One-dimensional western blotting coupled to LC-MS/MS analysis to identify chemical-adducted proteins in rat urine.

Matthew T. Labenski, Ashley A. Fisher, Terrence J. Monks, Serrine S. Lau

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

The environmental toxicant hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis via a combination of redox cycling and the covalent adduction of proteins within the S segment of the renal proximal tubules in the outer stripe of the outer medulla (OSOM). Following administration of 2-(glutathion-S-yl)HQ (MGHQ) (400 μmol/kg, i.v., 2 h) to Long Evans (wild-type Eker) rats, Western analysis utilizing an antibody specific for quinol-thioether metabolites of HQ revealed the presence of large amounts of chemical-protein adducts in both the OSOM and urine. By aligning the Western blot film with a parallel gel stained for protein, we can isolate the adducted proteins for LC-MS/MS analysis. Subsequent database searching can identify the specific site(s) of chemical adduction within these proteins. Finally, a combination of software programs can validate the identity of the adducted peptides. The site-specific identification of covalently adducted and oxidized proteins is a prerequisite for understanding the biological significance of chemical-induced posttranslational modifications (PTMs) and their toxicological significance.

Original languageEnglish (US)
Pages (from-to)327-338
Number of pages12
JournalMethods in molecular biology (Clifton, N.J.)
Volume691
StatePublished - 2011

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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