Cell-specific proteomic analysis in Caenorhabditis elegans

Abstract

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...

Nascent Proteome Remodeling following Homeostatic Scaling at Hippocampal Synapses

“…To gain selective access to particular cell classes, a recent development in which a mutant MetRS enables cell-type-specific labeling could be used in future experiments. This has already been employed in both C. elegans (Yuet et al., 2015) and Drosophila (Erdmann et al., 2015) to monitor protein synthesis in specific cell types. Applying this technology to homeostatic scaling and other forms of plasticity will allow us to discern the coordinated proteomic response in the different cell types that make up neuronal circuits.…”

Bioorthogonal Noncanonical Amino Acid Tagging (BONCAT) Enables Time-Resolved Analysis of Protein Synthesis in Native Plant Tissue

“…BONCAT has been used to study proteome-wide responses in mammalian and microbial culture (Dieterich et al, 2006;Zhang et al, 2014;Bagert et al, 2014) and to analyze subpopulations of cells in complex multicellular eukaryotes (Erdmann et al, 2015;Genheden et al, 2015;Yuet et al, 2015), but has yet to be employed in plant systems. Here, we applied BONCAT to Arabidopsis (Arabidopsis thaliana) to visualize proteins synthesized within 3 h after imposition of four significant abiotic perturbations: light stress (shift to darkness), high temperature, salt, and osmotic stress.…”

Phenylalanyl-tRNA synthetase

“…This study provides first-hand information that proteomic analysis of rare cells is possible that otherwise pose difficulty for enriching, identifying and quantifying proteins in complex biological systems. 134 Two recently published reports state that PheRS can be used as a counter selection marker in genome recombineering, genome mutagenesis, and plasmid curing experiments. Mayazaki develops two highly efficient variants (Thr251Ala/Ala294Gly and Thr251Ser/Ala294Gly) of E. coli PheRS that show improved incorporation efficiency of a toxic analog 4-chloro-phenylalanine over the single mutant (Ala294Gly) leading to the high lethality in common bacterial growth media.…”