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.

X-ray-initiated hydroxyl radical protein footprinting (XF) coupled to mass spectrometry (MS) is a well-characterized method for investigating protein and nucleic acid conformational changes in vitro over large timescales ranging from microseconds to milliseconds. For the past two decades, XF has been applied to a variety of structural biology studies, and now, there is growing interest in utilizing XF to probe more complex biological systems such as large macromolecular complexes, protein aggregates, and live cells, which are not easily prosecuted using other protein footprinting strategies. These difficult XF studies require increased X-ray flux density and greater protein sequence coverage, and the goal of this dissertation is to develop a high-flux X-ray beamline and a synergistic footprinting reagent, which provide improved beam power and extend the resolution of XF, respectively.The chapters herein describe the scientific commissioning and benchmarking of a new, high-flux X-ray beamline, XFP (Biological X-ray Footprinting and Spectroscopy beamline), at NSLS-II and the adaptation of a novel footprinting methodology, the trifluoromethylation of proteins, to XF. Results demonstrated XFP is capable delivering 10 times the flux density of its predecessor X28C, and trifluoromethylated radicals (•CF3) and hydroxyl radicals (•OH) were shown to label distinct subsets of amino acid side chains. The high-flux beamline and X-ray-initiated •CF3 footprinting strategy discussed in this dissertation provide a foundation for novel XF experiments on challenging systems in the next coming decades. An example of a recent, high impact XF study in which I played an integral role in the successful completion of the project is given in the concluding chapter.

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.

Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced increases the complexity of identifying and quantifying oxidation products. The current methods available for quantifying the extent of oxidation either involve manual analysis steps, or limit the number of searchable modifications or the size of sequence database. This creates a bottleneck which can result in a long and arduous analysis process, which is further compounded in a complex sample. In addition to the data complexity, the peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography. The recently published in cell FPOP method exemplifies where this field is headed - larger and more complex systems. This dissertation describes two new methodologies and one new technology for hydroxyl radical-mediated protein footprinting, expanding the applicability of the method. First is development of a new footprinting analysis method for both peptide and residue level analysis, allowing for faster quantification of results. This method utilizes a customized multilevel search workflow developed for an on-market search platform in conjunction with a quantitation platform developed using a free Excel add-in, expediting the analysis process. Second is the application of multidimensional protein identification technology (MudPIT) in combination with hydroxyl radical footprinting as a method to increase the identification of quantifiable peptides in these experiments. Last is the design and implementation of a flow system for in cell FPOP, which hydrodynamically focuses the cells, and when used yielded a 13-fold increase in oxidized proteins and 2 orders of magnitude increase in the dynamic range of the method.

Integral mass spectrometry (MS) has emerged as an important tool for protein structural characterization. It readouts are a broad range of structural information, including stoichiometry, interactions, conformations and conformation change, and dynamics. Protein footprinting is a pivotal component in the intergral MS toolkit.My dissertation centers around the development and application of protein footprinting to characterize protein structure. It is divided into seven chapters. Chapter 1 serves as the introduction for integral mass spectrometry in structural proteomic. In Chapter 2, we extended the fast-photochemical oxidation of proteins (FPOP) platform by adding the trifluoromethyl radical (·CF3) as a new reagent. We discovered that ·CF3 footprint proteins in a complementary way as hydroxide radicals. The ·CF3 radical has exceptional reactivity, modifying 18 amino acids out of 20. Further studies demonstrate that it can report the conformational change between holo-myoglobin and apo-myoglobin and can define the topology of the VKOR membrane protein. This work bridges trifluoromethylation chemistry in materials and medicinal chemistry to that in structural biology. In Chapter 3, collaborated with Dr. Mark Chance's laboratory in Case Western Reserve University (CWRU) to apply ·CF3 chemistry on the synchrotron platform, which is the first platform that uses ·OH for protein footprinting. Synchrotron radiolysis generates ·CF3 in water media by ionizing water molecules to give ·OH. The ·CF3 shows complementary chemical reactivity with canonical ·OH labeling yet results in higher reactivity coverage. The ·CF3 reagent is the second footprinting reagent enabled by synchrotron since 1999. This work serves as a proof-of-concept to demonstrate that synchrotron platform is adaptable to other novel chemistries that can increase footprinting coverage. Further, taking advantage of X-ray irradiation, we achieved direct protein trifluoromethylation in the absence of metal catalysis or peroxide for the first time, with the synchrotron platform. In Chapter 4, we devloped a laser-mediated radical method for integral membrane protein (IMP) footprinting. Classical footprinting methods use hydrophilic reagents to label protein surfaces. IN so doing, we generate structural information by measuring the solvent accessibility of the backbone or side chains in aqueous media. Owing to the amphipathic nature of IMP, this new approach exploits the highly hydrophobic nature of perfluoroalkyl iodine together with tip sonication to ensure efficient labeling of a transmembrane domain (TM). The chemistry yields 100% reactivity coverage for tyrosine, and complete IMP labeling in a fast fashion. The resulting protein modification, which is resistant to hydrolysis, compatible with proteolysis, and amenable of tandem mass analysis, is appropriate for footprinting by bottom-up analysis. (Collaboration with Dr. Weikai Li from Wash U Medical School). In Chapter 5, we investigated an array of digestion conditions by using different combination of protease and additives to optimize the coverage of IMP digestion. IMPs are under-represented in conventional bottom-up proteomic analysis that generally favors soluble, abundant and easy-to-digest proteins. The new protrocol of IMP digestion significantly decreases our workload for sample preparation, allows us to avoid common contaminants that impair LC-MS, and generally yields >90% sequence coverage by generating peptides suitable for structural proteomic studies. Further, the deep analysis enable us to identify a "sweet spot" in the digestion protocol that may provide guidance to choose a suitable protease in structural proteomics in future. In Chapter 6, apart from methodology development, we used hydrogen/deuterium exchange mass spectrometry (HDX-MS) to characterize the binding interface for Mxra8-immune complex and Mxra8-chikungunya virus protein complex. The cell adhesion molecule Mxra8 is identified as a receptor for multiple arthritogenic alphaviruses such as chikungunya virus. We identified putative binding sites for eight anti-Mxra8 monoclonal antibodies (mAbs). HDX-MS enables us to classify the novel mAbs, predict their competing binding interface with chikungunya virus, and provide a molecular level explanation for the observation that mAbs can block the Chikungunya virus infection. From the HDX kinetic curves, we also observe that the mAbs have higher affinity than do Chikungunya virus proteins when binding with Mxa8. Finally, the HDX data help to assign the orientation of Mxra8 on the Cryo-EM structure of Chikungunya virus complex (Collaboration with Dr. Daved H. Fremont and Dr. Michael s Diamond, Wash U School of Medicine). In Chapter 7, we provide a conclusion for my dissertation. We will discuss challenges and opportunities for protein footprinting, and its role in the expanding toolkit of structural proteomics.

The dissertation will be solely focused on using mass spectrometry to characterize protein high order structures (HOS), it emphasizes the use of hydroxyl radical footprinting (FPOP) coupled to bottom-up MS approach. A detailed background information about FPOP, and the corresponding method developments as well as applications will be covered.The first chapter will be a comprehensive review regarding the FPOP. Following this, chapter 2, 3, and 4 will be focused on the method developments. Chapter 2 describes an isotope dilution GC-MS method to quantitate OH radicals in FPOP; chapter 3 describes the incorporation of Leu-enkephalin as reporter peptide for a more quantitative FPOP platform; and chapter 4 introduces how R-programming can facilitate the MS-based structural proteomics. After this, chapter 5, 6, and 7 are mainly about the applications of FPOP to characterize the proteins. Chapter 5 talks about using FPOP to localize the dimer dissociation and local unfolding of G93A SOD1; chapter 6 describes how FPOP can be used to characterize an intrinsically disordered tail of EGF receptor protein; and chapter 7 demonstrates the feasibility of a marriage of FPOP and Nanodiscs to study the membrane-associated KRAS protein.