Cell-specific proteomic analysis in Caenorhabditis elegans

Kai P Yuet, Meenakshi K Doma, John T Ngo, Michael J Sweredoski, Robert L J Graham, Annie Moradian, Sonja Hess, Erin M Schuman, Paul W Sternberg, David A Tirrell

Research output: Contribution to journalArticle

51 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)2705-10
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume112
Issue number9
DOIs
Publication statusPublished - 03 Mar 2015
Externally publishedYes

Keywords

  • Animals
  • Caenorhabditis elegans/cytology
  • Caenorhabditis elegans Proteins/biosynthesis
  • Gene Expression Regulation/physiology
  • Isotope Labeling/methods
  • Mutation
  • Proteome/biosynthesis
  • Proteomics/methods

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  • Cite this

    Yuet, K. P., Doma, M. K., Ngo, J. T., Sweredoski, M. J., Graham, R. L. J., Moradian, A., Hess, S., Schuman, E. M., Sternberg, P. W., & Tirrell, D. A. (2015). Cell-specific proteomic analysis in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 112(9), 2705-10. https://doi.org/10.1073/pnas.1421567112