Nakaya_VGLL3-FLAG_IP-MS_QEplus

Cardiac myofibroblasts retrovirally transduced with FLAG-VGLL3 or control vector were fixed with 0.1% PFA and lysed in a HEPES-RIPA buffer (20 mM HEPES-NaOH [pH 7.5]/1 mM EGTA/1 mM MgCl2/150 mM NaCl/0.25% sodium deoxycholate/0.05% SDS/1% NP-40) containing protease inhibitors (1:100), phosphatase inhibitors (1:100), and 28 U/µL of Benzonase (1:500, Novagen) at 4 °C for 20 min. The supernatant of the cell lysate was incubated overnight at 4 °C with anti-FLAG magnetic beads. The beads were washed three times with HEPES-RIPA buffer and twice with 50 mM ammonium bicarbonate. Proteins on the beads were digested with 200 ng of Trypsin/Lysyl Endopeptidase mixture (Promega), and the digested peptides were reduced, alkylated, acidified, and desalted using GL-Tip SDB (GL Sciences). The eluates were evaporated in a SpeedVac concentrator and dissolved in 0.1% trifluoroacetic acid and 3% acetonitrile (ACN). LC-MS/MS analysis of the resultant peptides was performed on an EASY-nLC 1200 ultra-high-performance liquid chromatograph connected to a Q Exactive Plus mass spectrometer through a nanoelectrospray ion source (Thermo Fisher Scientific). 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 0–150 min followed by an increase to 80% ACN for 20 min and finally hold at 80% ACN for 10 min. The mass spectrometer was operated in a data-dependent acquisition mode with a top 10 MS/MS method. MS1 spectra were measured at a resolution of 70,000, an automatic gain control (AGC) target of 1e6, and a mass range from 350 to 1,500 m/z. MS/MS spectra were triggered at a resolution of 17,500, an AGC target of 5e4, an isolation window of 2.0 m/z, a maximum injection time of 60 ms, and a normalised collision energy of 27. Dynamic exclusion was set to 15 s. Raw data were directly analysed against the SwissProt database, restricted to Mus musculus, using Proteome Discoverer version 2.4 (Thermo Fisher Scientific) with the Sequest_HT search engine for identification and label-free precursor ion quantification. The search parameters were as follows: (i) trypsin as an enzyme with up to two missed cleavages; (ii) precursor mass tolerance of 10 ppm; (iii) fragment mass tolerance of 0.02 Da; (iv) carbamidomethylation of cysteine as a fixed modification; and (v) acetylation of the 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 the protein FDR validator node, respectively. Normalisation was performed such that the total sum of abundance values for each sample across all peptides was the same.