Proteomic identification of carbonylated proteins in the kidney of trichloroethene-exposed MRL+/+ mice

Abstract

Trichloroethene (TCE), a common environmental and occupational pollutant, is associated with multiorgan toxicity. Kidney is one of major target organs affected as a result of TCE exposure. Our previous studies have shown that exposure to TCE causes increased protein oxidation (protein carbonylation) in the kidneys of autoimmune-prone MRL+/+ mice, and suggested a potential role of protein oxidation in TCE-mediated nephrotoxicity. To assess the impact of chronic TCE exposure on protein oxidation, particularly to identify the carbonylated proteins in kidneys, female MRL+/+ mice were treated with TCE at the dose of 2 mg/ml via drinking water for 36 weeks and kidney protein extracts were analyzed for protein carbonyls and carbonylated proteins identified using proteomic approaches (2D gel, Western blot, MALDI TOF/TOF MS/MS, etc.). TCE treatment led to significantly increased protein carbonyls in the kidney protein extracts (20 000 g pellet fraction). Interestingly, among 18 identified carbonylated proteins, 10 were found only in the kidneys of TCE-treated mice, whereas other 8 were present in the kidneys of both control and TCE-treated mice. The identified carbonylated proteins represent skeletal proteins, chaperones, stress proteins, enzymes, plasma protein and proteins involved in signaling pathways. The findings provide a map for further exploring the role of carbonylated proteins in TCE-mediated nephrotoxicity.

Figure 1

Carbonyl content in the kidney protein extracts (fraction #2) of control and TCE-treated mice. The data is expressed as mean±SEM (n=6). *p < 0.05.

Figure 2

2D gel profile of kidney proteins (fraction #2) of a control mouse (CBB G250 stained) is shown in Fig. 2A, whereas carbonylated proteins identified by Western blot are shown in Fig. 2B (black circles) which were also matched to the 2D gel protein profile (Fig. 2A). The numbers used for the spots are the same as in Tables 2 and 3.

Figure 3

2D gel profile of kidney proteins (fraction #2) of a TCE-treated mouse (CBB G250 stained) shown in Fig. 3A. The carbonylated protein, identified by Western blot, is shown in Fig. 3B (black circles), which were also matched to the 2D gel protein profile (Fig. 3A). The numbers used for the spots are the same as in Tables 1–3.