Mapping New Residents of the Mitochondrial Nucleoid Laura Trinkle-Mulcahy  Cell Chemical Biology  Volume 24, Issue 3, Pages 250-251 (March 2017) DOI: 10.1016/j.chembiol.2017.03.006 Copyright © 2017 Terms and Conditions

Figure 1 Design of Quantitative Ratiometric APEX2-Based Mapping of the Mitochondrial Nucleoid Proteome The strategy utilized to highlight nucleoid-specific proteins was based on stable expression of the nucleoid protein Twinkle fused to APEX2 in HEK293 cells and the addition of biotin-phenol and H2O2 to catalyze biotinylation of neighboring proteins. Control conditions that were included to flag non-specific hits omitted either H2O2 treatment or expression of Twinkle-APEX2. An additional control that was added to highlight nucleoid-specific proteins above the background of other mitochondrial proteins was HEK293 cells stably expressing mito-APEX2, which targets the peroxidase throughout the entire mitochondrial matrix. Cells were harvested for lysis and extraction/capture of biotinylated proteins on streptavidin-coated magnetic beads. Trypsin digestion, tandem mass tag (TMT) labeling and LC-MS/MS were used to identify proteins and quantify their relative abundance in the three conditions. Cell Chemical Biology 2017 24, 250-251DOI: (10.1016/j.chembiol.2017.03.006) Copyright © 2017 Terms and Conditions