Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Ngo, John T.; Schuman, Erin M.; Tirrell, David A.

doi:10.1073/pnas.1216375110

Cited by 60 publications

(62 citation statements)

References 66 publications

(54 reference statements)

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“…Combining this methodology with cell type specific protein labeling 20,42 will offer a valuable tool to dissect proteome dynamics in different cell populations in complex organs such us the nervous system. Finally, by demonstrating the robustness of our method for in vivo applications, in this report and a recent study, 43 we show for the first time the direct detection of biotinylated labeled in intact animals after a short pulse of AHA labeling.…”

Section: Resultsmentioning

confidence: 99%

Direct Detection of Biotinylated Proteins by Mass Spectrometry

et al. 2014

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Mass spectrometric strategies to identify protein subpopulations involved in specific biological functions rely on covalently tagging biotin to proteins using various chemical modification methods. The biotin tag is primarily used for enrichment of the targeted subpopulation for subsequent mass spectrometry (MS) analysis. A limitation of these strategies is that MS analysis does not easily discriminate unlabeled contaminants from the labeled protein subpopulation under study. To solve this problem, we developed a flexible method that only relies on direct MS detection of biotin-tagged proteins called “Direct Detection of Biotin-containing Tags” (DiDBiT). Compared with conventional targeted proteomic strategies, DiDBiT improves direct detection of biotinylated proteins ∼200 fold. We show that DiDBiT is applicable to several protein labeling protocols in cell culture and in vivo using cell permeable NHS-biotin and incorporation of the noncanonical amino acid, azidohomoalanine (AHA), into newly synthesized proteins, followed by click chemistry tagging with biotin. We demonstrate that DiDBiT improves the direct detection of biotin-tagged newly synthesized peptides more than 20-fold compared to conventional methods. With the increased sensitivity afforded by DiDBiT, we demonstrate the MS detection of newly synthesized proteins labeled in vivo in the rodent nervous system with unprecedented temporal resolution as short as 3 h.

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Section: Resultsmentioning

confidence: 99%

Direct Detection of Biotinylated Proteins by Mass Spectrometry

et al. 2014

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“…showed that both E. coli and mammalian cells expressing a mutant MetRS are able to utilize ANL as a surrogate for methionine in protein synthesis, while wild-type cells are inert to ANL and proteins made in these cells are not labeled [42,43•]. In co-culture experiments, labeling of newly synthesized proteins with affinity reagents or fluorescent dyes is restricted to cells expressing the mutant MetRS, therefore enabling cell-specific enrichment, identification and visualization, even in mixtures of different cell types.…”

Section: New Developmentsmentioning

confidence: 99%

Teaching old NCATs new tricks: using non-canonical amino acid tagging to study neuronal plasticity

Hinz¹,

Dieterich²,

Schuman³

2013

Current Opinion in Chemical Biology

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The non-canonical amino acid labeling techniques BONCAT (bioorthogonal non-canonical amino acid tagging) and FUNCAT (fluorescent non-canonical amino acid tagging) enable the specific identification and visualization of newly synthesized proteins. Recently, these techniques have been applied to neuronal systems to elucidate protein synthesis dynamics during plasticity, identify stimulation-induced proteomes and subproteomes and to investigate local protein synthesis in specific subcellular compartments. The next generation of tools and applications, reviewed here, includes the development of new tags, the quantitative identification of newly synthesized proteins, the application of NCAT to whole animals, and the ability to genetically restrict NCAT labeling. These techniques will enable not only improved detection but also allow new scientific questions to be tackled.

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“…58 Interestingly, Anl replaces methionine selectively at N-terminal positions not at internal sites. Site-selectivity occurs because NLL-EcMetRS catalyzes aminoacylation only of the mammalian initiator tRNA Met , not the mammalian elongator tRNA Met .…”

Section: Spatially Resolved Proteomic Analysismentioning

confidence: 99%

Chemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics

Yuet

Tirrell

2013

Ann Biomed Eng

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Accurate measurements of the abundances, synthesis rates and degradation rates of cellular proteins are critical for understanding how cells and organisms respond to changes in their environments. Over the past two decades, there has been increasing interest in the use of mass spectrometry for proteomic analysis. In many systems, however, protein diversity as well as cell and tissue heterogeneity limit the usefulness of mass spectrometry-based proteomics. As a result, researchers have had difficulty in systematically identifying proteins expressed within specified time intervals, or low abundance proteins expressed in specific tissues or in a few cells in complex microbial systems. In this review, we present recently-developed tools and strategies that probe these two subsets of the proteome: proteins synthesized during well-defined time intervals – temporally resolved proteomics – and proteins expressed in predetermined cell types, cells or cellular compartments – spatially resolved proteomics – with a focus on chemical and biological mass spectrometry-based methodologies.

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Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Cited by 60 publications

References 66 publications

Direct Detection of Biotinylated Proteins by Mass Spectrometry

Direct Detection of Biotinylated Proteins by Mass Spectrometry

Teaching old NCATs new tricks: using non-canonical amino acid tagging to study neuronal plasticity

Chemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics

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