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Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms

Citation

Yuet, Kai P. (2016) Tools For Spatiotemporally Specific Proteomic Analysis In Multicellular Organisms. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9VD6WDH. https://resolver.caltech.edu/CaltechTHESIS:05252016-151033826

Abstract

The emergence of mass spectrometry-based proteomics has revolutionized the study of proteins and their abundances, functions, interactions, and modifications. However, in a multicellular organism, it is difficult to monitor dynamic changes in protein synthesis in a specific cell type within its native environment. In this thesis, we describe methods that enable the metabolic labeling, purification, and analysis of proteins in specific cell types and during defined periods in live animals. We first engineered a eukaryotic phenylalanyl-tRNA synthetase (PheRS) to selectively recognize the unnatural L-phenylalanine analog p-azido-L-phenylalanine (Azf). Using Caenorhabditis elegans, we expressed the engineered PheRS 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. Labeled proteins are therefore subject to "click" conjugation to cyclooctyne-functionalized affnity 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; spatiotemporal selectivity typically results from the combinatorial action of multiple regulators. To enhance spatiotemporal selectivity, we next developed a two-component system to drive overlapping -- but not identical -- patterns of expression of engineered PheRS, restricting labeling to cells that express both elements. Specifically, we developed a split-intein-based split-PheRS system for highly efficient PheRS-reconstitution through protein splicing. Together, these tools represent a powerful approach for unbiased discovery of proteins uniquely expressed in a subset of cells at specific developmental stages.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:click chemistry; protein engineering; proteomics; cell-specific protein expression; nematode pharyngeal muscle
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemical Engineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Tirrell, David A. (co-advisor)
  • Sternberg, Paul W. (co-advisor)
Thesis Committee:
  • Tirrell, David A. (co-chair)
  • Sternberg, Paul W. (co-chair)
  • Davis, Mark E.
  • Shapiro, Mikhail G.
Defense Date:24 May 2016
Record Number:CaltechTHESIS:05252016-151033826
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:05252016-151033826
DOI:10.7907/Z9VD6WDH
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1007/s10439-013-0878-3DOIChemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics (Adapted for Chapter 1)
http://dx.doi.org/10.1073/pnas.1421567112DOICell-Specific Proteomic Analysis in Caenorhabditis elegans (Adapted for Chapter 2)
ORCID:
AuthorORCID
Yuet, Kai P.0000-0002-1381-8923
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9758
Collection:CaltechTHESIS
Deposited By: Kai Yuet
Deposited On:26 May 2016 19:21
Last Modified:04 Oct 2019 00:13

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