Biochemistry Commons^™

Comparative Analysis Of In Situ Fibronectin Using Tof-Sims, Spi-Ms, And Dropdesi-Ms In A Microfluidic Reactor, Shannon Fasing, Xiao-Ying Yu, Juan Yao, Jiachao Yu

STAR Program Research Presentations

Fibronectin is an important biomolecule due to its role in cell differentiation, growth, kinesis, and adhesion. Such biological responses are mediated through membrane recognition and signaling; where fibronectin is found. Studying the outer molecular surface of fibronectin allows deeper insight into the microbiological reactions that occur during these processes. In situ mass spectrometry analysis in aqueous solution accurately represents fibronectin’s chemical components, made possible by a vacuum compatible microfluidic reactor, SALVI (System for Analysis at the Liquid Vacuum Interface). SALVI was paired with the analytical tools: time-of-flight secondary ion mass spectrometer (ToF-SIMS), single photon ionization mass spectrometer (SPI-MS) and drop …

The Development Of Peptide Ligands To Target H69 Rrna, Danielle Nicole Dremann

Wayne State University Dissertations

ABSTRACT

THE DEVELOPMENT OF PEPTIDE LIGANDS TO TARGET H69

by

DANIELLE NICOLE DREMANN

December 2015

Advisor: Prof. Christine S. Chow

Major: Chemistry (Biochemistry)

Degree: Doctor of Philosophy

In the development of peptide ligands to target H69, SPPS and ESI MS was used to determine if 1) peptides could bind to modified H69 and 2) if increased affinity for the target RNA could be enhanced with modification. An alanine and arginine scan was synthesized and tested for this determination. Selected peptides were then tested using biophysical techniques such as circular dichroism and isothermal titration calorimetry. An assay was also designed to …

Image-Charge Detection €“ Novel Instrumentation And Applications, Brandon Lee Barney

Theses and Dissertations

Image-charge detection is an analytical technique in which a highly-charged particle is detected by the magnitude of the image current that it generates in a detecting electrode. This current is represented as a voltage between the charged particle and the sensing electrode. It is a single particle detection method, ideal for the analysis of large, variable mass particles such as biological cells. Some of the physical properties of Bacillus subtilis spores were explored using different applications of image-charge detection. B. subtilis is a gram-negative spore-forming bacteria that has been shown to exhibit extremophile behavior. The particular extremophile behavior that was …

Distinguishing Macrophage Activation States By Mass Spectrometry, Matthias Manfred Knust

Graduate Theses and Dissertations

Macrophages are versatile and highly adaptive cells that are involved in a wide range of physiological processes including host defense, homeostasis or regeneration, as well as pathogenesis. They react to their microenvironment, assuming various roles based on chemical and/or physical cues, and can reversibly shift between these so-called activation states. Concurrently, the technique of immunohistochemistry is used to gain spatial information on activated macrophages on tissue sections. The aim of this work was to find mass spectral biomarkers that allow the differentiation of activation states, and establish conditions that can be used in imaging mass spectrometry (IMS) experiments to investigate …

Investigation Into The Biological Importance And Function Of Proinsulin C-Peptide, Christina L. Newsome

Theses, Dissertations and Capstones

The C-peptide of insulin was thought to be biologically inactive, but recent studies have shown that the C-peptide causes multiple molecular and physiological effects. Evidence has shown that C-peptide binds to a cell surface receptor, probably a G-protein coupled receptor, and that the COOH-terminal pentapeptide is essential for binding and constitutes an active site. For a further understanding of the detailed nature of the physiological effects of C-peptide, the receptor structure needs to be determined. We designed an affinity column using C-peptide to try and gain a better understanding of the biological effects by examining what proteins the affinity column …

Bioinformatic Solutions To Complex Problems In Mass Spectrometry Based Analysis Of Biomolecules, Ryan M. Taylor

Theses and Dissertations

Biological research has benefitted greatly from the advent of omic methods. For many biomolecules, mass spectrometry (MS) methods are most widely employed due to the sensitivity which allows low quantities of sample and the speed which allows analysis of complex samples. Improvements in instrument and sample preparation techniques create opportunities for large scale experimentation. The complexity and volume of data produced by modern MS-omic instrumentation challenges biological interpretation, while the complexity of the instrumentation, sample noise, and complexity of data analysis present difficulties in maintaining and ensuring data quality, validity, and relevance. We present a corpus of tools which improves …

Investigation Into The Cellular Actions Of Carnosine And C-Peptide, Emma H. Gardner

Theses, Dissertations and Capstones

Carnosine is a dipeptide composed of beta-alanine and histidine found exclusively in long-lived animal tissues. The cellular action of carnosine is still under extensive investigation; however, it has been proposed to have a role as an anti-oxidant and oxygen free radical scavenger, a physiological buffer, a heavy metal chelator, and has been implicated as an anti-aging agent.^2,4 Our lab has been studying the interaction between carnosine and heme by analyzing both the effect carnosine has on the glycation of the heme containing protein cytochrome c and the interaction of carnosine with free hemin. We have observed that the addition …

Monitoring Ligand-Induced Nucleic Acid Conformational Changes Using Ion Mobility Spectrometry-Mass Spectrometry, Bill Kenneth Redick

Legacy Theses & Dissertations (2009 - 2024)

Three-dimensional structures of biopolymers frequently dictate the biological role those molecules play. As such, investigation into structure of nucleic acids can provide important information pertaining to how those nucleic acids work. Many nucleic acid species, especially single-stranded RNA, fold into unique structures that allow them to function properly. Metals, and other cationic species, are often bound to the nucleic acid to make folding into the proper structure more favorable by neutralizing the negative charge on the nucleic acid imparted by the phosphate group. This investigation explores tertiary structure of nucleic acids that have been folded in the presence of ligands …

Gas-Phase Covalent And Non-Covalent Ion/Ion Chemistry Of Biological Macromolecules, John Robert Stutzman

Open Access Dissertations

Gas-phase ion/ion chemistry involves the interaction of oppositely charged ions inside of the mass spectrometer. During this gas-phase chemistry, particle transfer (i.e., proton and electron) or synthesis can occur at rapid reaction rates. Particle transfer represents a mature area of ion/ion chemistry, while selective covalent modification represents a fairly new area of gas-phase chemistry. Gas-phase covalent chemistry is based on traditional solution phase organic chemistry.

The work demonstrated in this dissertation greatly involves gas-phase covalent and non-covalent Schiff base chemistry on peptide and protein ions. The reagent dianion, 4-formyl 1,3-benzene disulfonic acid, has been used to covalently modify unprotonated primary …

Bacterial Spores Remain Viable After Electrospray Charging And Desolvation, Sara Nielson Pratt

Theses and Dissertations

The electrospray survivability of B. subtilis spores and E. coli was tested in atmospheric mobility experiments. E. coli did not survive electrospray charging and desolvation, but B. subtilis did. Experimental conditions ensured that any surviving bacteria were charged, desolvated, and de-agglomerated. B. subtilis was also found to survive both positive and negative electrospray and subsequent introduction into vacuum conditions. Attempts were made to measure the charge distribution of viable B. subtilis spores using electrostatic deflection. From those experiments, it was found that either the spores do not become highly charged under the electrospray conditions used or only spores in a …

A Novel Algorithm For Validating Peptide Identification From A Shotgun Proteomics Search Engine, Ling Jian, Xinnan Niu, Zhonghang Xia, Parimal Samir, Chiranthani Sumanasekera, Zheng Mu, Jennifer L. Jennings, Kristen L. Hoek, Tara Allos, Leigh M. Howard, Kathryn M. Edwards, P. Anthony Weil, Andrew J. Link

Chemistry Faculty Research

Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC–MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to …

Novel Ion Trap Made Using Lithographically Patterned Plates, Ying Peng

Theses and Dissertations

A new approach of making ion trap mass analyzers was developed in which trapping fields are created in the space between two ceramic plates. Based on microfabrication technology, a series of independently-adjustable electrode rings is lithographically patterned on the facing surfaces of each ceramic plate. The trapping field can be modified or fine-tuned simply by changing the RF amplitude applied to each electrode ring. By adjusting the potential function applied to the plates, arbitrary trapping fields can be created using the same set of ceramic plates. Unlike conventional ion traps, the electrodes of planar ion traps have a non-equipotential surface, …

Method Development And Applications To Screening And Characterization Of Rrna-Targeting Small Molecules, Papa Nii Asare-Okai

Wayne State University Dissertations

A series of single ring aminoglycoside analogues was tested for binding to a model RNA representing the A site using electrospray ionization mass spectrometry (ESI-MS). Several of the synthetic analogues with low molecular weights were found to bind to the RNA with affinities comparable to the parental aminoglycoside neamine, with apparent dissociation constants in the low micromolar range. Salt dependence of the affinity constants for the single ring analogues revealed a predominantly electrostatic binding mode. Footprinting experiments revealed that one of the compounds (DHR23) has a similar binding site as the antibiotic paromomycin. DMS chemical probing results also suggest that …

Targeted Identification Of Metastasis-Associated Cell-Surface Sialoglycoproteins In Prostate Cancer, Lifang Yang, Julius O. Nyalwidhe, Sigi Guo, Richard R. Drake, O. John Semmes

Bioelectrics Publications

Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC₄ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins …

Halo Ion Trap Mass Spectrometry: Design, Instrumentation, And Performance, Miao Wang

Theses and Dissertations

New ion trap mass spectrometry (ITMS) instrumentation, the toroidal IT and halo IT, were developed to meet the significant growth in on-site analysis applications. The miniature toroidal IT mass analyzer was operated with radio frequency (RF) trapping voltages of 3 kVp-p or less. Despite its reduced dimensions, it has roughly the same ion trapping capacity as conventional 3D quadrupole ITs. Unit-mass resolved spectra for n-butylbenzene, xenon, and naphthalene were obtained. The desired linear mass scale was obtained using conventional mass-selective instability scan combined with resonance ejection. The halo IT was also based on toroidal trapping geometry and microfabrication technology, consisting …

Glycopeptidome Of A Heavily N-Glycosylated Cell Surface Glycoprotein Of Dictyostelium Implicated In Cell Adhesion, Christa L. Feasley, Jennifer M. Johnson, Christopher M. West, Catherine P. Chia

School of Biological Sciences: Faculty Publications

Genetic analysis has implicated the cell surface glycoprotein gp130 in cell interactions of the social amoeba Dictyostelium, and information about the utilization of the 18 N-glycosylation sequons present in gp130 is needed to identify critical molecular determinants of its activity. Various glycomics strategies, including mass spectrometry of native and derivatized glycans, monosaccharide analysis, exoglycosidase digestion, and antibody binding, were applied to characterize a nonanchored version secreted from Dictyostelium. s-gp130 is modified by a predominant Man8GlcNAc4 species containing bisecting and intersecting GlcNAc residues and additional high-mannose N-glycans substituted with sulfate, methyl-phosphate, and/or core R3-fucose. Site mapping confirmed the occupancy …

Characterization Of Glycation Sites On Human Serum Albumin Using Mass Spectrometry, Omar S. Barnaby

Department of Chemistry: Dissertations, Theses, and Student Research

The modification of proteins by reducing sugars is a process that occurs naturally in the body. This process, which is known as glycation, has been linked to many of the chronic complications encountered during diabetes. Glycation has also been linked to changes in the binding of human serum albumin (HSA) to several drugs and small solutes in the body. While these effects are known, there is little information that  explains why these changes in binding occur. The goal of this project was to obtain qualitative and quantitative information about glycation that occurs on HSA. The first section of this dissertation …

Gas Phase Chiral Recognition, Characterization Of Porous Polymer Monolith Nanospray Ionization, And The Negative Mode Crafti Method Using Fourier Transform Ion Cyclotron Resonance Mass Spectrometry, Nannan Fang

Theses and Dissertations

Our group has been studying chiral recognition in gas phase using mass spectrometry for more than 10 years. We are interested in gas phase studies of fundamental interactions because the gas phase avoids complications and masking effects that may arise upon solvation. Therefore, the results of gas phase experiments can be directly compared with those of high-level computational studies. In chapter 2, I studied the roles of hydrogen bonding and pi stacking in gas phase chiral recognition between aromatic crown molecules and aromatic amines. High affinity between host and guest doesn't necessarily result in better recognition. If the affinity is …

A Top-Down Proteomic Approach For The Discovery Of Novel Serum Biomarkers Of Pregnancy-Related Disease, Karen Merrell

Theses and Dissertations

The serum fraction of blood is an ideal material in which to search for novel biomarkers for disease. It is easily obtained through relatively non-invasive means, routinely collected, and a rich treasure-trove of information about the health of an individual. Cells react to signal molecules, take up nutrients, and release waste products, fragments that are the result of proteolysis, and other molecules out into the bloodstream. If these components are unique to the cells in question, that part of the complex mixture that is the blood stream can potentially characterize the health of the tissue or organ those cells are …

Radiocarbon Evidence For The Importance Of Surface Vegetation On Fermentation And Methanogenesis In Contrasting Types Of Boreal Peatlands, J. P. Chanton, P. H. Glaser, L. S. Chasar, David J. Burdige, M. E. Hines, D. I. Seigel, L. B. Tremblay, W. T. Cooper

OES Faculty Publications

We found a consistent distribution pattern for radiocarbon in dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and methane replicated across spatial and temporal scales in northern peatlands from Minnesota to Alaska. The ¹⁴C content of DOC is relatively modern throughout the peat column, to depths of 3 m. In sedge-dominated peatlands, the ¹⁴C contents of the products of respiration, CH₄ and DIC, are essentially the same and are similar to that of DOC. In Sphagnum- and woody plant-dominated peatlands with few sedges, however, the respiration products are similar but intermediate between the ¹⁴C contents of …

A Conserved Cam- And Radial Spoke–Associated Complex Mediates Regulation Of Flagellar Dynein Activity, Erin E. Dymek, Elizabeth F. Smith

Dartmouth Scholarship

For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)- binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which …

Development Of Methods For The Study Of Phosphoproteins, Zhaoyuan Chen

Theses and Dissertations

Characterization of phosphoproteins-including detection, identification of phosphoproteins and identification of phosphorylation sites-is mostly done with radiolabeling and proteomic techniques. Three main topics related to phosphoprotein characterization are included in this dissertation. First, large-scale characterization of the CHO (Chinese hamster ovary) cell phosphoproteome was done using two dimensional gel electrophoresis (2DE) separation, visualization of phosphoproteins by radiolabeling or a phosphoprotein specific dye, followed by MALDI-TOF identification. Because radiolabeling of phosphoproteins is very sensitive and straightforward to quantify, such analysis can give a clear picture of the relative phosphosphorylation of proteins present in a sample. But there are also limitations to this …

Biochemical Investigations Of Macular Degeneration: The Significance Of Protein Oxidation Including Novel Methods For Its Study, Sarah Warburton

Theses and Dissertations

The retinal pigment epithelium (RPE) is a monolayer of cells located directly behind the photoreceptor cells in the retina. These cells are involved in a variety of functions that support the visual process in the eye, namely 1) they form a blood-retina barrier which separates the neural retina from the choroid's blood supply, 2) the apical processes of RPE cells diurnally phagocytose the outer segments of photoreceptor cells, and 3) they participate in the renewal of the photopigment 11-cis retinal. Age-related macular degneration (AMD) is the leading cause of blindness in people over the age of 50 years in North …

Enhanced Electrospray Ionization For Mass Spectrometry And Ion Mobility Spectrometry, Li Zhou

Theses and Dissertations

Electrospray ionization (ESI) has become one of the most commonly used ionization techniques for mass spectrometry (MS) and ion mobility spectrometry (IMS), and efforts continue to improve its performance. ESI-MS is most recognized for its wide application to biomacromolecules where high sensitivity is of paramount importance. However, the major limitation in sensitivity with ESI-MS is due to its low ion transmission efficiency from the ESI source into the sampling orifice and through any stages utilized for transfer of ions from atmosphere to vacuum in the MS. A series of atmospheric pressure ion focusing interfaces were designed and implemented to enhance …

Computational Protein Biomarker Prediction: A Case Study For Prostate Cancer, Michael Wagner, Dayanand N. Naik, Alex Pothen, Srinivas Kasukurti, Raghu Ram Devineni, Bao-Ling Adam, O. John Semmes, George L. Wright Jr.

Mathematics & Statistics Faculty Publications

Background: Recent technological advances in mass spectrometry pose challenges in computational mathematics and statistics to process the mass spectral data into predictive models with clinical and biological significance. We discuss several classification-based approaches to finding protein biomarker candidates using protein profiles obtained via mass spectrometry, and we assess their statistical significance. Our overall goal is to implicate peaks that have a high likelihood of being biologically linked to a given disease state, and thus to narrow the search for biomarker candidates.

Results: Thorough cross-validation studies and randomization tests are performed on a prostate cancer dataset with over 300 patients, obtained …