Orbitrap fusion tribid mass spectrometer

Cells were lysed, processed into tryptic peptides and analyzed by mass spectrometry using an Orbitrap Fusion Tribid mass spectrometer (ThermoFisher). Data were processed using the Maxquant computational platform essentially as described.42 The .raw MS files and search/identification files obtained with MaxQuant were deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD013857. A more detailed description of the mass spectrometry sample acquisition and data analysis is provided in Data S1.

Protein identification by LC-MS/MS analysis of peptides was performed using an Orbitrap Fusion Tribid mass spectrometer (Thermo Fisher Scientific, San Jose, CA) interfaced with an Ultimate 3000 Nano-HPLC apparatus (Thermo Fisher Scientific). Peptides were fractionated by reverse-phase HPLC on a Picofrit column (250 mm long, internal diameter 75 μm, tip internal diameter 15 μm, packed with BetaBasic 5-μm 300-Å particles; New Objective, Woburn, MA) using a 120-min linear gradient of 5%–35% acetonitrile in 0.1% formic acid at a flow rate of 300 nL/min. Survey scans of peptide precursors from 300 to 2,000 m/z were performed at 60K resolution. Tandem MS was performed with the quadrupole, HCD fragmentation with collision energy of 35, and rapid-scan MS analysis using an ion trap. The instrument was run in a top speed mode with 3-s cycles. This experiment was performed from biological replicates from one differentiation. Additional details on sample preparation and data analysis are provided online.