Protein quantification was carried out as described by Higgs et al. (2005). Briefly, after the raw files were acquired from the LTQ, all extracted ion chromatograms (X!C) were aligned by retention time, using the algorithm and procedure described by Higgs et al. (2005). Each aligned peak must match precursor ion, charge state, fragment ions (MS/MS data), and retention time (within a three-minute window). After alignment, the area-under-the-curve (AUC) for each individually aligned peak from each sample was measured, normalized, and compared for relative abundance. Peak intensities were transformed to a log2 scale before quantile normalization (Bolstad et al., 2003). When multiple peptides had the same protein identification, their quantile normalized log2 intensities were averaged to obtain log2 protein intensities. The log2 protein intensity was used for the Linear Mixed Model statistical analysis for each protein. For each protein, estimates of individual p-values and q-values (measure of False Discovery Rate, FDR) were determined. Fold changes were computed as the ratio of mean treated/mean control.
