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Characterization of Protein-Protein Interactions for Therapeutic Drug Design Utilizing Mass Spectrometry
No full textThe number of transferrin based therapeutics progressing to clinical trials remains disappointingly small despite promising capabilities of transporting therapeutic payloads to cancer cells and across the blood brain barrier. This meager success record is largely due to the complexity and heterogeneity of all protein conjugation products that generates difficulties for their analytical characterization. Discussed in this work, transferrin is conjugated to lysozyme as a model therapeutic to deliver this bacteriostatic protein to target central nervous system infections. In this work ESI- and MALDI-MS were used to characterize the modification sites at lysine residues in hopes of characterizing heterogeneity within the conjugate. Identification and quantization of modification sites using MS on tryptic digested samples proved difficult with poor signal to noise ratios and missing peptide fragments. The use of an 18O labeling method that exchanges both C-terminal oxygen atoms with 18O provided more reliable results, but still proved difficult to observe all needed peptide fragments. MALDI-MS allowed for verification of ESI-MS results, but was found unhelpful with full characterization due to abundant overlapping of isotopic labeled peaks. Hoping to create an ideal 1:1 binding ratio between the two proteins, a site-specific modification method using kinetically controlled conditions was used and was confirmed that the method, although capable of producing 1:1 conjugated species, actually created different isomers with separate binding frequencies at each lysine. Online-IEC helped with the identification of isomers and started the initial work of correlating modification sites with bioactivity of the proteins. It was determined that lysozyme has a high chance of being modified at lysine 33 and 116, with a possibility of also being highly modified at lysine 97. More work is needed to complete the characterization, especially with transferrin, but the experimental approaches developed in this work prove to be promising. This work aims at delivering an optimized framework for analytical characterization of protein and antibody conjugates to guide the development of future biopharmaceuticals.Master of Science (M.S.
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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NOVEL MASS SPECTROMETRY BASED STRATEGIES TO STUDY HEPARIN-PROTEIN INTERACTIONS
No full textHeparin and heparan sulfate (HS) are linear polyanions from the glycosaminoglycan (GAG) family. They are conceived to play critical roles in a variety of biophysiological processes by interacting with key proteins and regulating protein functions. Understanding their biological functions and exploit heparin’s unique versatility for therapeutic purposes critically depend on the characterization of their interactions with relevant proteins, which, however, is largely impeded by the structural heterogeneity of these polyanions. This work presented the development of novel mass spectrometry-based analytical strategies incorporated with liquid chromatography and ion mobility spectroscopy to investigate heparin-protein interactions and address their significance from different aspects.ChemistryDoctor of Philosophy (Ph.D.
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Kevin M.DownardMass Spectrometry of Protein Interactions2007John Wiley & Sons, Inc111 River Street, Hoboken, NJ 07030-5774, USAISBN: 978-0-471-79373-1, Copyright ©, Hardcover, $59.95 US, 137 pp
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Characterization of Highly Heterogeneous Heparin-Protein Complexes Using Novel Mass Spectrometry-Based Approaches
No full textHeparin-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 mass spectrometry, ion-mobility, gas-phase chemistry and chromatography, with success in characterizing protein-GAG interacting stoichiometry and deciphering the structural code related to protein-GAG affinity.ChemistryDoctor of Philosophy (Ph.D.
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CHARACTERIZATION OF BIODISTRIBUTION OF TRANSFERRIN AND RECEPTOR BINDING MECHANISM BY MASS SPECTROMETRY
No full textProtein-based therapeutics have emerged as a key driver of rapid growth in drug development pipelines. However, developing such protein drugs is not straightforward in most cases, the existence of physiological barriers greatly restricts the efficient delivery of many therapeutic molecules, and therefore limits their clinical applications. A promising way to address this challenge takes advantage of certain transport protein which can effectively across and enhance the permeability of these barriers, such as transferrin (Tf) which can be internalized by malignant cells and cross physiological barriers via transferrin receptor (TfR)-mediated endocytosis and transcytosis. However, developing such products is impossible without successfully understanding the molecular mechanisms governing Tf/TfR interactions and the ability to monitor the biodistribution of Tf. In this work, hydrogen/deuterium exchange mass spectrometry (HDX MS) is used to investigate TfR higher order structural and dynamic changes in different Tf/TfR models that mimic various stages encountered during endocytosis. Detailed characterizations of TfR gained by HDX MS reveal the regions located at the interdomain cleft exhibiting bimodal exchange patterns may be responsible for the loss of its enzyme function in the molecular evolution. At neutral pH, a movement at the TfR/TfR interface helps to stabilize the holoTf/TfR complexation. At acidic pH, the pH-induced conformational changes at the TfR helical domain trigger a series of movements that lead to specific binding properties for holo- and apoTf C-lobe. Obtaining this information greatly enhances our understanding of the pH-dependent Tf binding properties and how TfR facilitates iron release at acidic pH. Another aspect of this dissertation work is utilizing the ability of Tf to bind to noncognate metals to trace the biodistribution of Tf. Particularly, indium has been evaluated and demonstrated as an ideal tracer of exogenous Tf in complex biological matrices using inductively coupled plasma mass spectrometry (ICP MS) as a detection tool. In addition, combining laser ablation (LA) with ICP MS detection allows distribution of exogenous Tf to be mapped within animal tissue cross-sections. The high sensitivity and selectivity of this novel approach make it an ideal quantitation/imaging tool for in vivo studies of biodistribution of Tf and Tf-based therapeutics.ChemistryDoctor of Philosophy (Ph.D.
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