Isolation and Purification of Mitochondria from Cell Culture for Proteomic Analyses

In-depth analysis of the mitochondrial proteome can be greatly improved by analyzing isolated mitochondria instead of whole cells. However, isolation of sufficient amounts of mitochondria from cell culture has proven to be notoriously difficult due to small sample size. Thus, we have developed a reproducible, controllable, and highly customizable method to isolate high microgram to low milligram amounts of intact mitochondria from cell culture samples along with an optional density gradient purification. This chapter provides a methodological update of our approach and underlines the excellent quality and coverage of the mitochondrial proteome of crude and purified mitochondria from cultured liver cancer cell lines.