Reprosil pur 120 c18 aq 3.0 m beads

Peptides were analyzed with nano–high pressure liquid chromatography–tandem mass spectrometry (nano-LC-MS/MS). Briefly, the peptides were loaded onto an in-house packed column (40 cm long × 75 μm ID × 360 OD, Dr. Maisch GmbH ReproSil-Pur 120 C18-AQ 3.0-µm beads) analytical column (Thermo Scientific) using a Dionex nanoLC system (Thermo Scientific). A flow rate of 0.300 μl/min with a linear acetonitrile gradient from 8 to 27% in 0.1% formic acid for 120 min was used. The column output was connected to a Q Exactive Plus mass spectrometer (Thermo Scientific) through a nanoelectrospray ion source. The mass spectrometer was controlled by Xcalibur software (Thermo, 4.0.27.19) and operated in the data-dependent mode in which the initial MS scan recorded the mass-to-charge ratios (m/z) of ions over the range 350–1750 at a resolution of 70,000 with a target value of 1 × 10⁶ ions and a maximum injection time of 100 ms. The 10 most abundant ions were automatically selected for subsequent higher-­energy collision dissociation (HCD) with the energy set at 28 NCE. The MS/MS settings included a resolution of 35,000, a target value of 5 × 10⁵ ions, a maximum integration time of 108 ms, and an isolation window set at 3.0 m/z. Ions with undetermined charge, z = 1, and z > 7 were excluded.