Reprosil pur c18 material

LC-MS experiments were performed on an Orbitrap Fusion Lumos mass spectrometer interfaced with an Easy-nLC1200 nanoflow liquid chromatography system (both from Thermo Fisher Scientific). A total of 8 µl out of 15 μl of each peptide sample were trapped on an Acclaim Pepmap 100 C18 trap column (100 μm × 2 cm, particle size 5 μm, Thermo Fischer Scientific) and separated on an analytical column (75 μm × 35 cm) packed in-house with Reprosil-Pur C18 material (particle size 3 μm, Dr. Maisch, Germany) using a gradient with 0.2% formic acid in water as solvent A and 80% acetonitrile with 0.2% formic acid as solvent B at a flow rate of 300 nL/min. The elution profile was as follows: 5% to 33% B in 77 min, 33% to 100% B in 3 min, and 100% B for 10 min. Precursor ion scans were performed at 120,000 target resolution with an m/z range of 375–1500 and an AGC target of 4e5. The most abundant precursors with charges 2–7 were selected for fragmentation with a maximum duty cycle of 3 s and a dynamic exclusion duration of 45 s. Precursors were isolated with a 1.0 Da window and fragmented by higher energy collision-induced dissociation at 30% collision energy with a maximum injection time of 150 ms and an AGC target 5e4, and the MS² spectra were recorded at 30,000 resolution.

1 μg of each peptide mixture were separated on a 23 minute gradient using an EASY-nLC II (Proxeon, Odense, Denmark) equipped with a 200 mm*75μm ID pulled-emitter column, packed with 3μm Reprosil Pur C18 material (Dr. Maisch, Germany), operating at 250 nL/min. Samples were analyzed using a Q-Exactive HF mass spectrometer (Thermo Scientific, Bremen, Germany). Data was recorded in a Top 10 DDA manner with an MS1 resolution of 120 000 and an MS2 resolution of 15 000.