Data Independent Acquisition (DIA)
Each sample was analyzed by a second acquisition in data independent (DIA) mode. LC separation parameters and conditions were identical to those used for DDA but only MS2 spectra were acquired. The mass range for DIA 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 mProphet algorithm integrated into Skyline. The mass error threshold was set at 20 ppm for transitions, and the resolving power was 30,000. Randomly scrambled decoys were used in mProphet Q-value calculation. The R package MSstats 3.1 (Choi et al., 2014) was used for power analysis to calculate the fold-change (FC) cutoff that is appropriate for this 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% with protein quantity as the scope for each 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 and the minimal mProphet detection Q-value for peak quality was set to 0.01.