MaxQuant Data Analysis

The raw data from the MS were analysed in MaxQuant (version 1.5.6.5). The database search engine Andromeda was used to search the spectra against the UniProt database. The search settings were as follows: trypsin/P digestion, with up to 2 missed cleavages, fixed modification was carbamidomethyl (C), variable modifications were oxidation (M) and acetylation (Protein N-term), Label Free Quantification (LFQ) was performed with a minimum number of neighbours of 3 and average number of neighbour of 6. Peptide tolerance was set at 4.5 ppm and minimum peptide length was set at 7, amino acid maximum peptide mass was set at 4600 Da and Protein FDR was set at 0.01. Downstream analysis was performed in R version 3.4.0. The protein identification files were read in, results matching to a reverse sequence database and or those matching to a contaminant database were removed as were those with less than 2 unique peptides. The label-free intensities were then median-corrected for each sample and log2 transformed. Differential protein expression was calculated using Limma version 3.32.2. a repeated measures ANOVA to enable comparison to the microarray analysis (36).