Twenty-four proteome fractions were analyzed by LC–MS/MS using an Easy nLC 1200 coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Scientific). Samples were injected onto an Easy Spray PepMap C18 column (75 μm id × 25 cm, 2 μm particle size) (Thermo Scientific) and separated over a 120-min method. The gradient for separation consisted of 5–42% mobile phase B at a 250 nl/min flow rate, where mobile phase A was 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 80% ACN.
For the proteome fractions, the Lumos was operated in SPS-MS3 mode [119 (link)], with a 3-s cycle time. Resolution for the precursor scan (m/z 350–2000) was set to 120,000 with a AGC target set to standard and a maximum injection time of 50 ms. MS2 scans consisted of CID normalized collision energy (NCE) 30; AGC target set to standard; maximum injection time of 50 ms; isolation window of 0.7 Da. Following MS2 acquisition, MS3 spectra were collected in SPS mode (10 scans per outcome); HCD set to 65; resolution set to 50,000; scan range set to 100–500; AGC target set to 200% with a 150 ms maximum inject time.
For the proteome fractions, the Lumos was operated in SPS-MS3 mode [119 (link)], with a 3-s cycle time. Resolution for the precursor scan (m/z 350–2000) was set to 120,000 with a AGC target set to standard and a maximum injection time of 50 ms. MS2 scans consisted of CID normalized collision energy (NCE) 30; AGC target set to standard; maximum injection time of 50 ms; isolation window of 0.7 Da. Following MS2 acquisition, MS3 spectra were collected in SPS mode (10 scans per outcome); HCD set to 65; resolution set to 50,000; scan range set to 100–500; AGC target set to 200% with a 150 ms maximum inject time.
Free full text: Click here
