For cICAT labeling, protein extracts from the pure r 0 or r + mitochondria were prepared with lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM MgCl 2 , 0.5 mM dithiothreitol, 1 mM phenyl-methylsulfonyl fluoride [PMSF]). Proteins were concentrated, and other non-protein materials such as salts were removed by the acetone precipitation method. Precipitated proteins were denatured in labeling buffer (6 M urea, 0.05% SDS, 5 mM EDTA, 50 mM Tris-Cl, pH 8.3) for 30 min and reduced with 5.3 mM Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for 30 min at 37uC. After readjustment to pH 8.3 with 1 M Tris-Cl buffer (pH 8.3), the r + or r 0 protein samples were labeled with a 15-fold molar excess of cleavable cICAT light ( 12 C) or heavy ( 13 C) reagents (Applied Biosystems, Foster City, CA) relative to proteins for 2 h at 37uC. Each 110-mg aliquot of separately labeled samples was equally combined and digested by trypsin (Promega, Madison, WI). The cICAT-labeled peptides were selectively isolated by strong cation exchange (SCX) and avidin affinity chromatography on a manually programmed AKTA Explorer 100 system (Amersharm Pharmacia, Sweden). Biotin moieties from cICAT-labeled peptides were cleaved by incubation for 1.5 h with 95% trifluoroacetic acid at 37uC. Samples were then dried in a speed-vacuum dryer and dissolved with 0.4% acetic acid for LC-MS/MS analysis. A schematic summary of the cICAT analysis workflow is presented in Figure  S1 .