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Detection and identification of in vivo chemical‐protein adducts
Author(s) -
Labenski Matt Thomas,
Fisher Ashley Ann,
Tsaiprailis George,
Monks Terry,
Lau Serrine
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a440-a
Subject(s) - chemistry , adduct , biochemistry , thioether , morpholino , stereochemistry , zebrafish , organic chemistry , gene
Hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis by producing ROS and adducting proteins. GSHQ adducted proteins are localized in the S3 segment of renal proximal tubules within the outer stripe of the outer medulla, the target region for toxicity. Recent technological advancements in mass spectrometry permit the detection of chemical‐induced post‐translational modifications (PTM) that may either directly alter structures and functions of the modified proteins or indirectly alter signaling pathways. We have now identified such modified proteins following administration of 2‐GSHQ (400μmol/kg, iv, 2hr) to Long Evans rats. Urinary proteins were precipitated, 1D gel separated, trypsin digested and analyzed by MudPIT. P‐Mod was used to confirm adducted peptides identified by X!Tandem, followed by manual validation of each spectra. We identified 4 proteins adducted by 2‐(cystein‐S‐yl)HQ (CSHQ), a metabolite of 2‐GSHQ, and also 3 proteins adducted by HQ. The HQ adduct originates as either a CSHQ or NAC‐HQ adduct followed by post‐adduction hydrolysis of the thioether bond during protein digestion, resulting in a final adduct with a mass of 105 Da. The site‐specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical‐induced PTMs and the subsequent toxicological response. (GM39338, GM070890 , ES06694).