Co-IP lysates were prepared as above using 40 μL α-FLAG-conjugated magnetic beads. Following Co-IP, samples were resuspended in Co-IP buffer with reduced Triton X-100 (0.1%). To prepare samples for mass spectrometry, FLAG-tagged affinity purifications were eluted, reduced, and alkylated using 5% (w/v) sodium dodecyl-sulfate, 10 mM tris(2-carboxyethylphosphine), 40 mM 2-chloroacetamide, 50 mM Tris-HCl, pH 8.5, boiled for 10 min, and incubated shaking at 1000 rpm at 37°C for 30 min. Affinity-purified proteins were digested using the SP3 method with Sera-Mag™ carboxylate-functionalized SpeedBeads (Cytiva).54 (link) Cleaned-up peptides were then dried in a SpeedVac vacuum concentrator and stored at −20°C until analysis.
Tryptic peptides were suspended in 3% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and directly injected onto a reversed-phase C18 1.7 μm, 130 Å, 75 mm × 250 mm M-class column (Waters), using an Ultimate 3000 nanoUPLC (ThermoFisher). Peptides were eluted with an acetonitrile gradient and detected using a Q-Exactive HF-X mass spectrometer (ThermoFisher). Raw files were searched against the Uniprot Human database UP000005640 using MaxQuant v.1.6.14.0. All peptide and protein identifications were thresholded at a 1% false discovery rate. Statistical analysis was performed on log2-transformed iBAQ intensities using limma.
Tryptic peptides were suspended in 3% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and directly injected onto a reversed-phase C18 1.7 μm, 130 Å, 75 mm × 250 mm M-class column (Waters), using an Ultimate 3000 nanoUPLC (ThermoFisher). Peptides were eluted with an acetonitrile gradient and detected using a Q-Exactive HF-X mass spectrometer (ThermoFisher). Raw files were searched against the Uniprot Human database UP000005640 using MaxQuant v.1.6.14.0. All peptide and protein identifications were thresholded at a 1% false discovery rate. Statistical analysis was performed on log2-transformed iBAQ intensities using limma.