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Cell-Specific Proteomic Analysis in Caenorhabditis elegans

Yuet, Kai P. and Doma, Meenakshi K. and Ngo, John T. and Sweredoski, Michael J. and Graham, Robert L. J. and Moradian, Annie and Hess, Sonja and Schuman, Erin M. and Sternberg, Paul W. and Tirrell, David A. (2015) Cell-Specific Proteomic Analysis in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America, 112 (9). pp. 2705-2710. ISSN 0027-8424. PMCID PMC4352802. https://resolver.caltech.edu/CaltechAUTHORS:20150127-223214858

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1073/pnas.1421567112DOIArticle
http://www.pnas.org/content/112/9/2705PublisherArticle
http://www.pnas.org/content/112/9/2705/suppl/DCSupplementalPublisherSupporting Information
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4352802/PubMed CentralArticle
ORCID:
AuthorORCID
Yuet, Kai P.0000-0002-1381-8923
Sweredoski, Michael J.0000-0003-0878-3831
Moradian, Annie0000-0002-0407-2031
Hess, Sonja0000-0002-5904-9816
Schuman, Erin M.0000-0002-7053-1005
Sternberg, Paul W.0000-0002-7699-0173
Tirrell, David A.0000-0003-3175-4596
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.
Funders:
Funding AgencyGrant Number
NIHR01 GM062523
NSF Graduate FellowshipUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Gordon and Betty Moore FoundationGBMF775
Caltech Beckman InstituteUNSPECIFIED
NIH1S10RR029594-01A1
Subject Keywords:click chemistry; protein engineering; proteomics; cell-specific protein expression; nematode pharyngeal muscle
Issue or Number:9
PubMed Central ID:PMC4352802
Record Number:CaltechAUTHORS:20150127-223214858
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150127-223214858
Official Citation: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, and David A. Tirrell Cell-specific proteomic analysis in Caenorhabditis elegans PNAS 2015 112 (9) 2705-2710; published ahead of print February 17, 2015, doi:10.1073/pnas.1421567112
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54160
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:23 Feb 2015 20:47
Last Modified:09 Mar 2020 13:19

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