Physical Sciences and Mathematics Commons^™

Utilizing Ultra-Performance Chromatography High-Resolution Mass Spectrometry To Investigate Fatty Acid Mediated Antibiotic Tolerance, Brittni Woodall

Doctoral Dissertations

The lipid membrane is the first component necessary to sustain life. To maintain homeostasis, segregate cellular machinery, provide protection from the environment, and reproduce, an organism must establish a boundary in which the processes can occur. Throughout the last two decades, research has propelled our knowledge of lipid membranes much beyond original hypotheses. Once thought of to be static and uniform, the understanding of the lipid membrane has evolved to encompass a structure that is responsive, unique, and intricately constructed by the organism itself. By chance or by choice, organisms adapt the lipid membrane according to the environment for which …

Protein Detection And Structural Characterization By Mass Spectrometry Using Supramolecular Assemblies And Small Molecules, Bo Zhao

Doctoral Dissertations

Mass spectrometry (MS) has played an increasingly prominent role in proteomics and structure biology because it shows superior capabilities in identification, quantification and structural characterization of proteins. To realize its full potential in protein analysis, significant progress has been made in developing innovative techniques and reagents that can couple to MS detection. This dissertation demonstrates the use of polymeric supramolecular assemblies for enhanced protein detection in complex biological mixtures by MS. An amphiphilic random co-polymer scaffold is developed to form functional supramolecular assemblies for protein/ peptide enrichment. The influences of charge density and functional group pK_a on host-guest interactions …

Enhanced Mass Spectrometric Analysis Of Peptides And Proteins Using Polymeric Reverse Micelles, Mahalia Adelina Corazon Paningbatan Serrano

Doctoral Dissertations

Mass spectrometry (MS) has become a key and indispensable tool in the identification, characterization, and quantitative analysis of proteins owing to its universality, sensitivity, specificity, and its capability for multiplexed detection. Because biological samples containing these protein analytes are almost always complex systems, various techniques are employed in conjunction with MS to fully harness its analytical potential and enhance its detection capabilities. This dissertation explores the use of amphiphilic polymeric reverse micelles in enriching proteins and peptides from complex biological mixtures and in enhancing their mass spectrometric analysis. Fundamental studies that elucidate the molecular basis for the observed MS signal …

Characterization Of Highly Heterogeneous Heparin-Protein Complexes Using Novel Mass Spectrometry-Based Approaches, Yunlong Zhao

Doctoral Dissertations

Heparin-like glycosaminoglycan (GAG) is a family of polysaccharide involved in variety of physiological processes. They have potentials to interact with a broad range of proteins and many of them hold crucial values in regulation of protein functions. My dissertation addresses the significance and challenges in the field of heparin-mediated studies, with a focus on the questions in biological and analytical aspects, which are largely hindered by the structural heterogeneity and function diversity of heparin. My dissertation reports the efforts I made in the past few years with respect to the development of novel analytical strategies based on a combination of …

Analytical Methods To Support Design And Optimization Of Protein Drug Conjugate: Focusing On Haptoglobin-Hemoglobin Complex As A Drug Carrier, Shengsheng Xu

Doctoral Dissertations

Acquired immunodeficiency syndrome (AIDS) remains one of the most serious public health challenges and a significant cause of mortality for certain populations. Despite the large number of antiretrovirals (mostly nucleotide and nucleoside analogs) developed in the past two decades, the inability of small molecule therapeutics to target HIV reservoirs directly creates a significant obstacle to their effective utilization. Indeed, achieving the desired therapeutic effect in the absence of the effective targeted delivery must rely on dosage escalation, which frequently causes severe toxicity. This problem may be solved by conjugation of antiretroviral agents to endogenous proteins (e.g., hemoglobin haptoglobin complex) that …

New Mass Spectrometric Methods For Studying Proteins In Solution And In The Gas Phase, Zhe Zhang

Doctoral Dissertations

This dissertation focuses on the mass spectrometric based methods for studying protein structures in solution phase and in gas phase. Protein structure is important to study because it is related to many disease mechanism and related therapies. The relationship between protein structures in solution phase and in gas phase has been investigated. Especially, the electrostatic interaction is characterized by mass spectrometry (MS). A HDX method has been developed to study protein oligomer kinetics and applied to study β-2-microglobulin (β2m) oligomerization. This dissertation firstly focuses on the relationship between protein gas phase structure and solution phase structure. We studied the structures …

Analysis Of Gold Nanoparticles And Their Use With Laser Desorption/Ionization Mass Spectrometry, Alyssa Marsico

Doctoral Dissertations

Gold nanoparticles (AuNPs) have many unique properties that make them attractive for use in various biological applications. Laser desorption/ionization mass spectrometry (LDI-MS) has been used to monitor AuNPs in complex biological samples but there are still many ways in which AuNPs can be used with MS. In this dissertation, the use of AuNPs to assist in analyte ionization has been investigated. Their ability to enhance signal from biomolecules based on their surface chemistry, size, aggregation and method of deposition has been studied. The first use of an inkjet printer to create surfaces from which analytes can be sampled is discussed …

Novel Analytical Methods Combining Non-Denaturing Chromatography And Mass Spectrometry To Study Biopolymer Structure And Interactions, Khaja Muneeruddin

Doctoral Dissertations

Biotherapeutics, an emerging class of medicines containing biopolymers (e.g., proteins, peptide, and polysaccharides) have been developed for a variety of indications including cancer, autoimmune, genetic, and blood diseases. Among these biopolymers protein therapeutics have been the rapidly growing segment in the pharmaceutical industry. This trend combined with the complexity of proteins has necessitated the development of powerful and robust analytical methods to study their structure and interactions with physiological partners. Mass spectrometry (MS) has become an indispensable tool to analyze various attributes of protein drugs such as profiling of intact mass, amino acid sequencing, and post translational modifications (PTMs). In …

Transferrin-Based Therapeutics And Analytical Methods To Characterize Them, Son N. Nguyen

Doctoral Dissertations

Modern development of medicine requires detailed characterization by state-of-the art analytical techniques that can be used to analyze covalent structure, conformations and protein-receptor interaction to quantitatively measure biodistribution of protein therapeutics. Mass spectrometry has already become an indispensable tool facilitating all stages of protein drug development. Particularly, this work has demonstrated the tremendous potential of electrospray ionization (ESI) mass spectrometry (MS) in this arena by providing invaluable information beyond mass measurement that can be used to optimize protein drug conjugate structures during early stages of development, and to further catalyze drug design efforts. Additionally, a new sensitive and selective method …

Investigations Into Ambient Ionization Mechanisms: Electrospray Ionization And Direct Analysis In Real Time Mass Spectrometry, Stephen Colin Gibson

Doctoral Dissertations

Mass spectrometry (MS) is an important tool for chemical analyses. Despite the MS requirement for generation of analyte gas-phase ions, many ion source designs afford little-to-no fragmentation, allowing characterization of intact molecules. However, this does not assure that detected ions are representative of the analytes’ natural state. Ionization mechanisms are generally complex and rarely fully understood. Fundamental research into these mechanisms provides greater insight into the relationship between solution chemistry and mass spectra. Work herein addresses aspects of two ambient ionization mechanisms: electrospray ionization (ESI) and Direct Analysis in Real Time (DART).

Ions produced by ESI are dispersed into a …

Mass Spectrometry Based Experimental Strategies To Characterize Native And Non-Native Disulfide Bonds In Cysteine-Rich Protein Therapeutics, Adriana Z. Kita

Doctoral Dissertations

The impact post-translational modifications (PTMs) can have on the structure, function, and immunogenicity of protein therapeutics makes it especially important for these protein-based treatments to be well characterized. Mass spectrometry has become instrumental in the examination of enzymatic and non-enzymatic PTMs. Disulfide bonds fall into both of these categories in which native disulfide bonds are formed in the ER by disulfide mediated enzymes and non-native disulfide bonds are often formed by non-enzymatic reducing/oxidizing reactions. Disulfide bonds are particularly important for protein folding and reinforcing higher order structure, and are typically characterized by LC-MS of non-reduced peptides. However, characterizing the disulfide …

Structural Analysis Of Proteins By Covalent Labeling And Mass Spectrometric Detection, Yuping Zhou

Doctoral Dissertations

Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. This dissertation focuses on the improvement of diethylpyrocarbonate (DEPC)-basedcovalent labeling …

Advanced Techniques In Mass Spectrometry For Qualitative And Quantitative Protein Characterization, Andrew Boissy Dykstra

Doctoral Dissertations

Though mass spectrometry has earned a central role in the field of proteomics due to its versatility in a wide range of experiments, challenges and complications are still encountered when using mass spectrometry to characterize protein structures, post-translational modifications (PTMs), and abundances. In this dissertation, analytical methods utilizing mass spectrometry have been developed to address challenges associated with both qualitative and quantitative protein characterization. The effectiveness of using multiple pepsin-like proteases, both separately and in mixtures, combined with online proteolysis using a special triaxial probe has been demonstrated on an amyloid beta peptide related to the onset of Alzheimer’s disease. …

Spatially Resolved Laser And Thermal Desorption/Ionization Coupled With Mass Spectrometry, Olga Sergeevna Ovchinnikova

Doctoral Dissertations

The work discussed in this dissertation is aimed at creating novel approaches to chemical imaging that ultimately allow for submicron resolution. This goal has been approached from two direction using laser based desorption and coupling it with an AFM using apertureless tip-enhanced laser ablation/ionization. The second direction was through the development a new approach to thermal desorption based mass spectrometry experiments by using a proximal probe to spatially desorb the surface and ionizing the plume of neutrals using a secondary ionization source at atmospheric pressure. The thermal desorption approach allows for the easy scaling of the technique all the way …