Hydrogen deuterium exchange mass spectrometry hdxms is a powerful. Hydra hydrogen deuterium exchange a software package that supports both traditional and exploratory treatments of hdxms data. Hydrogendeuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between il23 and adnectin 2 and to determine the correlation between the two. Hydrogendeuterium hd exchange catalysis in alkanes. Any data in x,y format can be analyzed or spectral data can be directly imported from waters masslynx software. The resulting labeled product is characterized by 1 h nmr. Hydrogendeuterium and 16o18o exchange mass spectrometry can. Two cold zones one for labeling and one for quenching. Dual studies on a hydrogendeuterium exchange of resorcinol.
Hydrogendeuterium exchange coupled to top and middledown. The use of hydrogendeuterium exchange mass spectrometry. Hydrogen deuterium exchange hdx is an experimental technique to obtain structural data on proteins. Hydrogen or deuterium exchange principles and applications of nmr spectroscopy.
The labeling approach involves hydrogen deuterium exchange hdex of protein backbone amide hydrogens with rapid and sensitive detection by mass spectrometry ms. The use of hydrogendeuterium exchange mass spectrometry in. Proton exchange organic spectroscopy part7 duration. Hydrogen deuterium exchangemass spectrometry hdxms has emerged as a powerful technique for interrogating the. An efficient laboratory experiment has been developed for undergraduate students to conduct hydrogendeuterium hd exchange of resorcinol by electrophilic aromatic substitution using d 2o and a catalytic amount of h 2so 4. Spectral density function rotational diffusion in solution occurs at a range of frequencies 1.
Hydrogen deuterium exchange hdxms when diluted in heavy water d2o, backbone hydrogens of amino acids exchange with deuterium at varying kinetics rates depending upon their hydrogen bonding and solvent accessibility. Aug 12, 2014 an efficient laboratory experiment has been developed for undergraduate students to conduct hydrogendeuterium hd exchange of resorcinol by electrophilic aromatic substitution using d 2 o and a catalytic amount of h 2 so 4. Hydrogen deuterium exchange with mass spec and hdx ms. Spectra are acquired over a range of 3501,500 mz with a scanning rate. These studies helped elucidate the mechanisms by which alkane ch bonds interact with catalytic materials. The catalytic exchange of hydrogen and deuterium hd exchange in light alkanes has been studied for almost a century. Hydrogen deuterium exchange hdx coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. It was then determined that the basecatalyzed reaction dominates near or above physiological conditions ph 58, and the slowest exchange occurs at.
Hydrogen deuterium exchange hdx thermo fisher scientific us. Well established techniques, such as xray crystallography and nmr, determine protein structure. University of singapore, and a waters center of innovation program honoree. Frontiers applications of hydrogen deuterium exchange. Amide hydrogendeuterium hd exchange of proteins monitored by mass spectrometry has established itself as a powerful method for probing protein conformational dynamics and protein interactions. Discover a robust hydrogen deuterium exchange method for the analysis of. Deuterium has a natural abundance in earths oceans of about one atom in 6420 of hydrogen. Acquisition of pepsin and protein only spectra note. Integrated software platform for analyzing hydrogen. This project applies hydrogen deuterium exchange mass spectrometry hdxms for determining the dynamical structure of therapeutic antibodies, glycoproteins, and proteins.
Hydrogendeuterium exchange coupled to top and middle. Equipment for exchanging hydrogen and deuterium, thermal hotels manufactured by mecour. Creative biolabs has used hydrogendeuterium hd exchange on a research scale for many years and with the advancement of sample handling and data analysis software, now we are dedicated to offering our clients cremap amide hydrogendeuterium hd exchange platform for epitope mapping. The more accessible portions of the protein exchange rapidly compared to the less exposed parts which may be buried in the center of a protein or involved in hydrogen bonding. So im trying to explain why it is that the emission lines are shifted and how they are shifted. Hydrogen deuterium exchange also called hd or hd exchange is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa. Amide hydrogen atoms can readily exchange at this intrinsic rate with solvent deuterium if they are accessible to the solvent and free from intramolecular or intermolecular hydrogen bonding, as occurs in. However, bridging the gap between commercial hdxms software for. Any data in x, y format can be analyzed or spectral data can be directly imported from waters masslynx software. Acquity uplc mclass system whydrogen deuterium exchange hdx technology and highresolution ms identifies changes in protein conformation with. The atomic spectra of hydrogen and deuterium are similar however shifted in energies.
Performing hydrogendeuterium exchange with mass spectrometry. Hydrogendeuterium exchange also called hd or hd exchange is a chemical reaction in which a covalently bonded hydrogen atom is replaced by a deuterium atom, or vice versa. The advantages of a unified platform include code reuse, faster learning time, and integrated reports. Topics of particular interest include proteinligand interactions, structurefunction relationships between glycan structure and glycoprotein folding, and. Hydrogendeuterium exchange mass spectrometry springerlink.
Hdx as a technique was pioneered by lindstrom lang in the 1940s. Hydrogen deuterium exchange mass spectrometry hdx ms has become an important technique for the analysis of protein structure and dynamics. The rate of hydrogentodeuterium exchange within the amide hydrogen on the backbone of biotherapeutics provides solvent accessibility information and thus protein structure and. The rate of deuterium incorporation is characteristic of local structure and dynamics. Timeresolved electrospray ionization hydrogen deuterium exchange. Hydrogendeuterium exchange mass spectrometry hdx ms has become an important technique for the analysis of protein structure and dynamics.
A nondestructive infrared ir spectroscopy assay to measure hydrogen deuterium exchange hdx in surfaceadsorbed protein monolayers has been developed. Therefore, the ability of mass spectrometry msbased hydrogen deuterium exchange hdex methods to examine protein structure and dynamics in a nearly unlimited set of solution conditions and protein concentrations fills an existing gap in protein analysis methods. Evaluation of different hydrogen deuterium exchange data processing software by looking at conformational changes induced by mab aggregation terry zhang,1 shanhua lin,2 stephane houel,1 jennifer sutton,1 xiaodong liu2 and jonathan josephs1 1thermo fisher scienti. An automated approach for the rapid analysis of protein structure has been developed and used to study acidinduced conformational changes in human growth hormone. Timeresolved electrospray ionization hydrogendeuterium exchange acquisition of pepsin and protein only spectra note. Hydrogen deuterium exchange coupled to mass spectrometry hdxms is a technique used to monitor the structure and dynamics of proteins in solution. By following the isotopic labeling of amide protons, which largely depends on their solvent accessibility and dynamics, hydrogendeuterium exchange coupled to mass spectrometry hdxms is now an established method for investigating ligand binding, proteinprotein interactions, protein dynamics, conformational changes and foldingunfolding. Hdx workbench is an established feature rich software platform for the analysis of hydrogendeuterium exchange hdx mass spectrometry data from waters or thermo instruments. Nov 16, 2015 in highly dynamic unstructured regions, the exchange reaction proceeds in milliseconds to seconds, whereas hydrogen bonded amide hydrogens exchange much slower minutes to days. It can be applied most easily to exchangeable protons and deuterons, where such a transformation occurs in the presence of a suitable deuterium source, without any.
However, the analysis of hdx by matrixassisted laser. With over a decade specializing in mass spec characterization of protein complexes, covalx offers unique expertise in the. Perfect for universities and small labs wishing to get into hdx without a major investment. For higher order structure studies, hydrogen deuterium exchange with mass. Hydrogen deuterium exchangemass spectrometry hdxms has emerged as a powerful technique for interrogating the conformational dynamics of proteins and their complexes. An integrated solution for hydrogendeuterium exchange. Sep 05, 2016 hydrogen or deuterium exchange principles and applications of nmr spectroscopy. Optimization of a fully automated hydrogendeuterium exchange. Recommendations for performing, interpreting and reporting. Our approach is based on the characterization of compounds by the number of labile hydrogens and oxygens atoms in the molecule which can be measured using hydrogen deuterium and 16o18o exchange approaches. Timeresolved electrospray ionization hydrogendeuterium. Hxexpress2 is microsoft excelbased software for semiautomated analysis of hydrogen exchange mass spectrometry data. Label, quench, digest and separate all in one convenient package and all inline. Sophisticated computer analysis is required to scan through the voluminous ms output in order to find, identify, and validate many partially deuterated peptides, elicit the hdx information, and extend.
Integrated software platform for analyzing hydrogendeuterium. When the antigen is complexed with the antibody, this deuterium uptake rate is altered in the epitope regions. Hydras software architecture tolerates flexible data analysis procedures by allowing the addition of new algorithms without significant change to the underlying code base. Currently, analysis of hdxms data remains a laborious procedure, mainly due to the lack of streamlined software to process the large datasets. Hydrogen deuterium exchange mass spectrometry hdxms is a powerful and sensitive technique that can probe the higher order structure of proteins and has been used in the assessment and development of monoclonal antibodies mabs, antibodydrug conjugates adcs and biosimilar antibodies. Data analysis software has progressed markedly, increasing. Protein interactions probed by mass spectrometric techniques. Cremap epitope mapping service by hydrogendeuterium. By following the isotopic labeling of amide protons, which largely depends on their solvent accessibility and dynamics, hydrogen deuterium exchange coupled to mass spectrometry hdxms is now an established method for investigating ligand binding, proteinprotein interactions, protein dynamics, conformational changes and foldingunfolding. Hydrogen deuterium exchange hdx thermo fisher scientific jp. Hydrogen deuterium exchange hdx mass spec covalx offers unique analytical services for epitope mapping based on mass spectrometry. Hydrogen deuterium exchange hdx thermo fisher scientific ca.
Hd exchange coupled with mass spectrometry measures the deuteration rate. Since the nucleus of deuterium contains both a proton and a neutron its notably heavier than the nuclueus of hydrogen, which only contains a proton. Hydrogen deuterium exchange hdxor hdeis a mass spec technique which compares the amount of deuterium which remains bound to the backbone of protein fragments after. The nucleus of a deuterium atom, called a deuteron, contains one proton and one neutron, whereas the far more common protium has no neutron in the nucleus. With this technology, deuterium in a solution replaces the amide hydrogen in a biomolecules backbone, and ms measures these changes.
The rate of hydrogentodeuterium exchange within the amide hydrogen on the backbone of biotherapeutics provides solvent accessibility information and thus protein structure and conformation can be inferred. Development of software for deep analysis of protein dynamics. Hydrogen deuterium exchange with mass spec hdx ms the system. Hdxms reports on the local chemical environment and solvent accessibility of the protein backbone by monitoring the exchange of peptide bond amide protons with the deuterons of a d2o solvent. However, bridging the gap between commercial hdxms software for raw. Evaluation of different hydrogendeuterium exchange data. Applications of hydrogen deuterium exchange hdx for the characterization of conformational dynamics in lightactivated photoreceptors robert lindner 1, udo heintz 1 and andreas winkler 2 1 department of biomolecular mechanisms, max planck institute. The method uses isotope labeling to probe the rate at which protein backbone amide hydrogens undergo exchange. The byologic software platform was initially developed for sequence variant analysis, but has since been adapted for stress studies, biosimilarity, oxidative footprinting, and now hydrogendeuterium exchange. Hdxms reports on the local chemical environment and solvent accessibility of the protein backbone by monitoring the exchange of peptide bond amide.
Deuterium or hydrogen 2, symbol 2 h or d, also known as heavy hydrogen is one of two stable isotopes of hydrogen the other being protium, or hydrogen 1. Creative biolabs has used hydrogen deuterium hd exchange on a research scale for many years and with the advancement of sample handling and data analysis software, now we are dedicated to offering our clients cremap amide hydrogen deuterium hd exchange platform for epitope mapping. Hydrogendeuterium exchange mass spectrometry and computational methods were applied to probe the binding interactions between il23 and adnectin 2 and to. Hydrogen deuterium exchange hdx thermo fisher scientific cn.
Understanding hydrogen deuterium exchange hdx lcms. In an hdxms experiment, natively folded proteins are diluted into d 2 o, allowing accessible amide protons to be exchanged for deuterium atoms in the solvent. Exchange rates are particularly sensitive to changes in hydrogen bonding, secondary structure, solvent accessibility, and dynamics. The procedure can be used to measure hdx for different proteins on a variety of materials applied to goldcoated substrates. Labile hydrogens, such as those on primary amines and on the side chains, exchange nearly instantaneously.
Hydrogen deuterium exchange ms analysis for calmodulin. Obtained results can help understand how protein interacts with its ligand and elucidate the role of this complex in a living organism. The dynamics of protein changes is determined with the mz. Hydrogen deuterium exchange connected with mass spectrometry is increasingly applied for the interrogation of protein conformation, mapping protein dynamics, identification of proteinligand interaction sites, and allosteric conformation changes. Development of software for deep analysis of protein dynamics as determined by maldi mass spectrometrybased hydrogen deuterium exchange article december 2019 with. Jun 27, 2019 hydrogen deuterium exchange mass spectrometry hdxms is a powerful technique that can provide insights into protein behavior by serving as a link between structure, conformational dynamics and. From this measure, the higher order structure of the molecule, typically a protein, could be inferred. Although xray crystallography and other biochemical techniques are frequently used to study these interactions, there are limitations. Protein dynamics and conformational changes explored by. As depicted schematically above, when a protein is diluted 20fold in d 2 o buffer, labile hydrogen atoms on the molecule will exchange with deuterium.
The rate of hydrogen to deuterium exchange within the amide hydrogen on the backbone of biotherapeutics provides solvent accessibility information and thus protein structure and. Hydrogen deuterium exchange hdx mass spectrometry ms is a powerful tool for studying the dynamics of higher order structure of proteinbased therapeutics. Methods of screening small molecule fragments having a low binding affinity to a target protein, to identify fragments of interest or to identify binding sites of interest within a target protein, are described in which ligandpeptide binding index lpbi values are assigned to test peptides, and composite lpbi values are determined, by which small molecule fragments with composite lpbi value. An affordable automated hydrogen deuterium exchange for mass spectrometry used as inlet to any brand of mass spectrometer. While alkanes and their ch bonds are relatively inert, hd exchange studies have shown that a large number of materials can catalytically activate these bonds. It can be applied most easily to exchangeable protons and deuterons, where such a transformation occurs in the presence of a suitable deuterium source, without any catalyst.
Hydrogen deuterium exchange mass spectrometry hdxms is a powerful technique that can provide insights into protein behavior by serving as. How hydrogen exchange mass spectrometry can help investigate protein higher order structure. Data analysis remains a bottleneck in the workflow. May 16, 2018 hydrogen deuterium exchange hdx lcms analysis is a workflow that is increasingly used to increase the knowledge of biotherapeutics and biosimilars, find out how this analysis is performed, the. Rapid analysis of protein structure and dynamics by hydrogen. A microsoft excelbased software for semiautomated analysis of hydrogen exchange mass spectrometry data. The byologic software platform was initially developed for sequence variant analysis, but has since been adapted for stress studies, biosimilarity, oxidative footprinting, and now hydrogen deuterium exchange. Hydrogendeuterium exchange mass spectrometry hdxms is a powerful approach for mapping protein folding, proteinligand interactions, proteinprotein interactions, and conformational changes. Output is deuterium uptake curves and peakwidth plots. The hydrogen deuterium exchange hdx ms analysis is used to measure changes in protein conformation. The resulting labeled product is characterized by 1h nmr. Hydrogendeuterium exchange software tools omictools. Analysis and visualisation of hydrogendeuterium exchange mass spectrometry data.
Wo2012166056a1 fragmentbased approach for inhibitor design. Hydrogendeuterium exchange mass spectrometry laboratory. The goal of this application note is to determine the conformational changes between apo and holocalmodulin using hydrogen deuterium exchange combined with high resolution ms. Hydrogen deuterium exchange mass spectrometry hdxms is a structural technique which has garnered attention for its ability to assess proteinprotein and. Hdx mass spectrometry is confounded by deuterium exchange associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon. The process is accelerated by the addition of an acid or base. The unprecedented systemlevel design integrates all the steps necessary to accurately, reproducibly and most of all, easily perform protein conformation studies. Biopharma finder software automates previously timeconsuming manual. Development of software for deep analysis of protein. The samples can be sorted by sample evaluation of different hydrogendeuterium exchange data processing software by looking at conformational changes induced by mab aggregation terry zhang 1, shanhua lin2, scientific 4 ms. Wo2012166056a1 fragmentbased approach for inhibitor. Hydrogen deuterium exchange ms analysis for calmodulin protein conformation author. Hydrogen deuterium exchange hdxms solution for the budget conscious lab. Cremap epitope mapping service by hydrogendeuterium exchange.
Hydrogen deuterium exchange hdx ms works in many applications, including understanding how smallmolecule therapeutics bind to protein targets, and performing epitope mapping. Hydrogen deuterium exchange with mass spec hdx ms instrumentation. It relies on the accurate measurement of the degree of labeling of the protein by deuterated hydrogen during a precisely measured labeling interval. Finally, data is analyzed in hdexaminer software with full report generation using easy to understand hdx heat map descriptions and personal. Hydrogen deuterium exchange data are processed with selected software and finally quaternary structure of protein is visualized showing how different protein and ligand chains hook up with each other.
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