Digital Commons Network^™

Identification Of Early Diagnostic And Predictive Biomarkers Of Cisplatin-Induced Acute Kidney Injury Using Metabolomics, Yong Jin Lim

Electronic Thesis and Dissertation Repository

Cisplatin is an effective chemotherapeutic agent used for the treatment of a wide variety of solid tumors and hematologic cancers. Despite its effectiveness, cisplatin is associated with several toxic effects to non-cancerous tissues. Nephrotoxicity is the main dose-limiting toxicity in cisplatin therapy, manifesting as acute kidney injury (AKI) in approximately one-third of patients receiving cisplatin. AKI is defined as an abrupt decline in kidney function and is associated with several short- and long-term adverse outcomes, including chronic kidney disease, cardiovascular disease, and mortality. AKI is currently diagnosed by increased serum creatinine (SCr) concentrations. However, SCr is a marker of functional …

De Novo Sequencing Of Multiple Tandem Mass Spectra Of Peptide Containing Silac Labeling, Fang Han

Electronic Thesis and Dissertation Repository

The systematic studies of proteins has gradually become fundamental in the research related to molecular biology. Shotgun proteomics use bottom-up proteomics techniques in identifying proteins contained in complex mixtures using a combination of high performance liquid chromatography coupled with mass spectrometry technology. Current mass spectrometers equipped with high sensitivity and accuracy can produce thousands of tandem mass spectrometry (MS/MS) spectra in a single run. The large amount of data collected in a single LC-MS/MS run requires effective computational approaches to automate the process of spectra interpretation. De novo peptide sequencing from tandem mass spectrometry (MS/MS) has emerged as an important …

The Study Of The Structure And Dynamics Of Parkin Activation, Elaine Aisha Freeman

Electronic Thesis and Dissertation Repository

Parkin is an RBR E3 ubiquitin ligase that has been implicated in both sporadic and familial Parkinson’s disease. Upon mitochondrial damage, parkin is activated step-wise to recruit and ligate ubiquitin to a substrate on the outer mitochondrial membrane. Disruption of this activation and ligation cascade is hypothesized to result in neuronal death related to Parkinson’s disease.

While structures of parkin for a number of these activation states exist, it is important to note they are not of full-length human parkin. These structures are often truncated and come from various non-human species to eliminate important, yet hard to quantify structural elements. …

Uncovering The Ubiquitin Ligase Activity And Substrates Of The Human C-Terminal To Lish (Ctlh) Complex, Matthew E.R. Maitland

Electronic Thesis and Dissertation Repository

Ubiquitination is the transfer of a ubiquitin molecule to protein substrates by the sequential actions of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases. It is a post-translational modification that controls the fate and function of the substrate protein. Substrate specificity in the ubiquitination reaction is conferred by the E3 ligases. Sequence homology suggests the human C-terminal to LisH (CTLH) complex could be an E3 ligase; however, very little is known about this complex. In this thesis, I characterize the human CTLH complex as a multi-subunit E3 ligase and define its activity, structure, and substrates. I demonstrate that the …

A Workflow To Analyze Ethcd Mass Spectrometry Data For Studying Hiv Gp120 Glycosylation, Yingxue Sun

Electronic Thesis and Dissertation Repository

The great heterogeneity of HIV populations and richness of surface glycan clouds makes it difficult to locate a conserved and exposed protein epitope as an effective vaccine target. However, more than 80% new infections result from single transmitted founder (T/F) viruses. We set out to design a workflow to study the traits of T/Fs that allow for their superior infectivity, specifically, the glycosylation patterns of gp120, a subunit of HIV envelope protein responsible for binding to host cell receptors. Our main research methods include Western blot and mass spectrometry. Our current understanding of the mass spectrometry data indicates that our …

Effects Of Oxidative Modifications On The Structure And Non-Canonical Functions Of Cytochrome C Studied By Mass Spectrometry, Victor Yin

Electronic Thesis and Dissertation Repository

The peroxidase activity of the mitochondrial protein cytochrome c (cyt c) plays a critical role in triggering programmed cell death, or apoptosis. However, the native structure of cyt c should render this activity impossible due to the lack of open iron coordination sites at its heme cofactor. Despite its key biological importance, the molecular mechanisms underlying this structure-function mismatch remain enigmatic. The work detailed in this dissertation fills this knowledge gap by using mass spectrometry (MS) to decipher the central role that protein oxidative modifications and their associated structural changes play in activating the peroxidase function of cyt c …

Mass Spectrometry Identification Of Membrane-Type 1 Matrix Metalloproteinase (Mt1-Mmp) Binding Partners Following Co-Immunoprecipitation In Mcf-7 Cells, Bradley Bork

Electronic Thesis and Dissertation Repository

Membrane-type 1 matrix metalloproteinase (MT1-MMP) is an integral multidomain membrane protease involved in extracellular matrix remodelling. No longer recognized solely as a destructive enzyme, MT1-MMP proteolytic and non-proteolytic activities are involved in a variety of cellular processes. I hypothesized that the diverse functions of MT1-MMP are dependent on domain-specific binding partner interactions that elicit a cellular response. Using a combination of co-immunoprecipitation and mass spectrometry, 248 unique proteins were isolated in MT1-MMP variant expressing MCF-7 cells. Newly identified binding partners suggest potential roles of MT1-MMP in the nucleus, endoplasmic reticulum, cytoplasm, and plasma membrane. Additionally, the cytoplasmic domain of MT1-MMP …

Systematic Identification Of The Lysine Methylome Using Methyllysine Binding Domains, Wen Qin

Electronic Thesis and Dissertation Repository

Post-translational modifications (PTM) are vital regulators of protein function and homeostasis. The role of dynamic regulations of non-histone lysine methylated proteins (NHKMP) recently began to be recognized in DNA damage repair, apoptosis and transcriptional pathways. My goal was to identify components of the NHKMP network to understand its importance in a healthy versus diseased cellular state. I used membrane peptide arrays to systematically characterize nine naturally occurring lysine methyl binding domains (KMBD). Five KMBDs were chosen based on their overlapping specificities to achieve maximum coverage of lysine methylated peptides. These five KMBDs was used to enrich for methylated lysine peptides …

Mass Spectrometry-Based Proteomics Analysis Of Bioactive Proteins In Emd That Modulate Adhesion Of Gingival Fibroblast To Improve Bio-Integration Of Dental Implants, David Zuanazzi Machado Jr

Electronic Thesis and Dissertation Repository

Titanium implants are used in dental practice to replace damaged or lost teeth. The implant needs to integrate with the surrounding gingiva to protect it against bacterial invasion that leads to implant loss. The biointegration is dependent on the implant surface that interacts with proteins from biological fluids to modulate tissues response. Tailoring the surface with specific proteins from the enamel matrix derivative (EMD) would be beneficial to improve the implant-gingiva interface since EMD can affect various cells including gingival fibroblasts. A surface-affinity approach using three different titanium surfaces and saliva was utilized as a model in combination with tandem …

Proteomic Characterization Of Ovarian And Breast Cancer Microenvironments For Improved Diagnostics And Therapeutic Targeting, Dylan Z. Dieters-Castator

Electronic Thesis and Dissertation Repository

Cancers exist within complex microenvironments formed by heterogeneous cell types. This diversity creates significant challenges for detection, diagnosis and treatment. Mass spectrometry-based proteomics is a powerful approach capable of characterizing complex biological systems which are characteristic of cancer biology. In this thesis, proteomics was utilized to answer several questions related to ovarian cancer diagnosis and detection, and the effects of NODAL, an embryonic morphogen, on the breast cancer secretome and stromal cell recruitment. First, I compared multiple sample preparation techniques and found high-pH/low-pH fractionation to yield the greatest proteome coverage over commonly used approaches. Second, I compared the proteomes from …

Quantitative Proteomic Characterization Of Cx-4945, A Clinical Stage Inhibitor Of Protein Kinase Ck2, Adam J. Rabalski

Electronic Thesis and Dissertation Repository

Protein phosphorylation is controlled by protein kinases, and represents a critical signaling mechanism involved in the regulation of fundamental biological processes. Furthermore, the aberrant regulation of kinase activity is implicated in diseases such as cancer and has resulted in efforts to target kinases therapeutically. Protein kinase CK2, although frequently considered constitutively active, has emerged as a clinical target on the basis of its altered expression in different types of human cancers and its regulatory participation in multiple biological processes. In fact, CX-4945, a small molecule ATP-competitive inhibitor of CK2 has advanced to clinical trial and has been widely used to …

At The Interface Of Glycolipids And Glycoproteins In The Two Gastro-Intestinal Pathogens Campylobacter Jejuni And Helicobacter Pylori, Najwa Zebian

Electronic Thesis and Dissertation Repository

Campylobacter jejuni and Helicobacter pylori are phylogenetically related human gastro-intestinal pathogens. C. jejuni colonizes the intestine and is the leading cause of bacterial gastroenteritis worldwide. H. pylori colonizes the stomach of half the world’s population, causing gastritis, gastric ulcers and gastric cancer. Both pathogens express glycoproteins and glycolipids (capsule, lipooligosaccharide or lipopolysaccharide (LPS)) that are important in pathogenesis. Sugar units from capsule or LPS can modify proteins in several bacteria. We reasoned that this may occur in C. jejuni and H. pylori as means to diversify their surface glycosylation and aid in colonization, immune evasion and virulence.

The putative sugar …

Targeted Proteomics Of Human Pluripotent Stem Cells, Kevin Gregory Kania

Electronic Thesis and Dissertation Repository

Human pluripotent stem cells (hPSCs) exhibit two unique characteristics: pluripotency and self-renewal. These properties are maintained by a series of complex signaling pathways, however, quantitative data for the respective proteins is lacking. Selected reaction monitoring (SRM) is a targeted, quantitative technique in mass spectrometry that is highly sensitive in peptide detection. In this thesis, an SRM protocol was developed in order to detect and quantify a defined set of proteins responsible for maintaining stem cell pluripotency. Two hESC differentiation protocols were validated for use as model systems within which to measure differential protein expression by SRM. SRM assays were generated …

Mass Spectrometry-Based Proteomics Analysis Of The Matrix Microenvironment In Pluripotent Stem Cell Culture, Christopher Hughes

Electronic Thesis and Dissertation Repository

The stem cell microenvironment contains soluble factors, support cells, and components of the extracellular matrix (ECM) that combine to effect cellular behavior. Mass spectrometry based proteomics offers the opportunity to directly assay components of extracellular microenvironments, thereby providing a sensitive means for obtaining insight into the stem cell niche. In this study we present the generation and analysis of human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) matrix microenvironments using an MS-based proteomics approach.

One of the primary limitations in the proteomics analysis of hESCs and hiPSCs is the reproducible generation of sufficient cell numbers amenable …

Structural Characterization Of Protein Folding Intermediates By Oxidative Labeling And Mass Spectrometry, Bradley B. Stocks

Electronic Thesis and Dissertation Repository

A key challenge associated with protein folding studies is the characterization of short-lived intermediates that become populated en route to the native state. In this work, a covalent labeling method was developed that provides insights into the structures of these transient species. Hydroxyl radical (·OH) reacts with solvent-exposed side chains, whereas buried residues are protected. Mass spectrometry is used for monitoring the locations and the extent of labeling. Pulsed ·OH labeling of proteins at selected time points during folding results in high temporal and spatial resolution when compared to existing other labeling methods.

This novel technique was validated by studying …

Structure And Dynamics Of The Membrane Protein Bacteriorhodopsin Studied By Mass Spectrometry, Yan Pan

Electronic Thesis and Dissertation Repository

Membrane proteins continue to represent a major challenge for most analytical techniques. Using bacteriorhodopsin (BR) as model system, this work aims to develop mass spectrometry (MS)-based approaches for exploring the structure, dynamics and folding of membrane proteins.

As the first step, BR in its native lipid environment was exposed to hydroxyl radicals, which were produced by laser photolysis of hydrogen peroxide. It was found that the resulting methionine (Met) labeling pattern was consistent with the known BR structure. This finding demonstrates that laser-induced oxidative Met labeling can provide structural information on membrane proteins. In subsequent experiments, the effects of different …