Optimisation of bortezomib concentration for experiments in HFFF-TERTs
Bortezomib has been employed in a number of studies of human cell lines as a specific inhibitor of the proteasome. However, a wide range of concentrations have been used, from 0.1 to 20 µM (Price et al., 2011;Chui et al., 2019). To optimise conditions for proteomic analysis in HCMV-infected immortalized primary human foetal foreskin fibroblasts (HFFF-TERTs), a range of bortezomib concentrations were compared with 10 μM MG132, a concentration we previously showed to provide efficacious inhibition of protein degradation (Figures 1, S1 ) (Nightingale et al., 2018). TMT peptide labels and MS3 mass spectrometry enabled very precise protein quantitation, as well as multiplexed analysis of up to 11 samples in the same experiment.
For each protein, ratios of (HCMV with bortezomib) / HCMV and (HCMV with MG132) / HCMV were compared to quantify the relative efficacy of protein rescue. The trend of linear correlation and slope of the trend line both increased with increasing bortezomib concentration, with a gradient near to one for 2 µM bortezomib. At this concentration, the degree of rescue was most similar between MG132 and bortezomib, and 2 µM bortezomib at 12 h post infection (hpi) was therefore selected for detailed comparative analysis (Figure 1A-B )