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

Authors: Labenski, Matthew T; Fisher, Ashley A; Monks, Terrence J; Lau, Serrine S

Published In Methods Mol Biol, (2011)

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 S₃ 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.

PubMed ID: 20972763

MeSH Terms: Animals; Blotting, Western/methods*; Chromatography, Liquid/methods*; Electrophoresis, Gel, Two-Dimensional; Electrophoresis, Polyacrylamide Gel; Environmental Pollutants/metabolism*; Environmental Pollutants/toxicity; Hydroquinones/metabolism*; Hydroquinones/toxicity; Protein Processing, Post-Translational/drug effects*; Proteinuria/metabolism*; Rats; Statistics as Topic; Tandem Mass Spectrometry/methods*