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).