Q exactive exploris 480

Samples for label-free quantification were process as described above. Both phosphoproteome and proteome peptides were analyzed with a “20 samples per day” method on the EvoSep One instrument and analysed on Q-Exactive Exploris 480 instrument (Thermo Fisher Scientific) running a high-resolution MS1 data-independent acquisition method. Full MS spectra were collected at 120 000 with AGC target of 3x10ˆ6 or maximum injection time of 50 ms. Scan range from 400-1000 m/z was used. The MS2 spectra were obtained with 60,000 resolution, with a AGC target of 10e5. 75 windows windows of 8 m/z were used with a MS1 scan every 200 m/z. The raw files were the analysis with Spectronaut 16 software.

Samples were analyzed on a Q Exactive Exploris 480 (Thermo Scientific, Waltham, MA, USA) mass spectrometer equipped with a FAIMSpro differential ion mobility device that was coupled to an UltiMate 3000 nLC (all Thermo Scientific Waltham, MA, USA). Samples were loaded onto a 5 µm PepMap Trap cartridge precolumn (Thermo Scientific, Waltham, MA, USA) and reverse-flushed onto an in-house packed analytical pulled-tip column (30–75 µm I.D., filled with 2.7 µm Poroshell EC120 C18, Agilent, Santa Clara, CA, USA). Peptides were chromatographically separated at a constant flow rate of 300 nL/min and the following gradient: initial 2% B (0.1% formic acid in 80% acetonitrile), up to 6 and in 1 min, up to 32% B in 72 min, up to 55% B within 7.0 min and up to 95% solvent B within 2.0 min, followed by a 6 min column wash with 95% solvent B. The FAIMS pro was operated at −50 compensation voltage and electrode temperatures of 99.5 °C for the inner and 85 °C for the outer electrode.

For each fraction, peptides were analysed using the pre-set ’30 samples per day’ or ’15 samples per day’ method on the EvoSep One instrument. Global proteome peptides were eluted over a 44-min gradient, and analysed on a Q-Exactive Exploris 480 instrument (Thermo Fisher Scientific) running in a DD-MS2 method. Full MS spectra were collected at a resolution of 120,000, with an AGC target of 3×106 or maximum injection time of 50 ms and a scan range of 350–1500 m/z. The MS2 spectra were obtained at a resolution of 45,000, with an AGC target value of 1×105 or maximum injection time of 86 ms, a normalized collision energy of 32 and an intensity threshold of 1e5. First mass was set to 110 m/z to ensure capture of the TMT reporter ions. Dynamic exclusion was set to 60 s, and ions with a charge state <2, >6 and unknown were excluded. For phosphorylated peptides, the gradient length was doubled to 88mins. MS performance was verified for consistency by running complex cell lysate quality control standards, and chromatography was monitored to check for reproducibility.