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

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Science  15 Mar 2013:
Vol. 339, Issue 6125, pp. 1328-1331
DOI: 10.1126/science.1230593

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Mitochondrial Makeup Mapped

Because mass spectrometry (MS) cannot be performed on living cells, biologists currently recover spatial information indirectly, by purifying organelles or protein complexes prior to MS analysis. These purifications often yield false positives because of sample contamination and false negatives because of material loss. Rhee et al. (p. 1328, published online 31 January) present an approach that bridges microscopy and proteomics to produce a spatially and temporally resolved proteomic map of mitochondria from living cells. A nonspecific labeling enzyme (peroxidase) was genetically targeted to the mitochondria within live cells, where it tagged endogenous proteins in a spatially restricted manner within a 1-minute window, for subsequent identification and analysis by MS. This rapid and straightforward technology provides the ability to access otherwise inaccessible cellular regions and requires a very small amount of starting material.


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.

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