This monograph reviews all relevant technologies based on mass spectrometry that are used to study or screen biological interactions in general. Arranged in three parts, the text begins by reviewing techniques nowadays almost considered classical, such as affinity chromatography and ultrafiltration, as well as the latest techniques. The second part focusses on all MS-based methods for the study of interactions of proteins with all classes of biomolecules. Besides pull down-based approaches, this section also emphasizes the use of ion mobility MS, capture-compound approaches, chemical proteomics and interactomics. The third and final part discusses other important technologies frequently employed in interaction studies, such as biosensors and microarrays. For pharmaceutical, analytical, protein, environmental and biochemists, as well as those working in pharmaceutical and analytical laboratories.

This volume provides a cross-section of RNA exosome research protocols, applied to a diversity of model organisms. Chapters guide readers through methods that e.g. delineate eukaryotic exosomes’ origins in prokaryotes, probe its RNA substrates, adapter complexes and macromolecular interaction of networks, and establish critical structural-function relationships. 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, The Eukaryotic RNA Exosome: Methods and Protocols aims to ensure successful results in the further study of this vital field.

Introduction to Protein Mass Spectrometry provides a comprehensive overview of this increasingly important, yet complex, analytical technique. Unlike many other methods which automatically yield an absolutely unique protein name as output, protein mass spectrometry generally requires a deduction of protein identity from determination of peptide fragmentation products. This book enables readers to both understand, and appreciate, how determinations about protein identity from mass spectrometric data are made. Coverage begins with the technical basics, including preparations, instruments, and spectrometric analysis of peptides and proteins, before exploring applied use in biological applications, bioinformatics, database, and software resources. Citing the most recent and relevant work in the field of biological mass spectrometry, the book is written for researchers and scientists new to the field, but is also an ideal resource for those hoping to hone their analytical abilities. Offers introductory information for scientists and researchers new to the field, as well as advanced insight into the critical assessment of computer-analyzed mass spectrometric results and their current limitations Provides examples of commonly-used MS instruments from Bruker, Applied Biosystems, JEOL, Thermo Scientific/Thermo Fisher Scientific, IU, and Waters Includes biological applications and exploration of analytical tools and databases for bioinformatics

With the most comprehensive and up-to-date overview of structure-based drug discovery covering both experimental and computational approaches, Structural Biology in Drug Discovery: Methods, Techniques, and Practices describes principles, methods, applications, and emerging paradigms of structural biology as a tool for more efficient drug development. Coverage includes successful examples, academic and industry insights, novel concepts, and advances in a rapidly evolving field. The combined chapters, by authors writing from the frontlines of structural biology and drug discovery, give readers a valuable reference and resource that: Presents the benefits, limitations, and potentiality of major techniques in the field such as X-ray crystallography, NMR, neutron crystallography, cryo-EM, mass spectrometry and other biophysical techniques, and computational structural biology Includes detailed chapters on druggability, allostery, complementary use of thermodynamic and kinetic information, and powerful approaches such as structural chemogenomics and fragment-based drug design Emphasizes the need for the in-depth biophysical characterization of protein targets as well as of therapeutic proteins, and for a thorough quality assessment of experimental structures Illustrates advances in the field of established therapeutic targets like kinases, serine proteinases, GPCRs, and epigenetic proteins, and of more challenging ones like protein-protein interactions and intrinsically disordered proteins

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.

Native mass spectrometry (MS) is a structural biology tool that probes proteins and protein complexes in the gas phase. In native MS, electrospray ionization (ESI) of protein samples are prepared in nondenaturing conditions and generate "native-like" protein ions, which retain noncovalent interactions observed in solution. Therefore, native MS is useful to provide information about the stoichiometry, topology, and ligand binding of protein complexes. Native MS coupled with ion mobility (IM) provides the momentum transfer collision cross section (omega), which is indicative of the size and shape of ions. Collision-induced unfolding (CIU) is an energy-dependent IM-MS technique that probes the unfolding of protein structures monitored by omega as a function of energy. This dissertation explores the utility of native MS and IM-MS analysis to study protein complexes. First, native MS analysis is used to investigate the stoichiometry of iron and sulfur in the endogenous Fe-S clusters binding to the F-box and leucine-rich protein 5 (FBXL5) and Skp1 from the Skp1-Cul1-Fbox (SCF) ubiquitin ligase in Chapter 2. The thermal activation in solution prior to ESI in combination with instrumental activation effectively elucidates the binding of [2Fe-2S] to FBXL5-Skp1 by reducing the presence of nonspecifically binding adducts (NSA). The effects of polarity on native-like avidin tetramers are characterized using native MS and IM-MS analysis in Chapter 3. The native MS of native-like avidin tetramer shows that the average charge state distribution is different between the cations and the anions. The native IM-MS results show that the omega of native-like avidin tetramer is similar regardless of the charge state and polarity. However, the CIU results display differences due to the charge state and polarity. In particular, the differences between the CIU results of 14+ and 14- avidin ions indicate that CIU analysis of the avidin ions is sensitive to solely polarity. In order to quantitatively compare the CIU results of 14+ and 14- avidin ions, a similarity score is developed. Similarity score analysis comparing the 14+ and 14- ions indicates that the largest difference in omega is observed at near 800 eV, while the greatest similarity is observed at low energy range (56 to ~400 eV). The utility of native MS and IM-MS analysis is explored to characterize antibodies (Abs) using two IgG2 samples (SIgG2 and AIgG2) in Chapter 4. These two samples are from the same subclass (IgG2), have the kappa light chain and were each purified from human myeloma plasma, but were from different manufacturing origins. Native MS results of the two samples indicate that the two Abs display vastly different apparent mass (SIgG2: ~154 kDa and AIgG2: 157, 159 kDa respectively) and the relative mass heterogeneity based on the peak width and shape. The IM-MS analysis demonstrates that the omega of the native-like Abs depends on the z, which contrasts from the omega of most proteins. The strong dependence of omega on z may be due to large differences in structural populations and/or the presence of flexible hinge region. The CIU analysis of SIgG2 and AIgG2 demonstrates that the greatest difference in omega between the two Abs is present at low energy with greater difference for anions than for cations. Overall, these results indicate that anions and low energy may preferentially provide significant differences when comparing similar proteins using native IM-MS and CIU analysis. Collision-induced unfolding (CIU) is increasingly used to study the effects of ligand binding to proteins and protein complexes. In chapter 5, a workflow is developed to more accurately assess the effects of ligand binding on the CIU stability. Mass spectra of the quadrupole-selected precursor ions at varying collision-energy display signals indicate that the precursor ions for CIU analysis is interfered by the presence of NSA despite extensive buffer exchange. Therefore, Srelative method is developed to determine the minimum collision-energy threshold at which all of the apparent NSA are removed from the initial m/z window of the precursor ions. More generally, Srelative may be used for quality control of CIU analysis.

Written by recognized experts in the study of proteins, Proteomics for Biological Discovery begins by discussing the emergence of proteomics from genome sequencing projects and a summary of potential answers to be gained from proteome-level research. The tools of proteomics, from conventional to novel techniques, are then dealt with in terms of underlying concepts, limitations and future directions. An invaluable source of information, this title also provides a thorough overview of the current developments in post-translational modification studies, structural proteomics, biochemical proteomics, microfabrication, applied proteomics, and bioinformatics relevant to proteomics. Presents a comprehensive and coherent review of the major issues faced in terms of technology development, bioinformatics, strategic approaches, and applications Chapters offer a rigorous overview with summary of limitations, emerging approaches, questions, and realistic future industry and basic science applications Discusses higher level integrative aspects, including technical challenges and applications for drug discovery Accessible to the novice while providing experienced investigators essential information Proteomics for Biological Discovery is an essential resource for students, postdoctoral fellows, and researchers across all fields of biomedical research, including biochemistry, protein chemistry, molecular genetics, cell/developmental biology, and bioinformatics.