Presented in this dissertation are studies on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b2+ion from HAAAA is an oxazolone or diketopiperazine. The IRMPD spectrum showed bands that matched both a diketopiperazine and an oxazolone structure. H/D exchange and fragmentation studies further supported the presence of a mixture of species. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.

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.

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.

The book that highlights mass spectrometry and its application in characterizing proteins and peptides in drug discovery An instrumental analytical method for quantifying the mass and characterization of various samples from small molecules to large proteins, mass spectrometry (MS) has become one of the most widely used techniques for studying proteins and peptides over the last decade. Bringing together the work of experts in academia and industry, Protein and Peptide Mass Spectrometry in Drug Discovery highlights current analytical approaches, industry practices, and modern strategies for the characterization of both peptides and proteins in drug discovery. Illustrating the critical role MS technology plays in characterizing target proteins and protein products, the methods used, ion mobility, and the use of microwave radiation to speed proteolysis, the book also covers important emerging applications for neuroproteomics and antigenic peptides. Placing an emphasis on the pharmaceutical industry, the book stresses practice and applications, presenting real-world examples covering the most recent advances in mass spectrometry, and providing an invaluable resource for pharmaceutical scientists in industry and academia, analytical and bioanalytical chemists, and researchers in protein science and proteomics.

Leading practitioners authoritatively describe the newest and most effective spectrometric techniques for the analysis of proteins and peptides. The areas covered range from the elucidation of primary and secondary protein structure and the rapid identification of proteins using database techniques to methods for sequencing, as well as methods for the quantitative determination of peptides. Other chapters provide detailed information on the analysis of glycoproteins and glycopeptides and on the use of mass spectrometry to probe the interactions of proteins, both covalent and noncovalent.

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.

Mass spectrometry-based techniques have emerged as powerful analytical tools to investigate the structure of proteins from the primary to quaternary levels. The advancement of mass spectrometry instrumentation and methods has allowed researchers to go beyond just measuring an analyte’s mass-to-charge ratio, but to also probe gas-phase dissociation behaviors and conformations of peptides, proteins, and protein complexes. The primary structure of a protein refers to the linear sequence of amino acids linked together via peptide bonds. The presence, and the order, of specific amino acids in a peptide can strongly influence how a peptide fragments in the gas-phase. Particular amino acids can direct where along the peptide backbone fragmentation is favored and the structure of the fragment ions formed. One method for probing the structure of peptide fragment ions is infrared multiphoton dissociation (IRMPD) mass spectrometry coupled with theoretical quantum chemical calculations. This approach is used to investigate the role of peptide bond conformation on the structure of b2+ fragment ions formed from proline and dimethylproline-containing peptides (Chapter 3). Additionally, IRMPD is used to study the fragmentation patterns of proline containing pentapeptides into b3+ ions (Chapter 4). Native mass spectrometry (nMS) analyzes the intact structures of proteins and protein complexes and offers complementary information to traditional biophysical methods, such as NMR or cryo-EM. Tandem mass spectrometry, specifically surface-induced dissociation (SID), provides information on protein complex connectivity, stoichiometry, and gas-phase structural rearrangement. SID is utilized to monitor deviation from native structure for protein complexes generated from submicrometer nanoelectrospray capillaries (Chapter 5), as well as to provide insight into connectivity of protein complexes selected by trapped ion mobility spectrometry (Chapter 6). In addition to SID, ion mobility spectrometry provides information on the gas-phase shape or conformation of biomolecules. Here, ion mobility spectrometry is utilized to separate multiple conformers of proline-containing peptides (Chapter 3), compare the collision cross sections of protein complexes generated from submicrometer and micrometer sized nanoelectrospray capillaries (Chapter 5), and select protein complexes and isomeric peptides prior to dissociation on an ultrahigh resolution mass spectrometry platform (Chapter 6). Finally, the development and optimization of Trapped Ion Mobility Spectrometry (TIMS) for native mass spectrometry applications is applied to the widely available timsTOF Pro mass spectrometry platform to promote the dissemination of native ion mobility technology.