Q exactive plus high resolution quadrupole orbitrap mass spectrometer

Chromatography was performed on an Agilent 1260 quaternary HPLC with constant flow rate of ~600 nL min -1 achieved via a splitter. A Sutter P-2000 laser puller was used to generate sharp nanospray tips from 200 µm ID fused silica capillary. Columns were all inhouse packed on a Next Advance pressure cell using 200 µm ID capillary. All samples were loaded into a 10 cm capillary column packed with 5 μM Zorbax SB-C18 (Agilent)
and then connected to a 5 cm-long strong cationic exchange (SCX) column packed with 5µM PolySulfoethyl. The SCX column was then connected to a 20 cm long nanospray tip, packed with 2.5 µm C18 (Waters). For global protein abundance, 45 µg of labeled peptides were fractionated online using 27 ammonium acetate salt steps. For phosphoproteomics, 25 µg of enriched peptides and 14 salt steps were used. For MAKS, 30 µg of enriched peptides and 17 salt steps were used. Each salt step was then separated using a 150 min reverse-phase gradient (Zhang et al., 2019) .
Eluted peptides were analyzed using a Thermo Scientific Q-Exactive Plus high-resolution quadrupole Orbitrap mass spectrometer, which was directly coupled to the HPLC. Data Charge exclusion was set to unassigned, 1, 5-8, and >8. MS1 that triggered MS2 scans were dynamically excluded for 25 s for global proteome and 45 s for phosphoproteome.