PXD029736

PXD029736 is an original dataset announced via ProteomeXchange.

Title	LFQ of GFP-Trap IP-MS from hTBJ1 cells expressing EGFP-mSTING
Description	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.
HostingRepository	jPOST
AnnounceDate	2022-12-09
AnnouncementXML	Submission_2022-12-22_10:55:42.538.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Hidetaka Kosako
SpeciesList	scientific name: Homo sapiens (Human); NCBI TaxID: 9606;
ModificationList	S-carboxamidomethyl-L-cysteine; alpha-amino acetylated residue; L-methionine sulfoxide
Instrument	Orbitrap Fusion

Revision	Datetime	Status	ChangeLog Entry
0	2021-11-14 23:51:06	ID requested
1	2022-12-08 12:40:26	announced
⏵ 2	2022-12-22 10:55:43	announced	2022-12-23: Updated PubMed.

Motani K, Saito-Tarashima N, Nishino K, Yamauchi S, Minakawa N, Kosako H, The Golgi-resident protein ACBD3 concentrates STING at ER-Golgi contact sites to drive export from the ER. Cell Rep, 41(12):111868(2022) [pubmed]

submitter keyword: EGFP, STING, cGAMP, GFP-Trap

Hidetaka Kosako
lab head
Hidetaka Kosako
contact affiliation	Tokushima University
dataset submitter

jPOST dataset URI

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