Detecting protein carbonylation in adipose tissue and in cultured adipocytes.

Reactive oxygen species-mediated attack of the acyl chains of polyunsaturated fatty acids and triglycerides leads to the formation of lipid hydroperoxides. Lipid hydroperoxides are subject to nonenzymatic Fenton chemistry producing a variety of reactive aldehydes that covalently modify proteins in a reaction referred to as protein carbonylation. Given the significant content of triglycerides in fat tissue, adipose proteins are among the most heavily carbonylated. The laboratory has utilized two methodologies for the detection of protein carbonylation in tissue- and cell-based samples. The first utilizes biotin coupled to a hydrazide moiety and takes advantage of the numerous biotin detection systems. The second method utilizes an anti 4-hydroxy-trans-2,3-nonenal (4-HNE)-directed antibody that can detect both 4-HNE and the corresponding 4-oxo derivative when the samples are reduced. Using such methods, we have evaluated the profile of carbonylated proteins in epididymal white adipose tissue and 3T3-L1 adipocytes using both methods. In addition, we have investigated the effects of two antidiabetic drugs, pioglitazone and metformin, on protein carbonylation in 3T3-L1 adipocytes. Overall, the biotin hydrazide method is rapid, inexpensive, and easy to use, but its usefulness is limited because it detects a wide variety of carbonylated derivatives, which makes assignments of individual proteins difficult. Compared to the biotin hydrazide method, the anti-HNE antibody method detects specific proteins more readily but identifies only a subset of carbonylated proteins. As such, the combination of both methods allows for a comprehensive evaluation of protein carbonylation plus provides a means towards identification of specific carbonylation targets.