자료유형  

 학위논문

Control Number  

0017164233

International Standard Book Number  

9798346871316

Dewey Decimal Classification Number  

543

Main Entry-Personal Name  

Ryan, Jack P.

Publication, Distribution, etc. (Imprint  

[S.l.] : The University of North Carolina at Chapel Hill., 2024

Publication, Distribution, etc. (Imprint  

Ann Arbor : ProQuest Dissertations & Theses, 2024

Physical Description  

241 p.

General Note  

Source: Dissertations Abstracts International, Volume: 86-06, Section: B.

General Note  

Advisor: Baker, Erin S.

Dissertation Note  

Thesis (Ph.D.)--The University of North Carolina at Chapel Hill, 2024.

Summary, Etc.  

요약Mass spectrometry (MS) is a powerful analytical technique that allows for identification, quantification and elucidation of structures of a variety of diverse molecules. Commonly, front-end separation techniques such as gas or liquid chromatography or ion mobility spectrometry are coupled with MS platforms to facilitate more in-depth analyses. This is of great importance in complex matrices, as is often the case with biological samples. While the complexity of these biological samples presents a challenge to traditional analytical workflows, the molecular diversity found in each sample represents an opportunity to further understanding of molecular processes. To this end, the work presented in this dissertation makes use of a liquid chromatography - drift tube ion mobility spectrometry - mass spectrometry (LC-DTIMS-MS) platform to develop methods for improved analyses of a wide range of biomolecules including proteins and protein subunits, oligonucleotides, bacterial lipids, and opioids. IMS is a gas-phase separation technique wherein ions are separated based on their size, shape, and charge. When coupled with a mass spectrometer analyses provide measurements of both an analytes mass and gas-phase structure. Additional separation based on and characterization of the analyte's polarity is also achieved through either reversed-phase (RPLC) or hydrophilic interaction liquid chromatography (HILIC). In this dissertation, a HILIC chromatography method was developed and then coupled with IMS-MS to separate and characterize the three viral protein subunits (VPs) that comprise an intact adeno-associated virus (AAV) capsid. The characterization of the VP subunits was then coupled with analysis of intact AAV capsids using charge detection - mass spectrometry (CD-MS) offering a comprehensive workflow for a thorough and robust analysis of AAVs as they are continually being investigated vectors for gene therapeutic treatments.Following this work, the same LC-IMS-MS platform was utilized to enhance MS-based oligonucleotide sequencing, paired with an improved data analysis workflow based on Agilent Technologies' BioConfirm software. As existing MS-based sequencing isolates one single charge state at a time and then fragment that charge state at multiple collision energies, the number of injections (and therefore experimental time) is quite large. Additionally, analysis of these numerous MS/MS spectra can be time consuming, making MS-based oligonucleotide sequencing a daunting analytical technique. Herein, we leverage the gas-phase separation of IMS coupled with a drift time dependent collision induced dissociation (CID) ramp to ensure optimal fragmentation of all observed charge states in a single injection. Furthermore, data analysis is expedited using a novel workflow based on Agilent's BioConfirm software.Next, this platform was used to assess and characterize the unique lipidome of bacterial membrane proteins. Specifically, an RPLC method was developed to optimally separate lipopolysaccharide (LPS) and LPS-like lipids that are unique to gram negative bacteria. The LPS entities found in gram negative bacteria act as endotoxins, and induce adverse health effects such as fever, inflammation, and septic shock. Through investigating the lipids bound to the membrane protein MsbA, which facilitates endotoxin formation, we can gain insight into specific binding sites and mechanistic pathways that may be exploited in the therapeutic space for bacterial infections. Analysis of these lipids with an IMS-MS platform enables simultaneous measurement of mass and size of the analytes, enhancing our knowledge of this membrane protein-lipid complex.The final research chapter investigates an alternative IMS separation technique, traveling wave ion mobility spectrometry (TWIMS), and evaluates improvements in IMS separation relative to DTIMS experiments. Specifically, these experiments were performed on a Mobilion MOBIE 2.0 platform, which is a newer commercially available platform compared to the Agilent 6560 used in previous experiments. To assess separation capacity of the MOBIE platform, pairs of isomeric opioids were analyzed alone and as mixtures. Previous work on a DTIMS platform separated 3 of 11 isomer pairs initially, with an additional 5 achieving baseline separation through data post-processing. Using the structure for lossless ion manipulation (SLIM) based TWIMS platform, the same 8 isomer pairs were separated without requiring data post-processing. A primary concern for TWIMS based IMS experiments is adequate calibration in the mobility dimension, and by extension accurate collision cross section (CCS) measurements. Herein, we observed CCS agreement with gold standard DTIMS values with a percent CCS difference of 2% for all analytes in the experiment.

Subject Added Entry-Topical Term  

Analytical chemistry.

Subject Added Entry-Topical Term  

Physical chemistry.

Subject Added Entry-Topical Term  

Biochemistry.

Subject Added Entry-Topical Term  

Computational chemistry.

Index Term-Uncontrolled  

Biomolecules

Index Term-Uncontrolled  

Ion mobility

Index Term-Uncontrolled  

Mass spectrometry

Index Term-Uncontrolled  

TWIMS

Index Term-Uncontrolled  

DTIMS experiments

Added Entry-Corporate Name  

The University of North Carolina at Chapel Hill Chemistry

Host Item Entry  

Dissertations Abstracts International. 86-06B.

Electronic Location and Access  

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Control Number  

joongbu:657399