LFQ of Spot-Trap IP-MS from MEF cells expressing Irgb6-Spot

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.