(A) Design of two 3-state SILAC (Stable Isotope Labeling by Amino acids in in Cell culture) experiments. HEK 293T cells stably expressing APEX2-OMM or ERM-APEX2 were cultured in media containing heavy (H) isotopes of arginine and lysine. APEX2-NES stable cells were cultured in media with medium (M) isotopes of arginine and lysine, and negative control cells where either expression of APEX2 was omitted or ERM-APEX2 or APEX2-OMM stable cells were not treated with H2O2 were cultured in light (L) media. All dishes were then identically treated with biotin-phenol and H2O2 except for the negative controls mentioned before. The cells were separately lysed, and the three lysates in each replicate were combined 1:1:1 by protein mass. The mixture was then enriched using streptavidin beads and analyzed by mass spectrometry. (B) Scatter plot showing log2(H/M) versus log2(H/L) for Replicate 1 for the OMM proteomic experiment (left) and the ERM proteomic experiment (right). For each of the 5296 proteins detected in Replicate 1 of the OMM proteomic experiment, the log2(H/M) value was plotted against the log2(H/L) value. Known OMM proteins (members of our OMM true positive list, Supplementary file 2a) are shown in green; proteins without prior mitochondrial annotation according to MitoCarta (Pagliarini et al., 2008), Gene Ontology Cell Component (GOCC) (Ashburner et al., 2000) annotation, or the APEX-mapped mitochondrial matrix (Rhee et al., 2013) or mitochondrial intermembrane space (IMS) (Hung et al., 2014) proteomes are shown in red; all other proteins are shown in black. SILAC ratio cut-offs used to filter the OMM mass spectrometric data and obtain the final OMM proteome are shown by the dashed lines. On the right, a similar analysis was conducted for the 4799 proteins detected in Replicate 1 of the ERM proteomic experiment. Known ERM proteins (from our ERM true positive list, Supplementary file 2b) are shown in green; proteins with cytosolic annotation are shown in red; all others are shown in black. (C) Characterization of the specificity of the OMM and ERM proteomes. The first two columns show the percentage of proteins in the entire human proteome and our OMM proteome with prior mitochondrial annotation according to MitoCarta (Pagliarini et al., 2008), GOCC (Ashburner et al., 2000), and our APEX-mapped mitochondrial matrix (Rhee et al., 2013) and mitochondrial IMS (Hung et al., 2014) proteomes. The next two columns show the breakdown of proteins with GOCC sub-mitochondrial annotation available (mitochondrial matrix, IMM, IMS, or OMM). If a protein had more than one sub-mitochondrial annotation, we assigned it to one of the four categories in this priority: OMM>IMS>IMM>matrix. Columns 5 and 6 show the percentage of proteins with secretory pathway annotation according to GOCC (Ashburner et al., 2000) or Phobius (Käll et al., 2004, Käll et al., 2007) in the entire human proteome and our ERM proteome, respectively. The last two columns show the breakdown of proteins with GOCC sub-secretory annotation available. Protein assignments were based on the following priority: endoplasmic reticulum>Golgi apparatus>plasma membrane. (D) Fluorescence imaging of endogenous C2CD3, a mitochondrial orphan, in COS-7 cells. COS-7 cells were transfected with mitochondrially-targeted blue fluorescent protein (mito-BFP) and mCherry-KDEL (not shown). The cells were fixed and stained with an antibody against endogenous C2CD3. Scale bar, 10 µm.