Anton Simeonov Ph.D. National Institute of Health
Researchers introduce technology that offers spatially and temporally resolved proteomic maps of endogenous proteins within living cells.
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.
Anton Simeonov, Ph.D., works at the NIH.
ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Science titled “Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging”. Authors of the paper are Rhee HW, Zou P, Udeshi ND, Martell JD, Mootha VK, Carr SA, and Ting AY.