Research

Starting in 2022, the MitoTOL consortium aimed to build high-quality mitochondrial protein inventories (mitoproteomes) across diverse species by developing experimental and computational workflows.  After piloting many different experimental approaches (including cell lysis, density gradients, affinity purification, and proximity biotinylation), the consortium converged on a three step methodology that could be adapted for each organism:

1. Serial mitochondrial purification (using a combination of differential centrifugation, density gradient centrifugation, with or without affinity purification, and with or without Proteinase K shaving)
2. Data-independent acquisition (DIA) mass spectrometry applied to all fractions from the serial purification 
3. Data analysis by protein correlation profiling (PCP) whereby each protein’s relative abundance across purification steps was scored using Pearson correlation to a “bait profile” of three mitochondrial conserved marker proteins.

For each species, this methodology resulted in an experimentally defined inventory called “MitoViaPCP”, which was then augmented by combining this list with microscopy validation and literature evidence into a final “MitoCarta” inventory. 

We also used these new experimentally-defined inventories to retrain a computational tool for subcellular localization and predicted mitoproteomes across several hundred eukaryotic species.