Novel Mass Spectrometry-based Strategies for Biophysical Analysis of Proteins and Protein-ligand Complexes PDF Download
Are you looking for read ebook online? Search for your book and save it on your Kindle device, PC, phones or tablets. Download Novel Mass Spectrometry-based Strategies for Biophysical Analysis of Proteins and Protein-ligand Complexes PDF full book. Access full book title Novel Mass Spectrometry-based Strategies for Biophysical Analysis of Proteins and Protein-ligand Complexes by Liangjie Tang. Download full books in PDF and EPUB format.
Author: Liangjie Tang
Publisher: ProQuest
ISBN: 9780549685463
Category : Ligands (Biochemistry)
Languages : en
Pages : 308
Get Book Here
Book Description
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.
Author: Liangjie Tang
Publisher: ProQuest
ISBN: 9780549685463
Category : Ligands (Biochemistry)
Languages : en
Pages : 308
Get Book Here
Book Description
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.
Author: Richard Yu-Cheng Huang
Publisher:
ISBN:
Category :
Languages : en
Pages : 548
Get Book Here
Book Description
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.
Author: Igor A. Kaltashov
Publisher: John Wiley & Sons
ISBN: 0471705160
Category : Science
Languages : en
Pages : 320
Get Book Here
Book Description
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.
Author: Ke Li (Chemist)
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 189
Get Book Here
Book Description
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.
Author: Ying Zhang
Publisher:
ISBN:
Category :
Languages : en
Pages : 492
Get Book Here
Book Description
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.
Author: W. Andy Tao
Publisher: John Wiley & Sons
ISBN: 1118970217
Category : Science
Languages : en
Pages : 448
Get Book Here
Book Description
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.
Author: M. Chance
Publisher: John Wiley & Sons
ISBN: 0470258861
Category : Science
Languages : en
Pages : 325
Get Book Here
Book Description
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.
Author: Jing Li
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 198
Get Book Here
Book Description
Mass spectrometry (MS) has emerged as a powerful tool for epitope mapping, protein-ligand interaction, protein-protein interaction, aggregation, and effect of solution environment on protein conformation because they provide high-throughput data with relatively high structural resolution. Two popular MS-based approaches are hydrogen deuterium exchange-mass spectrometry (HDX-MS) and fast photochemical oxidation of proteins (FPOP), which complement classical biophysical and biochemical techniques in achieving higher structural resolution. The research presented in this dissertation is focused on the application of mass spectrometry-based footprinting techniques in characterizing the biophysical properties of Part I: pH-dependent conformation change of diphtheria toxin T domain (Chapters 2-4)); Part II: Ca2+ binding proteins and the role of Ca2+ regulation (Chapters 5-6); and Part III: protein-protein interaction including epitope mapping of IL-23 (Chapter 7) and Marburg virus protein VP24 (Chapter 8). Chapter 1 serves as an introduction to mass spectrometry instrumentation and standard LC-MS workflow. Two mass spectrometry based-footprinting techniques are introduced: (1) hydrogen deuterium exchange (HDX), and (2) fast photochemical oxidation of proteins (FPOP). Part I focuses on the development of pH-dependent HDX-MS for the conformation study of diphtheria toxin T domain. In Chapter 2, we describe the use pH-dependent HDX to study the pH-dependent conformation change of wild-type diphtheria toxin T domain monomer along its translocation pathway. In Chapter 3, we study the pH-dependent dissociation and reformation of T domain dimer. In Chapter 4, we apply the same method to a T domain mutant H223Q to further investigate the role of key histidine residues in triggering the conformation change. Part II focuses on the application of HDX mass spectrometry for the study of calcium binding proteins. Chapter 5 describes the Ca2+-binding property of ACaM and its Ca2+-regulated interaction with myosin VI. In chapter 6, HDX is also applied to an EF-hand Ca2+ binding protein, DREAM, for the study of its Ca2+ binding sites and stoichiometry. Part III of the dissertation focuses on the development and application of MS-based footprinting methods to investigate protein-protein interaction. Chapter 7 describes the methodology of fast photochemical oxidation of proteins (FPOP) for epitope mapping of IL-23 interacting a therapeutic antibody from Bristol-Myers Squibb. Chapter 8 discusses the use of HDX, FPOP, and NEM chemical labeling for the study of Marburg virus protein VP24 and its interaction with the host protein Keap1 Kelch domain. These seven studies on characterization of protein conformation dynamics, Ca2+ binding protein, and protein-protein interaction show the successful application of mass spectrometry in the structural study of large biomolecules.
Author: Julian Whitelegge
Publisher: Elsevier
ISBN: 0080932037
Category : Science
Languages : en
Pages : 563
Get Book Here
Book Description
This book is designed to be a central text for young graduate students interested in mass spectrometry as it relates to the study of protein structure and function as well as proteomics. It is a definite must-have work for:- libraries at academic institutions with Master and Graduate programs in biochemistry, molecular biology, structural biology and proteomics- individual laboratories with interests covering these areas - libraries and individual laboratories in the pharmaceutical and biotechnology industries.*Serves as an essential reference to those working in the field*Incorporates the contributions of prominent experts *Features comprehensive coverage and a logical structure
Author: Yining Huang
Publisher:
ISBN:
Category : Electronic dissertations
Languages : en
Pages : 193
Get Book Here
Book Description
Mass spectrometry (MS) is an essential tool to study proteins whose structures are of great importance in biological systems. The primary structures of proteins can be determined by the powerful sequencing capabilities of MS. The recent advancements in instrumentation and methodology have made MS increasingly valuable in probing secondary, tertiary and quaternary structures, as well as binding strength, interfaces and in solution dynamics of proteins and protein complexes. Various protein footprinting techniques, including hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), encode structural information onto the protein molecule in different forms of modifications, and then MS is utilized to interpret the mass shifts resulted from modifications and extract the structural information. Protein footprinting coupled with bottom-up proteomics, which utilizes front-end LC separation and tandem mass spectrometry, has gained a solid ground in protein biophysics. On the other hand, opportunities emerge as native MS, ion-mobility separation, gas-phase activation and fragmentation techniques allow new approaches to be developed. In the first part of this dissertation, we describe epitope mapping of three malaria antigens (Plasmodium vivax Duffy binding protein in Chapter 2, Plasmodium vivax and falciparum cell-traversal protein for ookinetes and sporozoites in Chapter 6) and one flavivirus antigen (West Nile virus envelope protein domain III (DIII) in Chapter 4) by HDX in combination with bottom-up MS. We also report epitope mapping of DIII antigen by FPOP (Chapter 5). Challenged by highly disulfide-linked antigens, sample complexity and discontinuous epitopes with only a few residues each, we implemented immunoprecipitation, non-canonical quenching and digestion protocols to achieve complete sequence coverage and map the epitopes with high confidence and spatial resolution. In the second part (Chapter 3), we describe the usage of native MS and ion mobility to characterize antigen-antibody complexes formed by the Duffy binding protein antigen with various antibodies targeting different epitopes. The last part (chapter 7 and 8) describes the development an on-line HDX, native-spray platform in conjunction with top-down MS. The strategy is validated by determining the amide hydrogen exchange rates of a model peptide at the residue level. With evidence for adequate mixing efficiency, high sequence coverage, low hydrogen scrambling and capable data analysis, we applied the platform to study solution-phase amyloid beta 1-40 monomer structure by continuous-labeling and monitoring exchange kinetics and to probe the dimerization interfaces of human insulin by pulse-labeling experiment. These seven studies demonstrate the applications of the mature bottom-up and promising top-down MS on characterizing protein conformation and protein-protein interactions.
