FIGURE 5.

Identification of glutathionylated mitochondrial proteins: regulation by respiratory substrates. A, immunoblotting of glutathionylated proteins in mitochondria isolated using a discontinuous Percoll gradient. Following isolation, brain mitochondria were incubated at 37 °C for 10 min in the presence or absence of either glutamate/malate (7.5 mm) plus ADP (50 μm) or DTT (1 mm). Mitochondria were lysed in a 2% CHAPS/nonreducing and reducing buffer and underwent a series (three times) of freezing and thawing to ensure maximal protein release. Then mitochondria were separated via SDS-PAGE, transferred to a nitrocellulose membrane, and probed using a Virogen GSH antibody (1:500). B, immunoprecipitation (IP) of glutathionylated mitochondrial proteins following mitochondria isolation using discontinuous Percoll gradient and identification using LC/MS/MS. Following isolation, brain mitochondria were treated with 2% CHAPS/nonreducing buffer and immunoprecipitated using GSH antibody (1:200). Immunoprecipitation proteins were then separated via SDS-PAGE. Protein bands stained by SYPRO® Ruby were excised and identified by LC/MS/MS.