Dataset Information

LFQ of GFP-Trap IP-MS from hTBJ1 cells expressing EGFP-mSTING

ABSTRACT: Permeabilised hTBJ1 cells expressing EGFP-mSTING were stimulated with or without 1 µM 2’3’-cGAMP at 10°C and cross-linked with 0.1 or 0.5% paraformaldehyde at 4°C for 1 h in three independent experiments. After quenching with 100 mM glycine-NaOH, pH 7.5 at 4°C for 15 min, the cells were lysed in RIPA buffer (20 mM HEPES-NaOH, pH7.5, 150 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.25% sodium deoxycholate, 0.05% SDS and 1% NP-40) supplemented with Complete protease inhibitor cocktail, PhosSTOP phosphatase inhibitor cocktail and 50 unit/ml Benzonase. After centrifugation at 20,000 g for 15 min at 4°C, the supernatants were incubated with a 2.5 μl slurry of GFP-Trap magnetic agarose for 3 h at 4°C. The beads were washed four times with RIPA buffer and then twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix at 37°C overnight. The digests were reduced, alkylated, acidified and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. Raw data were directly analysed against the SwissProt database restricted to Homo sapiens supplemented with mouse STING protein sequence using Proteome Discoverer version 2.4 with Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the Percolator node and Protein FDR Validator node, respectively. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalisation was performed such that the total sum of abundance values for each sample over all peptides was the same.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD029736 | JPOST Repository | Fri Dec 09 00:00:00 GMT 2022

REPOSITORIES: jPOST

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	EGFP_STING_CLIP_MS_Exp1.xlsx	Xlsx
	EGFP_STING_CLIP_MS_Exp2.xlsx	Xlsx
	EGFP_STING_CLIP_MS_Exp3.xlsx	Xlsx
	Exp1_EGFP_STING_cGAMP_05PFA.raw	Raw
	Exp1_EGFP_STING_minus_05PFA.raw	Raw

Items per page:

1 - 5 of 9

Publications

The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER.

Motani Kou K Saito-Tarashima Noriko N Nishino Kohei K Yamauchi Shunya S Minakawa Noriaki N Kosako Hidetaka H

Cell reports 20221201 12

STING, an endoplasmic reticulum (ER)-resident receptor for cyclic di-nucleotides (CDNs), is essential for innate immune responses. Upon CDN binding, STING moves from the ER to the Golgi, where it activates downstream type-I interferon (IFN) signaling. General cargo proteins exit from the ER via concentration at ER exit sites. However, the mechanism of STING concentration is poorly understood. Here, we visualize the ER exit sites of STING by blocking its transport at low temperature or by live-ce ...[more]

PMID: 36543137

Similar Datasets

Project description:Parental HeLa cells and HeLa cells stably expressing PINK1-3FLAG were treated with or without 10 μM CCCP for 3 h, incubated with 0.1% paraformaldehyde for 10 min at room temperature, and then quenched with 100 mM glycine-NaOH (pH7.5) for 4 min. After washing twice with 20 mM HEPES-NaOH (pH7.4) and 137 mM NaCl, the cells were solubilized on ice for 10 min in RIPA buffer (20 mM HEPES-NaOH [pH 7.5], 150 mM NaCl, 1mM MgCl2, 1 mM EGTA, 0.05% SDS, 0.25% sodium deoxycholate, 1% NP-40) supplemented with a protease inhibitor cocktail and 50 unit/ml Benzonase (Merck Millipore), and then subjected to centrifugation (20,000 x g) at 4°C for 15 min. The resultant supernatants were incubated at 4°C for 1 h with anti-FLAG (M2) mAb magnetic beads (Sigma-Aldrich). The beads were washed four times with RIPA buffer and then twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) at 37°C overnight. The digests were reduced, alkylated, acidified, and desalted with GL-Tip SDB (GL Sciences). The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher). The peptides were separated on a 75-μm inner diameter x 150 mm C18 reversed-phase column (Nikkyo Technos) with a linear 4%-32% acetonitrile gradient for 0-150 min followed by an increase to 80% acetonitrile for 15 min and finally held at 80% acetonitrile for 15 min. The mass spectrometer was operated in data-dependent acquisition mode with the top 10 MS/MS method. MS1 spectra were measured at a resolution of 70,000, an automatic gain control target of 1e6, and a mass range from 350 to 1500 m/z. HCD MS/MS spectra were triggered at a resolution of 17,500, an automatic gain control target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalized collision energy of 27. Dynamic exclusion was set to 10 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer 2.4 (Thermo Fisher) with the Sequest HT search engine. 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) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the Percolator node and Protein FDR Validator node, respectively. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Permeabilised hTBJ1 cells expressing APEX2-STING were stimulated with or without 1 μM cGAMP at 10°C and incubated for 1 min in the presence of 1 mM H2O2 and 50 µM biotin phenol. The reaction was then quickly stopped by washing three times with ice-cold 500 mM sodium ascorbate, followed by washing twice with ice-cold PBS. The cells were lysed in ice-cold 6 M guanidine-HCl containing 100 mM HEPES-NaOH, pH 7.5, 10 mM tris (2-carboxyethyl) phosphine and 40 mM chloroacetamide. The lysates were dissolved by heating and sonication and then centrifuged at 20,000 g for 15 min at 4°C. The supernatants were recovered, and proteins (400 μg) were purified by methanol–chloroform precipitation and solubilized with 8 M urea and 1% SDS in TBS (50 mM Tris-HCl, pH7.5 and 150 mM NaCl). After sonication and 8-fold dilution with TBS, biotinylated proteins were captured on a 15 μl slurry of NanoLink streptavidin magnetic beads by incubation for 5 h at 4°C. After washing four times with 1 M urea and 0.125% SDS in TBS and three times with 1 M urea in 50 mM ammonium bicarbonate, proteins on the beads were digested by adding 400 ng trypsin/Lys-C mix at 37°C overnight. The digests were acidified, desalted using GL-Tip SDB, evaporated and dissolved in 0.1% TFA and 3% ACN. LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. Raw data were directly analysed against the SwissProt database restricted to Homo sapiens supplemented with mouse STING protein sequence using Proteome Discoverer version 2.4 with Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides and proteins were filtered at a false discovery rate (FDR) of 1% using the Percolator node and Protein FDR Validator node, respectively. Label-free quantification was performed based on intensities of precursor ions using the Precursor Ions Quantifier node. Normalisation was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Irgb6-deficient MEFs stably expressing Spot-tagged Irgb6 were infected with or without T. gondii ME49 for 4 h and lysed in guanidine buffer (6 M guanidine-HCl, 100 mM Tris-HCl, pH 8.0, and 2 mM DTT). The lysates were diluted 8-fold with RIPA buffer (20 mM HEPES-NaOH, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 0.25% sodium deoxycholate, 0.05% SDS, and 1% NP-40) supplemented with cOmplete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche). After centrifugation at 20,000 × g for 15 min at 4 ºC, the supernatants were incubated with a 5-µL slurry of anti-Spot nanobody-coupled magnetic agarose beads (Spot-Trap, ChromoTek) for 3 h at 4 ºC. The beads were washed four times with RIPA buffer and then twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested with 200 ng trypsin (MS grade, Thermo Fisher Scientific) at 37 ºC overnight. The digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated and dissolved in 3% acetonitrile (ACN) and 0.1% trifluoroacetic acid. LC–MS/MS analysis of the resultant peptides 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 (Nikkyo Technos) with a linear 4–32% ACN gradient for 0–100 min, followed by an increase to 80% ACN for 10 min and finally held at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with the top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an automatic gain control target of 1e6, and a mass range from 350 to 1,500 m/z. HCD MS/MS spectra were acquired at a resolution of 17,500, an automatic gain control target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalized collision energy of 27. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus supplemented with amino acid sequences of Spot-tagged Irgb6 and T. gondii ME49 proteins (ToxoDB release 38) using Proteome Discoverer v2.5 with the Sequest HT search engine. 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) carbamidomethylation of cysteine as a fixed modification; (e) acetylation of the protein N-terminus, oxidation of methionine, and phosphorylation of serine, threonine, and tyrosine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node.

Project description:Control or GFP-WIPI2d expressing MEFs were treated with or without 0.5uM Apilimod for one hour, and then cross-linked with 0.1% formaldehyde for ten mins at room temperature. After cross-linking was quenched with 100mM glycine for four mins at room temperature, cells were washed with HEPES saline containing 20mM HEPES-NaOH buffer (pH 7.5) and 137 mM NaCl, and lysed in HEPES-RIPA buffer containing 20mM HEPES-NaOH buffer (pH7.5), 150mM NaCl, 1mM EGTA, 1mM MgCl2, 0.25% (w/v) sodium deoxycholate, 0.05% SDS, 1% (v/v) NP-40, 0.2% (v/v) Benzonase Nuclease, PhosSTOP, and Protease Inhibitor Cocktail on ice. After sonication and centrifugation at 20,380 × g at 4℃ for 15 mins, the supernatants were incubated with GFP-Trap magnetic agarose beads at 4℃ for 2 h. The beads were washed four times with HEPES-RIPA buffer and then twice with 50mM ammonium bicarbonate. Proteins on the beads were digested by adding a 200 ng trypsin/Lys-C mix (Promega) at 37℃ overnight. The resultant digests were reduced, alkylated, acidified, and desalted using a GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). Liquid chromatography-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a C18 reversed-phase column (75 μm×150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 mins, followed by an increase to 80% ACN for ten mins and a final hold at 80% ACN for ten mins. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of three secs. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375-1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 msecs, and a normalized collision energy of 30. Dynamic exclusion was set to 20 secs. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer 2.5 with the Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed on the basis of the intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Control or GFP-Rubicon expressing MEFs were treated with or without 0.5uM Apilimod for one hour, and then cross-linked with 0.1% formaldehyde for ten mins at room temperature. After cross-linking was quenched with 100mM glycine for four mins at room temperature, cells were washed with HEPES saline containing 20mM HEPES-NaOH buffer (pH 7.5) and 137 mM NaCl, and lysed in HEPES-RIPA buffer containing 20mM HEPES-NaOH buffer (pH7.5), 150mM NaCl, 1mM EGTA, 1mM MgCl2, 0.25% (w/v) sodium deoxycholate, 0.05% SDS, 1% (v/v) NP-40, 0.2% (v/v) Benzonase Nuclease, PhosSTOP, and Protease Inhibitor Cocktail on ice. After sonication and centrifugation at 20,380 × g at 4℃ for 15 mins, the supernatants were incubated with GFP-Trap magnetic agarose beads at 4℃ for 2 h. The beads were washed four times with HEPES-RIPA buffer and then twice with 50mM ammonium bicarbonate. Proteins on the beads were digested by adding a 200 ng trypsin/Lys-C mix (Promega) at 37℃ overnight. The resultant digests were reduced, alkylated, acidified, and desalted using a GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). Liquid chromatography-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source. The peptides were separated on a C18 reversed-phase column (75 μm×150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 mins, followed by an increase to 80% ACN for ten mins and a final hold at 80% ACN for ten mins. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of three secs. MS1 spectra were measured with a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375-1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 msecs, and a normalized collision energy of 30. Dynamic exclusion was set to 20 secs. Raw data were directly analyzed against the SwissProt database restricted to Mus musculus using Proteome Discoverer 2.5 with the Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; and (e) acetylation of the protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label-free quantification was performed on the basis of the intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:Cells stably expressing full-length HKDC1-mNeonGreen or △N20 HKDC1-mNeonGreen proteins were grown in a 10-cm dish and were treated with or without antimycin/oligomycin for 3h, and then cross-linked with 0.1% formaldehyde for 10min at room temperature. After cross-linking was quenched with 100mM glycine for 10min at room temperature, cells were washed with HEPES-saline containing 20mM HEPES-NaOH (pH7.5) and 137mM NaCl, and lysed on ice in HEPES-RIPA buffer containing 20mM HEPES-NaOH (pH7.5), 150mM NaCl, 1mM EGTA, 1mM MgCl2, 0.25% (w/v) Na-deoxycholate, 0.05% SDS, 1% (v/v) NP-40, 0.2% (v/v) Benzonase Nuclease (70746, Millipore), PhosSTOP (4906837001, Roche) and Protease Inhibitor Cocktail. After sonication and centrifugation at 15,000rpm, 4°C for 15min, the supernatants were incubated with mNeonGreen-Trap magnetic agarose beads (ntma-20, ChromoTek) for 2 h at 4°C. The beads were washed four times with HEPES-RIPA buffer and then twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) at 37°C overnight. The resulting digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 µm x 150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 min, followed by an increase to 80% ACN for 10 min and final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an AGC target of 4e5, and a mass range of 375-1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label free quantification was performed on the basis of the intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.

Project description:RD3-KO Y-79 cells and RD3-SPOT-expressing cells were fixed by adding 27 µl of 37% formaldehyde (Wako) (final: 0.1%), incubated at RT for 10 min, quenched with 1 ml of 1 M Glycine-NaOH pH 7.5 (Nacalai) (final: 100 mM) at RT for 4 min, washed with 10 ml DPBS, and stocked at -80℃ as a cell pellet. For immunoprecipitation, the cell pellet was lysed by 500 µl RIPA buffer containing 5 µl Protease Inhibitor Cocktail (Nacalai) and 1 µl Benzonase Nuclease (Sigma), incubated at 4℃ for 1 hr and centrifuged at 4℃, 20000 G for 10 min to extract the supernatant. Subsequently, the supernatant was incubated with 10 µl ChromoTek Spot-Trap Magnetic Agarose (Chromotek) at 4℃ for 2 hr. The slurry was washed three times with 500 µl RIPA buffer before the reaction. After the incubation, the immunoprecipitated product was washed three times with 300 µl RIPA buffer including 3 µl of Protease Inhibitor Cocktail (Nacalai), transferred into a new 1.5 ml tube to remove non-specific binding proteins and additionally washed with 300 µl of 50 mM Ammonium Bicarbonate (ABC) buffer. The final products were resuspended with 50 µl of ABC buffer and transferred into a new 1.5ml tube. Proteins on the beads were digested by adding 200 ng trypsin/Lys-C mix (Promega) at 37°C overnight. The resulting digests were reduced, alkylated, acidified, and desalted using GL-Tip SDB. The eluates were evaporated and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 UHPLC connected to an Orbitrap Fusion mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a C18 reversed-phase column (75 µm x 150 mm; Nikkyo Technos) with a linear 4-32% ACN gradient for 0-100 min, followed by an increase to 80% ACN for 10 min and final hold at 80% ACN for 10 min. The mass spectrometer was operated in data-dependent acquisition mode with a maximum duty cycle of 3 s. MS1 spectra were measured with a resolution of 120,000, an AGC target of 4e5, and a mass range of 375-1,500 m/z. HCD MS/MS spectra were acquired in the linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 35 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 20 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer 2.5 (Thermo Fisher Scientific) with Sequest HT search engine. 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 0.6 Da; (d) carbamidomethylation of cysteine as a fixed modification; (e) acetylation of protein N-terminus and oxidation of methionine as variable modifications. Peptides were filtered at a false discovery rate of 1% using the Percolator node. Label free quantification was performed on the basis of the intensities of precursor ions using the Precursor Ions Quantifier node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.