title: 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.
authors.affiliation: Divisions of Chemistry and Chemical Engineering and.
authors.forename: Kai P
authors.initials: KP
authors.lastname: Yuet
authors.affiliation: Biology and Biological Engineering, Howard Hughes Medical Institute, and.
authors.forename: Meenakshi K
authors.initials: MK
authors.lastname: Doma
authors.affiliation: Divisions of Chemistry and Chemical Engineering and.
authors.forename: John T
authors.initials: JT
authors.lastname: Ngo
authors.affiliation: Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125; and.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Sweredoski
authors.affiliation: Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125; and.
authors.forename: Robert L J
authors.initials: RL
authors.lastname: Graham
authors.affiliation: Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125; and.
authors.forename: Annie
authors.initials: A
authors.lastname: Moradian
authors.affiliation: Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA 91125; and.
authors.forename: Sonja
authors.initials: S
authors.lastname: Hess
authors.affiliation: Max Planck Institute for Brain Research, D-60528 Frankfurt am Main, Germany.
authors.forename: Erin M
authors.initials: EM
authors.lastname: Schuman
authors.affiliation: Biology and Biological Engineering, Howard Hughes Medical Institute, and.
authors.forename: Paul W
authors.initials: PW
authors.lastname: Sternberg
authors.affiliation: Divisions of Chemistry and Chemical Engineering and tirrell@caltech.edu.
authors.forename: David A
authors.initials: DA
authors.lastname: Tirrell
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D007553:Isotope Labeling / Q000379:methods; D009154:Mutation; D020543:Proteome / Q000096:biosynthesis* / Q000235:genetics; D040901:Proteomics / Q000379:methods*
chemical_list: D029742:Caenorhabditis elegans Proteins; D020543:Proteome
keywords: cell-specific protein expression; click chemistry; nematode pharyngeal muscle; protein engineering; proteomics