Orbitrap fusion lumos ms system

The digested peptides were dissolved in 0.1% formic acid and loaded on a Michrom Peptide Captrap column (MW 0.5–50 kD, 0.5 × 2 mm; Michrom Bioresources, Auburn, CA, United States). The eluent was transferred to a reversed-phase microcapillary column (0.1 × 150 mm, packed with Magic C18, 3 μm, 200Å; Michrom Bioresources, Auburn, CA, United States) by a Waters Ultra-performance EASY-nLC1200 HPLC system. Elution was performed over a gradient of 5–28% buffer B (buffer A: 0.1% formic acid, 99.9% H₂O; buffer B: 0.1% formic acid, 79.9% ACN, 20% H₂O; flow rate, 0.3 μL/min) for 60 min. The eluted peptides were analyzed using Orbitrap Fusion Lumos MS system (Thermo Fisher Scientific, Bremen, Germany). The MS data were acquired using an ion spray voltage of 2.1 kV, curtain gas of 20 PSI, nebulizer gas of 30 PSI, and an interface heater temperature of 300°C. The precursors were acquired in 500 ms ranging from 350 to 1,550 m/z with the resolution set to 120,000, and the product ion scans were acquired in 50 ms ranging from 250 to 1800 m/z with the resolution set to 60,000. Dynamic exclusion was employed with a 60-s window to prevent the repetitive selection of the same peptide. Each sample was analyzed at least three times.

The total peptide concentrations were measured by NanoPhotometer (Implen, Munich, Germany). The peptide mixtures were loaded into a reverse−phase microcapillary column (0.1 × 150 mm, packed with Magic C18, 100 μm, 100Å; Michrom Bioresources). The eluted peptides were analyzed using the Orbitrap Fusion Lumos MS system (Thermo Fisher Scientific). The instrument was run with peptide recognition mode enabled. The full MS scans were acquired at a resolution of 120,000 at m/z 200 and 60,000 at m/z 200 for MS/MS scan. The maximum injection time was set to 500 ms for MS and 50 ms for MS/MS. The normalized collision energy was 27, and the isolation window was set to 1.6 Th. The dynamic exclusion duration was 60 s. Each sample was analyzed at least three times.