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Mass spectrometry-based methods for protein-ligand identification have expanded classical techniques for the bioanalytical characterization of small molecule target engagement and their modes of action. In the last decade, a series of techniques have coupled mass spectrometry readout, structure-function framework, and thermodynamic stability to expand the suite of proteomics techniques for protein-ligand interactions. Although these methods have proven powerful, due to the complex nature of these large-scale studies, having multiple avenues of assessment is critical for the proper evaluation of clinical value. In this work, the interfacing of these protein-denaturation experimental designs with cross-linking mass spectrometry sample workflows is investigated to better understand the protein topologies in these protein-ligand large-scale analyses. The developed method, protein-denaturation and quantitative cross-linking mass spectrometry, offers another strategy in the unbiased assessment of protein target engagement studies. Additionally, from a basic science perspective, this method also provides data in understanding the molecular principles of protein folding in structure-(dys)function studies. First, I validated a proof-of-concept of protein-denaturation with quantitative cross-linking mass spectrometry in a standard protein and known ligand. Then, I adapted and assessed the viability of this method on the proteome-level scale. Although this method has much room for optimization for tackling large-scale studies, its data provides promise with smaller complex proteomes. Overall, quantitative cross-linking mass spectrometry during protein unfolding is a reliable assay that can be used alone or provide complementary information to the current generation of protein-denaturation mass-spectrometry methods for generating target-engagement atlases.
Presents a wide variety of mass spectrometry methods used to explore structural mechanisms, protein dynamics and interactions between proteins. Preliminary chapters cover mass spectrometry methods for examining proteins and are then followed by chapters devoted to presenting very practical, how-to methods in a detailed way. Includes footprinting and plistex specifically, setting this book apart from the competition.
Hydrogen exchange mass spectrometry is widely recognized for its ability to probe the structure and dynamics of proteins. The application of this technique is becoming widespread due to its versatility for providing structural information about challenging biological macromolecules such as antibodies, flexible proteins and glycoproteins. Although the technique has been around for 25 years, this is the first definitive book devoted entirely to the topic. Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Methods and Applications brings into one comprehensive volume the theory, instrumentation and applications of Hydrogen Exchange Mass Spectrometry (HX-MS) - a technique relevant to bioanalytical chemistry, protein science and pharmaceuticals. The book provides a solid foundation in the basics of the technique and data interpretation to inform readers of current research in the method, and provides illustrative examples of its use in bio- and pharmaceutical chemistry and biophysics In-depth chapters on the fundamental theory of hydrogen exchange, and tutorial chapters on measurement and data analysis provide the essential background for those ready to adopt HX-MS. Expert users may advance their current understanding through chapters on methods including membrane protein analysis, alternative proteases, millisecond hydrogen exchange, top-down mass spectrometry, histidine exchange and method validation. All readers can explore the diversity of HX-MS applications in areas such as ligand binding, membrane proteins, drug discovery, therapeutic protein formulation, biocomparability, and intrinsically disordered proteins.
This book highlights current approaches and future trends in the use of mass spectrometry to characterize protein therapies. As one of the most frequently utilized analytical techniques in pharmaceutical research and development, mass spectrometry has been widely used in the characterization of protein therapeutics due to its analytical sensitivity, selectivity, and specificity. This book begins with an overview of mass spectrometry techniques as related to the analysis of protein therapeutics, structural identification strategies, quantitative approaches, followed by studies involving characterization of process related protein drug impurities/degradants, metabolites, higher order structures of protein therapeutics. Both general practitioners in pharmaceutical research and specialists in analytical sciences will benefit from this book that details step-by-step approaches and new strategies to solve challenging problems related to protein therapeutics research and development.
The authoritative guide to analyzing protein interactions by mass spectrometry Mass spectrometry (MS) is playing an increasingly important role in the study of protein interactions. Mass Spectrometry of Protein Interactionspresents timely and definitive discussions of the diverse range of approaches for studying protein interactions by mass spectrometry with an extensive set of references to the primary literature. Each chapter is written by authors or teams of authors who are international authorities in their fields. This leading reference text: * Discusses the direct detection of protein interactions through electrospray ionization (ESI-MS); ion mobility analysis; and matrix-assisted laser desorption/ionization (MALDI-MS) * Covers the indirect analysis of protein interactions through hydrogen-deuterium exchange (HX-MS); limited proteolysis; cross-linking; and radial probe (RP-MS) * Guides researchers in the use of mass spectrometry in structural biology, biochemistry, and protein science to map and define the huge number and diversity of protein interactions * Reviews the latest discoveries and applications and addresses new and ongoing challenges This is a comprehensive reference for researchers in academia and industry engaged in studies of protein interactions and an excellent text for graduate and postgraduate students.
Chemical cross-linking mass spectrometry elucidates protein structures and protein-protein interactions by establishing distance constraints between residues pairs. Interpreting the mass spectra of covalently linked peptide pairs is more challenging than identifying peptides in proteomics. This dissertation presents improvements in methodology for identifying cross-linked peptides and their application to probing protein interacting surfaces.
Cross-linking mass spectrometry maps the structural topology of protein complexes by using the distance between linked residues as spatial constraints, complementing other structural biology techniques. However, the identification of cross-linked peptides scales poorly with the number of proteins analyzed. Our lab has previously developed MS-cleavable cross-linkers to enable the separation of cross-linked peptides prior to sequencing, enabling peptide identifica- tion using standard peptide search databases. We describe the design and implementation of platform and application named XLTools for the automated identification of MS-cleavable cross-linked peptides. XLTools supports open and proprietary data formats and common peptide search databases, facilitating its integration into existing workflows. Furthermore, we developed peak-picking and validation algorithms to enable the accurate quantitation of cross-linked peptides in complex samples. We demonstrate the application of XLTools to the quantitative analysis of the 26S proteasome cross-linked in vivo and in vitro.
Proteins are the most active molecules in living bodies. They catalyze chemical reactions, provide structural support for cells and allow organisms to move. Their function is intrinsically linked to their folded structure. Resolving the structures of proteins and protein complexes is crucial for our understanding of basic biological processes and diseases. Cross-Linking Mass Spectrometry (XL-MS) is a method to gain structural insights into protein complexes. The field of XL-MS data analysis software is not yet as established as many other methods in proteomics. XL-MS analysis software has significant room for improvement in terms of sensitivity, efficiency and standardization of file formats and workflows to facilitate interoperability and reproducibility. In this thesis we present a new XL-MS search engine, OpenPepXL. We develop an algorithm that scores all candidate cross-linked peptide pairs and is efficient enough to be used on a standard desktop PC for most applications. OpenPepXL supports the standardized XL-MS identification file format defined as a part of the MzIdentML 1.2 specifications that were developed in collaboration with the Proteomics Standards Initiative. We benchmark OpenPepXL against other state-of-the-art XL-MS identification tools on multiple datasets that allow cross-link validation through structures or other means. We show that our exhaustive approach, although not the quickest one, is superior in sensitivity to other tools. We suggest this is due to some tools improving their processing time by discarding too many candidates in early steps of the data analysis. We apply XL-MS analysis with OpenPepXL to multiple protein complexes related to meiosis and the type III secretion system. The first project involved several proteins with unknown structures, some of which are expected to be at least partially intrinsically disordered and therefore difficult to investigate using most traditional structural research methods. Unfortunately, we could not find cross-links between the interaction sites we were interested in the most, but we were able to identify many others in these complexes and gained some structural insights. In the second project we used the photo-cross-linking amino acid pBpa to test very specific hypotheses about interactions within the type III secretion system. We were not able to gain any new structural information yet. However, we could confirm that this is a viable approach. It is possible to identify cross-links between a pBpa residue incorporated into a protein sequence and a residue it cross-links to on a residue level resolution.
This book is designed to be a central text for young graduate students interested in mass spectrometry as it relates to the study of protein structure and function as well as proteomics. It is a definite must-have work for:- libraries at academic institutions with Master and Graduate programs in biochemistry, molecular biology, structural biology and proteomics- individual laboratories with interests covering these areas - libraries and individual laboratories in the pharmaceutical and biotechnology industries. *Serves as an essential reference to those working in the field*Incorporates the contributions of prominent experts *Features comprehensive coverage and a logical structure
Protein assemblies represent the workhorses of the cell, forming the basis of all cellular processes. Their biological roles are intimately associated with their topologies, making the structural elucidation of proteins and protein complexes a critical requirement to understanding their function. While traditional structural biology approaches have greatly contributed to our current understanding of protein structure, they are ill-suited for analyzing the conformational dynamics associated with heterogenous protein complexes and their protein-protein interactions (PPIs). As a result, there is a growing demand for the development of new structural approaches to elucidate the impact of protein dynamics on the regulation of integral biological processes required for cell homeostasis. In particular, cross-linking mass spectrometry (XL-MS) has arisen in recent years as a popular hybrid structural technique for the topological determination of conformationally and compositionally heterogenous protein complexes. However, most studies utilizing cross-linking thus far have been limited to the determination of static protein structures.Here, I focus on the development and application of quantitative cross-linking mass spectrometry (QXL-MS) strategies to determine how conformational dynamics of cullin-RING ligases (CRLs) dictate and regulate their ubiquitinating activity. Proteasomal dysregulation has been associated with a wide range of human pathologies, from diabetes and various forms of cancer to autoimmune and neurodegenerative disorders. As a result, the PPIs associated with CRL assemblies represent potential targets for therapeutic intervention. A comprehensive understanding of E3 ligase structure and conformational dynamics is critical for the development of pharmacological drugs that selectively inhibit or upregulate their function. However, such heterogenous assemblies are notoriously difficult to study using traditional structural biology techniques.While this thesis focuses on the application of quantitative XL-MS strategies to study cullin-RING ligase machinery, these platforms represent versatile methodologies that can be universally employed for structural studies on a wide range of protein systems.