LFQ of streptavidin pulldown from hTBJ1 cells expressing APEX2-STING

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.