ABSTRACT: Proteins are vital macromolecules of every living cell. They undertake a variety of tasks that are essential for life. Most proteins function in assemblies rather than individually. Protein function is strongly correlated to structure and elucidation of both aspects of the protein is indispensable to understand its mechanism of action. Among several structural characterization techniques, solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has emerged in the last 25 years as a powerful tool to study protein-protein interaction and dynamics. In the first half of this dissertation, we describe several aspects of method development that allowed tackling of previously inconceivable biological problems. In the second half, we demonstrate how this tool is applied to study muscle biochemistry; particularly, the troponin complex and myosin. In Chapter 1, we introduce the method, its mechanism, each of its sequential steps improvements which overcame multiple limitations and permitted its application to wide variety of biological problems. In Chapter 2, we describe the optimization of the separation step to minimize back-exchange, a major drawback of the technique. Chapter 3. demonstrates how isotopic depletion of proteins could be advantageous for peptide fragment identification, reduction of the mass spectral complexity, and the increase of the signal-to-noise ratio. In a different aspect of method development, we present a global analysis algorithm in Chapter 4, to manage the enormous amount of data, increase throughput, and most importantly, take advantage of peptide fragment overlap to increase sequence resolution. On the other hand, the second half of the dissertation is dedicated to muscle biochemistry. Troponin is a heterotrimeric complex involved in muscle regulation. The structure of a truncated version of the cardiac isoform of the complex was only solved in the calcium-saturated state. In Chapter 5, we apply hydrogen/deuterium exchange on full length troponin in the calcium-saturated and the calcium-free states to understand how the troponin subunits fit together and what are the conformational changes induced by calcium. Moreover, Chapter 6 describes the allosteric changes induced by phosphorylation the inhibitory subunit N-terminal extension, specific to the cardiac isoform of the complex. Finally, in Chapter 7, we elucidate the conformational changes that accompany the transition of myosin V (a molecular motor) from the post-rigor (ATP-analog bound) to the rigor-like (nucleotide-free) states, thus characterizing new features of the powerstroke.

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.

Biophysical Characterization of Proteins in Developing Biopharmaceuticals, Second Edition, presents the latest on the analysis and characterization of the higher-order structure (HOS) or conformation of protein based drugs. Starting from the very basics of protein structure, this book explains the best way to achieve this goal using key methods commonly employed in the biopharmaceutical industry. This book will help today's industrial scientists plan a career in this industry and successfully implement these biophysical methodologies. This updated edition has been fully revised, with new chapters focusing on the use of chromatography and electrophoresis and the biophysical characterization of very large biopharmaceuticals. In addition, best practices of applying statistical analysis to biophysical characterization data is included, along with practical issues associated with the concept of a biopharmaceutical's developability and the technical decision-making process needed when dealing with biophysical characterization data. - Presents basic protein characterization methods and tools applicable to (bio)pharmaceutical research and development - Highlights the capabilities and limitations of each technique - Discusses the underlining science of each tool - Empowers industrial biophysical chemists by providing a roadmap for applying biophysical tools - Outlines the needs for new characterization and analytical tools in the biopharmaceutical industry

Issues in Analysis, Measurement, Monitoring, Imaging, and Remote Sensing Technology: 2011 Edition is a ScholarlyEditions™ eBook that delivers timely, authoritative, and comprehensive information about Analysis, Measurement, Monitoring, Imaging, and Remote Sensing Technology. The editors have built Issues in Analysis, Measurement, Monitoring, Imaging, and Remote Sensing Technology: 2011 Edition on the vast information databases of ScholarlyNews.™ You can expect the information about Analysis, Measurement, Monitoring, Imaging, and Remote Sensing Technology in this eBook to be deeper than what you can access anywhere else, as well as consistently reliable, authoritative, informed, and relevant. The content of Issues in Analysis, Measurement, Monitoring, Imaging, and Remote Sensing Technology: 2011 Edition has been produced by the world’s leading scientists, engineers, analysts, research institutions, and companies. All of the content is from peer-reviewed sources, and all of it is written, assembled, and edited by the editors at ScholarlyEditions™ and available exclusively from us. You now have a source you can cite with authority, confidence, and credibility. More information is available at http://www.ScholarlyEditions.com/.

Facilitates the discovery and development of new, effective therapeutics With coverage of the latest mass spectrometry technology, this book explains how mass spectrometry can be used to enhance almost all phases of drug discovery and drug development, including new and emerging applications. The book's fifteen chapters have been written by leading pharmaceutical and analytical scientists. Their contributions are based on a thorough review of the current literature as well as their own experience developing new mass spectrometry techniques to improve the ability to discover and develop new and effective therapeutics. Mass Spectrometry for Drug Discovery and Drug Development begins with an overview of the types of mass spectrometers that facilitate drug discovery and development. Next it covers: HPLC high-resolution mass spectrometry for quantitative assays Mass spectrometry for siRNA Quantitative analysis of peptides Mass spectrometry analysis of biological drugs Applications that support medicinal chemistry investigations Mass spectrometry imaging and profiling Throughout the book, detailed examples underscore the growing role of mass spectrometry throughout the drug discovery and development process. In addition, images of mass spectra are provided to explain how results are interpreted. Extensive references at the end of each chapter guide readers to the primary literature in the field. Mass Spectrometry for Drug Discovery and Drug Development is recommended for readers in pharmaceutics, including medicinal chemists, analytical chemists, and drug metabolism scientists. All readers will discover how mass spectrometry can streamline and advance new drug discovery and development efforts.

Covers all major modifications, including phosphorylation, glycosylation, acetylation, ubiquitination, sulfonation and and glycation Discussion of the chemistry behind each modification, along with key methods and references Contributions from some of the leading researchers in the field A valuable reference source for all laboratories undertaking proteomics, mass spectrometry and post-translational modification research

During the last two decades there has been considerable growth in the development of electrospray ionization mass spectrometry (ESI-MS) as a practical method in the study of reaction mechanisms. This method allows the interception and characterization of key intermediates, either as transient species or as protonated/deprotonated forms of neutral species by API-MS. The outstanding features and advantages of ESI-MS make it one of the most suitable tools for the fast screening of intermediates directly from solution, providing hitherto unavailable chemical information to organic chemists. This monograph provides an overview of the mechanisms involved in ESI-MS, the historical perspectives before looking further in-depth at specific reactions and intermediates. Written by researchers in the field, this book is an unique resource for the understanding of this cutting-edge technique.