Affinity Purification and Mass Spectrometry

Eluates from biotin pull-down were transferred to fresh microfuge tubes. NuPAGE sample loading buffer was added to the beads and heated at 90 °C for 5 min. A magnetic rack was used to separate the beads from the proteins. The supernatant was then run on an SDS–PAGE gel (Bis-Tris, 4–12%) enough to get the sample into the gel. Gel sections were excised, washed, reduced with DTT, alkylated with iodoacetamide and digested overnight with trypsin at 37 °C (ref. ^{54 (link)}). Homemade C18 StageTips were prepared as described previously^{55 (link)} and preconditioned with a 50 μl wash of methanol, 50 μl wash of 70% acetonitrile/0.1% trifluoroacetic acid and two 50 μl washes of 0.1% trifluoroacetic acid at 1,000g. Peptides were then loaded onto StageTips and washed with 50 μl of 0.1% formic acid and were eluted with 60 μl of 70% acetonitrile/0.1% formic acid. The samples were then vacuum centrifuged using the SpeedVac and reconstituted in 0.1% formic acid for LC–MS/MS and were analysed by microcapillary LC–MS/MS using the nanoAcquity system (Waters) with a 100 μm inner-diameter × 10 cm length C18 column (1.7 μm BEH130, Waters) configured with a 180 μm × 2 cm trap column coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were eluted at 300 nl min⁻¹ using a 4 h acetonitrile gradient (0.1% formic acid). The Q-Exactive Plus mass spectrometer was operated in automatic, data-dependent MS/MS acquisition mode with one MS full scan (380–1,600 m/z) at 70,000 mass resolution and up to ten concurrent MS/MS scans for the ten most intense peaks selected from each survey scan. Survey scans were acquired in profile mode and MS/MS scans were acquired in centroid mode at 17,500 resolutions with an isolation window of 1.5 amu and normalized collision energy of 27; AGC was set to 1 × 10⁶ for MS1 and 5 × 10⁴ and 50 ms max IT for MS2; charge exclusion of unassigned, +1 and greater than 6 was enabled with dynamic exclusion of 15 s.