ABSTRACT: HEK293T cells stably expressing AGIA-AirID-DCAF, -SKP2, and -IκBα were cultured in a 10-cm dish in biological replicates (n = 2) and treated with 10 µM MG132 for 6 h in the presence of 10 µM biotin for 24 h. The cell pellet was then lysed in 300 µL of a guanidine buffer (6 M guanidine-HCl, 100 mM HEPES-NaOH [pH 7.5], 10 mM TCEP, 40 mM chloroacetamide). The cell 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 were purified by methanol-chloroform precipitation and solubilized in 150 µL of PTS buffer (12 mM SDC, 12 mM SLS, 100 mM Tris-HCl, pH 8.0). After sonication and heating, the protein solution was diluted 5-fold with 100 mM Tris-HCl (pH 8.0) and digested with trypsin (MS grade, Thermo Fisher Scientific) at 37°C overnight. The resulting peptide solutions were diluted 2-fold with TBS (50 mM Tris-HCl [pH 7.5] and 150 mM NaCl). Biotinylated peptides were captured on a 15 µL slurry of MagCapture HP Tamavidin 2-REV magnetic beads (#133-18611, FUJIFILM Wako) via incubation for 3 h at 4°C. After washing with TBS five times, the biotinylated peptides were eluted with 100 µL of 1 mM biotin in TBS for 15 min at 37°C twice. The combined eluates were desalted using GL-Tip SDB (#7820-11200, GL Sciences), evaporated in a SpeedVac concentrator (Thermo Fisher Scientific), and redissolved in 0.1% TFA and 3% acetonitrile (ACN). LC-MS/MS analysis of the biotinylated peptides was performed using an EASY-nLC 1200 UHPLC system connected to an Orbitrap Fusion mass spectrometer using a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 150-mm C18 reversed-phase column with an inner diameter of 75 µm (Nikkyo Technos) with a linear ACN gradient (4–32%) for 0–60 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 the data-dependent acquisition mode with a maximum duty cycle of 3 s. The MS1 spectra were measured at a resolution of 120,000, an automatic gain control (AGC) target of 4e5, and a mass range of 375–1500 m/z. HCD MS/MS spectra were acquired using a linear ion trap with an AGC target of 1e4, an isolation window of 1.6 m/z, a maximum injection time of 200 ms, and a normalized collision energy of 30. Dynamic exclusion was set to 10 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.5 (Thermo Fisher Scientific) 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 0.6 Da, (d) carbamidomethylation of cysteine as a fixed modification, and (e) acetylation of protein N-terminus, oxidation of methionine, and biotinylation of lysine as variable modifications. Peptides were filtered at a false discovery rate (FDR) of 1% using a percolator node. Label-free quantification was performed based on the intensity of the precursor ions using a precursor-ion quantifier node. Normalization was performed so that the sum of the abundance values for each sample was the same for all peptides.
