Hydrogen-Deuterium Exchange

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John R Engen - One of the best experts on this subject based on the ideXlab platform.

  • recommendations for performing interpreting and reporting hydrogen deuterium Exchange mass spectrometry hdx ms experiments
    Nature Methods, 2019
    Co-Authors: Glenn R Masson, John E Burke, Walter S Englander, John R Engen, Natalie G Ahn, Ganesh S Anand, Christoph H Borchers, Sebastien Brier, George M Bouassaf, Johan H Faber
    Abstract:

    Hydrogen deuterium Exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

  • kras g12c drug development discrimination between switch ii pocket configurations using hydrogen deuterium Exchange mass spectrometry
    Structure, 2017
    Co-Authors: Rane A Harrison, John R Engen, Mei Zeng, Sudershan R Gondi, David L Scott, Nathanael S Gray, Kenneth D Westover
    Abstract:

    KRAS G12C, the most common RAS mutation found in non-small-cell lung cancer, has been the subject of multiple recent covalent small-molecule inhibitor campaigns including efforts directed at the guanine nucleotide pocket and separate work focused on an inducible pocket adjacent to the switch motifs. Multiple conformations of switch II have been observed, suggesting that switch II pocket (SIIP) binders may be capable of engaging a range of KRAS conformations. Here we report the use of hydrogen/deuterium-Exchange mass spectrometry (HDX MS) to discriminate between conformations of switch II induced by two chemical classes of SIIP binders. We investigated the structural basis for differences in HDX MS using X-ray crystallography and discovered a new SIIP configuration in response to binding of a quinazoline chemotype. These results have implications for structure-guided drug design targeting the RAS SIIP.

  • hydrogen deuterium Exchange mass spectrometry for probing higher order structure of protein therapeutics methodology and applications
    Drug Discovery Today, 2014
    Co-Authors: Hui Wei, Li Tao, Reb J Russell, Adrienne A Tymiak, Guodong Chen, Roxana E Iacob, John R Engen
    Abstract:

    The higher order structure of protein therapeutics can be interrogated with hydrogen/deuterium Exchange mass spectrometry (HDX-MS). HDX-MS is now a widely used tool in the structural characterization of protein therapeutics. In this review, HDX-MS based workflows designed for protein therapeutic discovery and development processes are presented, focusing on the specific applications of epitope mapping for protein/drug interactions and biopharmaceutical comparability studies. Future trends in the application of HDX-MS in protein therapeutics characterization are also described.

  • analysis of overlapped and noisy hydrogen deuterium Exchange mass spectra
    Journal of the American Society for Mass Spectrometry, 2013
    Co-Authors: Miklos Guttman, John R Engen, David D Weis, Kelly K Lee
    Abstract:

    Noisy and overlapped mass spectrometry data hinder the sequence coverage that can be obtained from hydrogen deuterium Exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have addressed these limits, but as the complexity of the biological samples under investigation increases, these problems are re-encountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining Exchange data for 42 additional peptides, improving the sequence coverage by 11 %. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2.

  • the utility of hydrogen deuterium Exchange mass spectrometry in biopharmaceutical comparability studies
    Journal of Pharmaceutical Sciences, 2011
    Co-Authors: Steven A Berkowitz, Damian Houde, John R Engen
    Abstract:

    The function, efficacy, and safety of protein biopharmaceuticals are tied to their three-dimensional structure. The analysis and verification of this higher-order structure are critical in demonstrating manufacturing consistency and in establishing the absence of structural changes in response to changes in production. It is, therefore, essential to have reliable, high-resolution and high sensitivity biophysical tools capable of interrogating protein structure and conformation. Here, we demonstrate the use of hydrogen/deuterium Exchange mass spectrometry (H/DX-MS) in biopharmaceutical comparability studies. H/DX-MS measurements can be conducted with good precision, consume only picomoles of protein, interrogate nearly the entire molecule with peptide level resolution, and can be completed in a few days. Structural comparability or lack of comparability was monitored for different preparations of interferon-β-1a. We present specific graphical formats for the display of H/DX-MS data that aid in rapidly making both the qualitative (visual) and quantitative assessment of comparability. H/DX-MS is capable of making significant contributions in biopharmaceutical characterization by providing more informative and confidant comparability assessments of protein higher-order structures than are currently available within the biopharmaceutical industry.

Miklos Guttman - One of the best experts on this subject based on the ideXlab platform.

  • imidazolium compounds as internal Exchange reporters for hydrogen deuterium Exchange by mass spectrometry
    Analytical Chemistry, 2020
    Co-Authors: Taylor A Murphree, Clint Vorauer, Marie Brzoska, Miklos Guttman
    Abstract:

    Hydrogen-Deuterium Exchange mass spectrometry (HDX-MS) is a powerful tool for protein structure analysis that is well suited for biotherapeutic development and characterization. Because HDX is strongly dependent on solution conditions, even small variations in temperature or pH can have a pronounced effect on the observed kinetics that can manifest in significant run-to-run variability and compromise reproducibility. Recent attention has been given to the development of internal Exchange reporters (IERs), which directly monitor changes to Exchange reaction conditions. However, the currently available small peptide IERs are only capable of sampling a very narrow temporal window and are understood to exhibit complex solution dependent Exchange behavior. Here we demonstrate the use of imidazolium carbon acids as superior IERs for HDX-MS. These compounds exhibit predictable Exchange behavior under a wide variety of reaction conditions, are highly stable, and can be readily modified to Exchange over a broad temporal window. The use of these compounds as IERs for solution based HDX-MS could considerably extend the utility of the technique by allowing for more robust empirical Exchange correction, thereby improving reproducibility.

  • high precision gas phase hydrogen deuterium Exchange kinetics by mass spectrometry enabled by Exchange standards
    Analytical Chemistry, 2020
    Co-Authors: Sanjit S Uppal, Abhigya Mookherjee, Rick Harkewicz, Sarah E Beasley, Matthew F Bush, Miklos Guttman
    Abstract:

    Mass spectrometry (MS) has become a primary tool for identifying and quantifying biological molecules. In combination with other orthogonal techniques, such as gas-phase hydrogen/deuterium Exchange...

  • gas phase hydrogen deuterium Exchange for distinguishing isomeric carbohydrate ions
    Analytical Chemistry, 2017
    Co-Authors: Sanjit S Uppal, Sarah E Beasley, Michele Scian, Miklos Guttman
    Abstract:

    The structural diversity of carbohydrates presents a major challenge for glycobiology and the analysis of glycoconjugates. Mass spectrometry has become a primary tool for glycan analysis thanks to its speed and sensitivity, but the information content regarding the glycan structure of protonated glycoconjugates is hindered by the inability to differentiate linkage and stereoisomers. Here, we examine a variety of protonated carbohydrate structures by gas-phase hydrogen/deuterium Exchange (HDX) to discover that the Exchange rates are distinct for isomeric carbohydrates with even subtle structural differences. By incorporating an internal Exchange standard, HDX could effectively distinguish all linkage and stereoisomers that were examined and presents a mass spectrometry-based approach for glycan structural analysis with immense potential.

  • analysis of overlapped and noisy hydrogen deuterium Exchange mass spectra
    Journal of the American Society for Mass Spectrometry, 2013
    Co-Authors: Miklos Guttman, John R Engen, David D Weis, Kelly K Lee
    Abstract:

    Noisy and overlapped mass spectrometry data hinder the sequence coverage that can be obtained from hydrogen deuterium Exchange analysis, and places a limit on the complexity of the samples that can be studied by this technique. Advances in instrumentation have addressed these limits, but as the complexity of the biological samples under investigation increases, these problems are re-encountered. Here we describe the use of binomial distribution fitting with asymmetric linear squares regression for calculating the accurate deuterium content for mass envelopes of low signal or that contain significant overlap. The approach is demonstrated with a test data set of HIV Env gp140 wherein inclusion of the new analysis regime resulted in obtaining Exchange data for 42 additional peptides, improving the sequence coverage by 11 %. At the same time, the precision of deuterium uptake measurements was improved for nearly every peptide examined. The improved processing algorithms also provide an efficient method for deconvolution of bimodal mass envelopes and EX1 kinetic signatures. All these functions and visualization tools have been implemented in the new version of the freely available software, HX-Express v2.

Patrick R. Griffin - One of the best experts on this subject based on the ideXlab platform.

  • differential isotopic enrichment to facilitate characterization of asymmetric multimeric proteins using hydrogen deuterium Exchange mass spectrometry
    Analytical Chemistry, 2015
    Co-Authors: Devrishi Goswami, Bruce D. Pascal, Steve Tuske, Joseph D Bauman, D Patel, Eddy Arnold, Patrick R. Griffin
    Abstract:

    Hydrogen/deuterium Exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein–ligand and protein–protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched (15N-labeled) p51 and unlabeled p66. To enable detection of 15N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100%...

  • differential hydrogen deuterium Exchange mass spectrometry analysis of protein ligand interactions
    Expert Review of Proteomics, 2011
    Co-Authors: Michael J. Chalmers, Bruce D. Pascal, Scott A Busby, Graham M West, Patrick R. Griffin
    Abstract:

    Functional regulation of ligand-activated receptors is driven by alterations in the conformational dynamics of the protein upon ligand binding. Differential hydrogen/deuterium Exchange (HDX) coupled with mass spectrometry has emerged as a rapid and sensitive approach for characterization of perturbations in conformational dynamics of proteins following ligand binding. While this technique is sensitive to detecting ligand interactions and alterations in receptor dynamics, it also can provide important mechanistic insights into ligand regulation. For example, HDX has been used to determine a novel mechanism of ligand activation of the nuclear receptor peroxisome proliferator activated receptor-γ, perform detailed analyses of binding modes of ligands within the ligand-binding pocket of two estrogen receptor isoforms, providing insight into selectivity, and helped classify different types of estrogen receptor-α ligands by correlating their pharmacology with the way they interact with the receptor based solely on hierarchical clustering of receptor HDX signatures. Beyond small-molecule–receptor interactions, this technique has also been applied to study protein–protein complexes, such as mapping antibody–antigen interactions. In this article, we summarize the current state of the differential HDX approaches and the future outlook. We summarize how HDX analysis of protein–ligand interactions has had an impact on biology and drug discovery.

  • hydrogen deuterium Exchange reveals distinct agonist partial agonist receptor dynamics within vitamin d receptor retinoid x receptor heterodimer
    Structure, 2010
    Co-Authors: Jun Zhang, Michael J. Chalmers, Bruce D. Pascal, Keith R Stayrook, Lorri L Burris, Ruben D Garciaordonez, Thomas P Burris, Jeffery A Dodge, Patrick R. Griffin
    Abstract:

    Regulation of nuclear receptor (NR) activity is driven by alterations in the conformational dynamics of the receptor upon ligand binding. Previously, we demonstrated that hydrogen/deuterium Exchange (HDX) can be applied to determine novel mechanism of action of PPARγ ligands and in predicting tissue specificity of selective estrogen receptor modulators. Here, we applied HDX to probe the conformational dynamics of the ligand binding domain (LBD) of the vitamin D receptor (VDR) upon binding its natural ligand 1α,25-dihydroxyvitamin D3 (1,25D3), and two analogs, alfacalcidol and ED-71. Comparison of HDX profiles from ligands in complex with the LBD with full-length receptor bound to its cognate receptor retinoid X receptor (RXR) revealed unique receptor dynamics that could not be inferred from static crystal structures. These results demonstrate that ligands modulate the dynamics of the heterodimer interface as well as provide insight into the role of AF-2 dynamics in the action of VDR partial agonists.

  • P.R.: Dynamics of the β2-Adrenergic G-Protein Coupled Receptor Revealed by Hydrogen–Deuterium Exchange
    2010
    Co-Authors: Xi Zhang, Ellen Y. T. Chien, Michael J. Chalmers, Bruce D. Pascal, Jovylyn Gatchalian, Raymond C. Stevens, Patrick R. Griffin
    Abstract:

    To examine the molecular details of ligand activation of G-protein coupled receptor (GPCRs), emphasis has been placed on structure determination of these receptors with stabilizing ligands. Here we present the methodology for receptor dynamics characterization of the GPCR human β2 adrenergic receptor bound to the inverse agonist carazolol using the technique of amide hydrogen/ deuterium Exchange coupled with mass spectrometry (HDX MS). The HDX MS profile of receptor bound to carazolol is consistent with thermal parameter observations in the crystal structure and provides additional information in highly dynamic regions of the receptor and chemical modifications demonstrating the highly complementary nature of the techniques. Following optimization of HDX experimental conditions for this membrane protein, better than 89 % sequence coverage was obtained for the receptor. The methodology presented paves the way for future analysis of β2AR bound to pharmacologically distinct ligands as well as analysis of other GPCR family members. Keywords β2-adrenergic receptor; G-protein coupled receptor; H/D Exchange; mass spectrometry; membrane protein

  • probing protein ligand interactions by automated hydrogen deuterium Exchange mass spectrometry
    Analytical Chemistry, 2006
    Co-Authors: Michael J. Chalmers, Bruce D. Pascal, Scott A Busby, Christopher L Hendrickson, Alan Marshall G And, Patrick R. Griffin
    Abstract:

    Amide hydrogen/deuterium Exchange is a powerful biophysical technique for probing changes in protein dynamics induced by ligand interaction. The inherent low throughput of the technology has limited its impact on drug screening and lead optimization. Automation increases the throughput of H/D Exchange to make it compatible with drug discovery efforts. Here we describe the first fully automated H/D Exchange system that provides highly reproducible H/D Exchange kinetics from 130 ms to 24 h. Throughput is maximized by parallel sample processing, and the system can run H/D Exchange assays in triplicate without user intervention. We demonstrate the utility of this system to differentiate structural perturbations in the ligand-binding domain (LBD) of the nuclear receptor PPARγ induced upon binding a full agonist and a partial agonist. PPARγ is the target of glitazones, drugs used for treatment of insulin resistance associated with type II diabetes. Recently it has been shown that partial agonists of PPARγ have ...

John E Burke - One of the best experts on this subject based on the ideXlab platform.

  • recommendations for performing interpreting and reporting hydrogen deuterium Exchange mass spectrometry hdx ms experiments
    Nature Methods, 2019
    Co-Authors: Glenn R Masson, John E Burke, Walter S Englander, John R Engen, Natalie G Ahn, Ganesh S Anand, Christoph H Borchers, Sebastien Brier, George M Bouassaf, Johan H Faber
    Abstract:

    Hydrogen deuterium Exchange mass spectrometry (HDX-MS) is a powerful biophysical technique being increasingly applied to a wide variety of problems. As the HDX-MS community continues to grow, adoption of best practices in data collection, analysis, presentation and interpretation will greatly enhance the accessibility of this technique to nonspecialists. Here we provide recommendations arising from community discussions emerging out of the first International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX; 2017). It is meant to represent both a consensus viewpoint and an opportunity to stimulate further additions and refinements as the field advances.

  • an overview of hydrogen deuterium Exchange mass spectrometry hdx ms in drug discovery
    Expert Opinion on Drug Discovery, 2017
    Co-Authors: Glenn R Masson, Meredith L Jenkins, John E Burke
    Abstract:

    Introduction: Hydrogen deuterium Exchange mass spectrometry (HDX-MS) is a powerful methodology to study protein dynamics, protein folding, protein-protein interactions, and protein small molecule i...

  • probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium Exchange ms hdx ms
    Biochemical Society Transactions, 2015
    Co-Authors: Oscar Vadas, John E Burke
    Abstract:

    Many cellular signalling events are controlled by the selective recruitment of protein complexes to membranes. Determining the molecular basis for how lipid signalling complexes are recruited, assembled and regulated on specific membrane compartments has remained challenging due to the difficulty of working in conditions mimicking native biological membrane environments. Enzyme recruitment to membranes is controlled by a variety of regulatory mechanisms, including binding to specific lipid species, protein–protein interactions, membrane curvature, as well as post-translational modifications. A powerful tool to study the regulation of membrane signalling enzymes and complexes is hydrogen deuterium Exchange–MS (HDX–MS), a technique that allows for the interrogation of protein dynamics upon membrane binding and recruitment. This review will highlight the theory and development of HDX–MS and its application to examine the molecular basis of lipid signalling enzymes, specifically the regulation and activation of phosphoinositide 3-kinases (PI3Ks). * ABD, : adaptor-binding domain; ECD, : electron capture dissociation; ETD, : electron transfer dissociation; Gβγ, : G protein βγ heterodimers; GPCR, : G-protein-coupled receptor; HDX–MS, : hydrogen deuterium Exchange–MS; PI3K, : phosphoinositide 3 kinases; PIK3CA, : gene encoding for the p110α catalytic subunit of PI3K; PIP3, : phosphatidylinositol 3,4,5 trisphosphate; PLA2, : phospholipase A2; RTK, : receptor tyrosine kinase; SH2, : Src-homology 2, inter SH2 coiled coil (iSH2), n-terminal SH2 (nSH2), c-terminal SH2, phosphatase and tensin homolog (PTEN)

  • using hydrogen deuterium Exchange mass spectrometry to define the specific interactions of the phospholipase a2 superfamily with lipid substrates inhibitors and membranes
    Journal of Biological Chemistry, 2013
    Co-Authors: Jian Cao, John E Burke, Edward A Dennis
    Abstract:

    The phospholipase A2 (PLA2) superfamily consists of 16 groups and many subgroups and constitutes a diverse set of enzymes that have a common catalytic activity due to convergent evolution. However, different PLA2 types have unique three-dimensional structures and catalytic residues as well as specific tissue localization and distinct biological functions. Understanding how the different PLA2 enzymes associate with phospholipid membranes, specific phospholipid substrate molecules, and inhibitors on a molecular basis has advanced in recent years due to the introduction of hydrogen/deuterium Exchange mass spectrometry. Its theory, practical considerations, and application to understanding PLA2/membrane interactions are addressed.

Sanjit S Uppal - One of the best experts on this subject based on the ideXlab platform.

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