The LC–MS/MS analysis of pre-fractionated samples was performed using UltiMate 3000 RSLCnano system coupled with a Thermo Scientific Orbitrap Q Exactive HF. Mobile phase A consisted of 0.2% formic acid (FA), 3% DMSO in water and mobile phase B consisted of 0.2% FA, 3% DMSO in 67% acetonitrile. Peptides were loaded into a CoAnn C18 column (75 μm × 20 cm, 1.9 μm particles) at a flow rate of 250 nL/min with the following gradient: 12–22% mobile phase B over 30 min, 22–40% mobile phase B in 30 min, 40–60% mobile phase B in 7 min, 60–95% mobile phase B in 3 min, stay at 95% mobile phase B for 3 min, followed by 15 min equilibration (44 (link)).
MS data were acquired using a ‘high-high’ acquisition method (high resolution on both MS1 and MS2). MS1 scans were detected in the Orbitrap at 120K resolution in the m/z range 400–2000 and AGC target of 1 × 106 with a maximum injection time of 50 ms. Ions with charge states from 2+ to 8+ were selected for fragmentation by stepped high energy collision dissociation (HCD) at 27%, 30% 33% with AGC of 1 × 105, maximum injection time of 100 ms, and detected in the Orbitrap at 60K resolution.
MS data were acquired using a ‘high-high’ acquisition method (high resolution on both MS1 and MS2). MS1 scans were detected in the Orbitrap at 120K resolution in the m/z range 400–2000 and AGC target of 1 × 106 with a maximum injection time of 50 ms. Ions with charge states from 2+ to 8+ were selected for fragmentation by stepped high energy collision dissociation (HCD) at 27%, 30% 33% with AGC of 1 × 105, maximum injection time of 100 ms, and detected in the Orbitrap at 60K resolution.
