Presentation/Session Information

Session Information

Session Title: Plenary Session 3 Session Type: Plenary
Session Location: Royce Hall Session Time: Fri, Jun 26 1:30PM - 4:30PM

Presentation Information

Program Number: 132 Presentation Time: 3:36PM

Presentation Content

The C. elegans Cell-Specific Proteomics Toolkit.Kai P. Yuet 1, Meenakshi K. Doma 2,3, Paul W. Sternberg 2,3, David A. Tirrell 1. 1)Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America; 2)Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, United States of America; 3)Howard Hughes Medical Institute, California Institute of Technology, Pasadena, California, United States of America

In a multicellular animal like C. elegans, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. To address these challenges, we engineered a C. elegans phenylalanyl-tRNA synthetase (CePheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). We expressed the engineered CePheRS in a cell type of choice (i.e. body wall muscles, intestinal epithelial cells, neurons, pharyngeal muscles), permitting proteins in those cells – and only those cells – to be labeled with azides upon feeding Azf-labeled E. coli to the worms. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affinity probes, separation from the rest of the protein pool and identification by mass spectrometry. By coupling our methodology with heavy isotopic labeling, we successfully identified proteins – including proteins with previously unknown expression patterns – expressed in targeted subsets of cells. While cell types like body wall or pharyngeal muscles can be targeted with a single promoter, many cells cannot; cell-specificity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we also developed a two-component system to drive overlapping – but not identical – patterns of expression of engineered CePheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-CePheRS system for highly efficient CePheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells.




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