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Jun 28, 2013

Literature Review: A Snapshot Taken from the Inside

Researchers introduce technology that offers spatially and temporally resolved proteomic maps of endogenous proteins within living cells.

Literature Review: A Snapshot Taken from the Inside

Using the approach described below, the researchers were able to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. [© zozulinskyi - Fotolia.com]

  • Click Image To Enlarge +
    Figure. Labeling the mitochondrial matrix proteome in living cells. (A) Labeling scheme. The APEX peroxidase was genetically targeted to the mitochondrial matrix via fusion to a 24–amino acid targeting peptide (Martel et al., Nat Biotechnol 2012;30:1143–1148). Labeling was initiated by the addition of biotin-phenol and H2O2 to live cells for 1 min. Cells were then lysed, and biotinylated proteins were recovered with streptavidin-coated beads, eluted, separated on a gel, and identified by MS. The peroxidase-generated phenoxyl radical is short-lived and membrane-impermeant and, hence, covalently tags only neighboring and not distant endogenous proteins. (B) Confocal fluorescence imaging of biotinylated proteins (stained with neutravidin) after live labeling of HEK cells expressing mito-APEX as in (A). Controls were performed with either biotin-phenol or H2O2 omitted. (C) Superresolution stochastic optical reconstruction microscopy (STORM) images showing streptavidin and APEX (AF405/AF647) localization patterns at 22-nm resolution in U2OS cells. Samples were reacted as in (B). (D) Gel analysis of biotinylated mitochondrial matrix proteins, before (lanes 1 to 3) and after (lanes 4 to 6) streptavidin bead enrichment. Samples were labeled as in (B). Substrates are biotin-phenol and H2O2. Mammalian cells have four endogenously biotinylated proteins, three of which were observed in the negative control lanes (2 and 3) of the streptavidin blot. (E) Electron microscopy of HEK cells expressing mito-APEX. EM contrast was generated by treating fixed cells with H2O2 and diaminobenzidine. APEX catalyzes the polymerization of diaminobenzidine into a local precipitate, which is subsequently stained with electron dense OsO4 (Martel et al.). Dark contrast is apparent in the mitochondrial matrix, but not the intermembrane space. B, biotin; DIC, differential interference contrast.

    The approach reported by Rhee and colleagues* promises to address a major deficiency of current proteomics platforms—their inability to report on protein distribution within the very small confines of discrete cell organelles and compartments. Here, the team uses a genetically targetable enzyme that catalyzes the generation of short-lived, highly reactive radicals in live cells; in turn, these radicals covalently react with electron-rich amino acids such as Tyr, Trp, His, and Cys. The enzyme selected was ascorbate peroxidase (APEX), chosen for its stability and activity in all cellular compartments.

    In initial studies, APEX was targeted to the mitochondrial matrix of human embryonic kidney (HEK) cells and labeling was started by adding biotin-phenol and H2O2 to the cell medium; the phenol portion of the reagent served as the source of radicals to tag the nearby proteins, while the biotin unit was intended for use in streptavidin-mediated microscopy analysis. Fixing the cells 1 min after labeling initiation served to also stop the APEX reaction (see Figure). Analysis of the experiment revealed that the radicals produced did indeed have a very short range of action (few tens of nanometers), allowing for good spatial resolution of the labeling reaction, and that labeling occurred in a strict APEX- and H2O2-dependent manner. The proof-of-concept work was extended to include various cytosol-oriented APEX fusions, as well as a mitochondrial matrix facing variant. Analysis of the proteome labeled in the latter case revealed a number of proteins previously unknown to be associated with the mitochondria.

    The new technique appears to be well-validated through the present work and should be extendable to nearly all systems in which fusing APEX to an organelle- or compartment-targeting signal is available.

  • *Abstract from Science 2013, Volume 339: 1328–1331

    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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