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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
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.
Glyco-engineering is being developed as a method to control the composition of carbohydrates and to enhance the pharmacological properties of monoclonal antibodies (mAbs) and other proteins. In Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols, experts in the field provide readers with production and characterization protocols of glycoproteins and glyco-engineered biopharmaceuticals with a focus on mAbs. The volume is divided in four complementary parts dealing with glyco-engineering of therapeutic proteins, glycoanalytics, glycoprotein complexes characterization, and PK/PD assays for therapeutic antibodies. Written in the highly successful Methods in Molecular BiologyTM series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Glycosylation Engineering of Biopharmaceuticals: Methods and Protocols serves as an ideal guide for scientists striving to push forward the exciting field of engineered biopharmaceuticals.
This volume details the importance of multiple experimental techniques and computational methods needed to obtain the comprehensive picture of protein complex structure, dynamics and assembly afforded by the emerging field of integrative structural biology. Chapters guide readers through the broad spectrum of approaches required for a complete representation of protein complexes, including expression and purification, experimental characterization of structure and assembly, and computational methods for identifying protein complexes and modelling their assembly. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Protein Complex Assembly: Methods and Protocols aims to ensure successful results in the further study of this vital field.
PROVIDES STRATEGIES AND CONCEPTS FOR UNDERSTANDING CHEMICAL PROTEOMICS, AND ANALYZING PROTEIN FUNCTIONS, MODIFICATIONS, AND INTERACTIONS—EMPHASIZING MASS SPECTROMETRY THROUGHOUT Covering mass spectrometry for chemical proteomics, this book helps readers understand analytical strategies behind protein functions, their modifications and interactions, and applications in drug discovery. It provides a basic overview and presents concepts in chemical proteomics through three angles: Strategies, Technical Advances, and Applications. Chapters cover those many technical advances and applications in drug discovery, from target identification to validation and potential treatments. The first section of Mass Spectrometry-Based Chemical Proteomics starts by reviewing basic methods and recent advances in mass spectrometry for proteomics, including shotgun proteomics, quantitative proteomics, and data analyses. The next section covers a variety of techniques and strategies coupling chemical probes to MS-based proteomics to provide functional insights into the proteome. In the last section, it focuses on using chemical strategies to study protein post-translational modifications and high-order structures. Summarizes chemical proteomics, up-to-date concepts, analysis, and target validation Covers fundamentals and strategies, including the profiling of enzyme activities and protein-drug interactions Explains technical advances in the field and describes on shotgun proteomics, quantitative proteomics, and corresponding methods of software and database usage for proteomics Includes a wide variety of applications in drug discovery, from kinase inhibitors and intracellular drug targets to the chemoproteomics analysis of natural products Addresses an important tool in small molecule drug discovery, appealing to both academia and the pharmaceutical industry Mass Spectrometry-Based Chemical Proteomics is an excellent source of information for readers in both academia and industry in a variety of fields, including pharmaceutical sciences, drug discovery, molecular biology, bioinformatics, and analytical sciences.
This book collects up-to-date advanced protocols and advice from leading experts in the area of membrane protein biology that can be applied to structural and functional studies of any membrane protein system. The contents explore methods for cloning and expression of membrane proteins and membrane protein complexes in prokaryotic and eukaryotic systems, approaches for protein purification, nanobody applications, as well as biophysical characterization and much more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and thorough, Expression, Purification, and Structure Biology of Membrane Proteins serves to guide and encourage young researchers and newcomers to the field to tackle bold new studies on membrane proteins. Chapter 11 is available open access under a CC-BY 4.0 license via link.springer.com.
The first systematic summary of biophysical mass spectrometrytechniques Recent advances in mass spectrometry (MS) have pushed the frontiersof analytical chemistry into the biophysical laboratory. As aresult, the biophysical community's acceptance of MS-based methods,used to study protein higher-order structure and dynamics, hasaccelerated the expansion of biophysical MS. Despite this growing trend, until now no single text has presentedthe full array of MS-based experimental techniques and strategiesfor biophysics. Mass Spectrometry in Biophysics expertly closesthis gap in the literature. Covering the theoretical background and technical aspects of eachmethod, this much-needed reference offers an unparalleled overviewof the current state of biophysical MS. Mass Spectrometry inBiophysics begins with a helpful discussion of general biophysicalconcepts and MS-related techniques. Subsequent chaptersaddress: * Modern spectrometric hardware * High-order structure and dynamics as probed by various MS-basedmethods * Techniques used to study structure and behavior of non-nativeprotein states that become populated under denaturingconditions * Kinetic aspects of protein folding and enzyme catalysis * MS-based methods used to extract quantitative information onprotein-ligand interactions * Relation of MS-based techniques to other experimental tools * Biomolecular properties in the gas phase Fully referenced and containing a helpful appendix on the physicsof electrospray mass spectrometry, Mass Spectrometry in Biophysicsalso offers a compelling look at the current challenges facingbiomolecular MS and the potential applications that will likelyshape its future.
This volume explores strategies and detailed protocols for the preparation of macromolecular complexes and their characterization in view of structural analysis. The chapters in this book are separated into three parts: Part One focuses on sample preparation, and covers strategies for recombinant expression of multiprotein complexes in prokaryotic and eukaryotic hosts, for genome engineering using the CRISPR/Cas9 system and for production of specific binders such as reformatted antibodies and artificial binding proteins. Part Two looks at the biophysical methods that can provide useful indicators for sample optimization, and often complement structural information obtained with core technologies for structure determination—x-ray crystallography and cryo-electron microscopy—by quantitative solution data. Part Three discusses the characterization of multiprotein complexes in a cellular environment using the latest technologies and in vivo approaches. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Cutting-edge and authoritative, Multiprotein Complexes: Methods and Protocols is a valuable resource for structural and molecular biologists who need to prepare multi-components for their applications, and for other scientists working on macromolecular assemblies from other angles that need to know the latest approaches that the field has to offer.
The activity of many biopharmaceutical polymers is dependent on conformation, and the next several years will see increased interest in the conformational analysis of these polymers resulting from the development of biosimilar or "follow-on" biological products. While a wide variety of approaches to analysis exists, finding the most viable ones wou