Data Independent Acquisition (DIA)
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 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 (1 m/z overlap, 2.5 sec scan interval). Quantitative analyses and visualization of DIA data was performed using Skyline 20.0 (Pino et al., 2017). At least four (generally six) transition peaks were detected for each peptide and scored using the integrated Skyline algorithm mProphet. The mass error threshold was set to 20 ppm for transitions, and resolving power was 30,000. Randomly scrambled decoys were used in mProphet Q-value calculation. MSstats 3.1 (Choi et al., 2014) 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 (Benjamini & Hochberg, 1995) 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. Tukey’s median polish was used as the summary method for MSstats analyses to weight each transition and each peptide of a given protein equally. The minimal mProphet detection Q-value for peak quality was set to 0.01. All DIA data can be accessed at PanoramaPublic (https://panoramaweb.org/lr03.url) and ProteomeXchange (PXD029254).