LC-MS/MS Analysis of Peptide Digests

LC-MS/MS analysis was performed on an UltiMate 3000 RSLC (Dionex/Thermo Fisher Scientific, Idstein, Germany) coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The peptide digest was enriched on a 2-cm by 100-μm Acclaim PepMap 100 trap column (100-Å pore size, 5-μm C18 particles; Thermo Fisher Scientific) and separated on a 50-cm by 75-μm Accucore 150-C18 analytical column (150-Å pore size, 2.6-μm particles; Thermo Fisher Scientific) with a flow rate of 200 nl/min or a 25-cm by 75-μm Accucore 150-C18 analytical column (150-Å pore size, 2.6-μm particles; Thermo Fisher Scientific) with a flow rate of 300 nl/min at a constant temperature of 40°C. Reversed-phase chromatography was performed with a binary buffer system consisting of 0.1% acetic acid-5% ACN in water (buffer A) and 100% ACN in 0.1% acetic acid (buffer B). The peptides were separated by applying a linear gradient from 2% to 25% buffer B over a time of 120 min and 180 min for the 25-cm and the 50-cm analytical column, respectively. Eluting peptides were ionized using the chip-based TriVersa NanoMate ion source (Advion Biosciences, Norwich, United Kingdom). For the data-independent mode, the parameters from the work of Bruderer and coworkers (67 (link)) were used.