Hydrogen Deuterium Exchange Mass Spectrometry For Protein Protein Interaction And Structural Dynamics

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Hydrogen Deuterium Exchange Mass Spectrometry for Protein-protein Interaction and Structural Dynamics

Author : Harsimran Singh
Publisher :
ISBN 13 :
Total Pages : 159 pages
Book Rating : 4.:/5 (87 download)

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Book Synopsis Hydrogen Deuterium Exchange Mass Spectrometry for Protein-protein Interaction and Structural Dynamics by : Harsimran Singh

Download or read book Hydrogen Deuterium Exchange Mass Spectrometry for Protein-protein Interaction and Structural Dynamics written by Harsimran Singh and published by . This book was released on 2013 with total page 159 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hydrogen deuterium exchange mass spectrometry has emerged as an important technique to probe protein structure and conformational dynamics. The rate of exchange of hydrogen with deuterium by the peptide backbone is dependent on the solvent accessibility, extent of hydrogen bonding in secondary structural elements and protein dynamics. The extent and the rate of deuterium incorporation are affected by changes in protein structure, interaction with ligand, protein-protein interaction and environmental factors such as pH and temperature. These conformational changes can be global and/or local. The increase in the mass is used to localize the deuterium incorporation after pepsin digestion of the protein and analysis by electrospray ionization mass spectrometry. In this dissertation traditional HDX-MS and a new deuterium trapping assay were used to probe the interaction sites between E. coli cysteine desulfurase SufS and acceptor protein SufE. SufS and SufE form an important part of the SUF pathway, essential for the biosynthesis of Fe-S clusters under oxidative stress and iron depletion conditions. In addition, SufE is known to stimulate SufS cysteine desulfurase activity, but the mechanism is unknown. The HDX-MS results show that the regions affected by the SufS-SufE interaction are dependent on the catalytic intermediate states of the two proteins. HDX-MS was also used to probe the conformational changes resulting upon persulfuration of SufS of Cys364 in the active site. The persulfuration of SufS not only affected regions in the active site cavity, but also had other conformational changes in more distal regions. Based on our findings a model for the interaction SufS and SufE was proposed. A mechanism for the enhancement of SufS cysteine desulfurase activity upon interaction with SufE was also postulated. In all this work demonstrates that hydrogen deuterium exchange mass spectrometry and the deuterium trapping methodology optimized for this system can be easily and effectively used to study the protein-protein interactions and the accompanying changes in structural dynamics for other proteins. Deuterium trapping was demonstrated to be fast, sensitive and reliable method to deduce the changes in solvent accessibility between two or more states of a protein. Both techniques can easily be applied to large number of protein complexes to determine the regions of interaction as well as gain mechanistic information not available through traditional methods such as X-ray crystallography and NMR.

Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics

Author : M. Chance
Publisher : John Wiley & Sons
ISBN 13 : 0470258861
Total Pages : 325 pages
Book Rating : 4.4/5 (72 download)

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Book Synopsis Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics by : M. Chance

Download or read book Mass Spectrometry Analysis for Protein-Protein Interactions and Dynamics written by M. Chance and published by John Wiley & Sons. This book was released on 2008-09-22 with total page 325 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Application of Hydrogen Deuterium Exchange Mass Spectrometry in Protein-ligand and Protein-protein Interactions

Author : Siqi Guan
Publisher :
ISBN 13 :
Total Pages : 322 pages
Book Rating : 4.:/5 (131 download)

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Book Synopsis Application of Hydrogen Deuterium Exchange Mass Spectrometry in Protein-ligand and Protein-protein Interactions by : Siqi Guan

Download or read book Application of Hydrogen Deuterium Exchange Mass Spectrometry in Protein-ligand and Protein-protein Interactions written by Siqi Guan and published by . This book was released on 2016 with total page 322 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are not static objects. They have a great variety of internal motions with different amplitudes and different timescales. These internal motions play an important role in catalytic processes. Therefore, the existence of an intimate relationship between protein dynamics and protein function is widely accepted. Due to the significance of protein dynamics, techniques have been developed to study protein dynamics including nuclear magnetic resonance (NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and mass spectrometry (MS). Compared with NMR and EPR spectroscopy, MS has less stringent sample requirements, including protein concentration and protein size. Moreover, the mass accuracy, sensitivity, and faster data analysis also have contributed to the rapid growth of MS based techniques. Hydrogen-deuterium exchange mass spectrometry (HDX-MS), a combination of HPLC and MS, has become a common and sensitive tool to probe protein structural flexibility and solution dynamics. In this dissertation, HDX-MS was applied to study dynamic changes of proteins due to substrate binding and protein-protein interactions. The GT-A glycosyltransferase glucosyl-3-phosphoglycerate synthase from Mycobacterium tuberculosis (MtGpgS) catalyzes the first step of biosynthesis of 6-O-methylglucose lipopolysaccharides (MGLPs), which are essential to growth and existence of mycobacterium. The HDX-MS data revealed that the two substrates UDP-glucose (UDPG) and 3-phosphoglycerate (3PGA) can bind to MtGpgS independently, disagreeing with the previous proposal that 3PGA can only bind to MtGpgS after UDPG. Moreover, 3PGA was found to bind to or allosterically affect the UDPG binding site. Furthermore, the HDX-MS data revealed that MtGpgS may provide a necessary conformation for UDPG binding, while it goes through a large conformational change on 3PGA binding. The GT-B glycosyltransferase MshA from Corynebacterium glutamicum (CgMshA) catalyzes the initial step of mycothiol biosynthesis. A large conformational change was observed in CgMshA on nucleotide binding by superimposing APO structure of CgMshA and complex structure with UDP. HDX-MS was utilized to study conformational changes of CgMshA on substrate binding from the aspect of dynamics, providing a complementary to static structures. The HDX-MS data showed that both substrates uridine diphosphate glucose-N-acetylglucosamine (UDP-GlcNAc) and 1-L-myo-inositol-1-phosphate (I1P) can bind to CgMshA independently, but the I1P binding is not productive since it binds to an uncorrect site. Moreover, the I1P binding can lead to dynamic changes of CgMshA, while only UDP-GlcNAc can induce the major conformational change of CgMshA. Furthermore, the 3PGA binding cannot induce further dynamic changes of CgMshA in the presence of UDP. HDX-MS was also employed to study dynamic changes of protein complex SufBC2D from Escherichia coli on ADP/Mg2+ binding. This complex is responsible for Fe-S cluster assembly under oxidative stress. The crystal structure of SufBC2D complex has been determined, while little dynamic information is known. So HDX-MS was applied to study dynamic changes of the SufBC2D complex. The HDX-MS data revealed that SufC has a significant conformational change, which may be required by ATP binding and hydrolysis. Moreover, SufB and SufD are detected to have dynamic changes due to SufC conformational changes. These dynamic changes suggest that SufB-SufD protomer may have a conformational change in order to provide a suitable conformation for Fe-S cluster assembly. This work demonstrates that HDX-MS can be effectively used to study protein-ligand and protein-protein interactions, as well as the accompanying changes in structural dynamics. HDX-MS data detects substrate binding mechanism and conformational changes that are not available through x-ray crystallography. With these advantages, HDX-MS has been applied in study of protein structure and dynamics, studying protein-ligand and protein-protein interactions, protein folding, as well as protein therapeutics discovery and development.

Hydrogen Exchange Mass Spectrometry of Proteins

Author : David D. Weis
Publisher : John Wiley & Sons
ISBN 13 : 1118616499
Total Pages : 422 pages
Book Rating : 4.1/5 (186 download)

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Book Synopsis Hydrogen Exchange Mass Spectrometry of Proteins by : David D. Weis

Download or read book Hydrogen Exchange Mass Spectrometry of Proteins written by David D. Weis and published by John Wiley & Sons. This book was released on 2016-03-21 with total page 422 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hydrogen exchange mass spectrometry is widely recognized for its ability to probe the structure and dynamics of proteins. The application of this technique is becoming widespread due to its versatility for providing structural information about challenging biological macromolecules such as antibodies, flexible proteins and glycoproteins. Although the technique has been around for 25 years, this is the first definitive book devoted entirely to the topic. Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Methods and Applications brings into one comprehensive volume the theory, instrumentation and applications of Hydrogen Exchange Mass Spectrometry (HX-MS) - a technique relevant to bioanalytical chemistry, protein science and pharmaceuticals. The book provides a solid foundation in the basics of the technique and data interpretation to inform readers of current research in the method, and provides illustrative examples of its use in bio- and pharmaceutical chemistry and biophysics In-depth chapters on the fundamental theory of hydrogen exchange, and tutorial chapters on measurement and data analysis provide the essential background for those ready to adopt HX-MS. Expert users may advance their current understanding through chapters on methods including membrane protein analysis, alternative proteases, millisecond hydrogen exchange, top-down mass spectrometry, histidine exchange and method validation. All readers can explore the diversity of HX-MS applications in areas such as ligand binding, membrane proteins, drug discovery, therapeutic protein formulation, biocomparability, and intrinsically disordered proteins.

Hydrogen Exchange Mass Spectrometry for Studying Protein-ligand Interactions

Author : Modupeola A. Sowole
Publisher :
ISBN 13 :
Total Pages : 354 pages
Book Rating : 4.:/5 (16 download)

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Book Synopsis Hydrogen Exchange Mass Spectrometry for Studying Protein-ligand Interactions by : Modupeola A. Sowole

Download or read book Hydrogen Exchange Mass Spectrometry for Studying Protein-ligand Interactions written by Modupeola A. Sowole and published by . This book was released on 2015 with total page 354 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hydrogen deuterium exchange (HDX) coupled with mass spectrometry is widely used for probing protein structure and dynamics. Protein-ligand interactions usually induce a reduction in the measured HDX rates an effect that may be ascribed to stabilization of the protein structure. This work aims to improve the general understanding of the changes in HDX patterns associated with ligand binding. We initially applied HDX for studying differences between oxy -hemoglobin (Oxy- Hb) and aquomet-hemoglobin (Chapter 2). The results show that the and subunits respond differently to the oxy to aquomet transition with the heme binding pocket being destabilized in both cases. The results suggest that enhanced structural dynamics in the heme binding pocket may have adverse effects on heme-protein interactions. Chapter 3 focuses on the different scenarios that can be encountered in an HDX experiment upon ligand binding. Myoglobin and hemoglobin were used as model systems, focusing on the oxy and deoxy states of both proteins. Our results demons trate that ligand binding can be stabilizing or destabilizing, leading to decreased or increased HDX rates respectively. In Chapters 4 HDX was used to probe the changes in structural dynamics of caseinolytic protease P (ClpP), an antibiotic drug target, after binding ADEP antibiotics. The mechanism of ADEP binding and the N-terminal structure of ClpP is not well understood with conflicting x-ray structures reported in literature. Our findings demonstrate that the N- terminus of ClpP remains quite unstructured after ADEP binding, while belt region undergoes tightening. Pin 1, a peptidyl prolyl isomerase, binding to a cyclic peptide inhibitor was studied in Chapter 5. Characterization of Pin1-CRYPEVEIC interactions by ot her techniques has been difficult. This study demonstrates that binding of the inhibitor triggers an overall stabilization of Pin 1. We identify a loop that interacts with basic sites of the ligand and that becomes destabilized upon ligand binding. This destabilization is ascribed to steric clashes between the peptide inhibitor and the protein.

Mass Spectrometry of Protein Interactions

Author : Kevin Downard
Publisher : John Wiley & Sons
ISBN 13 : 047014632X
Total Pages : 153 pages
Book Rating : 4.4/5 (71 download)

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Book Synopsis Mass Spectrometry of Protein Interactions by : Kevin Downard

Download or read book Mass Spectrometry of Protein Interactions written by Kevin Downard and published by John Wiley & Sons. This book was released on 2007-08-24 with total page 153 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Protein Structural Dynamics

Author : Simon Mysling
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (9 download)

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Book Synopsis Protein Structural Dynamics by : Simon Mysling

Download or read book Protein Structural Dynamics written by Simon Mysling and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Protein-protein Interactions and Dynamics Probed by Hydrogen/deuterium Exchange

Author : Carrie A. Hughes
Publisher :
ISBN 13 :
Total Pages : 320 pages
Book Rating : 4.:/5 (318 download)

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Book Synopsis Protein-protein Interactions and Dynamics Probed by Hydrogen/deuterium Exchange by : Carrie A. Hughes

Download or read book Protein-protein Interactions and Dynamics Probed by Hydrogen/deuterium Exchange written by Carrie A. Hughes and published by . This book was released on 2003 with total page 320 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry

Author : Diana Resetca
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (13 download)

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Book Synopsis Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry by : Diana Resetca

Download or read book Characterizing Protein Dynamics of Protein-Ligand Interactions by Hydrogen-Deuterium Exchange Mass Spectrometry written by Diana Resetca and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:

Hydrogen/deuterium Exchange Mass Spectrometry as a Technology Platform for Studying Conformational Dynamics in Large Protein Complexes

Download Hydrogen/deuterium Exchange Mass Spectrometry as a Technology Platform for Studying Conformational Dynamics in Large Protein Complexes PDF Online Free

Author : Sasidhar N. Nirudodhi
Publisher :
ISBN 13 :
Total Pages : 199 pages
Book Rating : 4.:/5 (866 download)

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Book Synopsis Hydrogen/deuterium Exchange Mass Spectrometry as a Technology Platform for Studying Conformational Dynamics in Large Protein Complexes by : Sasidhar N. Nirudodhi

Download or read book Hydrogen/deuterium Exchange Mass Spectrometry as a Technology Platform for Studying Conformational Dynamics in Large Protein Complexes written by Sasidhar N. Nirudodhi and published by . This book was released on 2013 with total page 199 pages. Available in PDF, EPUB and Kindle. Book excerpt: Proteins are essential to all biological systems. Proteins participate in numerous cellular processes by interacting with other proteins, other metabolites and membranes in a dynamic environment. Studying the structural and conformational properties of proteins in the solution phase is necessary to understand their protein folding and interaction dynamics. This research project focused on the development and application of hydrogen deuterium exchange mass spectrometry (HDX-MS) technology for studying the conformational dynamics of large multi-subunit protein systems. HDX-MS studies were conducted on representative proteins of two much researched protein families, namely Peroxiredoxins (Prxs) and Cullin Ring Ligases (CRLs). As part of this research we implemented tandem mass spectrometry in the data independent acquisition (MS[supserscript E]) mode for the HDX-MS analysis. We also used ion mobility as a second and orthogonal dimension of separation to overcome the spectral crowdedness. Peroxiredoxins are ubiquitous antioxidant enzymes present in many organisms. Their catalytic activity is regulated by redox dependent oligomerization and their sensitivity to overoxidation is related to the flexibility of the active site loop to undergo partial unfolding. In this research we conducted HDX-MS experiments for determining to what extent the flexibility of the active site loop governs the sensitivity of peroxiredoxins to overoxidation. As example of a robust peroxiredoxin we studied initially the conformational properties of Salmonella typhimurium AhpC wild-type protein by HDX-MS. Subsequently, we conducted comparative HDX-MS analysis on the reduced form of the wild-type protein, and two single point mutants, T77V, and T77I, with the objective to decipher to what extent the stability of the dimer-dimer (A)interface affects the conformational dynamics of the active site loop. Differential HDX-MS results of the wild-type, disulfide reduced wild-type protein have exhibited a decrease in the motility of the active site loop and the C-terminal end of the protein upon disulfide reduction. The Thr77 single point mutation by valine enhanced the dimer-dimer interaction thereby stabilizing the decamer interface and increasing the motility of the active site loop. Whereas, the substitution of T77 by isoleucine increased the motility of the interfacial region which forms the dimer-dimer interface thereby promoting the dissociation of the decamer to dimers. A technically more advanced HDX-MS experimental setup was used to study the exchange-in properties of two robust peroxiredoxins, namely the wild-type StAhpC and the C46S mutant of StAhpC, which mimicks the reduced wild-type StAhpC, in comparison to human Prx2, a peroxiredoxin which is considered as sensitive to overoxidation. When differential deuterium uptake of wild-type StAhpC, C46S mutant StAhpC were compared, increased conformational rigidity was observed in the C46S mutant protein compared to the wild-type Prx. The peptide with highest deuterium incorporation levels in the human Prx2 is much lower compared to the bacterial wild type and C46S mutant Prxs. These comparative HDX-MS studies have fostered our understanding of the underlying conformational dynamics that lead to robust and sensitive Prxs. The second protein system that was studied was a representative of the Cullin Ring Ligases (CRLs), the largest family of RING-type E3 ligases that catalyze ubiquitylation of substrates. Protein ubiquitination is a post-translational modification that regulates several important biological processes in eukaryotic cells. It involves a three enzyme enzymatic cascade consisting of an ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligases (E3). In this study focus was directed toward the Cullin scaffold protein, which adopts an elongated structure that allows substrate receptor binding at the N-terminal domain (NTD) via adaptor proteins. Its C-terminal domain (CTD) binds to E2-ubiquitin through the RBX ring subdomain. Covalent attachment of the ubiquitin-like protein Nedd8 to the conserved lysine residue of the CTD stimulates the transfer of ubiquitin to substrate proteins thereby promoting ubiquitination. The HDX-MS studies of CUL1-RBX1 protein and its neddylated form highlighted that neddylation induces significant flexibility in the conformational dynamics of the CUL1 and RBX1 protein. The HDX-MS results support a mechanistic model in which conformational flexibility in the C-terminal domain of CUL1 and a concomitant opening of the RBX1 protein is necessary to allow the ubiquitin-bound E2 to be placed in close proximity to the protein substrates thereby facilitating the CRL activity.

Mass Spectrometry-based Strategies for Protein Biophysics

Author : Yining Huang
Publisher :
ISBN 13 :
Total Pages : 193 pages
Book Rating : 4.:/5 (973 download)

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Book Synopsis Mass Spectrometry-based Strategies for Protein Biophysics by : Yining Huang

Download or read book Mass Spectrometry-based Strategies for Protein Biophysics written by Yining Huang and published by . This book was released on 2016 with total page 193 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Protein Mass Spectrometry

Author : Julian Whitelegge
Publisher : Elsevier
ISBN 13 : 0080932037
Total Pages : 563 pages
Book Rating : 4.0/5 (89 download)

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Book Synopsis Protein Mass Spectrometry by : Julian Whitelegge

Download or read book Protein Mass Spectrometry written by Julian Whitelegge and published by Elsevier. This book was released on 2008-10-09 with total page 563 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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

Mass Spectrometry-based Strategies for Protein Footprinting

Author : Jing Li
Publisher :
ISBN 13 :
Total Pages : 198 pages
Book Rating : 4.:/5 (958 download)

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Book Synopsis Mass Spectrometry-based Strategies for Protein Footprinting by : Jing Li

Download or read book Mass Spectrometry-based Strategies for Protein Footprinting written by Jing Li and published by . This book was released on 2016 with total page 198 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Solution-Phase Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Author : George Michel Bou-Assaf
Publisher :
ISBN 13 :
Total Pages : pages
Book Rating : 4.:/5 (859 download)

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Book Synopsis Solution-Phase Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry by : George Michel Bou-Assaf

Download or read book Solution-Phase Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry written by George Michel Bou-Assaf and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: ABSTRACT: Proteins are vital macromolecules of every living cell. They undertake a variety of tasks that are essential for life. Most proteins function in assemblies rather than individually. Protein function is strongly correlated to structure and elucidation of both aspects of the protein is indispensable to understand its mechanism of action. Among several structural characterization techniques, solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has emerged in the last 25 years as a powerful tool to study protein-protein interaction and dynamics. In the first half of this dissertation, we describe several aspects of method development that allowed tackling of previously inconceivable biological problems. In the second half, we demonstrate how this tool is applied to study muscle biochemistry; particularly, the troponin complex and myosin. In Chapter 1, we introduce the method, its mechanism, each of its sequential steps improvements which overcame multiple limitations and permitted its application to wide variety of biological problems. In Chapter 2, we describe the optimization of the separation step to minimize back-exchange, a major drawback of the technique. Chapter 3. demonstrates how isotopic depletion of proteins could be advantageous for peptide fragment identification, reduction of the mass spectral complexity, and the increase of the signal-to-noise ratio. In a different aspect of method development, we present a global analysis algorithm in Chapter 4, to manage the enormous amount of data, increase throughput, and most importantly, take advantage of peptide fragment overlap to increase sequence resolution. On the other hand, the second half of the dissertation is dedicated to muscle biochemistry. Troponin is a heterotrimeric complex involved in muscle regulation. The structure of a truncated version of the cardiac isoform of the complex was only solved in the calcium-saturated state. In Chapter 5, we apply hydrogen/deuterium exchange on full length troponin in the calcium-saturated and the calcium-free states to understand how the troponin subunits fit together and what are the conformational changes induced by calcium. Moreover, Chapter 6 describes the allosteric changes induced by phosphorylation the inhibitory subunit N-terminal extension, specific to the cardiac isoform of the complex. Finally, in Chapter 7, we elucidate the conformational changes that accompany the transition of myosin V (a molecular motor) from the post-rigor (ATP-analog bound) to the rigor-like (nucleotide-free) states, thus characterizing new features of the powerstroke.

Mass Spectrometry in Structural Biology and Biophysics

Author : Igor A. Kaltashov
Publisher : John Wiley & Sons
ISBN 13 : 1118232119
Total Pages : 312 pages
Book Rating : 4.1/5 (182 download)

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Book Synopsis Mass Spectrometry in Structural Biology and Biophysics by : Igor A. Kaltashov

Download or read book Mass Spectrometry in Structural Biology and Biophysics written by Igor A. Kaltashov and published by John Wiley & Sons. This book was released on 2012-03-02 with total page 312 pages. Available in PDF, EPUB and Kindle. Book excerpt: The definitive guide to mass spectrometry techniques in biology and biophysics The use of mass spectrometry (MS) to study the architecture and dynamics of proteins is increasingly common within the biophysical community, and Mass Spectrometry in Structural Biology and Biophysics: Architecture, Dynamics, and Interaction of Biomolecules, Second Edition provides readers with detailed, systematic coverage of the current state of the art. Offering an unrivalled overview of modern MS-based armamentarium that can be used to solve the most challenging problems in biophysics, structural biology, and biopharmaceuticals, the book is a practical guide to understanding the role of MS techniques in biophysical research. Designed to meet the needs of both academic and industrial researchers, it makes mass spectrometry accessible to professionals in a range of fields, including biopharmaceuticals. This new edition has been significantly expanded and updated to include the most recent experimental methodologies and techniques, MS applications in biophysics and structural biology, methods for studying higher order structure and dynamics of proteins, an examination of other biopolymers and synthetic polymers, such as nucleic acids and oligosaccharides, and much more. Featuring high-quality illustrations that illuminate the concepts described in the text, as well as extensive references that enable the reader to pursue further study, Mass Spectrometry in Structural Biology and Biophysics is an indispensable resource for researchers and graduate students working in biophysics, structural biology, protein chemistry, and related fields.

Facilitation of Protein 3-D Structure Determination Using Enhanced Peptide Amide Deuterium Exchange Mass Spectrometry (DXMS)

Author : Dennis Peter Pantazatos
Publisher :
ISBN 13 :
Total Pages : 248 pages
Book Rating : 4.:/5 (712 download)

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Book Synopsis Facilitation of Protein 3-D Structure Determination Using Enhanced Peptide Amide Deuterium Exchange Mass Spectrometry (DXMS) by : Dennis Peter Pantazatos

Download or read book Facilitation of Protein 3-D Structure Determination Using Enhanced Peptide Amide Deuterium Exchange Mass Spectrometry (DXMS) written by Dennis Peter Pantazatos and published by . This book was released on 2006 with total page 248 pages. Available in PDF, EPUB and Kindle. Book excerpt: Three dimensional structure determination and analysis of proteins is necessary for the understanding of how proteins participate in human disease, and are critical for the effective design of therapeutics for clinically important targets. Current efforts for determining protein structures are centered on novel high-throughput (HT) approaches. These include high throughput (HT) crystallization efforts and global structure prediction efforts monitored through the Critical Assessment of Structure Prediction (CASP) experiments where progress has been incremental at best. Protein structure analysis of conformational changes and protein-proteins interactions can be monitored by biophysical methods which include fluorescence spectroscopy, differential scanning calorimetry, circular dichroism and ultra centrifugation. These methods provide adequate low resolution information on global changes in secondary and tertiary structure but are limited in providing detailed information on protein structure, protein conformational changes and protein-protein interactions. Therefore, there is a great need for improvements in the speed and ease of determining and analyzing protein structures and protein dynamics. Hydrogen/Deuterium (H/D) exchange rates are highly dependent on protein structure and amide hydrogen solvent accessibility. Exchange rates can report structure stability at the individual amino acid scale and provide important information on the secondary and tertiary structure. The dissertation is arranged as follows: Chapter 1 is an introduction to Hydrogen/Deuterium exchange mass spectrometry and also reports my studies on the thrombin-Lepirudin complex. Chapter 2 is in preparation for submission and reports the application of DXMS for characterizing the molecular dynamics of spectrin. It also presents the development and validation studies for a computational method for generating amide exchange rate maps from DXMS data, a critical component of the structure determination method described in Chapters six and seven. Chapter 3 reports the application of DXMS for structural analysis of drug-protein interactions. Chapter 4 reports methods for using DXMS to improve the crystallizability of protein constructs for 3D structure determination by x ray crystallography. Chapter 5 reports the detailed 3-D structures of the first two proteins that were successfully studied with the DXMS- guided construct design method. Chapter 6 outlines the development of a hybrid computational-experimental method for high-throughput protein 3-D structure determination: DXMS-Rosetta-COREX engine. Chapter 7 summarizes my conclusions from the foregoing studies and outlines future directions of these studies.

Hydrogen Exchange Mass Spectrometry of Proteins

Author : David D. Weis
Publisher : John Wiley & Sons
ISBN 13 : 1118703693
Total Pages : 376 pages
Book Rating : 4.1/5 (187 download)

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Book Synopsis Hydrogen Exchange Mass Spectrometry of Proteins by : David D. Weis

Download or read book Hydrogen Exchange Mass Spectrometry of Proteins written by David D. Weis and published by John Wiley & Sons. This book was released on 2016-01-11 with total page 376 pages. Available in PDF, EPUB and Kindle. Book excerpt: Hydrogen exchange mass spectrometry is widely recognized for its ability to probe the structure and dynamics of proteins. The application of this technique is becoming widespread due to its versatility for providing structural information about challenging biological macromolecules such as antibodies, flexible proteins and glycoproteins. Although the technique has been around for 25 years, this is the first definitive book devoted entirely to the topic. Hydrogen Exchange Mass Spectrometry of Proteins: Fundamentals, Methods and Applications brings into one comprehensive volume the theory, instrumentation and applications of Hydrogen Exchange Mass Spectrometry (HX-MS) - a technique relevant to bioanalytical chemistry, protein science and pharmaceuticals. The book provides a solid foundation in the basics of the technique and data interpretation to inform readers of current research in the method, and provides illustrative examples of its use in bio- and pharmaceutical chemistry and biophysics In-depth chapters on the fundamental theory of hydrogen exchange, and tutorial chapters on measurement and data analysis provide the essential background for those ready to adopt HX-MS. Expert users may advance their current understanding through chapters on methods including membrane protein analysis, alternative proteases, millisecond hydrogen exchange, top-down mass spectrometry, histidine exchange and method validation. All readers can explore the diversity of HX-MS applications in areas such as ligand binding, membrane proteins, drug discovery, therapeutic protein formulation, biocomparability, and intrinsically disordered proteins.