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Nat Commun. 2015 Jul 3;6:7521. doi: 10.1038/ncomms8521.

Cell-selective labelling of proteomes in Drosophila melanogaster.

Author information

1
1] Research Group Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg 39120, Germany [2] Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg 39118, Germany.
2
Department of Neurochemistry and Molecular Biology, Leibniz Institute for Neurobiology, Magdeburg 39118, Germany.
3
1] Molecular Neurogenetics Laboratory, Max Planck Institute for Molecular Biomedicine, Münster 48149, Germany [2] Faculty of Medicine, University of Münster, Münster 48149, Germany.
4
Smoler Proteomics Center, Faculty of Biology, Technion, Haifa 32000, Israel.
5
1] Research Group Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg 39120, Germany [2] Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg 39118, Germany [3] Center for Behavioral Brain Sciences, Magdeburg 39118, Germany.

Abstract

The specification and adaptability of cells rely on changes in protein composition. Nonetheless, uncovering proteome dynamics with cell-type-specific resolution remains challenging. Here we introduce a strategy for cell-specific analysis of newly synthesized proteomes by combining targeted expression of a mutated methionyl-tRNA synthetase (MetRS) with bioorthogonal or fluorescent non-canonical amino-acid-tagging techniques (BONCAT or FUNCAT). Substituting leucine by glycine within the MetRS-binding pocket (MetRS(LtoG)) enables incorporation of the non-canonical amino acid azidonorleucine (ANL) instead of methionine during translation. Newly synthesized proteins can thus be labelled by coupling the azide group of ANL to alkyne-bearing tags through 'click chemistry'. To test these methods for applicability in vivo, we expressed MetRS(LtoG) cell specifically in Drosophila. FUNCAT and BONCAT reveal ANL incorporation into proteins selectively in cells expressing the mutated enzyme. Cell-type-specific FUNCAT and BONCAT, thus, constitute eligible techniques to study protein synthesis-dependent processes in complex and behaving organisms.

PMID:
26138272
PMCID:
PMC4507001
DOI:
10.1038/ncomms8521
[Indexed for MEDLINE]
Free PMC Article

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