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Identifying site‐specific chemical modifications of urinary excreted proteins using a proteomics approach
Author(s) -
Labenski Matthew Thomas,
Fisher Ashley A,
Tsaprailis George,
Monks Terrence J.,
Lau Serrine S
Publication year - 2006
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.20.4.a66-c
Subject(s) - chemistry , glutathione , biochemistry , cysteine , metabolite , hydroquinone , proteomics , mercapturic acid , enzyme , gene
Hydroquinone (HQ) and its glutathione conjugates (GSHQs) cause renal cell necrosis and 2,3,5‐ tris ‐(glutathion‐ S ‐yl)hydroquinone is nephrocarcinogenic in the Eker rat. Previous studies have shown that HQGSH adducted proteins were localized in the S 3 segment of the renal proximal tubules in the outer stripe of the outer medulla, which is the target region for toxicity. Recent technological advancements allow for the detection of chemical‐induced post‐translational modifications (PTMs) that may either indirectly alter cell signaling pathways or alter the structure and function of the modified proteins. We have now identified such modified proteins following administration of 2‐(glutathion‐ S ‐yl)HQ (GSHQ) (400mmol/kg, iv, 2hr) to Long Evans (wild‐type Eker) rats. Excreted urinary proteins were acetone precipitated, separated on a 1D gel, in‐gel digested and analyzed by MudPIT analysis. pMOD and SALSA, in addition to DeNovo sequencing were utilized to identify site‐specific chemical‐induced PTMs of at least 40 proteins. 2‐(Cystein‐ S ‐yl)HQ (CSHQ), a metabolite of GSHQ, was identified as the post‐translational adduct on several selective solvent‐exposed E, K, and R residues on various proteins, including g‐actin and hemopexin. GSHQ is adducted through the carboxyl group of E but with the e‐amino group of K and R. Oxidative modifications on M residues were also identified in >30 proteins. The site‐specific identification of covalently adducted and oxidized proteins is a prerequisite for understanding the biological significance of chemical‐induced PTMs and the subsequent toxicological response. (GM39338, GM070890, ES06694).