A replicate of the phosphopeptides was run using a SPS-MS3 method. LC conditions were the same as above. The mass spectrometer method was operated in positive ion mode with 3 sec cycle time data-dependent acquisition method with advanced peak determination and Easy-IC (internal calibrant). Precursor scans (m/z 400–1750) were done with an orbitrap resolution of 120000, RF lens% 30, maximum inject time 50 ms, standard AGC target, including charges of 2 to 7 for fragmentation with 60 sec dynamic exclusion. Data-Dependent MS2 scan properties included selection m/z 400–1200, exclusion mass width of 5 m/z, isobaric tag loss exclusion of TMTpro, an isolation window of 0.7 CID collision energy fixed at 35%, neutral loss mass 97.9673, detector: orbitrap, orbitrap resolution: 30000. MS3 data dependent scans in the scan range 100–500, based on 10 synchronous precursor selection (SPS) with 2 m/z isolation window, isolation offset off, HCD activation 65% collision energy, detector orbitrap, orbitrap resolution 50000. Data were recorded using Thermo Fisher Scientific Xcalibur (4.3) software (Thermo Fisher Scientific Inc.).
