Mass spectrometry analysis of biotinylated peptides

The proximity-dependent biotin identification method using AirID was performed according to a previous report (Kim et al., 2016). Briefly, confluent HEK293T cells stably expressing AirID or AirID-IκBα fused at the N-terminus with an AGIA tag in a 6 cm dish were incubated with 50 μM biotin for 6 hr before harvesting using ice-cold PBS. Cell pellets were lysed and digested with trypsin. The digested peptides were incubated with Tamavidin2-Rev magnetic beads (FUJIFILM) before eluting with 2 mM biotin. Detailed procedures will be described elsewhere (Motani K and Kosako H, in preparation).

LC-MS/MS analysis of the resulting peptides was performed on an EASY-nLC 1200 UHPLC connected to a Q Exactive Plus mass spectrometer using a nanoelectrospray ion source (Thermo Fisher Scientific). The peptides were separated on a 75-µm inner diameter ×150 mm C18 reverse-phase column (Nikkyo Technos) with a linear gradient from 4–28% acetonitrile for 0–40 min followed by an increase to 80% acetonitrile during 40–50 min. The mass spectrometer was operated in a data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured with a resolution of 70,000, an AGC target of 1 × 10⁶, and a mass range from 350 to 1500 m/z. HCD MS/MS spectra were acquired at a resolution of 17,500, an AGC target of 5 × 10⁴, 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 10 s. Raw data were directly analyzed against the SwissProt database restricted to Homo sapiens using Proteome Discoverer version 2.3 (Thermo Fisher Scientific) for identification and label-free precursor ion quantification. 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; and (e) protein N-terminal acetylation, methionine oxidation, and lysine biotinylation as variable modifications. Peptides were filtered at a false-discovery rate of 1% using the percolator node. Normalization was performed such that the total sum of abundance values for each sample over all peptides was the same.