Top Down Proteomics of Human Membrane Proteins from Enriched Mitochondrial Fractions

The interrogation of intact integral membrane proteins has long been a challenge for biological mass spectrometry. Here, we demonstrate the application of top down mass spectrometry to whole membrane proteins below 60 kDa with up to 8 transmembrane helices. Analysis of enriched mitochondrial membrane preparations from human cells yielded identification of 83 integral membrane proteins, along with 163 membrane-associated or soluble proteins, with a median q value of 3 x 10(-10). An analysis of matching fragment ions demonstrated that significantly more fragment ions were found within transmembrane domains than would be expected based upon the observed 1 protein sequence. In total, 46 proteins from the complexes of oxidative phosphorylation were identified which exemplifies the increasing ability of top down proteomics to provide extensive coverage in a biological network.