Data Independent Acquisition (DIA) and statistical analysis
Each sample was analyzed by a second acquisition run using data independent (DIA) mode. LC separation parameters and conditions were identical to those used for DDA but only MS2 spectra were acquired. The DIA mass range was set to 390 – 1015 m/z at 25 Hz scan rate with an isolation width of 10 m/z (0.5 m/z overlap, 2.5 sec scan interval). Quantitative analyses and visualization of DIA data was performed using Skyline 20.0 [30]. At least four (generally six) transition peaks were detected for each peptide and their Q value scored using mProphet and decoys generated by Skyline. The minimal mProphet detection Q-value for peak quality was set to 0.01. MSstats 3.1 [33] was used for power analysis to calculate the fold-change (FC) cutoff that was appropriate for each experiment, as well as statistical significance of differences between treatment and control. The cutoff for multiple testing adjusted p-values [34] was set to p<0.05. MSstats analyses were normalized by equalizing medians at a minimum confidence interval of 95% using protein quantity as the scope for the analysis. All DIA data can be accessed at PanoramaPublic (https://panoramaweb.org/lr03.url) and ProteomeXchange (PXD029254).