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Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging


Bagert, John David (2015) Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9QC01D9.


Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:time-resolved proteomics, BONCAT, sRNA, quorum sensing
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Tirrell, David A.
Thesis Committee:
  • Tirrell, David A. (chair)
  • Arnold, Frances Hamilton
  • Phillips, Robert B.
  • Leadbetter, Jared R.
Defense Date:20 May 2015
Record Number:CaltechTHESIS:05272015-123731363
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for ch. 2 adapted for Appendix B DOIArticle adapted for Appendix C adapted for Appendix D
Bagert, John David0000-0001-7768-2853
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8891
Deposited By: John Bagert
Deposited On:29 May 2015 20:53
Last Modified:08 Nov 2023 00:37

Thesis Files

PDF (Thesis) - Final Version
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[img] MS Excel (Table 2.S2) - Supplemental Material
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[img] MS Excel (Table 2.S4) - Supplemental Material
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[img] MS Excel (Table 3.S1) - Supplemental Material
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[img] MS Excel (Table 4.S1) - Supplemental Material
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