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Hydrogen/deuterium Exchange (deuterium + exchange)
Selected AbstractsHydrogen/deuterium exchange on protein solutions containing nucleic acids: utility of protamine sulfateRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2008Anton Poliakov Obtaining global hydrogen/deuterium (H/D) exchange data on proteins is an important first step in amide proton exchange experiments. Important information such as the mode of exchange, the cooperativity of folding/unfolding reactions, and the effects of ligand binding can be readily obtained in global exchange experiments. Many interesting biological systems are complexes containing both proteins and nucleic acids. The low pH conditions required to quench H/D exchange reactions result in the formation of stable protein/nucleic acid precipitates which interfere with the liquid chromatography step of the experiment and preclude obtaining mass spectrometric data. In this work we show that the precipitation of proteins and nucleic acids is electrostatic in nature and can be prevented by high ionic strength and by removing nucleic acids by protamine sulfate. Using protamine sulfate in quenching solution, we were able to obtain global H/D data with protein samples containing large amounts of DNA or RNA. Copyright © 2008 John Wiley & Sons, Ltd. [source] Mass spectrometry techniques for detection of ligand-dependent changes in the conformational flexibility of cellular retinol-binding protein type I localized by hydrogen/deuterium exchangeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2006M. Careri Hydrogen/deuterium exchange, measured by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), was used as a means to probe and map differences in conformational flexibility between the ligand-free and ligand-bound forms of cellular retinol-binding protein type I. Labelled fragments were obtained by digestion of the protein with pepsin. The differences in space-resolved time courses of deuterium incorporation identified regions that exhibit a remarkably higher degree of flexibility in the apo-protein than in the holo-protein. These segments encompass residues that are thought, on the basis of structural homology of the retinol carrier with other members of the intracellular lipid-binding proteins family, to belong to the dynamic portal through which all- trans retinol can access its high-affinity, solvent-shielded, binding site. Copyright © 2006 John Wiley & Sons, Ltd. [source] Ligand effects upon deuterium exchange in arenes mediated by [Ir(PR3)2(cod)]+.BF4,JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 1 2004George J. Ellames Abstract A series of complexes of general form [Ir(PR3)2(cod)]+ has been prepared and used, without isolation, to mediate deuteration of a range of model substrates. The data suggest that, with many substrates, basicity of the phosphine ligands bound to iridium is an important factor influencing substrate selectivity and the efficiency of deuteration. In addition, the spectrum of activity of iridium complexes bearing pure donor ligands is different in many cases to that of complexes where the ligands are known to be ,-acids. Copyright © 2003 John Wiley & Sons, Ltd. [source] The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain bindingPROTEIN SCIENCE, Issue 4 2007Shugui Chen Abstract The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis -interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain. [source] Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved proteinPROTEIN SCIENCE, Issue 6 2002Abel Baerga-Ortiz Abstract The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen. [source] The determination of high-affinity protein/inhibitor binding constants by electrospray ionization hydrogen/deuterium exchange mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006Lee Frego Recently, a hydrogen/deuterium exchange method termed SUPREX (Stability of Unpurified Proteins from Rates of hydrogen/deuterium EXchange), capable of measuring protein/ligand binding constants, which utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), has been reported. Unlike more conventional approaches, SUPREX is inherently capable of measuring Kd values of tight binding ligands. Here we present a SUPREX-based method, incorporating automation and electrospray ionization (ESI)-MS, to measure Kd values for very potent inhibitors of the kinase PKC,. The use of ESI offers an alternative to MALDI, with the advantages of improved mass measurement precision for larger proteins, and amenability to automation. Kd values generated by this method are in good agreement with those generated by a molecular protein kinase assay. Copyright © 2006 John Wiley & Sons, Ltd. [source] Structural identification of trace level enol tautomer impurity by on-line hydrogen/deuterium exchange HR-LC/MS in a LTQ-Orbitrap hybrid mass spectrometerJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 7 2007Dr Guodong Chen Guodong Chen [source] Noncovalent dimerization of paclitaxel in solution: Evidence from electrospray ionization mass spectrometryJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 9 2002Sarah A. Lorenz Abstract Paclitaxel, a unique antimitotic chemotherapy agent that inhibits cell division by binding to microtubules and prevents them from "depolymerizing," has received widespread interest because of its efficacy in fighting certain types of cancer, including breast and ovarian cancer. Paclitaxel undergoes aggregation at millimolar concentrations in both aqueous media and solvents of low polarity (mimicking hydrophobic environments). Its aggregation may have impact on its aqueous stability and its ability to stabilize microtubules. Here, we investigated the dimerization phenomenon of paclitaxel by electrospray ionization mass spectrometry (ESI-MS). Paclitaxel dimers were stable in solutions of acetonitrile/aqueous ammonium acetate (80/20) and aqueous sodium acetate/acetonitrile (92/8 or 95/5) at various pH values. Additional experiments using solution-phase hydrogen/deuterium exchange were employed to ascertain whether or not the observed dimers were formed in solution or as an artifact of the ESI process by ion,molecule reaction. The evidence supports formation of the dimer in solution, and the approach used can be extended to investigation of other types of drug,drug interactions. © 2002 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2057,2066, 2002 [source] A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulationPROTEIN SCIENCE, Issue 4 2007Ole H. Olsen Abstract Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365,374{216,225}) of the so-called activation domain and the 170-loop (residues 313,322{170A,175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S1 and S3 substrate pockets, the activation pocket, and N-terminal insertion. [source] Epitope mapping of a monoclonal antibody against human thrombin by H/D-exchange mass spectrometry reveals selection of a diverse sequence in a highly conserved proteinPROTEIN SCIENCE, Issue 6 2002Abel Baerga-Ortiz Abstract The epitope of a monoclonal antibody raised against human thrombin has been determined by hydrogen/deuterium exchange coupled to MALDI mass spectrometry. The antibody epitope was identified as the surface of thrombin that retained deuterium in the presence of the monoclonal antibody compared to control experiments in its absence. Covalent attachment of the antibody to protein G beads and efficient elution of the antigen after deuterium exchange afforded the analysis of all possible epitopes in a single MALDI mass spectrum. The epitope, which was discontinuous, consisting of two peptides close to anion-binding exosite I, was readily identified. The epitope overlapped with, but was not identical to, the thrombomodulin binding site, consistent with inhibition studies. The antibody bound specifically to human thrombin and not to murine or bovine thrombin, although these proteins share 86% identity with the human protein. Interestingly, the epitope turned out to be the more structured of two surface regions in which higher sequence variation between the three species is seen. [source] Real-time reaction monitoring by probe electrospray ionization mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2010Zhan Yu Probe electrospray ionization (PESI) is a modified version of the electrospray ionization (ESI), where the capillary for sampling and spraying is replaced by a solid needle. High tolerance to salts and direct ambient sampling are major advantages of PESI compared with conventional ESI. In this study, PESI-MS was used to monitor some biological and chemical reactions in real-time, such as acid-induced protein denaturation, hydrogen/deuterium exchange (HDX) of peptides, and Schiff base formation. By using PESI-MS, time-resolved mass spectra and ion chromatograms can be obtained reproducibly. Real-time PESI-MS monitoring can give direct and detailed information on each chemical species taking part in reactions, and this is valuable for a better understanding of the whole reaction process and for the optimization of reaction parameters. PESI-MS can be considered as a potential tool for real-time reaction monitoring due to its simplicity in instrumental setup, direct sampling with minimum sample preparation and low sample consumption. Copyright © 2010 John Wiley & Sons, Ltd. [source] Structural elucidation of metabolites of ginkgolic acid in rat liver microsomes by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry and hydrogen/deuterium exchangeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2009Z. H. Liu Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the invitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague-Dawley rat liver microsomes. The metabolites were analyzed by ultra-performance liquid chromatography coupled with photodiode array detector/negative-ion electrospray ionization tandem mass spectrometry (UPLC-PDA/ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids. Copyright © 2009 John Wiley & Sons, Ltd. [source] Assessment of the repeatability and reproducibility of hydrogen/deuterium exchange mass spectrometry measurements,RAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 23 2008William Burkitt A system to perform automated hydrogen/deuterium exchange mass spectrometry measurements was constructed using an XYZ robotic autosampler that was capable of performing solvent manipulations and a 4.7 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The system included features such as the first demonstration of a ,dual column' high-performance liquid chromatography (HPLC) setup, and a novel digestion strategy. The performance of the system, in terms of the repeatability and reproducibility of the measurement of protein hydrogen/deuterium exchange, was assessed over a 2-month period. The sensitivity of the measurement of hydrogen exchange towards several parameters was assessed, which allowed their impact on the reproducibility to be discussed. The parameters assessed were the temperature of the HPLC columns and switching valves, the temperature of the quench solutions, the pH of the mobile phase, the pH of the quenched solution, the acid used in the mobile phase and the analytical column used. Copyright © 2008 John Wiley & Sons, Ltd. [source] Mass spectrometry techniques for detection of ligand-dependent changes in the conformational flexibility of cellular retinol-binding protein type I localized by hydrogen/deuterium exchangeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 13 2006M. Careri Hydrogen/deuterium exchange, measured by electrospray ionization with orthogonal quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS) and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), was used as a means to probe and map differences in conformational flexibility between the ligand-free and ligand-bound forms of cellular retinol-binding protein type I. Labelled fragments were obtained by digestion of the protein with pepsin. The differences in space-resolved time courses of deuterium incorporation identified regions that exhibit a remarkably higher degree of flexibility in the apo-protein than in the holo-protein. These segments encompass residues that are thought, on the basis of structural homology of the retinol carrier with other members of the intracellular lipid-binding proteins family, to belong to the dynamic portal through which all- trans retinol can access its high-affinity, solvent-shielded, binding site. Copyright © 2006 John Wiley & Sons, Ltd. [source] Fragmentation of mycosporine-like amino acids by hydrogen/deuterium exchange and electrospray ionisation tandem mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 2 2006Karina H. M. Cardozo The determination and identification of mycosporine-like amino acids (MAAs) from algae remain a major challenge due to the low concentration. Mass spectrometry (MS) can make an invaluable contribution in the search and identification of MAAs because of its high sensitivity, possibility of coupling with liquid chromatography, and the availability of powerful tandem mass spectrometric techniques. However, the unequivocal determination of the presence and location of important functional groups present on the basic skeleton of the MAAs is often elusive due to their inherent instability under MS conditions. In this study, the use of hydrogen/deuterium (H/D) exchange and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) for characterisation of four MAAs (palythine, asterina, palythinol and shinorine) isolated from the macroalgae Gracilaria tenuistipitata Chang et Xia was investigated. The accurate-mass confirmation of the protonated molecules was performed on a Q-TOF instrument. We demonstrate that employing deuterium labelling in ESI-MS/MS analysis provides a convenient tool for the determination of new MAAs. Although the fragmentation patterns of MAAs were discussed earlier, to our knowledge, this is the first time that mechanisms are proposed. Copyright © 2005 John Wiley & Sons, Ltd. [source] Denaturant sensitive regions in creatine kinase identified by hydrogen/deuterium exchangeRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 11 2005Hortense Mazon The GdmHCl-induced unfolding of creatine kinase (CK) has been studied by hydrogen/deuterium (H/D) exchange combined with mass spectrometry. MM-CK unfolded for various periods in different denaturant concentrations was pulsed-labeled with deuterium to identify different conformational intermediate states. For all denaturation times or GdmHCl concentrations, we observed variable proportions of only two species. The low-mass envelope of isotope peaks corresponds to a species that has gained about 10 deuteriums more than native CK, and the high-mass envelope to a completely deuterated species. To localize precisely the unfolded regions in the states highly populated during denaturation, the protein was digested with two proteases (pepsin and type XIII protease) after H/D exchange and rapid quenching of the reaction. The two sets of fragments obtained were analyzed by liquid chromatography coupled to mass spectrometry to determine the deuterium level in each fragment. Bimodal distributions of deuterium were found for most peptides, indicating that these regions were either folded or unfolded. This behavior is consistent with cooperative, localized unfolding. However, we observed a monomodal distribution of deuterium in two regions (1,12 and 162,186). We conclude that the increment of mass observed in the low-mass species of the intact protein (+10,Da) has its origin in these two segments. These regions, which are very sensitive to low GdmHCl concentrations, are involved in the monomer,monomer interface of CK and their perturbation is likely to weaken the dimeric structure. At higher denaturant concentration, this would induce dissociation of the dimer. Copyright © 2005 John Wiley & Sons, Ltd. [source] Use of different proteases working in acidic conditions to improve sequence coverage and resolution in hydrogen/deuterium exchange of large proteinsRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 21 2003Laetitia Cravello The combination of hydrogen exchange and mass spectrometry has been widely used in structural biology, providing views on protein structure and protein dynamics. One of the constraints is to use proteases working at low pH and low temperature to limit back-exchange during proteolysis. Although pepsin works in these conditions and is currently used in such experiments, sequence coverage is not always complete especially for large proteins, and the spatial resolution of the exchange rate is limited by the size of the resulting peptides. In this study we tried two other proteases, protease type XIII from Aspergillus saitoi and protease type XVIII from Rhizhopus species. The penicillin-binding protein X (PBP-2X*), a 77-kDa protein, was selected as a model. Like pepsin, neither of these proteases is really specific, but we found very good reproducibility in the digestion pattern. Compared with using pepsin alone, combining the results of the three independent proteolyses increased the coverage for the peptide mapping, thus avoiding missing some potentially interesting regions of the protein. Furthermore, we obtained a better spatial resolution for deuterium incorporation data, specifying accurately the deuterated regions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Investigation of Protein,Ligand Interactions by Mass SpectrometryCHEMMEDCHEM, Issue 4 2007Andrea Sinz Prof. Abstract The rate of drug discovery is greatly dependent on the development and improvement of rapid and reliable analytical methods that allow screening for protein,ligand interactions. The solution-based methods for investigating protein,ligand interactions by mass spectrometry (MS), which are discussed in this paper, are hydrogen/deuterium exchange of protein backbone amide hydrogens, and photoaffinity labeling. Moreover, MS analysis of intact noncovalent protein,ligand complexes is described. Fourier transform ion cyclotron resonance mass spectrometry (FTICR,MS) with its ultra-high resolution and excellent mass accuracy is also considered herein as it is gaining increasing popularity for a mass spectrometric investigation of protein,ligand interactions. [source] Syntheses of deuterated jasmonates for mass spectrometry and metabolism studiesJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 11 2005Patrycja W. Galka Abstract Jasmonic acid and its metabolites play an essential role in the regulation of plant development and systemic defense responses. Isotopically labeled standards are required to quantify plant hormones for metabolism studies using mass spectrometry. A convenient method for the preparation of deuterated analogs of jasmonates is demonstrated. Modification of commercially available methyl jasmonate by base-catalyzed proton/deuterium exchange or Wittig reaction introduces either two or three heavy atoms into a molecule. Copyright © 2005 John Wiley & Sons, Ltd. [source] | ||||||||||