The ATP production (oxidative phosphorylation) involves five complexes embedded in the inner membrane of mitochondria. The yeast Saccharomyces cerevisiae is mainly used as a model for the study of oxidative phosphorylation; mutants are easy to produce and are still viable due to their ability to grow using the fermentation pathway. Here, we present a process for analyzing mitochondrial respiratory complexes using native electrophoresis (BN-PAGE) coupled to LC-MS/MS. BN-PAGE (1) permits the separation of functional respiratory complexes, thus allowing in-gel activity detection of most of the respiratory complexes and (2) provides convenient samples for bottom-up proteomics. Combining BN-PAGE and LC-MS/MS leads to the identification of the subunit composition of membrane complexes and offers the possibility of highlighting potential interacting proteins.
Keywords: BN-PAGE; Mass spectrometry; OXPHOS; Respiratory complexes; S. cerevisiae.