Acclaim pepmap rslc 50

Each sample was separated using a nanoliter flow rate Easy nLC system. Buffer
A is a 0.1% formic acid aqueous solution, and B is a 0.1% formic acid
acetonitrile aqueous solution (80% acetonitrile). The column was
equilibrated with 100% liquid A. The sample was separated from the
autosampler onto an analytical column (Thermo Fisher Scientific, Acclaim
PepMap RSLC 50 µm × 15 cm, nano viper, P/N164943), and the flow rate was
300 nl/min.
The samples were separated by chromatography and analyzed by Q Exactive Plus
mass spectrometer. The analysis time was 60 min, the detection method was
positive ion, the scanning range of the parent ion was 350–1800 m/z, the
resolution of the mass spectrometry was 70,000, the AGC target was 3e6, and
the maximum IT was 50 ms. The mass-to-charge ratio of peptides and fragments
was collected according to the following methods: 10 fragment spectra (MS2
scan) were collected after full scan, MS2 Activation Type was HCD, isolation
window was 2 m/z, secondary mass spectrometry resolution was 17,500,
microscans was 1, secondary maximum IT was 45 ms, and normalized collision
energy was 27 eV.

A 2 μg sample was injected and separated using nano-LC, followed by an online electrospray tandem mass spectrometry analysis. The complete LC-MS set consisted of a liquid phase system (Easy nLC system, Thermo Fisher Scientific) and a mass spectrometry system (Q-Exactive Plus, Thermo Fisher Scientific). Solution A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile in water (80% in acetonitrile). Sample was separated using a nonlinearly growing gradient solution in an analytical column (Thermo Fisher Scientific, Acclaim PepMap RSLC 50 µm × 15 cm, nano viper, P/N164943) at a flow rate of 300 nL/min. LC cycle conditions were as follows: 0 to 1 min, the linear gradient of solution B increased from 2% to 8%; 1 to 46 min, the linear gradient of solution B increased from 8% to 28%; 46 to 56 min, the linear gradient of solution B increased from 28% to 40%; 56 to 57 min, the linear gradient of solution B increased from 40% to 90%; 57 to 60 min, the linear gradient of solution B maintained at 90%. The MS parameters were set as follows: the scan range (m/z) was 350 to 1500; the resolution was 60,000; the AGC target was 1e6; and the maximum injection time was 50 ms. For PRM, the resolution was 15,000, the AGC target was 1e5, the maximum injection time was 50 ms, the loop count was 10, the isolation window was 1.6 m/z, and the NCE was 27%.