Peptidomic analysis of HEK293T cells: Effect of the proteasome inhibitor epoxomicin on intracellular peptides

Lloyd D. Fricker, Julia S. Gelman, Leandro M. Castro, Fabio C. Gozzo, Emer S. Ferro

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1981-1990
Number of pages10
JournalJournal of Proteome Research
Volume11
Issue number3
DOIs
StatePublished - Mar 2 2012

Fingerprint

Proteasome Inhibitors
Peptides
Proteasome Endopeptidase Complex
epoxomicin
Tissue Extracts
Mitochondrial Proteins
Nuclear Proteins
Proteolysis
Mass spectrometry
Sequence Analysis
Mass Spectrometry
Animals
Proteins
Cells
Tissue
Cell Line
Degradation

Keywords

  • peptidase
  • peptides
  • protease
  • proteasome

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

Cite this

Peptidomic analysis of HEK293T cells : Effect of the proteasome inhibitor epoxomicin on intracellular peptides. / Fricker, Lloyd D.; Gelman, Julia S.; Castro, Leandro M.; Gozzo, Fabio C.; Ferro, Emer S.

In: Journal of Proteome Research, Vol. 11, No. 3, 02.03.2012, p. 1981-1990.

Research output: Contribution to journalArticle

Fricker, Lloyd D. ; Gelman, Julia S. ; Castro, Leandro M. ; Gozzo, Fabio C. ; Ferro, Emer S. / Peptidomic analysis of HEK293T cells : Effect of the proteasome inhibitor epoxomicin on intracellular peptides. In: Journal of Proteome Research. 2012 ; Vol. 11, No. 3. pp. 1981-1990.
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