One-dimensional western blotting coupled to LC-MS/MS analysis to identify chemical-adducted proteins in rat urine.
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.
Department of Pharmacology and Toxicology, Southwest Environmental Health Sciences Center, Center for Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.
Matthew T
MT
Labenski
Ashley A
AA
Fisher
Terrence J
TJ
Monks
Serrine S
SS
Lau
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002853:Chromatography, Liquid / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004785:Environmental Pollutants / Q000378:metabolism* / Q000633:toxicity; D006873:Hydroquinones / Q000378:metabolism* / Q000633:toxicity; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D011507:Proteinuria / Q000378:metabolism*; D051381:Rats; D013223:Statistics as Topic; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004785:Environmental Pollutants; D006873:Hydroquinones; C031927:hydroquinone