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.

Ultimately, the work outlined within this dissertation demonstrates the development of SID, IM, and UVPD instrumentation and methods, expanding the tools available within native MS to perform more in-depth characterization of peptide, protein, and protein complex structures.

Abstract: There is a growing interest in application of mass spectrometry as a high throughput technique for quaternary structure studies of protein complexes. One way to study protein complexes by mass spectrometry is to specifically label peptides segments that carry critical structural information, and after protein digestion subsequently identify the labeled peptides using liquid chromatography - mass spectrometry. A chemical crosslinker forms covalent bonds at specific amino acid sidechains that are in proximity in the protein structure. This approach is used to probe the binding interface of LexA/RecA proteins in Escherichia coli (Chapter 3). In contrast, intact noncovalent protein complexes can be directly transferred into the gas phase, while retaining memory of their solution structures. Accurate molecular weight measurement by mass spectrometry can be used for stoichiometry determination of protein-protein and protein-ligand systems, as manifested by the two examples of stoichiometry determination of differently treated adiponectin oligomers (Chapter 4), and the silver binding properties of the N-terminal region of a bacterial protein CusB (Chapter 5).

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.

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.

Chiral Analysis covers an important area of analytical chemistry of relevance to a wide variety of scientific professionals. The target audience is scientific professionals with an undergraduate background in chemistry or a related discipline, specifically organic chemists, researchers in drug discovery, pharmaceutical researchers involved with process analysis or combinatorial libraries, and graduate students in chemistry. Chapters have been written with the nonspecialist in mind so as to be self-contained.* Broad coverage - spectroscopic and separation methods covered in a single volume* Up-to-date and detailed review of the various techniques available and/or under development in this field* Contributions from leading experts in the field