Membrane protein complexes are involved in essential cellular processes such as cell-cell communication, cell adhesion, transport, and energy homeostasis. Our current understanding of their function in health and disease is often limited despite their importance. Insight into the chemical structure of membrane complexes is fundamental to understand their biogenesis, function, and regulation. Analysis of the diverse proteoforms within a complex can be achieved by studying purified complexes via intact protein tandem mass spectrometry or “top-down proteomics”. The hydrophobic or fragile nature of these complexes makes the task more difficult. We have developed a highly efficient method for the reproducible and unbiased extraction of intact subunits from excised clear native gel bands for top-down proteomics.
This top-down proteomics approach was validated by studying the five protein complexes of the mitochondrial oxidative phosphorylation (OXPHOS) system in Bos taurus. The OXPHOS enzymes are hydrophobic heteromeric complexes in the range of 0.2 – 1 megadalton that generate adenosine triphosphate to fuel cellular processes. A mitochondrial fraction was prepared from bovine heart,extracted protein complexes were subjected to high resolution clear native gel electrophoresis, and gel slices were cut from the gel to isolate OXPHOS complexes. The reproducibility and recovery of the extraction method was evaluated by denaturing gel electrophoresis and top-down proteomics using liquid chromatography with online ultra high resolution quadrupole time-of-flight tandem mass spectrometry. Denaturing gel electrophoresis results showed that all subunits from each complex were extracted without any bias. The vast majority of subunits for each complex could be detected by top-down proteomics except for some large or hydrophobic subunits. Various post-translational modifications were found such as mitochondrial import sequence cleavage, acetylation, formylation, and phosphorylation. In-depth characterization of proteoforms by electron transfer dissociation and collision induced dissociation tandem mass spectrometry will be presented.