N-terminal COFRADIC - a powerful method to analyze mitochondrial protein processing

Due to their role in synthesis of ATP, mitochondria are called the power plants of the cell. Since >99% of mitochondrial proteins are encoded in the nucleus and post-translationally imported into the four compartments of the mitochondrion, malfunctioning of the mitochondrial import machinery has dramatic consequences for cell and organism vitality. Mitochondrial matrix proteins carry a targeting N-terminal pre-sequence which is cleaved-off by the mitochondrial processing peptidase (MPP). Since the specificity of MPP cleavage was controversially discussed, we conducted a large-scale analysis of mature protein N-termini in purified mitochondria. Thus, we were able to identify 615 N-termini, finally leading to a refined understanding of protein processing after import and to the identification of a novel peptidase, ICP55, which removes N-terminal amino acids after MPP cleavage, according to the N-end rule.

We further optimized the N-terminal COFRADIC approach by incorporating a targeted quality control: since the efficiency of specific derivatization reaction is crucial, we now control those reactions by selected reaction monitoring (SRM). Thus, in case of inefficient derivatization, the time and cost-intense LC-MS/MS analyses and data evaluation can be omitted and the experiment has to be repeated.

The improved workflow will be utilized to further improve our understanding of mitochondrial protein processing.