Analysis of Overlapped and Noisy Hydrogen/Deuterium Exchange Mass Spectra

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.

A new method for the accurate determination of the isotopic state of single amide hydrogens within peptides using Fourier transform ion cyclotron resonance mass spectrometry

A new method is presented to accurately determine the probability of having a deuterium or hydrogen atom on a specific amide position within a peptide after deuterium/hydrogen (D/H) exchange in solution. Amide hydrogen exchange has been proven to be a sensitive probe for studying protein structures and structural dynamics. At the same time, mass spectrometry in combination with physical fragmentation methods is commonly used to sequence proteins based on an amino acid residue specific mass analysis. In the present study it is demonstrated that the isotopic patterns of a series of peptide fragment ions obtained with capillary-skimmer dissociation, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, can be used to calculate the isotopic state of specific amide hydrogens. This calculation is based on the experimentally observed isotopic patterns of two consecutive fragments and on the isotopic binomial distributions of the atoms in the residue constituting the difference between these two consecutive fragments. The applicability of the method is demonstrated by following the sequence-specific D/H exchange rate in solution of single amide hydrogens within some peptides.     

Factors affecting gas-phase deuterium scrambling in peptide ions and their implications for protein structure determination

In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and proteins. This methodology, in which partly deuterated peptide ions are subjected to collision-induced dissociation in the vacuum of a mass spectrometer, has emerged as a useful tool in structural biology. It may potentially provide quantitatively the extent of deuterium incorporation per individual amino acid in peptides and proteins, thus providing detailed structural information related to protein structure and folding. We report that this general methodology has limitations caused by the fact that the incorporated deuterium atoms migrate prior or during the CID MS analysis. Our data are focused on a variety of transmembrane peptides, incorporated in a lipid bilayer, in which the near-terminal amino acids that anchor at the lipid-water interface are systematically varied. Our findings suggest that, under the experimental conditions we use, the extent of intramolecular hydrogen scrambling is strongly influenced by experimental factors, such as the exact amino acid sequence of the peptide, the nature of the charge carrier, and therefore most likely by the gas-phase structure of the peptide ion. Moreover, the observed scrambling seems to be independent of the nature of the peptide fragment ions (i.e., protonated B and Y' ' ions, and sodiated A and Y' ions). Our results strongly suggest that scrambling may be reduced by using alkali metal cationization instead of protonation in the ionization process.     

Cyclic peptide as reference system for b ion structural analysis in the gas phase

Infrared multiple photon dissociation spectroscopy and hydrogen/deuterium exchange methods are used to confirm the macrocylic structure of a b(6) peptide fragment by direct comparison with a synthetically made cyclic peptide. The acetylation of the peptide N-terminus results in the inhibition of the macrocyclic formation, supporting the "head-to-tail" cyclization mechanism. Differences in hydrogen/deuterium exchange rates for macrocyclic and oxazalone structure peptide fragments are interpreted to be a result of the complex interplay of multiple basic sites in the peptide fragment, supporting the relay mechanism for deuterium exchange with CH(3)OD.     

HDXFinder: automated analysis and data reporting of deuterium/hydrogen exchange mass spectrometry

Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data. This application relies on the high resolution of mass spectrometers to extract all isotopic envelopes before correlating these envelopes with individual peptides. Although a fully automatic analysis is possible, a variety of graphical tools are included to aid in the verification of correlations and rankings of the isotopic peptide envelopes. As a demonstration, the rate constants for H/D exchange of peptides from rabbit muscle pyruvate kinase are mapped onto the structure of this protein.     

High-resolution H/D exchange studies on the HET-s218-295 prion protein

In a search for improved resolution of hydrogen/deuterium (H/D) exchange experiments analyzed by mass spectrometry (HXMS), we evaluated two methodologies for a detailed structural study of solvent accessibility in the case of the HET-s(218-295) prion protein. For the first approach, after incubation in the deuterated solvent, aggregated HET-s(218-295) was digested with pepsin and the generated peptides were analyzed by nanospray mass spectrometry in an ion trap, with and without collision-induced dissociation (CID). We compared deuterium incorporation in peptides as determined on peptide pseudomolecular ions and on b and y fragments produced by longer peptides under CID conditions. For both b and y fragment ions, an extensive H/D scrambling phenomenon was observed, in contrast with previous studies comparing CID-MS experiments and (1)H NMR data. Thus, the spatial resolution of HXMS experiments could not be improved by means of MS/MS data generated by an ion trap mass spectrometer. In a second approach, the incorporation of deuterium was analyzed by MS for 76 peptides of the HET-s(218-289) peptide mass fingerprint, and the use of shared boundaries among peptic peptides allowed us to determine deuteration levels of small regions ranging from one to four amino acids. This methodology led to evidence of highly protected regions along the HET-s(218-295) sequence.     

Modeling the electrophoretic mobility and diffusion of weakly charged peptides

A bead model to determine the electrophoretic mobilities and translational diffusion constants of weakly charged peptides is developed that is based on a approximate structural model of peptides and is also grounded in electrohydrodynamic theory. A peptide made up of X amino acids is modeled as N=2X beads with 2 beads representing each amino acid in the chain. For the two beads representing a particular amino acid in a peptide, the radius of one bead is set to one-half the nearest neighbor Calpha-Calpha distance, and the radius of the other bead is chosen on the basis of the diffusion constant of the free amino acid. Peptide conformations, which are defined by a set of psi-phi dihedral angles, are randomly generated by using the transformation matrix approach of Flory (Flory, P. Statistical Mechanics of Chain Molecules; John Wiley: New York, 1969) and rejecting conformations which result in bead overlap. The mobility and diffusion constants are computed for each conformation and at least 100 independent conformations are examined for each peptide. In general, the mobility is found to depend only weakly on peptide conformation. Model and experimental mobilities are compared by examining the data of Janini and co-workers (Janini, G.; et al. J. Chromatogr. 1999, 848, 417-433). A total of 58 peptides consisting of from 2 to 39 amino acids are considered. The average relative error between experimental and model mobilities is found to be 1.0% and the rms relative error 7.7%. In specific cases, the discrepancy can be substantial and possible reasons for this are discussed. It should be emphasized that the input parameters of the peptide model are totally independent of experimental mobilities. It is hoped that the peptide model developed here will be useful in the prediction of peptide mobility as well as in using peptide mobilities to extract information about peptide structure, conformation, and charge. Finally, we show how simultaneous measurements of translational diffusion and mobility can be used to estimate peptide charge.     

Improved algorithm for accurate computation of molecular solid angles

A new algorithm involving the calculation of the solid angle of a molecule about a point as a measure of steric size has been developed. The algorithm calculates the total solid angle in a stepwise fashion, summing all regions of individual atom solid angles and overlapped atoms, taking into account all orders of possible overlap of multiple atoms. The results for several molecular fragments have been compared to previous solid and cone angle calculations and improved correlations were observed. © 1996 by John Wiley & Sons, Inc.     

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.

Extraction of local hydrogen exchange data from HDX CAD MS measurements by deconvolution of isotopic distributions of fragment ions

Hydrogen/deuterium exchange (HDX) coupled to protein fragmentation either in solution (by means of proteolysis) or in the gas phase (using collisional activation of protein ions) and followed by mass spectral measurements of deuterium content of individual fragments has become one of the major experimental tools to probe protein structure and dynamics. One difficulty, which often arises in the course of interpretation of HDX MS data, is a need to separate deuterium contribution to the observed isotopic patterns from that of naturally occurring isotopes. Another frequently encountered problem, especially when HDX in solution is followed by protein ion fragmentation in the gas phase, is a need to determine the deuterium content of an internal protein segment based on the measured isotopic distributions of overlapping fragments. While several algorithms were developed in the past several years to address the first problem, the second one did not enjoy as much attention. Here we report a new algorithm based on a maximum entropy principle, which is capable of extracting local exchange data form the isotope distribution of overlapping fragments, as well as subtracting the background due to the presence of natural isotopes and residual deuterium in exchange buffers. The new method is tested with several proteins and appears to generate stable solutions even under unfavorable circumstances, e.g., when the resolving power of a mass analyzer is not sufficient to avoid signal interference or when the isotopic distributions of individual fragments are complex and cannot be approximated with simple binomial distributions. The latter feature makes the algorithm particularly useful when the exchange in solution is correlated or semicorrelated, paving the way to precise structural characterization of non-native protein states in solution.     

Fast reversed-phase liquid chromatography to reduce back exchange and increase throughput in H/D exchange monitored by FT-ICR mass spectrometry

In solution-phase hydrogen/deuterium exchange (HDX), it is essential to minimize the back-exchange level of H for D after the exchange has been quenched, to accurately assign protein conformation and protein-protein or protein-ligand interactions. Reversed-phase HPLC is conducted at low pH and low temperature to desalt and separate proteolytic fragments. However, back exchange averages roughly 30% because of the long exposure to H₂O in the mobile phase. In this report, we first show that there is no significant backbone amide hydrogen back exchange during quench and digestion; backbone exchange occurs primarily during subsequent liquid chromatography separation. We then show that a rapid reversed-phase separation reduces back exchange for HDX by at least 25%, resulting from the dramatically reduced retention time of the peptide fragments on the column. The influence of retention time on back exchange was also evaluated. The rapid separation coupled with high-resolution FT-ICR MS at 14.5 T provides high amino acid sequence coverage, high sample throughput, and high reproducibility and reliability.     

ExMS: Data Analysis for HX-MS Experiments

A previous paper considered the problems that presently limit the hydrogen exchange - mass spectrometry (HX-MS) method for studying the biophysical and functional properties of proteins. Many of these problems can be overcome by obtaining and analyzing hundreds of sequentially overlapping peptide fragments that cover the protein many times over (Mayne et al. J. Am. Soc. Mass Spectrom. 2011: ). This paper describes a computer program called ExMS that furthers this advance by making it possible to efficiently process crowded mass spectra and definitively identify and characterize these many peptide fragments. ExMS automatically scans through high resolution MS data to find the individual isotopic peaks and isotopic envelopes of a list of peptides previously identified by MS/MS. It performs a number of tests to ensure correct identification in spite of peptide overlap in both chromatographic and mass spectrometric dimensions and possible multi-modal envelopes due to static or dynamic structural heterogeneity or HX EX1 behavior. The program can automatically process data from many sequential HX time points with no operator intervention at the rate of ~2 sec per peptide per HX time point using desktop computer equipment, but it also provides for rapid manual checking and decision when ambiguity exists. Additional subroutines can provide a step by step report of performance at each test along the way and parameter adjustment, deconvolute isotopic envelopes, and plot the time course of single and multi-modal H-D exchange. The program will be available on an open source basis at:     

Photoionisation mass spectra of volatile derivatives of short peptides and appearance potentials of their characteristic ions

Mass spectra of the volatile derivatives of short peptides were studied by the photoionisation method with the use of monochromatic photons. The dependence of the intensity of the main peaks on the photon energy was analysed from 7·5 to 13·0 eV. The data obtain reveal the influence of the chemical structure of amino acid residues on the relative probability of the decomposition of peptide molecular ions at the CH? CO and CO? NH bonds, resulting in the formation of positively charged aldimine and amino acid N-terminal fragments, respectively. These data may be used as a basis for the application of the photoionisation technique to mass spectrometric sequential studies in peptides. In peptides containing residues of aliphatic amino acid the decomposition results mainly in the formation of aldimine ions, the stability of which increase with the increase of the alkyl chain size. In peptides containing residues of aromatic amino acids the decomposition is usually observed leading to formation of the amino acid ions. Ionisation potentials, as well as photoionisation efficiency curves and appearance potentials were determined for characteristic ions. Comparison was made of the values of the appearance potentials of different fragments formed upon decomposition of molecular ions. It has been shown that for peptides containing aliphatic amino acid moieties the appearance potentials of aldimine fragments are always lower than those inherent to peptides containing aromatic amino acid residues. The larger the size of an aliphatic chain, the lower is the energy of formation of these fragments. For all the compounds studied, including the peptides containing aromatic amino acid residues, the appearance potentials of the aldimine ions did not exceed those of the amino acid ions. These data indicate that, contrary to the experiments with electron-impact with energy of about 70 eV, upon ionisation with photons with energy from 7·5 to 13·0 eV, the aldimine fragments appear directly due to primary decomposition of molecular ions, independent of the formation of the amino acid fragments. The photoionisation efficiency curves for peptides containing different types of amino acid residues facilitate the choice of an optimal photon energy providing unequivocal information on the amino acid sequence in the peptide under study.     

The complexation of protonated peptides with saccharides in the gas phase decreases the rates of hydrogen/deuterium exchange reactions

Gas-phase noncovalently bound complexes are probed by hydrogen/deuterium exchange. The complexes, composed of a protonated amino acid and a monosaccharide, are investigated to observe the effects of complexation on the rates of exchange. Rate constants are determined and compared for complexed and uncomplexed amino acids. The overall rate constant, which corresponds to exchange of a specific number of hydrogens, is deconvoluted to yield site-specific rate constants. Complexation of amino acids with saccharides significantly decreases the rate constants of the exchange. Results of molecular orbital calculations are provided to explain the decrease in the rates.     

Visualization and application of amino acid retention coefficients obtained from modeling of peptide retention

We introduce a method for data inspection in liquid separations of peptides using amino acid retention coefficients and their relative change across experiments. Our method allows for the direct comparison between actual experimental conditions, regardless of sample content and without the use of internal standards. The modeling uses linear regression of peptide retention time as a function of amino acid composition. We demonstrate the pH dependency of the model in a control experiment where the pH of the mobile phase was changed in controlled way. We introduce a score to identify the false discovery rate on peptide spectrum match level that corresponds to the set of most robust models, i.e. to maximize the shared agreement between experiments. We demonstrate the method utility in reversed‐phase liquid chromatography using 24 datasets with minimal peptide overlap. We apply our method on datasets obtained from a public repository representing various separation designs, including one‐dimensional reversed‐phase liquid chromatography followed by tandem mass spectrometry, and two‐dimensional online strong cation exchange coupled to reversed‐phase liquid chromatography followed by tandem mass spectrometry, and highlight new insights. Our method provides a simple yet powerful way to inspect data quality, in particular for multidimensional separations, improving comparability of data at no additional experimental cost.     

New protonation microequilibrium treatment in the case of some amino acid and peptide derivatives containing a bis(imidazolyl)methyl group

We present a microequilibrium analysis of a series of amino acid and peptide derivatives containing the chelating bis(imidazol-2-yl)methyl group (BIP, Gly-BIMA, His-BIMA, alpha-Glu-BIMA, gamma-Glu-BIMA, and Phe-His-BIMA). NMR measurements were performed in D2O to follow the deprotonation steps. The software PSEQUAD and a specialized program written in MATLAB were used to determine the macroscopic and microscopic constants. The method assumes that the effect of pH on the chemical shift of an NMR-active nucleus can be interpreted by adding the independent effects of the protonation of individual sites. For derivatives containing histidine (His-BIMA and Phe-His-BIMA), the deprotonation steps of the second imidazole and the His-imidazole significantly overlap. In the Glu derivatives (alpha-Glu-BIMA and gamma-Glu-BIMA), the amino and the second imidazole pK values are separate; the deprotonation processes of the first imidazole nitrogen and the side-chain carboxyl group, however, significantly overlap. In gamma-Glu-BIMA, the deprotonation sequence is carboxylate-imidazole1-imidazole2-amino, while in the case of alpha-Glu-BIMA, it changes to imidazole1-carboxylate-imidazole2-amino, according to the microscopic pk values. The main advantage of the method is that it does not require the synthesis and NMR microequilibrium analysis of substances modeling the individual parts of the target ligand, in contrast to the methods used by others. The method presented here demands slightly more mathematical and computational power, which is readily available today.     

Peptide mapping by reversed-phase high-performance liquid chromatography employing silica rod monoliths

In this paper, a general procedure is described for the generation of peptide maps of proteins with monolithic silica-based columns. The peptide fragments were obtained by tryptic digestion of various cytochrome c species with purification of the tryptic fragments achieved by reversed-phase high-performance liquid chromatographic methods. Peak assignment of the various peptides was based on evaluation of the biophysical properties of the individual peptides and via mass spectrometric identification. The performance of several different monolithic sorbents prepared as columns of identical cross-sectional dimensions were investigated as part of these peptide mapping studies and the data evaluated by applying solvent strength theory. These studies revealed curvilinear dependencies in the corresponding relative resolution maps. These findings directly impact on the selection of specific sorbent types or column configurations for peptide separations with silica rod monoliths. Moreover, the influence of variations in the amino acid sequence of the cytochrome cs were evaluated with respect to their effect on intrinsic hydrophobicity, the number of experimental observed tryptic cleavage sites, detection limits of the derived fragments in relation to their molecular size, and the chromatographic selectivity and resolution of the various peptides obtained following enzymatic fragmentation of the parent protein. Finally, the scope of these approaches in method development was examined in terms of robustness and efficiency.     

Gas phase hydrogen deuterium exchange reactions of a model peptide: FT-ICR and computational analyses of metal induced conformational mutations

We utilized gas phase hydrogen/deuterium (H/D) exchange reactions and ab initio calculations to investigate the complexation between a model peptide (Arg-Gly-AspRGD) with various alkali metal ions. The peptide conformation is drastically altered upon alkali metal ion complexation. The associated conformational changes depend on both the number and type of complexing alkali metal ions. Sodium has a smaller ionic diameter and prefers a multidentate interaction that involves all three amino acids of the peptide. Conversely, potassium and cesium form different types of complexes with the RGD. The [RGD + 2Cs − H]⁺ species exhibit the slowest H/D exchange reactivity (reaction rate constant of 6 × 10⁻¹³ cm³molecule⁻¹s⁻¹ for the fastest exchanging labile hydrogen with ND₃). The reaction rate constant of the protonated RGD is two orders of magnitude faster than that of the [RGD + 2Cs − H]⁺. Addition of the first cesium to the RGD reduces the H/D exchange reaction rate constant (i.e., D₀) by a factor of seven whereas sodium reduces this value by a factor of thirty. Conversely, addition of the second alkali metal ions has the opposite effect; the rate of D₀ disappearance for all [RGD + 2Met − H]⁺ species (MetNa, K, and Cs) decreases with the alkali metal ion size.     

Protein-peptide affinity determination using an H/D exchange dilution strategy: Application to antigen-antibody interactions

A new methodology using hydrogen/deuterium amide exchange (HDX) to determine the binding affinity of protein-peptide interactions is reported. The method, based on our previously established approach, protein ligand interaction by mass spectrometry, titration, and H/D exchange (PLIMSTEX) [J. Am. Chem. Soc. 2003, 125, 5252–5253], makes use of a dilution strategy (dPLIMSTEX) for HDX, using the mass of the peptide ligand as readout. We employed dPLIMSTEX to study the interaction of calcium-saturated calmodulin with the opioid peptide β-endorphin as a model system; the affinity results are in good agreement with those from traditional PLIMSTEX and with literature values obtained by using other methods. We show that the dPLIMSTEX method is feasible to quantify an antigen-antibody interaction involving a 3-nitrotyrosine modified peptide in complex with a monoclonal anti-nitrotyrosine antibody. A dissociation constant in the low nanomolar range was determined, and a binding stoichiometry of antibody/peptide of 1:2 was confirmed. In addition, we determined that the epitope in the binding interface contains a minimum of five amino acids. The dPLIMSTEX approach is a sensitive and powerful tool for the quantitative determination of peptide affinities with antibodies, complementary to conventional immuno-analytical techniques.     

Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the alpha-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the alpha-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c*, and they also show c*-1 peaks in the deuterated peptides only. The presence of the c*-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H* and -CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.