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.