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Science DOI:10.1126/science.1230593

Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging.

Publication TypeJournal Article
Year of Publication2013
AuthorsRhee, H-W, Zou, P, Udeshi, ND, Martell, JD, Mootha, VK, Carr, SA, Ting, AY
JournalScience
Volume339
Issue6125
Pages1328-31
Date Published2013 Mar 15
ISSN1095-9203
KeywordsAnimals, Ascorbate Peroxidases, Biotinylation, Cercopithecus aethiops, COS Cells, Gene Targeting, Genetic Engineering, HEK293 Cells, Humans, Mass Spectrometry, Mitochondria, Proteomics
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.

URLhttp://www.sciencemag.org/cgi/pmidlookup?view=long&pmid=23371551
DOI10.1126/science.1230593
Pubmed

http://www.ncbi.nlm.nih.gov/pubmed/23371551?dopt=Abstract

Alternate JournalScience
PubMed ID23371551
PubMed Central IDPMC3916822
Grant ListDP1 OD003961 / OD / NIH HHS / United States
P30 CA014051 / CA / NCI NIH HHS / United States
R01 GM077465 / GM / NIGMS NIH HHS / United States