IP-MS of ASXL1 without fractionation using 293T cells and THP-1 cells

The cell lysates were mixed with anti-FLAG-antibody-crosslinked Dynabeads. The beads-lysate mix was incubated at 4°C for 45 min. After washing with PBS-T, proteins were eluted with Laemmli’s SDS ample buffer by incubating at 37°C for 30 min. The collected supernatants were frozen at -80°C as samples for LC-MS. After reduction with 10 mM TCEP at 100°C for 10 min and alkylation with 50 mM iodoacetamide at ambient temperature for 45 min, the protein samples were subjected to digestion with Trypsin/Lys-C Mix (Promega) at 47°C for 2 h on S-Trap columns (ProtiFi, NY, USA).
The resulting peptides were extracted from gel fragments and analysed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) combined with UltiMate 3000 RSLC nano-flow HPLC (Thermo Scientific). The peptides were enriched with μ-Precolumn (0.3 mm i.d. × 5 mm, 5 μm, Thermo Scientific) and separated on an AURORA column (0.075 mm i.d. × 250 mm, 1.6 μm, Ion Opticks Pty Ltd, Australia) using the two-step gradient 2-40% acetonitrile for 110 min, followed by 40-95% acetonitrile for 5 min in the presence of 0.1% formic acid. The compensation voltages for the gas-phase fractionation by FAIMS Pro (Thermo Scientific) were set at -40, -60 and -80 V. The analytical parameters of the Orbitrap Fusion Lumos were set as follows: resolution of full scans = 50,000, scan range (m/z) = 350-1500, maximum injection time of full scans = 50 ms, AGC target of full scans = 4 × 10^5, dynamic exclusion duration = 30 s, cycle time of data dependent MS/MS acquisition = 2 s, activation type = HCD, detector of MS/MS = ion trap, maximum injection time of MS/MS = 35 ms, and AGC target of MS/MS = 1 × 10^4.
The MS/MS spectra were searched against the Homo sapiens protein sequence database in SwissProt using Proteome Discoverer 3.0 software (Thermo Scientific), in which peptide identification filters were set at “false discovery rate < 1%”. Label-free relative quantification analysis for proteins was performed with the default parameters of Minora Feature Detector node, Feature Mapper node, and Precursor Ions Quantifier node in Proteome Discoverer 3.0 software.