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Development of TMTpro Complementary Ion Quantification and its Application to Protein Degradation Rates
Development of TMTpro Complementary Ion Quantification and its Application to Protein Degradation Rates
상세정보
- 자료유형
- 학위논문 서양
- 최종처리일시
- 20250211151100
- ISBN
- 9798382806785
- DDC
- 660
- 서명/저자
- Development of TMTpro Complementary Ion Quantification and its Application to Protein Degradation Rates
- 발행사항
- [Sl] : Princeton University, 2024
- 발행사항
- Ann Arbor : ProQuest Dissertations & Theses, 2024
- 형태사항
- 88 p
- 주기사항
- Source: Dissertations Abstracts International, Volume: 85-12, Section: B.
- 주기사항
- Advisor: Wuhr, Martin.
- 학위논문주기
- Thesis (Ph.D.)--Princeton University, 2024.
- 초록/해제
- 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.
- 일반주제명
- Chemical engineering
- 일반주제명
- Microbiology
- 일반주제명
- Biochemistry
- 키워드
- Proteomics
- 키워드
- Peptides
- 키워드
- Escherichia coli
- 기타저자
- Princeton University Chemical and Biological Engineering
- 기본자료저록
- Dissertations Abstracts International. 85-12B.
- 전자적 위치 및 접속
- 로그인 후 원문을 볼 수 있습니다.
MARC
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■00520250211151100
■006m o d
■007cr#unu||||||||
■020 ▼a9798382806785
■035 ▼a(MiAaPQ)AAI31142883
■040 ▼aMiAaPQ▼cMiAaPQ
■0820 ▼a660
■1001 ▼aJohnson, Alexander.
■24510▼aDevelopment of TMTpro Complementary Ion Quantification and its Application to Protein Degradation Rates
■260 ▼a[Sl]▼bPrinceton University▼c2024
■260 1▼aAnn Arbor▼bProQuest Dissertations & Theses▼c2024
■300 ▼a88 p
■500 ▼aSource: Dissertations Abstracts International, Volume: 85-12, Section: B.
■500 ▼aAdvisor: Wuhr, Martin.
■5021 ▼aThesis (Ph.D.)--Princeton University, 2024.
■520 ▼aMultiplexed 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.
■590 ▼aSchool code: 0181.
■650 4▼aChemical engineering
■650 4▼aMicrobiology
■650 4▼aBiochemistry
■653 ▼aProteomics
■653 ▼aPeptides
■653 ▼aEscherichia coli
■653 ▼aProtein degradation
■653 ▼aNitrogen limitation
■690 ▼a0542
■690 ▼a0410
■690 ▼a0487
■71020▼aPrinceton University▼bChemical and Biological Engineering.
■7730 ▼tDissertations Abstracts International▼g85-12B.
■790 ▼a0181
■791 ▼aPh.D.
■792 ▼a2024
■793 ▼aEnglish
■85640▼uhttp://www.riss.kr/pdu/ddodLink.do?id=T17160684▼nKERIS▼z이 자료의 원문은 한국교육학술정보원에서 제공합니다.
