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.
Labenski
Matthew T
MT
Department of Pharmacology and Toxicology, Southwest Environmental Health Sciences Center, Center for Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ, USA.
Fisher
Ashley A
AA
Monks
Terrence J
TJ
Lau
Serrine S
SS
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