Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a well established method for the measurement of solution-phase
deuterium incorporation into proteins, which can provide insight into protein conformational mobility. However, most HDX measurements
are constrained to regions of the protein where pepsin proteolysis allows detection at peptide resolution. Recently, single-amide
resolution deuterium incorporation has been achieved by limiting gas-phase scrambling in the mass spectrometer. This was accomplished
by employing a combination of soft ionization and desolvation conditions coupled with the radical-driven fragmentation technique
electron transfer dissociation (ETD). Here, a hybrid LTQ-Orbitrap XL is systematically evaluated for its utility in providing
single-amide deuterium incorporation for differential HDX analysis of a nuclear receptor upon binding small molecule ligands.
We are able to show that instrumental parameters can be optimized to minimize scrambling and can be incorporated into an established
and fully automated HDX platform making differential single-amide HDX possible for bottom-up analysis of complex systems.
We have applied this system to determine differential single amide resolution HDX data for the peroxizome proliferator activated
receptor bound with two ligands of interest. 相似文献
Two tools are described for integrating LC elution position with mass-based data in hydrogen-deuterium exchange (HDX) experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry (nanoLC/MALDI-MS, a novel approach to HDX-MS). The first of these, 'TOF2H-Z Comparator', highlights peptides in HDX experiments that are potentially misidentified on the basis of mass alone. The program first calculates normalized values for the organic solvent concentration responsible for the elution of ions in nanoLC/MALDI HDX experiments. It then allows the solvent gradients for the multiple experiments contributing to an MS/MS-confirmed peptic peptide library to be brought into mutual alignment by iteratively re-modeling variables among LC parameters such as gradient shape, solvent species, fraction duration and LC dead time. Finally, using the program, high-probability chromatographic outliers can be flagged within HDX experimental data. The role of the second tool, 'TOF2H-XIC Comparator', is to normalize the LC chromatograms corresponding to all deuteration timepoints of all HDX experiments of a project, to a common reference. Accurate normalization facilitates the verification of chromatographic consistency between all ions whose spectral segments contribute to particular deuterium uptake plots. Gradient normalization in this manner revealed chromatographic inconsistencies between ions whose masses were either indistinguishable or separated by precise isotopic increments. 相似文献
Hydrogen deuterium exchange measured by mass spectrometry (HDX-MS) is a commonly used technique for studying the structural dynamics of proteins in solution. The first part of any bottom-up HDX-MS experiment is to identify the peptides generated from a digestion step. This requires manual inspection of the identified peptides to determine their use for HDX-MS analysis, which is a time-consuming task. Throughout the literature, there have been different approaches for removing peptides that do not yield quantifiable HDX information. This includes using validity scores from the software used in the generation of the peptide map and that the peptide should be found in two out of three technical replicate experiments. Here, we analyze the previously available methods for filtering the identified peptides in regard to their ability to predict whether a peptide will provide quantifiable HDX-MS data or not. We also present a new score-based system relying on a combination of MS/MS parameters that offers an improved method for separating quantifiable peptides from the nonquantifiable. Using this score-based method reduces the number of peptide spectra that needs to be manually inspected and thereby the time spent curating HDX-MS data.
Hydrogen/deuterium exchange (HDX) mass spectrometry has been widely applied to the characterization of protein dynamics. More recently, differential HDX has been shown to be effective for the characterization of ligand binding. Previously we have described a fully automated HDX system for use as a ligand screening platform. Here we describe and validate the required data analysis workflow to facilitate the use of HDX as a robust approach for ligand screening. Following acquisition of HDX data at a single on-exchange time point (n ≥ 3), one way analysis of variance in conjunction with the Tukey multiple comparison procedure is used to establish the significance of any measured difference. Analysis results are graphed with respect to a single peptide, ligand or group of ligands, or displayed as an overview within a heat map. For the heat map display, only Δ%D values with a Tukey-adjusted P value less than 0.05 are colored. Hierarchical clustering is used to bin compounds with highly similar HDX signatures. The workflow is evaluated with a small data set showing the ligand binding domain (LDB) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) screened against 10 functionally selective ligands. More significantly, data for the vitamin D receptor (VDR) in complex with 87 ligands are presented. To highlight the robustness and precision of our automated HDX platform we analyzed the data from 4191 replicate HDX measurements acquired over an eight month timeframe. Ninety six percent of these measurements were within 10 percent of the mean value. Work has begun to integrate these analysis and graphing components within our HDX software suite. 相似文献
In this study, the effects of lead ions(Pb2+) on the conformations of leucine encephalin(LE) and methionine encephalin(ME) in gas phase were studied using hydrogen/deuterium exchange mass spectrometry(HDX-MS) and quantum chemistry theoretical calculations at the molecular level. The HDX-MS result revealed that the complexes with the monovalent compounds [LE+Pb–H]+ and [ME+Pb–H]+had a 1:1 stoichiometric ratio, and different HDX reactivates were observed in a follow of [ME+H]+>[LE+H]+>[LE+Pb–H]+> [ME+Pb–H]+. Combining the collision-induced dissociation energies of the complexes and their HDX results, it was found that the more stable the complex, the harder it was for HDX. In addition, the favo-rable conformations of the complexes were obtained by theoretical calculations, revealing that the similar coordination type with diffe-rent bond lengths was obtained. Then, the proton affinity(PA) values of the optimized complexes were calculated to interpret the HDX observations, indicating that the higher the PA values, the more difficult it was for HDX. Overall, the experiments and theoretical calculations revealed that Pb2+ could induce conformational changes of LE and ME, and generate ME into a more rigid conformation than LE. The results will prompt further fundamental investigations on the conformational properties of LE/ME in coordination with Pb2+. 相似文献
Here we describe an integrated software platform titled HD Desktop designed specifically to enhance the analysis of hydrogen/deuterium
exchange (HDX) mass spectrometry data. HD Desktop integrates tools for data extraction with visualization components within
a single web-based application. The interface design enables users to navigate from the peptide view to the sample and experiment
levels, tracking all manipulations while updating the aggregate graphs in real time. HD Desktop is integrated with a relational
database designed to provide performance enhancements, as well as a robust model for data storage and retrieval. Additional
features of the software include retention time determination, which is achieved with the use of theoretical isotope fitting;
here, we assume that the best theoretical fit will occur at the correct retention time for any given peptide. Peptide data
consolidation for the rendering of data in 2D was realized by automating known and novel approaches. Designed to address broad
needs of the HDX community, the platform presented here provides an efficient and manageable workflow for HDX data analysis
and is freely available as a web tool at the project home page http://hdx.florida.scripps.edu. 相似文献
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.
Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing D2O, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments.
Characterization of therapeutic drugs is a crucial step in drug development in the biopharmaceutical industry. Analysis of protein therapeutics is a challenging task because of the complexities associated with large molecular size and 3D structures. Recent advances in hydrogen/deuterium-exchange mass spectrometry (HDX-MS) have provided a means to assess higher-order structure of protein therapeutics in solution. In this review, the principles and procedures of HDX-MS for protein therapeutics characterization are presented, focusing on specific applications of epitope mapping for protein–protein interactions and higher-order structure comparison studies for conformational dynamics of protein therapeutics. Figure
PEGylation is the covalent attachment of polyethylene glycol to proteins, and it can be used to alter immunogenicity, circulating
half life and other properties of therapeutic proteins. To determine the impact of PEGylation on protein conformation, we
applied hydrogen/deuterium exchange mass spectrometry (HDX MS) to analyze granulocyte colony stimulating factor (G-CSF) upon
PEGylation as a model system. The combined use of HDX automation technology and data analysis software allowed reproducible
and robust measurements of the deuterium incorporation levels for peptic peptides of both PEGylated and non-PEGylated G-CSF.
The results indicated that significant differences in deuterium incorporation were induced by PEGylation of G-CSF, although
the overall changes observed were quite small. PEGylation did not result in gross conformational rearrangement of G-CSF. The
data complexity often encountered in HDX MS measurements was greatly reduced through a data processing and presentation format
designed to facilitate the comparison process. This study demonstrates the practical utility of HDX MS for comparability studies,
process monitoring, and protein therapeutic characterization in the biopharmaceutical industry. 相似文献
Hydrogen deuterium exchange mass spectrometry (HDX‐MS) is a powerful technique for studying protein dynamics, which is an important factor governing protein functions. However, the process of hydrogen/deuterium exchange (HDX) of proteins is highly complex and the underlying mechanism has not yet been fully elucidated. Meanwhile, molecular dynamics (MD) simulation is a computational technique that can be used to elucidate HDX behaviour on proteins and facilitate interpretation of HDX‐MS data. This article aims to summarize the current understandings on the mechanism of HDX and its correlation with MD simulation, to discuss the recent developments in the techniques of HDX‐MS and MD simulation and to extend the perspectives of these two techniques in protein dynamics study. 相似文献
Application of typical HDX methods to examine intrinsically disordered proteins (IDP), proteins that are natively unstructured and highly dynamic at physiological pH, is limited because of the rapid exchange of unprotected amide hydrogens with solvent. The exchange rates of these fast exchanging amides are usually faster than the shortest time scale (10 s) employed in typical automated HDX-MS experiments. Considering the functional importance of IDPs and their association with many diseases, it is valuable to develop methods that allow the study of solution dynamics of these proteins as well as the ability to probe the interaction of IDPs with their wide range of binding partners. Here, we report the application of time window expansion to the millisecond range by altering the on-exchange pH of the HDX experiment to study a well-characterized IDP; the activation domain of the nuclear receptor coactivator, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α). This method enabled mapping the regions of PGC-1α that are stabilized upon binding the ligand binding domain (LBD) of the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). We further demonstrate the method’s applicability to other binding partners of the IDP PGC-1α and pave the way for characterizing many other biologically important ID proteins.
The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.
The structural stability of hen egg white lysozyme in solution and adsorbed to small colloidal silica particles at various surface concentrations was investigated using hydrogen-deuterium (H/D) exchange in combination with mass spectrometry (HDX-MS) and differential scanning calorimetry (DSC). The combination of HDX-MS and DSC allows a full thermodynamic analysis of the lysozyme structure as both the enthalpy and the Gibbs free energy can be derived from the various measurements. Moreover, both HDX-MS and DSC provide information on the relative structural heterogeneity of lysozyme in the adsorbed state compared to that in solution. Results demonstrated that at high surface coverage, the structural stability of lysozyme was only marginally affected by adsorption to silica particles whereas the unfolding enthalpy decreased by more than 10%, meaning that the entropy of lysozyme increased with a similar value upon adsorption. Furthermore, the structural heterogeneity increased considerably. At lower surface concentrations, the structural heterogeneity increased further whereas the enthalpy of unfolding decreased. Further analyses of the HDX-MS experiments clearly indicated that folding/unfolding of lysozyme occurs through a two-domain process. These two domains had a similar amount of structural elements and a difference in stabilization energy of 8 kJ/mol, regardless if lysozyme was in solution or adsorbed to silica. 相似文献
Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract.
An algorithm for retention time alignment of mass shifted hydrogen-deuterium exchange (HDX) data based on an iterative distance minimization procedure is described. The algorithm performs pairwise comparisons in an iterative fashion between a list of features from a reference file and a file to be time aligned to calculate a retention time mapping function. Features are characterized by their charge, retention time and mass of the monoisotopic peak. The algorithm is able to align datasets with mass shifted features, which is a prerequisite for aligning hydrogen-deuterium exchange mass spectrometry datasets. Confidence assignments from the fully automated processing of a commercial HDX software package are shown to benefit significantly from retention time alignment prior to extraction of deuterium incorporation values. 相似文献
A Fourier transform infrared (FTIR) spectroscopy assay to measure hydrogen–deuterium exchange (HDX) in surface‐adsorbed protein monolayers is developed to provide information on protein tertiary structure, because the typical secondary structural analysis of our surface and solution protein samples proved to be very similar. Adsorbed protein HDX is quantified by exposing the protein to a 50% deuterated NaPO4 buffer solution and then measuring the normalized intensity change of the amide II band in the FTIR reflection spectrum. When collected as a function of exchange time, this intensity follows the kinetics of the exposure of the protein amides to solvent. HDX kinetics have been obtained for bovine serum albumin (BSA) in solution and adsorbed to gold surfaces. Using experiments designed to allow comparisons between protein in solution and on surfaces, the extent of HDX was found to increase over that observed for BSA in solution, consistent with an increase in the exposure of albumin amide groups and protein unfolding upon adsorption. We also show that BSA adsorbs to the surface of gold in multilayers and that the increase in amide exposure is present only in the first adsorbed monolayer. Published in 2009 by John Wiley & Sons, Ltd. 相似文献
The collision-induced dissociation (CID) products b2-b4 from Leu-enkephalin are examined with infrared multiple-photon dissociation (IR-MPD) spectroscopy and gas-phase hydrogen/deuterium
exchange (HDX). Infrared spectroscopy reveals that b2 exclusively adopts oxazolone structures, protonated at the N-terminus and at the oxazolone ring N, based on the presence
and absence of diagnostic infrared vibrations. This is correlated with the presence of a single HDX rate. For the larger b3 and b4, the IR-MPD measurements display diagnostic bands compatible with a mixture of oxazolone and macrocycle structures. This
result is supported by HDX experiments, which show a bimodal distribution in the HDX spectra and two distinct rates in the
HDX kinetic fitting. The kinetic fitting of the HDX data is employed to derive the relative abundances of macrocycle and oxazolone
structures for b3 and b4, using a procedure recently implemented by our group for a series of oligoglycine b fragments (Chen et al. J. Am. Chem. Soc.2009, 131(51), 18272–18282. doi: 10.1021/ja9030837). In analogy to that study, the results suggest that the relative abundance
of the macrocycle structure increases as a function of b fragment size, going from 0% for b2 to ∼6% for b3, and culminating in 31% for b4. Nonetheless, there are also surprising differences between both studies, both in the exchange kinetics and the propensity
in forming macrocycle structures. This indicates that the chemistry of “head-to-tail” cyclization depends on subtle differences
in the sequence as well as the size of the b fragment. 相似文献
When highly concentrated, an antibody solution can exhibit unusual behaviors, which can lead to unwanted properties, such as increased levels of protein aggregation and unusually high viscosity. Molecular modeling, along with many indirect biophysical measurements, has suggested that the cause for these phenomena can be due to short range electrostatic and/or hydrophobic protein–protein interactions. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a useful tool for investigating protein conformation, dynamics, and interactions. However, “traditional” continuous dilution labeling HDX-MS experiments have limited utility for the direct analysis of solutions with high concentrations of protein. Here, we present a dialysis-based HDX-MS (di-HDX-MS) method as an alternative HDX-MS labeling format, which takes advantage of passive dialysis rather than the classic dilution workflow. We applied this approach to a highly concentrated antibody solution without dilution or significant sample manipulation, prior to analysis. Such a method could pave the way for a deeper understanding of the unusual behavior of proteins at high concentrations, which is highly relevant for development of biopharmaceuticals in industry.
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