2.5.4. Label‐free quantitative proteomics pipeline

Statistically relevant protein expression changes between the DTX and control mouse TIF samples were identified using the default workflow in the R package Proteus (version 0.2.10) where quantitation was performed at the peptide level, with some minor differences. 36 Only unique and razor peptides were considered for quantification with intensity values present in at least two out of three replicates per group. Missing values were replaced by values drawn from a normal distribution of 1.8 standard deviations and a width of 0.3 for each sample (Perseus‐type). Peptides were assigned to their leading razor protein and peptide intensities were aggregated to protein intensities using the aggregateHifly function based on the high‐flyer method. Peptides were assigned to their leading razor protein and peptide intensities were aggregated to protein intensities using the aggregateHifly function based on the high‐flyer method. 37 Protein intensities were normalized according to the normalize Quantiles function from the limma Bioconductor package. 38 Differential protein expression was performed using the limmaDE function which uses the empirical Bayes moderated t tests using the limma package. Protein intensities were log2 transformed. Proteus corrects for multiple testing using the Benjamini‐Hochberg FDR procedure.