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 )