Cell-selective metabolic labeling of proteins

Ngo, John T.; Champion, Julie A.; Mahdavi, Alborz; Tanrikulu, I. Caglar; Beatty, Kimberly E.; Connor, Rebecca E.; Yoo, Tae Hyeon; Dieterich, Daniela C.; Schuman, Erin M.; Tirrell, David A.

doi:10.1038/nchembio.200

Cited by 164 publications

(163 citation statements)

References 14 publications

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“…(Figure 8). 75 Recently, additional MetRS mutant screening strategies have been employed to further improve efficiency and selectivity for NLL. 76 Overall, the simplicity of the expression protocols for incorporating these analogs, as well as practical modified protein yields, make this a 16 suitable method for fluorescent protein labeling either in vitro or in vivo.…”

Section: Probing Interactions With Solvatochromic Flaasmentioning

confidence: 99%

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

Krueger

Imperiali

2013

ChemBioChem

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Section: Probing Interactions With Solvatochromic Flaasmentioning

confidence: 99%

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

Krueger

Imperiali

2013

ChemBioChem

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“…This strategy has been used to selectively enrich microbial proteins from mixtures of bacterial and mammalian cells. For example, Ngo et al (5) found that proteins made in an E. coli strain outfitted with a mutant MetRS could be labeled with Anl in coculture with murine alveolar macrophages, which were not labeled. Using similar approaches, Grammel et al (6) identified virulence factors from Salmonella typhimurium that were expressed in the course of infection of murine macrophages, and Mahdavi et al (7) profiled Yersinia enterocolitica proteins that were injected into HeLa cells.…”

mentioning

confidence: 99%

Cell-specific proteomic analysis in Caenorhabditis elegans

Yuet¹,

Doma

Ngo³

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.click chemistry | protein engineering | proteomics | cell-specific protein expression | nematode pharyngeal muscle I n a complex eukaryote like Caenorhabditis elegans, cell heterogeneity restricts the usefulness of large-scale, mass spectrometry-based proteomic analysis. Enriching for specific cells is challenging, and researchers cannot systematically identify low-abundance proteins expressed in specific cells from wholeorganism lysates. Cell-selective bioorthogonal noncanonical amino acid tagging (cell-selective BONCAT) offers a way to overcome these limitations (1, 2). We have previously engineered a family of mutant Escherichia coli methionyl-tRNA synthetases (MetRSs) capable of appending the azide-bearing L-methionine (Met) analog L-azidonorleucine (Anl) to its cognate tRNA in competition with Met (3, 4). Because Anl is a poor substrate for any of the natural aminoacyl-tRNA synthetases, it is excluded from proteins made in wild-type cells but is incorporated readily into proteins made in cells that express an appropriately engineered MetRS. Controlling expression of mutant MetRSs by expression only in specific cells restricts Anl labeling to proteins produced in those cells. Tagged proteins can be distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to alkynyl or cyclooctynyl probes that permit facile detection, isolation, and visualization of labeled proteins. This strategy has been used to selectively enrich microbial proteins from mixtures of bacterial and mammalian cells. For example, Ngo et al. (5) found that proteins made in an E. coli strain outf...

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“…Using this approach FUNCATwas used to observe protein synthesis in individual dendrites that was diminished on addition of anisomycin, a translation inhibitor, and stimulated on addition of brain-derived neurotropic factor (BDNF), which induces translation-dependent enhancement of synaptic strength (Kang and Schuman 1996). The engineering of a mutant MetRS that is capable of charging the noncanonical amino acid azidonorleucine, which is not recognized by the cell's endogenous MetRS, can be used to restrict the incorporation of azidonorleucine to a defined population of cells (Ngo et al 2009). …”

Section: Fluorescent Noncanonical Amino Acid Tagging (Funcat)mentioning

confidence: 99%

Imaging Translation in Single Cells Using Fluorescent Microscopy

Chao

Yoon

Singer

2012

Cold Spring Harbor Perspectives in Biology

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The regulation of translation provides a mechanism to control not only the abundance of proteins, but also the precise time and subcellular location that they are synthesized. Much of what is known concerning the molecular basis for translational control has been gleaned from experiments (e.g., luciferase assays and polysome analysis) that measure average changes in the protein synthesis of a population of cells, however, mechanistic insights can be obscured in ensemble measurements. The development of fluorescent microscopy techniques and reagents has allowed translation to be studied within its cellular context. Here we highlight recent methodologies that can be used to study global changes in protein synthesis or regulation of specific mRNAs in single cells. Imaging of translation has provided direct evidence for local translation of mRNAs at synapses in neurons and will become an important tool for studying translational control.

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Cell-selective metabolic labeling of proteins

Cited by 164 publications

References 14 publications

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

Fluorescent Amino Acids: Modular Building Blocks for the Assembly of New Tools for Chemical Biology

Cell-specific proteomic analysis in Caenorhabditis elegans

Imaging Translation in Single Cells Using Fluorescent Microscopy

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