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Protein-Protein Interaction Analysis: Expanded Hydrogen/Deuterium Exchange Tandem Mass Spectrometry and Host Cell Protein Characterization
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| 자료유형 | 학위논문 |
|---|---|
| 서명/저자사항 | Protein-Protein Interaction Analysis: Expanded Hydrogen/Deuterium Exchange Tandem Mass Spectrometry and Host Cell Protein Characterization. |
| 개인저자 | Wang, Qingyi. |
| 단체저자명 | University of Michigan. Chemistry. |
| 발행사항 | [S.l.]: University of Michigan., 2019. |
| 발행사항 | Ann Arbor: ProQuest Dissertations & Theses, 2019. |
| 형태사항 | 195 p. |
| 기본자료 저록 | Dissertations Abstracts International 81-02B. Dissertation Abstract International |
| ISBN | 9781085669429 |
| 학위논문주기 | Thesis (Ph.D.)--University of Michigan, 2019. |
| 일반주기 |
Source: Dissertations Abstracts International, Volume: 81-02, Section: B.
Advisor: Hakansson, Kristina I. |
| 이용제한사항 | This item must not be sold to any third party vendors.This item must not be added to any third party search indexes. |
| 요약 | Elucidating protein structures, dynamics, and interactions is critical for understanding their roles in health and disease. In the biopharmaceutical industry, bioanalytical science allows, e.g., insights into how these complex molecules interact, and enables their characterization to assure safety and consistency of biotherapeutic manufacturing. Mass spectrometry (MS)-based methods, such as hydrogen/deuterium exchange (HDX) MS, are powerful tools for structural proteomics. This dissertation presents analytical strategies for incorporating gas-phase fragmentation for improved structural resolution in HDX MS workflows in both positive and negative ion mode, and for improving host cell protein characterization in biopharmaceuticals.HDX is typically performed in positive ion mode. In Chapter 2, the feasibility of several negative ion mode tandem mass spectrometry (MS/MS) techniques for HDX coupling is explored. Regio-selectively deuterated peptide anions were fragmented by negative-ion collision induced dissociation (nCID), negative-ion free radical initiated peptide sequencing (nFRIPS), electron detachment dissociation (EDD), and negative-ion electron capture dissociation (niECD) to determine the extent of H/D scrambling in each MS/MS technique. nCID induces extensive H/D scrambling involving histidine C-2 and C棺-hydrogen atoms for histidine-containing peptides. nFRIPS proceeds with complete hydrogen scrambling but without histidine participation, whereas EDD and niECD demonstrate moderate scrambling with slightly lower levels in niECD. However, improved ionization efficiency, ion transmission, and fragmentation efficiency under HDX quenching conditions are needed for routine application of niECD in HDX MS workflows.Due to the acidic HDX quenching conditions, the acid protease pepsin is employed for protein digestion. However, resulting peptic peptides may not contain basic residues and therefore carry less average charge than typical tryptic peptides. In Chapter 3, supercharging strategies are explored for combination with electron capture/transfer dissociation (ECD/ETD)-based HDX MS/MS in positive ion mode. These MS/MS techniques require analytes to carry at least two positive charges. The supercharging reagent m-NBA was found to enhance the average charge state for a variety of pepsin-derived peptides, thus increasing ECD/ETD fragmentation efficiency and peptide sequence coverage in bottom-up HDX liquid chromatography (LC)/MS workflows. Retention time shifts in the presence of m-NBA were avoided by injecting m-NBA through a mixing tee following the analytical column.During these experiments, b-type ions were observed in ECD spectra of supercharged peptides. Such fragment ions are atypical in ECD but have been found at increased levels for peptides containing few or no basic residues. In Chapter 4, we find that such peptides also show abundant hydrogen atom loss from the charge-reduced radical species. We show that, upon ECD of supercharged peptides, the number and abundance of b ions increases with increasing charge state |
| 일반주제명 | Chemistry. |
| 언어 | 영어 |
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: 이 자료의 원문은 한국교육학술정보원에서 제공합니다. |
