First, we applied the isotope-coded affinity tag (cICAT) quantitative analysis [29] to compare protein abundances in mitochondria isolated from normal (r + ) and mtDNA-depleted (r 0 ) osteosarcoma cells. MtDNA-depleted (r 0 ) osteosarcoma cell has been used as an important tool to investigate dysfunctional mitochondria. The r 0 cell was established by long-term treatment of ethidium bromide (EtBr) which intercalated into mtDNA without any detectable effect on nuclear DNA division [30] and led to the inhibition of mtDNA replication and transcription [31] . Thus, we examined a set of nuclear-encoded mt proteins as mt proteome. To quantify protein abundance ratios of mt proteins in r 0 versus r + mitochondria, we labeled mt proteins with cICAT containing light ( 12 C) and heavy ( 13 C) isotope signatures, which react with thiol groups of cysteines in proteins. The experimental scheme is summarized in Figure S1 (see Materials and Methods for details). From the cICAT analysis, we identified 1,121 proteins (Table S1 ). According to the mt protein abundance ratios (r 0 /r + ), we classified all proteins into three classes: up-regulated, downregulated, and not significantly changed proteins in r 0 mitochondria. The number of up-regulated proteins with r 0 /r + $1.5 was 201, while the number of down-regulated proteins with r 0 / r + #0.67 was 313.The thresholds of 1.5 represents the 1.5-fold increase, whereas the threshold of 0.67 represents 1.5-fold decrease under dysfunctional r 0 state. Meanwhile, 607 not significantly changed proteins were present in both r 0 and r + mitochondria with similar abundances (0.67,r 0 /r + ,1.5).