Dataset Information

LFQ of streptavidin pulldown from hTBJ1 cells expressing APEX2-STING

ABSTRACT: 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.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Hidetaka Kosako

PROVIDER: PXD029735 | JPOST Repository | Fri Dec 09 00:00:00 GMT 2022

REPOSITORIES: jPOST

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Dataset's files

Source:

			Action	DRS
	1_hTBJ1_APEX2_STING_StrA_minus.raw	Raw
	2_hTBJ1_APEX2_STING_StrA_cGAMP.raw	Raw
	3_hTBJ1_APEX2_STING_StrA_minus.raw	Raw
	4_hTBJ1_APEX2_STING_StrA_cGAMP.raw	Raw
	5_hTBJ1_APEX2_STING_StrA_minus.raw	Raw

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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

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