Mass spectrometric identification of mitochondrial oxidative phosphorylation subunits separated by two-dimensional blue-native polyacrylamide gel electrophoresis.

Abstract

Blue-native polyacrylamide gel electrophoresis is a powerful tool for the separation of intact membrane protein complexes mainly applied to the analysis of the enzymes of the mitochondrial oxidative phosphorylation system (OXPHOS). Combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), it reveals a two-dimensional pattern showing the individual subunits of the five OXPHOS multi-enzyme complexes. This pattern is useful in the diagnostic analysis of several diseases related to disorders in the oxidative phosphorylation system. However, in order to use this method for systematic diagnostic purposes and to be able to link disease with absence or reduced expression of specific subunits, an unambiguous identification of the individual subunits is necessary. In this study, we completed this task, implementing peptide mass fingerprinting and mass spectrometric sequence analysis. In the course of these analyses, we discovered a novel variant of a cytochrome c oxidase subunit VIc.