Research Paper Volume 16, Issue 19 pp 12726—12768

Figure 5. Experimental design for proteomics loading experiment. (A) 20 female C57BL/6N mice (5 mice/day/age) were subjected to daily in vivo axial tibial compression for either 1 (Day 1) or 5 (Day 5) bouts and sacrificed 6 hours after their final bout of loading. (B) The loaded (right) and non-loaded (left) tibial mid-diaphyses were isolated, removed of marrow, and snap frozen. Proteins were extracted in 4% SDS. Proteins for all 40 samples were then analyzed by proteomics using a tandem mass tag (TMT)-11 design. (C) Proteomics raw data were analyzed, and differential expression analysis was performed using ProteoQ. An unadjusted p-value cutoff of 0.05 and a fold-change threshold of 1.1 were used to identify loading-regulated proteins for downstream analyses, which included gene ontology (GO), PANTHER pathways, COMPBIO, and correlation with previously published RNA-seq data [11]. (D) At Day 1, multidimensional scaling (MDS) showed that the strongest differences were between ages rather than with loading status. (E) A Day 5, MDS showed slightly better separation between loaded and non-loaded samples, but the strongest separation was still between ages.