This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected. / <p>QC 20130821</p>

In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.

In the human genome, 43 BTB (Bric-à-brac, Tramtrack, and Broad Complex) containing BTB-Zinc Finger proteins have been identified, many of which are transcription factors involved in cancer and development. These BTB domains have been shown to form homodimers and heterodimers which raise DNA binding affinity and specificity for transcription factors. This project was to develop an efficient assay to systematically identify interactions between BTB domains. It combined a co-expression system, fluorescent protein tagging and Ni-NTA plate retention. It was concluded that fourteen analyzed BTB domains formed homodimers, but only certain BTB pairs formed heterodimers, such as BCL6 with Miz1 and Miz1 with RP58. To further understand the specificity of BTB domain interactions, more structural and sequence information is still needed. In conclusion, this assay provided a comprehensive detection method for BTB domain interaction mapping. The information generated provides candidates for further functional and structural studies.

<div> <div> <div> <p>The cosmos of noncoding RNAs (ncRNAs) has been thriving in recent years; so much so that researchers are discovering them much faster than they can uncover their functions. The subset of these RNAs that have been characterized have been noted to perform and regulate a plethora of remarkably diverse and essential biological functions. This diversity in function is accompanied by a large array of dynamic and elegantly folded 3-dimensional structures. In this collection of work, we will journey through the discovery of the first catalytic noncoding RNAs (ribozymes), explore a new method for identifying uniquely structured ribozymes, and detail the design of a technique to select for highly structured RNAs with a high affinity for an RNA binding partner. Although these topics vary widely within the field of RNA, this work strives to showcase the integral relationship between intricate macromolecular structures with their chemical and cellular functions. </p> </div> </div> </div>

Biomarkers are biological measures used for clinical assessment, whether an individual has a particular medical condition or to monitor and predict health states in individuals. Sensitive detection and quantification of various biomarkers are essential for disease diagnostics. The majority of biomarker-based diagnostics examines the presence and quantity of a single biomarker. Since the symptoms of many diseases are alike, multiplexed biomarker tests are highly desirable. Furthermore, detection of multiple biomarkers would improve the accuracy of diagnosis as well as providing additional information about the prognosis. Microarray platforms have the potential for higher level of multiplexing for biomarker detection. However, conventional microarray technologies are limited by the sensitivity of assays. This dissertation describes how single-particle interferometric reflectance imaging sensor (SP-IRIS) overcomes the sensitivity issues in biomarker detection and its applications to biomolecular and cellular biomarker detection assays. SP-IRIS provides optical detection of individual nanoparticles when they are captured onto a simple reflecting substrate, providing single-molecule sensitivity. This technique can be used to detect natural nanoparticles (such as viruses) without labels as well as molecular analytes (proteins and nucleic acids) that are labeled with metallic nanoparticles. Moreover, the advancements in technology make SP-IRIS ideal for the detection of low abundance biomarkers. Utilization of light polarization in combination with plasmonic gold nanorods as labels enhances the signal-to-noise ratio in nanoparticle detection allowing for the use of low numerical aperture optics increasing the field-of-view, hence, the throughput and sensitivity. Additionally, the integration of a disposable microfluidic flow cell and dynamic particle tracking in kinetic measurements provide a robust, ultra-sensitive and automated diagnostic platform. This dissertation focuses on the development of biological assays demonstrating effective use of SP-IRIS as a clinical diagnostic platform. We discuss the development of protein, nucleic acid and biological nanoparticle detecting SP-IRIS microarrays. We demonstrate four digital detection platforms for Hepatitis B, microRNA, rare mutations in an oncogene, KRAS, and virus-like particle detection with ultra-high sensitivity. / 2022-09-28T00:00:00Z

<p>  </p> <p>Sulfonation is a widespread biological reaction catalyzed by a supergene family of enzymes called sulfotransferases (SULTs). SULTs utilize 3’-phosphoadenosine-5’-phospho-sulfate (PAPS) as the universal sulfonate donor to conjugate with a diverse range of endo- and xenobiotic substrates, including neurotransmitters, hormones, and drugs resulting in altering their biological activity. This reaction serves as a major detoxification pathway as conjugation with a sulfonate group renders substrates more hydrophilic and facilitates excretion. Therefore, this process is responsible for reducing the bioavailability of some drugs. In some cases, sulfo-conjugation causes the bio-activation of pro-mutagens and pro-carcinogens, leading to SULTs being risk factors in some cancers. Despite the biological relevance, understanding of this family of enzymes is still scarce. One SULT member that is the focus of the studies described herein is human sulfotransferase 2B1b (SULT2B1b), which had been identified as a potential drug target in prostate cancer. However, the inconsistency in reported kinetic data obtained using radiolabeled assays and the lack of robust assays have become significant limitations for SULT2B1b-targeted drug discovery studies. A label-free assay was developed to bridge this knowledge gap that directly quantifies SULT2B1b sulfonated products. This novel assay utilized high-throughput technology based on Desorption Electrospray Ionization Mass Spectrometry (DESI-MS). Results obtained from the DESI-MS-based assay were compared with those from a fluorometric, coupled-enzyme assay already developed in the Mesecar lab. Both methods provided consistent kinetic data for the reaction of SULT2B1b. Therefore, this novel assay is promising for the application of drug discovery efforts aiming at identifying SULT2B1b inhibitors. The other SULT member studied and described herein is human sulfotransferase 1A1 (SULT1A1), one of humans' most vital detoxifying and drug-metabolizing SULT isoforms that can also be a potential drug target in some cancers. The detailed kinetic mechanism of SULT1A1 was elucidated using steady-state kinetic, product inhibition, dead-end inhibition, and X-crystallographic studies. to gain insights into the role of this enzyme in detoxification, drug metabolism, and the development of inhibitors.</p>

<p dir="ltr">Protein phosphorylation is a ubiquitous post-translational modification controlled by the opposing activities of protein kinases and phosphatases, which regulate diverse biological processes in all kingdoms of life. One of the key challenges to a complete understanding of phosphoregulatory networks is the unambiguous identification of kinase and phosphatase substrates. Liquid chromatography-coupled mass spectrometry (LC-MS/MS) and associated phosphoproteomic tools enable global surveys of phosphoproteome changes in response to signaling events or perturbation of phosphoregulatory network components. Despite the power of LC-MS/MS, it is still challenging to directly link kinases and phosphatases to specific substrate phosphorylation sites in many experiments. Here we described two methods for the LC-MS/MS-based characterization of protein phosphatases and kinases. The first is an <i>in-vitro</i> method designed to probe the inherent substrate specificity of kinase or phosphatases. This method utilizes an enzyme reaction with synthetic peptides, serving served as substrate proxies, coupled with LC-MS/MS for rapid, accurate high-throughput quantification of the specificity constant (<i>k</i>_<em>cat</em><i>/K</i>_<em>M</em>) for each substrate in the reaction and amino acid preference in the enzyme active site, providing insight into their cellular roles. The second couple’s auxin-inducible degradation system (AID) with phosphoproteomics for protein functional characterization. AID is a surrogate for specific chemical inhibition, which minimizes non-specific effects associated with long-term target perturbation. Using this system, we demonstrate-PP2A in complex with its B-subunit Rox Three Suppressor 1 (PP2A^Rts1) contributes to the phosphoregulation of a conserved fungal-specific membrane protein complex called the eisosome. By maintaining eisosomes in their hypophosphorylated state, PP2A^Rts1 aids fungal cells in preserving metabolic homeostasis. This work demonstrates the power of mass spectrometry as a critical tool for protein functional characterization.</p>

Cell surface RNA (csRNA) is a recent discovery in the field of RNA biology and has been implicated in playing important roles in many biological processes due to its extracellular properties. To understand the biogenesis, regulation, and function of csRNA, it is critical to develop methods to detect, isolate, and confidently characterize membrane-bound csRNA. Previously, csRNA has been profiled using methods based on cell membrane isolation that are expensive, laborious, and with unsatisfactory specificity and sensitivity . In this study, we use metabolic labeling and chemical cross-linking techniques to specifically label csRNA with biotin handles. We intended to use this technique for separating biotin-labeled csRNA from total RNA samples for characterization purposes. The primary materials that were used to label such csRNAs are 4-Thiouridine (4sU), an unnatural nucleotide analogue, and S-(2-aminoethyl)-ester-methanesulfonothioic-acid-biotin (MTSEA-biotin), a crosslinker designed specifically to label 4sU. By deploying these tools to cell lines such as HEK293T and HeLa, csRNA is detectable by Enhanced Chemiluminescent detection via Dot Blot. Furthermore, to separate biotin-labeled csRNA from total RNA, streptavidin-coated magnetic bead separation procedures could be used as a promising method for purifying csRNA from total RNA, for RNAseq characterization. This study highlights the processes of establishing the csRNA detection protocol and describes the current status and issues with developing the streptavidin-coated magnetic beads separation method. / Master of Science in Life Sciences / The 'central dogma' is a term that describes the process of DNA (a template-like molecule that holds all genetic coding within cells) transcribing into mRNA (a messenger molecule that transports this message to the ribosome) which is then translated into proteins (large, complex molecular machinery that is responsible for many biochemical functions within the body). However, RNA has been found to have a much wider range of functions than just being an intermediate messenger between DNA and proteins. Recently, short snippets of single nucleotide RNA strands have been discovered to be present on the outer cell membrane of certain mammalian cell types. The function of cell surface RNA (csRNA) is largely undiscovered, however, csRNA are likely involved in cell-cell interactions similar to outer membrane proteins, lipids, and carbohydrates. Currently, methods involved in detecting and characterizing csRNA are laborious, time extensive, and with unsatisfactory specificity and sensitivity. This study aims to develop novel methods to detect csRNA on different cell types in an undemanding and trustworthy manner to speed up research timelines while maintaining high confidence in results. Our design is to use metabolic labeling and click-chemistry to 'label' the csRNA. In this study, we describe early signs of detecting csRNA and how this was achieved. Additionally, the current status for separating and profiling csRNA sequences is discussed.

Mycobacterium species, which contain the causative agent for human tuberculosis (TB), produce inositol derivatives including mycothiol (MSH).  MSH is a unique and dominant cytosolic thiol that protects mycobacterial pathogens against the damaging effects of reactive oxygen species and is involved in antibiotic detoxification.  Therefore, MSH is considered a potential drug target.  The deacetylase MshB catalyzes the committed step in MSH biosynthesis by converting N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcNAc-Ins) to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins).  In this dissertation, we present detailed functional analysis of MshB.  Our work has shown that MshB is activated by divalent metal ions that can switch between Zn2+ and Fe2+ depending on environmental conditions, including  metal ion availability and oxidative conditions.  MshB employs a general acid-base catalyst mechanism wherein the Asp15 functions as a general base to activate the metal-bound water nucleophile for attack of the carbonyl carbon on substrate.  Proton-transfer from a general acid catalyst facilitates breakdown of the tetrahedral intermediate and release of products.  A dynamic tyrosine was identified that regulates access to the active site and participates in catalysis by stabilizing the oxyanion intermediate.  Molecular docking simulations suggest that the GlcNAc moiety on GlcNAc-Ins is stabilized by hydrogen bonding interactions with active site residues, while a hydrophobic stacking interaction between the inositol ring and Met98 also appears to contribute to substrate affinity for MshB.  Additional binding interactions with side chains in a hydrophobic cavity adjacent to the active site were suggested when the docking experiments were carried out with large amidase substrates.  Together the results from this study provide groundwork for the rational design of specific inhibitors against MshB, which may circumvent current challenges with TB treatment. / Ph. D.

The diagnosis and monitoring of AML relies predominantly on the identification of blast cells in the bone marrow and peripheral blood. While at the time of diagnosis the identification of leukemic cells is relatively easy, during remission the identification of small numbers of blasts is problematic. This is most evident by the fact that patients who achieve complete remission frequently relapse, despite pathologic examination indicating a marked reduction in leukemic cell burden. In this thesis I have explored the potential of using serum proteins secreted by leukemic cells as a means of monitoring disease in patients. To identify proteins that might be useful for monitoring, I took advantage of published gene expression arrays and looked into online bioinformatics databases. Using specific characteristics, I was able to identify approximately 107 candidate proteins secreted by AML cells. RT-PCR analysis and ELISA assays were performed to evaluate the variability of expressions and serum level differences of twelve different proteins in the list.