Peptidomic Analysis of HEK293T Cells: Effect of the Proteasome Inhibitor Epoxomicin on Intracellular Peptides

Peptides derived from cytosolic, mitochondrial, and nuclear
proteins
have been detected in extracts of animal tissues and cell lines. To
test whether the proteasome is involved in their formation, HEK293T
cells were treated with epoxomicin (0.2 or 2 μM) for 1 h and
quantitative peptidomics analysis was performed. Altogether, 147 unique
peptides were identified by mass spectrometry sequence analysis. Epoxomicin
treatment decreased the levels of the majority of intracellular peptides,
consistent with inhibition of the proteasome beta-2 and beta-5 subunits.
Treatment with the higher concentration of epoxomicin elevated the
levels of some peptides. Most of the elevated peptides resulted from
cleavages at acidic residues, suggesting that epoxomicin increased
the processing of proteins through the beta-1 subunit. Interestingly,
some of the peptides that were elevated by the epoxomicin treatment
had hydrophobic residues in P1 cleavage sites. Taken together, these
findings suggest that, while the proteasome is the major source of
intracellular peptides, other peptide-generating mechanisms exist.
Because intracellular peptides are likely to perform intracellular
functions, studies using proteasome inhibitors need to be interpreted
with caution, as it is possible that the effects of these inhibitors
are due to a change in the peptide levels rather than inhibition of
protein degradation.