Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging

TLDR

Microscopy provides spatiotemporal data for a few proteins, whereas mass spectrometry detects thousands of proteins but only in lysed samples. The study introduces a technology that merges microscopy and mass spectrometry to generate spatially and temporally resolved proteomic maps of endogenous proteins in living cells. The method uses a genetically targetable peroxidase to biotinylate nearby proteins, which are purified and identified by MS, enabling identification of 495 mitochondrial matrix proteins, 31 of which were novel. The labeling proved highly specific, distinguishing matrix‑facing inner membrane proteins from intermembrane space proteins, reassigning several proteins previously thought to be in the IMS or outer membrane to the matrix, and demonstrating that peroxidase‑mediated proteomic mapping in live cells is both precise and user‑friendly, offering a powerful tool for studying cellular composition.

Abstract

Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.