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).