The third protocol developed here is a SUPREX-like protocol that employs a chemical modification reaction (i.e., oxidation reaction) in place of the amide H/D exchange reaction in SUPREX. This SUPREX-like protocol, termed SPROX (Stability of Proteins from Rates of Oxidation) was developed and tested to detect and quantify protein-ligand binding using cyclophilin A and BCAII as model systems.

Two important biophysical characteristics of proteins, their interaction with ligands and their post-translational modifications, modulate various biological processes including signal transduction, chemical synthesis, and cell function. Protein-ligand interactions include the interactions with protein, peptide, DNA, and metal. Characterization of the physical properties of these interactions (binding interfaces, binding affinities, and the protein conformational changes due to the binding) is essential in understanding the mechanism of related diseases and, more importantly, in future drug design. Mass spectrometry, with its own revolution and improvement, becomes a powerful tool in protein and peptide analysis. In this thesis, we applied two mass spectrometry-based strategies, proteomics and protein footprinting, to characterize these biophysical properties of three disease-related proteins, connexin 43 (Cx43), troponin, and apolipoprotein E (ApoE), and Fenna-Matthews-Olson protein (FMO), which is the key factor in energy transfer of the photosynthetic system of green sulfur bacteria. By the combination of standard proteomics workflow and two fragmentation methods, collision-induced dissociation (CID) and electron transfer dissociation (ETD), we successfully identified 15 serine residues, including one novel site, in the Cx43-CT that are phosphorylated by CaMKII, the activity of which may be important in regulating Cx43 in normal and diseased hearts. We further utilized hydrogen/deuterium exchange (H/DX), one mass spectrometry-based protein footprinting strategy, to examine the binding affinities of troponin C (TnC), a cardiac disease-related protein, with its four metal binding ligands (Ca2+), and their binding order. We then expanded this approach to elucidate the dynamics of TnC within the complex and its interactions with other subunits (TnT and TnI) at peptide-level resolution. This same approach was also applied to two protein-ligand complexes: (1) the interaction of FMO and its binding partner, CsmA baseplate protein, in which the orientation of FMO between chlorosome and membrane can be confirmed, and (2) the interaction of ApoE and Abeta 40, which are both key factors in Alzheimer's disease. Moreover, we improved the spatial resolution of H/DX to residue-level by conducting ETD fragmentation in the study of ApoE oligomerization. Our results reveal, for the first time, the amino acid residues involved in its self-oligomerization. These six applications of mass spectrometry-based approaches show their potential in the characterization of different protein biophysical properties. The investigation of a more complex protein system can then be pursued.

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 (MS)-based protein footprinting characterizes protein structure and protein-ligand interactions by interrogating protein solvent-accessible surfaces by using chemical reagents as probes. The method is highly applicable to protein or protein-ligand complexes that are difficult to study by conventional means such as X-ray crystallography and nuclear magnetic resonance. In this dissertation, we describe the development and application of MS-based protein footprinting from three perspectives, including I) protein aggregation and amyloid formation (Chapter 2-3), II) protein-ligand interactions (Chapter 4-5), and III) in-cellulo structures and dynamic motion of membrane proteins (Chapter 6). Fast Photochemical Oxidation of Proteins (FPOP) is the main methodology implemented in the work presented in this dissertation. Chapter 1 provides an overview of FPOP and discusses its fundamentals as well as its important applications in both academic research and biotechnology drug development. In Part I, Chapter 2 covers the early method development of FPOP for monitoring amyloid beta (A[beta]) aggregation. In this work, we demonstrated the high sensitivity and spatial resolution of the method in probing the solvent accessibility of A[beta] at global, sub-regional, and some amino-acid residue levels as a function of its aggregation, and revealed A[beta] species at various oligomeric states identified by their characteristic modification levels. In Chapter 3, we extended the application of the platform to assess the effect of a putative polyphenol inhibitor on amyloid formation and to provide insights into the mechanism of action of the inhibitor in remodeling A[beta] aggregation pathways. In Part II, we evaluated different protein footprinting techniques, including FPOP, hydrogen-deuterium exchange (HDX), and carboxyl group footprinting, for probing protein-ligand (drug candidates) interaction in the context of a therapeutic development. Chapter 4 focused on protein-protein interaction by investigating the epitope of IL-6 receptor for two adnectins that have similar apparent biophysical properties. In Chapter 5, we probed the hydrophobic binding cavity of bromodomain protein for a small molecule inhibitor. This study serves as an example of interrogating protein-small molecule interactions. The two studies in Part II demonstrate the unique capabilities and limitations of protein footprinting methods in protein structural characterization. In Part III, we pushed the boundary of MS-based protein footprinting by applying the method to footprint live cells and investigate the dynamic structures/motion of membrane-transport proteins in their native cellular environment. We employed protein engineering, suspension cell expression and isotopic-encoded carboxyl group footprinting to identify salt bridges in two proteins, GLUT1 and GLUT5, that control their alternating access motions for substrate translocation. With functional analysis and mutagenesis, live-cell footprinting provides new insights into the transport mechanism of proteins in the major facilitator superfamily. The five studies in the dissertation demonstrate the powerful capability of MS-based protein footprinting in protein structural biology and biophysics research. The method also holds great potential in studying more complicated biological systems and solving demanding problems related to protein structure and properties.

Mass spectrometry (MS)-based biophysical approaches are new "tools" for protein characterization owing to its capability to analyze proteins and protein complexes that range in molecular weight from kDa to MDa. Protein characterization requires more than identifying the primary structure. More importantly, protein high order structures (i.e., secondary, tertiary and quaternary structures) are needed for biological studies. MS has become the major tool in studies of protein primary structure and post translational modifications (PTMs) over the past two decades. Because MS has high sensitivity and fast turnaround, more and more biophysical approaches rely on MS to generate information for protein higher order structures. One of the emerging biophysical approaches is MS-based protein footprinting. Protein surface regions can be covalently labeled by chemical reagents in a biologically relevant environment. These chemical labels can be read out by MS through either bottom-up or top-down MS proteomics analysis. The outcome provides protein conformational information. Among various chemical labeling strategies, hydrogen deuterium exchange (HDX) is one of the most commonly used approaches in MS-based protein biophysical studies. HDX-MS is introduced in Chapter 1 by covering the early developments and new applications especially in measuring interaction affinities. Although HDX-MS has been developed for decades, there are still many challenges in protein characterization that require new or improved HDX method development. One such challenge is characterization of protein aggregation. Protein aggregation leads to loss of protein function, and protein aggregates are implicated in several neurodegenerative diseases like Alzheimer's and Parkinson's diseases. A key issue in studies of protein aggregation is real-time monitoring under biologically relevant condition. We developed an HDX-MS-based approach by studying Alzheimer's disease related A[beta] aggregation, and we described this development in Chapter 2. A[beta] proteins are labeled by deuterium in a pulsed way during A[beta] aggregation. The extents of aggregations are monitored by MS as deuterium uptake. This pulsed HDX platform provides peptide-level information about A[beta] aggregation. Ligands (drug candidates) were also evaluated with this platform to determine how the drug candidates affect oligomerization (Chapter 3). Ligand interactions can induce protein conformational changes, which are required in various protein functions like signaling, enzyme activity. Such interactions are fundamental to all biological processes. One of the often used ligands in cells is calcium. Calcium interacts with a variety of calcium-binding proteins, most of which have conserved sequence that form EF-hand motifs to bind calcium. MS-HDX has been an important tool in studies of these typical calcium-binding proteins. Many proteins without an EF-hand motif also require calcium for their function. For example, protein-arginine deiminase (PAD) is an enzyme for arginine citrullination and binds calcium without EF-hand motif. We conducted differential HDX studies on PAD2 protein (Chapter 4). Multiple and cooperative calcium binding of PAD2 are detected by HDX. HDX was further extended by applying protein-ligand titration in an HDX experiment (i.e., Protein-ligand interactions by mass spectrometry, titration and H/D exchange, PLIMSTEX). The calcium binding affinity of each binding site can be elucidated by PLIMSTEX (Chapter 5). Protein aggregation or ligand-binding induced conformational changes can also be detected by MS-HDX. One significant question in MS-based biophysical studies is how to generate structural information for proteins in the absence of a high resolution structure. In a newly developed platform, we combined a traditional structural biology approach, homology modeling, and MS-HDX to generate a structural model for diheme cytochrome c (DHCC) from Heliobacterium (Chapter 6), a protein for which solvent accessibility information from HDX experiment was used as the guide for homology modeling and used to generate a refined structural model of DHCC by using various computational approaches. In summary, we describe in this thesis development and application of several new, refined approaches of HDX and analyze protein aggregation, protein-ligand binding and unknown protein structures. We hope other scientists can apply these approaches to solve complicated and demanding biological problems that are difficult to investigate using traditional biophysical methods.

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.

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.