Quantitation using PRM was performed on an Orbitrap Q Exactive Plus mass spectrometer with an EASY-Spray source coupled to a Nano-LC system (EASY-nLC 1000, Thermo Fisher Scientific, Waltham, MA). Samples were loaded and separated by reversed-phase chromatography. Upon LC direct injection, peptides were resolved with a 125-min HPLC gradient [linear gradient from 2 to 35% HPLC buffer B (0.1% formic acid in acetonitrile) in 110 min and then to 90% buffer B in 15 min]. The eluate was examined by MS using Q Exactive, which was coupled to the Nano-LC online. After a full-scan event, the MS/MS scans in the PRM mode were triggered by an inclusion list. A full mass spectrum was detected in the Orbitrap at a resolution of 70,000 [automatic gain control (AGC) target was set to 3E6; the maximum injection time was 50 ms; and the mass/charge ratio (m/z) range was 400 to 1350], followed by 20 MS/MS scans on the Orbitrap at a resolution of 17,500 (AGC target was 1E5, and the maximum injection time was 100 ms) in a data-independent procedure. The mass window for precursor ion selection was 1.6 m/z. The isolation window for MS/MS was set at 2.0 m/z. The normalized collision energy (NCE) was 27% with higher-energy collisional dissociation (HCD). Three biological replicates were performed. The PRM data were analyzed using Skyline daily.

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