Acclaim pepmap easy spray analytical column

Samples were analyzed on an EASY-nLC1000 system (ThermoFisher Scientific) coupled to an LTQ-Orbitrap Velos pro (ThermoFisher Scientific, Sunnyvale, CA, USA). Peptides for analysis were loaded via an Acclaim PepMap 100 trap column (100 µm × 2 cm, nanoViper, C18, 5 µm, 100 Å, Thermo Scientific), and subsequent peptide separation was performed on an Acclaim PepMap EASY-Spray analytical column (75 µm × 15 cm, nanoViper, C18, 2 µm, 100 Å, Thermo Scientific). For each liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis, peptides were loaded on a precolumn with microliter pickup. Peptides were separated at a flow rate of 250 nL/min using mobile phase acetonitrile with the addition of 0.1% formic acid in a gradient of 5–40% over 120 min. Eluted peptides were examined with an Orbitrap mass spectrometer using a spray voltage of +1.8 kV. A full scan from m/z 300 to 2000 at a resolution of 60,000 was acquired. Top N mode followed by a ten data-dependent acquisition of MS/MS scan method was applied using CID with normalized energy of 35 eV. Dynamic exclusion for previously fragmented precursor ions was used with the following parameters: exclusion time 180 s, repeat count 1, repeat duration 30 s, exclusion mass width 10 ppm, exclusion size 500 m, signal threshold 5000, and isolation width 1 m/z. Singly charged species were excluded from fragmentation.