Hydrogen-deuterium Exchange Methods Research Articles

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Developments in rapid hydrogen-deuterium exchange methods.

Biological macromolecules, such as proteins, nucleic acids, and carbohydrates, contain heteroatom-bonded hydrogens that undergo exchange with solvent hydrogens on timescales ranging from microseconds to hours. In hydrogen-deuterium exchange mass spectrometry (HDX-MS), this exchange process is used to extract information about biomolecular structure and dynamics. This minireview focuses on millisecond timescale HDX-MS measurements, which, while less common than 'conventional' timescale (seconds to hours) HDX-MS, provide a unique window into weakly structured species, weak (or fast cycling) binding interactions, and subtle shifts in conformational dynamics. This includes intrinsically disordered proteins and regions (IDPs/IDRs) that are associated with cancer and amyloidotic neurodegenerative disease. For nucleic acids and carbohydrates, structures such as isomers, stems, and loops, can be elucidated and overall structural rigidity can be assessed. We will provide a brief overview of technical developments in rapid HDX followed by highlights of various applications, emphasising the importance of broadening the HDX timescale to improve throughput and to capture a wider range of function-relevant dynamic and structural shifts.

Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c.

Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrome c (cyt c). Compared to the Fe(II) protein, Fe(III) cyt c shows enhanced deuteration on both the distal and proximal sides of the heme. Previous studies have attributed the enhanced dynamics of Fe(III) cyt c to the facile and reversible rupture of the distal M80-Fe(III) bond. Using molecular dynamics (MD) simulations, we conducted a detailed analysis of various cyt c conformers. Our MD data confirm that rupture of the M80-Fe(III) contact triggers major reorientation of the distal Ω loop. Surprisingly, this event takes place with only miniscule H-bonding alterations. In other words, the distal loop dynamics are almost "HDX-silent". Moreover, distal loop movements cannot account for enhanced dynamics on the opposite (proximal) side of the heme. Instead, enhanced deuteration of Fe(III) cyt c is attributed to sparsely populated conformers where both the distal (M80) and proximal (H18) coordination bonds have been ruptured, along with opening of numerous H-bonds on both sides of the heme. We conclude that there can be major structural fluctuations that are only weakly coupled to changes in H-bonding, making them virtually impossible to track by HDX-MS. In such cases, HDX-MS may provide an incomplete view of protein dynamics.

Utilization of Hydrogen/Deuterium Exchange in Biopharmaceutical Industry

The development of biopharmaceutics is the fastest growing segment of the present pharmaceutical industry. The analysis of proteins therapeutics is a challenging task due to their large size and complexity of spatial structure. Any changes in the primary, secondary, tertiary or quaternary protein structure can have huge impact on their function, efficiency and toxicity. Mass spectrometry proved itself to be a powerful tool for analysis of primary protein structure (amino acid sequence) and thanks to the development of new techniques in last years it is able to analyse higher order protein structures. One of these new techniques is hydrogen/deuterium exchange (HDX). HDX is based on exchange of amid protons with deuterium from solution on the protein backbone chain. Protons on the surface of protein are exchanging with deuterium much faster than protons buried inside of protein. HDX results could provide information about spatial protein structure and also about protein-protein interactions and protein-ligand interactions. Furthermore, by analysing of deuterium exchange in different time points this method could give information about dynamic changes of protein structure and dynamics of proteins interactions. Because of possibilities of this method, HDX become attractive method for characterization of protein biopharmaceuticals. This review article is focused on the utilization of mass spectrometry in biopharmaceutical industry and mainly on HDX method and its applications.Key words: mass spectrometry - proteomics - protein conformation - drug discovery - drug industry - hydrogen/deuterium exchangeThe work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 23. 5. 2016Accepted: 10. 6. 2016.

Dual-spray hydrogen/deuterium exchange (HDX) reactions: A new method of probing protein structure.

Traditionally, hydrogen/deuterium exchange (HDX) reactions are done in the solution phase. This usually involves incubating the protein with a suitable deuterating agent then acidifying the solution to quench the reaction. A more efficient method may be to conduct the reaction within the ion source of a mass spectrometer and subsequently analyze the products. Using the two electrospray emitters equipped on the Waters Synapt G1 mass spectrometer, HDX reactions were conducted within the ion source region in a controlled fashion ('dual-spray'). Peptide and protein solutions were electrosprayed through one emitter and the deuterating agent D2 O through the secondary electrospray emitter. For the relatively small peptides, Phe-Leu-Glu-Glu-Leu and oxytocin, the yield of products was calculated using deconvolution functions. Electrospray ionization (ESI) charge-state distributions and average number of deuterium exchanges were used to probe secondary and tertiary structures of ubiquitin, lysozyme, and cytochrome c in their native and unfolded states. Clear shifts in isotope distributions indicated HDX occurring within the ion source. By ion mobility, simultaneous deuterium exchange for two isobaric species, the oxytocin monomer and dimer, was observed. For denatured ubiquitin, the 12+ and 13+ charge states have a lower average number of exchanges relative to the lower charge states which indicates that these charge states have segments which restrict the access of D2 O. Lysozyme has a linear relationship between the charge state and the average number of exchanges, indicating that lysozyme becomes increasingly unfolded as the charge state increases. The dual-spray HDX method was paired to high-performance liquid chromatography (HPLC) to demonstrate the applicability of the technique for probing gas-phase structures in protein mixtures. ESI droplets formed from a secondary emitter penetrate primary ESI droplets and change the solvent composition. Dual-spray HDX is demonstrated to be a more efficient method for probing the structure of proteins than solution-phase HDX since the acid quenching step can be surpassed. Copyright © 2016 John Wiley & Sons, Ltd.

Computational analyses of cryptic intermediates in the native unfolding pathways of barnase and thioredoxin

AbstractOwing to various experimental constraints, detection and characterization of cryptic intermediates (CIs) presumably existing in the unfolding kinetics of barnase and thioredoxin were not successful by native-state hydrogen-deuterium exchange method at pH 6.4, 306 K and pH 7.0, 298 K, respectively. We have herein demonstrated possible existence of CIs under the solution conditions in native unfolding of the proteins by means of OneG-Vali. Structure and stability of each CI detected for the two proteins by the computational tool have also been delineated and the results were in good agreement with folding intermediates detected in the energetic landscapes of the proteins by variety of methods as reported in the literature. Moreover, we have also discussed unique merits of the computational tool to addressing extent of contributions of cis/trans proline isomerization and CIs on estimating overall thermodynamic stabilities (by various types of equilibrium unfolding experiments) of the proteins.

Subzero temperature chromatography and top-down mass spectrometry for protein higher-order structure characterization: method validation and application to therapeutic antibodies.

Characterization of the higher-order structure and structural dynamics of proteins is crucial for in-depth understanding of their functions. Amide hydrogen/deuterium exchange (HDX), monitored by mass spectrometry (MS), is now a popular technique for measuring protein higher-order structural changes. Although the proteolysis-based HDX-MS approach is most commonly used, the "top-down" approach, which fragments intact proteins directly using electron-based dissociation, is becoming an important alternative and has several advantages. However, the commonly used top-down strategies are direct-infusion based and thus can only be used with volatile buffers. This has meant that the "top-down" approach could not be used for studying proteins under physiological conditions-the very conditions which are often very important for preserving a protein's native structure and function. More complex proteins such as those with disulfide bonds present another challenge. Therefore, there is significant interest in developing novel top-down HDX methods that are applicable to all types of protein samples. In this paper, we show how top-down electron capture dissociation and subzero temperature HPLC can be combined and used for this purpose. This method keeps the back-exchange level as low as 2% and has no limitations in terms of protein type and sample solution conditions. Close to single-residue level protein structural information can be generated. The new method is validated through comparison with NMR data using calmodulin as a model protein. Its capability of determining structural changes in therapeutic antibodies (Herceptin) is also demonstrated.

Peptide–Column Interactions and Their Influence on Back Exchange Rates in Hydrogen/Deuterium Exchange-MS

Hydrogen/deuterium exchange (HDX) methods generate useful information on protein structure and dynamics, ideally at the individual residue level. Most MS-based HDX methods involve a rapid proteolytic digestion followed by LC/MS analysis, with exchange kinetics monitored at the peptide level. Localizing specific sites of HDX is usually restricted to a resolution the size of the host peptide because gas-phase processes can scramble deuterium throughout the peptide. Subtractive methods may improve resolution, where deuterium levels of overlapping and nested peptides are used in a subtractive manner to localize exchange to smaller segments. In this study, we explore the underlying assumption of the subtractive method, namely, that the measured back exchange kinetics of a given residue is independent of its host peptide. Using a series of deuterated peptides, we show that secondary structure can be partially retained under quenched conditions, and that interactions between peptides and reversed-phase LC columns may both accelerate and decelerate residue HDX, depending upon peptide sequence and length. Secondary structure is induced through column interactions in peptides with a solution-phase propensity for structure, which has the effect of slowing HDX rates relative to predicted random coil values. Conversely, column interactions can orient random-coil peptide conformers to accelerate HDX, the degree to which correlates with peptide charge in solution, and which can be reversed by using stronger ion pairing reagents. The dependency of these effects on sequence and length suggest that subtractive methods for improving structural resolution in HDX-MS will not offer a straightforward solution for increasing exchange site resolution.

Hydrogen Exchange Mass Spectrometry of Bacteriorhodopsin Reveals Light-Induced Changes in the Structural Dynamics of a Biomolecular Machine

Many proteins act as molecular machines that are fuelled by a nonthermal energy source. Examples include transmembrane pumps and stator-rotor complexes. These systems undergo cyclic motions (CMs) that are being driven along a well-defined conformational trajectory. Superimposed on these CMs are thermal fluctuations (TFs) that are coupled to stochastic motions of the solvent. Here we explore whether the TFs of a molecular machine are affected by the occurrence of CMs. Bacteriorhodopsin (BR) is a light-driven proton pump that serves as a model system in this study. The function of BR is based on a photocycle that involves trans/cis isomerization of a retinal chromophore, as well as motions of transmembrane helices. Hydrogen/deuterium exchange (HDX) mass spectrometry was used to monitor the TFs of BR, focusing on the monomeric form of the protein. Comparative HDX studies were conducted under illumination and in the dark. The HDX kinetics of BR are dramatically accelerated in the presence of light. The isotope exchange rates and the number of backbone amides involved in EX2 opening transitions increase roughly 2-fold upon illumination. In contrast, light/dark control experiments on retinal-free protein produced no discernible differences. It can be concluded that the extent of TFs in BR strongly depends on photon-driven CMs. The light-induced differences in HDX behavior are ascribed to protein destabilization. Specifically, the thermodynamic stability of the dark-adapted protein is estimated to be 5.5 kJ mol(-1) under the conditions of our work. This value represents the free energy difference between the folded state F and a significantly unfolded conformer U. Illumination reduces the stability of F by 2.2 kJ mol(-1). Mechanical agitation caused by isomerization of the chromophore is transferred to the surrounding protein scaffold, and subsequently, the energy dissipates into the solvent. Light-induced retinal motions therefore act analogously to an internal heat source that promotes the occurrence of TFs. Overall, our data highlight the potential of HDX methods for probing the structural dynamics of molecular machines under "engine on" and "engine off" conditions.

A comparison of mass spectrometry based hydrogen deuterium exchange methods for probing the cyclophilin A cyclosporin complex

Direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques provide an increasingly popular route to determine quantitative information on protein–protein and protein–ligand interactions. When combined with hydrogen deuterium exchange (HDX), details on protein stability and complex conformation can be obtained; however, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting gas phase results. Zhu et al. [1] and Powell and Fitzgerald [2] have outlined LC–MS based techniques to probe the solution phase properties of the protein–ligand system in question. We here have taken the well characterised soluble immunophilin protein cyclophilin A, and examined it in complex with its endogenous ligand cyclosporin A. This ligand is widely used as an immunosuppressant following organ transplant, and the complex provides a basis for drug discovery efforts. We have used direct infusion, coupled with HDX, gas phase HDX and also the LC-HDX techniques PLIMSTEX and SUPREX. Results from each of these four HDX methodologies are presented here and discussed critically. From our direct infusion we find that there are 2 observable hydrogen populations in the protein, a very fast exchanging population, and a slower group. The exchange rate of both is lowered in the presence of the ligand. For PLIMSTEX we find a K d for ligand binding of 321 ± 128 nM, which is within one order of magnitude of values previously reported. SUPREX under a variety of conditions provides a range of K d values, but when we average these for experimental error we obtain a K d of 7.11 ± 0.29 nM which agrees well with measurements from other studies including via SUPREX. Finally gas phase HDX of the native complex shows more than 3 distinct populations of exchangeable hydrogens, for both the apo- and the holo protein consistent with an unfolding and refolding of the protein in the gas phase. The different techniques are compared with respect to the advantages and disadvantages they bring to the study of this protein–ligand system.

Coupled Motions in the SH2 and Kinase Domains of Csk Control Src Phosphorylation

The C-terminal Src kinase (Csk) phosphorylates and down-regulates Src family tyrosine kinases. The Csk-binding protein (Cbp) localizes Csk close to its substrates at the plasma membrane, and increases the specific activity of the kinase. To investigate this long-range catalytic effect, the phosphorylation of Src and the conformation of Csk were investigated in the presence of a high-affinity phosphopeptide derived from Cbp. This peptide binds tightly to the SH2 domain and enhances Src recognition (lowers K(m)) by increasing the apparent phosphoryl transfer rate in the Csk active site, a phenomenon detected in rapid quench flow experiments. Previous studies demonstrated that the regulation of Csk activity is linked to conformational changes in the enzyme that can be probed with hydrogen-deuterium exchange methods. We show that the Cbp peptide impacts deuterium incorporation into its binding partner (the SH2 domain), and into the SH2-kinase linker and several sequences in the kinase domain, including the glycine-rich loop in the active site. These findings, along with computational data from normal mode analyses, suggest that the SH2 domain moves in a cantilever fashion with respect to the small lobe of the kinase domain, ordering the active site for catalysis. The binding of a small Cbp-derived peptide to the SH2 domain of Csk modifies these motions, enhancing Src recognition.

Usefulness of the hydrogen–deuterium exchange method in the study of drug metabolism using liquid chromatography-tandem mass spectrometry

The usefulness of the hydrogen–deuterium (H-D) exchange method in the study of drug metabolism was investigated. Metabolite samples of denopamine and promethazine prepared in vitro were introduced to a triple stage quadrupole tandem mass spectrometer via a high performance liquid chromatography (HPLC) system using a deuterated mobile phase. Mass spectra by various ionization modes and collisionally induced dissociation (CID) mass spectra were obtained. A metabolite of denopamine was observed to have a shift of 7 mass units by the H-D exchange method, and this shift proved to be a glucuronidated metabolite. Discrimination between N- or S-oxide formation and hydroxylation observed in the metabolism of promethazine was also easily accomplished by this method. In this manner, the structures of the metabolites were elucidated unequivocally by determining the number of labile hydrogen atoms by the use of the H-D exchange method, since various reactions in drug metabolism are accompanied by an increase or a decrease in the number of labile hydrogen atoms. Additional information on the structures was obtained by analysis of the CID spectra of the molecular ion species. Thus, the combination of the H-D exchange method and tandem mass spectrometry was found to be very useful for the study of drug metabolism.

Determination of water adsorption sites in wood by a hydrogen–deuterium exchange

IT is important in the field of forest products science to know the sorption mechanism of water in wood. Adsorption of water onto hygroscopic natural polymers, such as wood and cellulose, is thought to depend entirely on the hydroxyl groups on the surface1. The mechanism of water adsorption in wood can therefore be studied by evaluating the quantity and the state of the adsorption sites, hydroxyl groups. Until now, in order to measure the number of adsorption sites of water in wood, it has been usual to draw a sorption isotherm from which the adsorbed area can be calculated assuming monomolecular layer adsorption according to the BET theory2. Little is known of the sorption mechanism on the molecular level, however. We report here the use of a hydrogen–deuterium exchange method to determine the adsorption sites of water in wood. Hydrogen–deuterium exchange is a well established method which has been widely used to determine hydroxyl group accessibility3,8,9.

Hydrogen‐Exchange Study of the Conformational Stability of Human Carbonic‐Anhydrase B and Its Metallocomplexes

In the range of pH 4.6--8.8, 25 degrees C, the apoenzyme of carbonic anhydrase B shows no evidence of any gross conformational changes, as studied by the hydrogen-deuterium exchange method. At pH 4.6 the addition of Co(II), Cd(II) or Mn(II) to the apoenzyme results in a destabilization of the native protein conformation, but in the range of pH 5.5--8.8 these metal ions, and Zn(II), slightly increase the conformational stability of the protein, in so far as they reduce the probability phi of solvent exposure of the peptide groups. In comparison with other proteins studied, native carbonic anhydrase is characterized by a rather compact conformation; for half of the peptide groups the probability of solvent exposure is less than 10(-4), corresponding to changes in standard free energy larger than 5.5 kcal mol-1 (23 kJ mol-1) following the conformational transitions by which these groups are exposed to the solvent.