Graduate Thesis Or Dissertation
 

Revealing sequences and modifications of intact proteins using electron fragmentation

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/qz20t1979

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  • People turn to mass spectrometry to answer some of life’s most important questions. From carbon dating of archeological finds to newborn blood screening tests, mass spectrometry allows us to measure molecules which helps advance our knowledge of life and the world we live in. One area of mass spectrometry that has seen particularly rapid development is the study of protein sequence and structure. While initial efforts focused on analyzing small peptide mixtures, advancements in instrumentation have now extended the size limit for protein research with mass spectrometry, allowing for the measurement of whole proteins and even entire viruses. This thesis explores the use of electron capture dissociation (ECD) to fragment intact proteins, revealing their sequence and modifications. The findings of this research push the boundaries of ECD for whole protein fragmentation, demonstrate the potential of ECD in elucidating disease mechanisms, and advance the characterization of protein therapeutics. Throughout my PhD program, the majority of my research was conducted at e-MSion, Inc., an OSU-derived start up that is responsible for developing a technology for electron-based fragmentation, called the ExD cell. The new capabilities enabled by the ExD cell played a pivotal role in the success of my experiments. Cu, Zn superoxide dismutase (SOD1) is an antioxidant enzyme that relies on copper and zinc atoms for its normal redox activity. Mutations in SOD1 lead to structural changes that cause the protein to lose one of its metals, resulting in toxicity to motor neurons and the development of amyotrophic lateral sclerosis (ALS). At the intact protein level, distinguishing between Cu-deficient and Zn-deficient SOD1 is challenging due to the overlapping isotopes of copper and zinc. However, ECD offers an opportunity to retain metal cofactors during fragmentation, allowing for the identification of metal-deficient SOD1 based on the detection of metal-containing fragments. Chapter 2 presents a published methods paper describing the analysis of SOD1 metalation using ECD fragmentation. These methods represent a significant step toward directly analyzing SOD1 metalation status in ALS-associated tissues. To highlight the importance of the ExD cell technology for protein research, Chapter 3 presents a review of the development of the ExD cell from its conception to its current state, highlighting notable applications enabled by ExD technology. The remaining chapters all utilized the ExD cell for the characterization of whole proteins to uncover the sequences and modifications that drive their biological functions. Chapter 4 provides insights into the hyper-phosphorylation of the SARS-CoV nucleocapsid protein, which is an essential step in viral replication. Hyper-phosphorylation of the nucleocapsid occurs in a flexible serine/arginine rich region that has challenged measurement with traditional peptide-centric techniques. However, efficient measurement was achieved by employing electron-based fragmentation of the entire serine/arginine-rich region enabling the identification of up to 9 phosphorylation sites. Furthermore, these results provide proteoform-specific information, confirming expected and revealing unexpected details about nucleocapsid phosphorylation by GSK-3β. The production of therapeutic antibodies often leads to undesired modifications that significantly affect their efficacy, immunogenicity, and stability. Therefore, accurate characterization of antibodies is crucial for pharmaceutical development. While mass spectrometry-based techniques are considered the gold standard for antibody characterization, current methods require extensive sample digestion with multiple proteases and increases the risk of introducing artifacts. Therefore, Chapter 5 describes a fast method for fragmenting an entire antibody at once, enabling sequencing of both chains without the need for complex sample preparation. Importantly, pre-activation of antibodies before ECD facilitated the sequencing of disulfide-bonded regions. This method greatly simplifies and speeds the analysis of antibodies in minutes, which are the major focus of Biopharma.
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