The dynamic and unstable nature of protein nitrosothiols (PSNOs) derived from complex biological matrices (like cell lysates) make them unsuitable for proteomic/biochemical analysis in vitro. In an attempt to increase the stability of cell-derived PSNOs, scientists have devised methods to derivatize thiols undergoing nitrosylation, with a suitable molecule, to yield a stable adduct that could easily be detected using appropriate antibodies. The Biotin Switch Assay (BTSA) is currently the most widely used method for tagging PSNOs; however, the error-prone and cumbersome nature of the BTSA protocol prompted the development of alternative mechanisms of tagging cell-derived PSNOs. One such method is the immuno-spin trapping method using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), which effectively overcomes the shortcomings of the BTSA and proves to be a promising alternative. Here we describe the protocol for DMPO-based PSNO labeling and subsequent proteomic analysis by western blotting with an anti-DMPO antibody.
- immune-spin trapping
- thiyl radical