotated proteins from the strain OM5 including all the annotated plasmid-encoded proteins. Cysteine carbamidomethylation and methionine oxidation (single and double) were included in the search strategy. We used the reverse database functionality in Bioworks 3.2 and searched MS2 data against a reversed OM5 database using identical search criteria.
2012-05-23 | PRD000150 | Pride
Project description:The STING-null Raw264.7 cells expressing STING-BirA* were treated with DMSO or DMXAA for 15 min, and then biotin was added to the medium. After 8-h incubation, the cells were lysed and digested with trypsin. Biotinylated peptides were captured with tamavidin 2-REV magnetic beads and eluted with 2 mM biotin. The eluates were analysis by LC-MS/MS. Label-free quantification was performed using Proteome Discoverer 2.2 Software.
2023-04-17 | PXD009873 | JPOST Repository
Project description:Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).
2012-12-10 | PXD000004 | Pride
Project description:We tried purification of biotinylated peptides using SDB-SCX StageTip to reduce interference ions. We compared this method with the original and optimized workflows. We assessed reproducibility of each enrichment process by three technical replicates.
Original workflow with SDB-SCX: A 15-µL slurry of MagCapture HP Tamavidin 2-REV magnetic beads per sample was washed three times with TBS2 (50 mM Tris-HCl, pH 7.5, and 150 mM NaCl). The digested peptides containing 0.1% RapiGest SF were diluted 5-fold with TBS2 and incubated with the Tamavidin 2-REV beads in the presence of 1 mg/mL Pefabloc SC for 3 h at 4 °C. After washing five times with TBS2, biotinylated peptides were eluted with 100 μL of 1 mM biotin in TBS2 for 15 min at 95 °C twice. The combined eluates were subjected to GL-Tip SDB-SCX. After desalting with the first-stage SDB, biotinylated peptides were eluted from the second-stage SCX with 500 mM ammonium acetate and 30% ACN, evaporated in a SpeedVac concentrator, and redissolved in 0.1% TFA and 3% ACN.
2022-08-25 | PXD035229 | JPOST Repository