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Published March 3, 2015 | Published + Supplemental Material
Journal Article Open

Cell-Specific Proteomic Analysis in Caenorhabditis elegans


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.

Additional Information

© 2015 National Academy of Sciences. Edited by Carolyn R. Bertozzi, University of California, Berkeley, CA, and approved January 22, 2015 (received for review November 10, 2014). Published online before print February 17, 2015. We thank past and present members of the P.W.S. and D.A.T. laboratories for fruitful discussions and suggestions, and Roxana Eggleston-Rangel and Geoffrey T. Smith (Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology) for technical assistance. This work was supported by National Institutes of Health (NIH) Grant R01 GM062523. K.P.Y. was supported in part by a National Science Foundation graduate fellowship. P.W.S. is an Investigator of the Howard Hughes Medical Institute, which supported this work. The Proteome Exploration Laboratory is supported by Gordon and Betty Moore Foundation Grant GBMF775, the Beckman Institute, and NIH Grant 1S10RR029594-01A1. Author contributions: K.P.Y., M.K.D., J.T.N., E.M.S., P.W.S., and D.A.T. designed research; K.P.Y., M.K.D., and J.T.N. performed research; M.J.S., R.L.J.G., A.M., and S.H. contributed new reagents/analytic tools; K.P.Y., M.J.S., P.W.S., and D.A.T. analyzed data; and K.P.Y., P.W.S., and D.A.T. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The vectors generated in this study have been deposited in the Addgene database, www.addgene.org (Addgene nos. 62598 and 62599). This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1421567112/-/DCSupplemental.

Attached Files

Published - PNAS-2015-Yuet-2705-10.pdf

Supplemental Material - pnas.1421567112.sapp.pdf


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