Recently, proximity labelling methods have been developed to map spatially localized proteome in live cells. Usually, these methods employ enzymatic biotinylation of the proximal proteins with short-lived reactive biotin species. The labeled proteins may contain biotinylated modifications, which can be detected by mass spectrometry after enrichment by streptavidin bead affinity purification. Using the direct detection method of biotinylated proteins by mass spec (Spot-ID), we could reveal the in vivo topological direction of 135 inner-mitochondrial membrane (IMM) proteins by an in situ-generated radical probe with genetically targeted peroxidase (APEX) in live cells. Furthermore, we could identify the global topological information of the porous outer mitochondrial membrane proteome in live cells by using isotope-coded probes. From these results, we could see that our methods may have a great potential to reveal other sub-compartmental membrane proteomic architecture in live cells. Currently, we further extend this method to reveal in vivo mitochondrial proteome of different tissues and organs of the animal model. |