Combined Analysis DataFilter_Script for experiment 1 & 2

To determine how DEX treatment in WT and MKO mice leads to muscle atrophy, we measured muscle protein fractional synthesis rates using shot-gun LC-MS/MS analysis after in vivo 2H2O labeling. Mice were treated with PBS or DEX for 10 days. During the final 7 days, mice were also labeled with 2H2O. The sample size for wild type controls treated with PBS (WT-PBS, n=6), wild type treated with DEX (WT-DEX, n=6), MKO treated with PBS (MKO-PBS, n=5) and MKO treated with DEX (MKO-DEX, n=5). The rational for biological replicates of n=5-6 were used to ensure accurate comparison among each group. Biological replicates were chosen to asses biological variability rather than sole reliance on technical replicates. Controls included WT-PBS treated mice (n=6). Two separate dynamic proteomic experiments were completed and combined for final data analysis. This consisted of experiment 1 that included WT-PBS (n=4) and WT-DEX (n=4). Experiment 2 included the final biological replicates of WT-PBS (n=2), WT-DEX (n=2), MKO-PBS (n=5) and MKO-DEX (n=5). The data sets were combined and annotated as described by the following. We used several filtering criteria for inclusion of protein kinetic data that were included in the comparisons: more than one peptide had to be present for any protein; each peptide had to meet analytic accuracy criteria for fractional mass isotopomer abundances and for reproducibility; a protein had to be present in at least 3 animals per group; and these criteria had to be met for the protein in all 4 groups. The number of proteins meeting these criteria was 81 in WT-PBS, 88 in WT-DEX, 124 in MKO-PBS and 134 in MKO-DEX and 57 proteins met these criteria in gastrocnemius muscle in all 4 groups. To determine the difference in protein fractional synthesis rates (f), four groups were categorized as the following: WT-PBS, WT-DEX, MKO-PBS and MKO-DEX. The mean, medium, and standard deviation for each protein (n≥3 for each group) were calculated, and a 2X2 ANOVA analysis (InfernoRDN) was performed to compare the treatment, genotype, and interaction effects. Protein fractional synthesis were averaged within groups and the percent changes were compared across each group. An increase or decrease in fractional synthesis was assessed as ± 0.0 %. A binomial distribution statistical analysis was used to calculate the significance of the relative percent increase or decrease in GA protein fractional synthesis. Average protein fractional synthesis was also assessed by ANOVA followed by the Benjamini and Hochberg test for multiple comparisons (FDR=0.05, p ≤0.05) using GraphPad Prism version 8.0 for Mac, GraphPad Software, La Jolla California USA.

This is the combined data filter for protein kinetics data for experiment 1 and 2.