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Mol Cell. 2014 Jul 17;55(2):332-41. doi: 10.1016/j.molcel.2014.06.003. Epub 2014 Jul 4.

Proteomic mapping of the human mitochondrial intermembrane space in live cells via ratiometric APEX tagging.

Author information

1
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
2
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Korea.
3
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
4
Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
5
Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Molecular Biology, Howard Hughes Medical Institute, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
6
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address: ating@mit.edu.

Abstract

Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.

PMID:
25002142
PMCID:
PMC4743503
DOI:
10.1016/j.molcel.2014.06.003
[Indexed for MEDLINE]
Free PMC Article

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