Identification of nuclear partners of STING

Published: 21 July 2021| Version 1 | DOI: 10.17632/xgwb62nznh.1
Contributors:
Jose de las Heras,

Description

Human HEK293T cells were transfected with a STING-GFP or a NLS-GFP (referred to as 'mock' control) expression plasmid, and the nuclear envelope protein fraction isolated and analysed by mass spectrometry to identify the nuclear pool of STING interactors. Two runs were performed for each condition. These data are described in Dixon et al. (2021) iScience (accepted for publication - will update with PMID after publication).

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RAW, DAT and MGF files: A LTQ-Orbitrap mass spectrometer (Thermofisher Scientific) was coupled on-line to an Agilent 1100 binary nanopump and an HTC PAL autosampler (CTC). The peptides were separated using an analytical column with a self-assembled particle frit 87 and C18 material (ReproSil-Pur C18-AQ 3 μm; Dr. Maisch, GmbH) was packed into a spray emitter (100-μm ID, 8-μm opening, 80-mm length; New Objective) using an air-pressure pump (Proxeon Biosystems). Mobile phase A consisted of water, 5% acetonitrile, and 0.5% acetic acid; mobile phase B, consisted of acetonitrile and 0.5% acetic acid. The gradient used was 98 min. The peptides were loaded onto the column at a flow rate of 0.7uL/min and eluted at a flow rate of 0.3uL/min according to the gradient. 0% to 5%B in 5 min, 5% to 20% buffer B in 80 min and then to 80% B in 13 min. FTMS spectra were recorded at 30,000 resolution and the six most intense peaks of the MS scan were selected in the ion trap for MS2, (normal scan, wideband activation, filling 7.5E5 ions for MS scan, 1.5E4 ions for MS2, maximum fill time 150 msec, dynamic exclusion for 150s sec). Searches were conducted using Mascot software (Version 2.2.0) against a database containing Human sequences (sprothuman201106). The search parameters were: MS accuracy, 6 ppm; MS/MS accuracy, 0.6 Da; enzyme, trypsin; allowed number of missed cleavages, 2; fixed modification, carbamidometylation on Cysteine; variable modification, oxidation on Methionine. Table of identified proteins: Peptides with a detection score of 25 or above were used for downstream analysis. Spectral counts were normalised to the mass of each protein detected, and indicated as a relative abundance per sample (dnormS). 220 proteins were identified with >2-fold enrichment in the STING.GFP sample, comparing relative abundances and are highlighted at the top of the table.

Categories

Mass Spectrometry, Cell Nucleus, Innate Immunity

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