A biotinylated analog of the spin-trap DMPO opens new frontiers in detection of low abundance protein radicals by mass spectrometry

摘要

Methods using mass spectrometry in combination with spin-trapping are among the most powerful approaches for the identification of protein-based radicals. Herein, we present a variant to this approach, which combines spin-trapping with Bio- SS-DMPO, a biotinylated analog of the spin-trap DMPO, and a pull-down assay with streptavidin-coated magnetic beads. This method adds a dimension of selectivity prior to the MS analyses, thereby facilitating the location of DMPO nitrone adducts for the unambiguous structural determination of modified sites in the polypeptide chains. In addition, we show that Bio-SS-DMPO is accumulated inside intact metabolizing cells. The use of biotinylated spin trap analogs should allow for the identification of proteins as well as the site(s) of protein radical spin trapping from complex mixtures and/or proteomic-like studies.