자료유형  

 학위논문

Control Number  

0017160684

International Standard Book Number  

9798382806785

Dewey Decimal Classification Number  

660

Main Entry-Personal Name  

Johnson, Alexander.

Publication, Distribution, etc. (Imprint  

[S.l.] : Princeton University., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

88 p.

General Note  

Source: Dissertations Abstracts International, Volume: 85-12, Section: B.

General Note  

Advisor: Wuhr, Martin.

Dissertation Note  

Thesis (Ph.D.)--Princeton University, 2024.

Summary, Etc.  

요약Multiplexed proteomics is a powerful tool to assay cell states in health and disease, but quantification of protein abundance changes is distorted by interference from co-isolated peptides. One approach to reduce interference is quantification by complementary ions, the balancer group-peptide conjugates, which allows accurate and precise multiplexed quantification at the MS2 level and is compatible with most proteomics instruments. In this dissertation, I will discuss our development of complementary ion quantification for the isobaric TMTpro tag (TMTproC), and the application of this method to study protein degradation in the model organism Escherichia coli. First, we evaluate and optimize complementary ion quantification for TMTpro, which increases complementary ion plexing capacity to ninechannels. The beneficial fragmentation properties of TMTpro increase sensitivity for TMTproC resulting in ~65% more proteins quantified compared to TMTpro-MS3, and ~18% more when compared to real-time-search TMTPro-MS3 (RTS-SPS-MS3). Next, we implemented a super-resolution mass spectrometry approach using the least-squares fitting (LSF) method for processing Orbitrap transients. The LSF algorithm resolves the 6.32 mDa spaced doublets for all TMTproC channels in the standard mass range with transients as short as ~108 ms. This advance demonstrates that expansion of the TMTproC 9 plex to a 21 plex is theoretically achievable. Finally, we quantify the turnover rates of ~3.2k E. coli proteins under 13 conditions by combining heavy isotope labeling with TMTproC. We find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. We also provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to decipher the control of proteostasis. This body of work presents a broadly applicable technology for quantifying protein abundance changes and advances our understanding of protein degradation on a global scale.

Subject Added Entry-Topical Term  

Chemical engineering.

Subject Added Entry-Topical Term  

Microbiology.

Subject Added Entry-Topical Term  

Biochemistry.

Index Term-Uncontrolled  

Proteomics

Index Term-Uncontrolled  

Peptides

Index Term-Uncontrolled  

Escherichia coli

Index Term-Uncontrolled  

Protein degradation

Index Term-Uncontrolled  

Nitrogen limitation

Added Entry-Corporate Name  

Princeton University Chemical and Biological Engineering

Host Item Entry  

Dissertations Abstracts International. 85-12B.

Electronic Location and Access  

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Control Number  

joongbu:658085