Automated Hydrogen/Deuterium Exchange Electron Transfer Dissociation High Resolution Mass Spectrometry Measured at Single-Amide Resolution

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.     

ETD in a Traveling Wave Ion Guide at Tuned Z-Spray Ion Source Conditions Allows for Site-Specific Hydrogen/Deuterium Exchange Measurements

The recent application of electron transfer dissociation (ETD) to measure the hydrogen exchange of proteins in solution at single-residue resolution (HX-ETD) paves the way for mass spectrometry-based analyses of biomolecular structure at an unprecedented level of detail. The approach requires that activation of polypeptide ions prior to ETD is minimal so as to prevent undesirable gas-phase randomization of the deuterium label from solution (i.e., hydrogen scrambling). Here we explore the use of ETD in a traveling wave ion guide of a quadrupole-time-of-flight (Q-TOF) mass spectrometer with a “Z-spray” type ion source, to measure the deuterium content of individual residues in peptides. We systematically identify key parameters of the Z-spray ion source that contribute to collisional activation and define conditions that allow ETD experiments to be performed in the traveling wave ion guide without gas-phase hydrogen scrambling. We show that ETD and supplemental collisional activation in a subsequent traveling wave ion guide allows for improved extraction of residue-specific deuterium contents in peptides with low charge. Our results demonstrate the feasibility, and illustrate the advantages of performing HX-ETD experiments on a high-resolution Q-TOF instrument equipped with traveling wave ion guides. Determination of parameters of the Z-spray ion source that contribute to ion heating are similarly pertinent to a growing number of MS applications that also rely on an energetically gentle transfer of ions into the gas-phase, such as the analysis of biomolecular structure by native mass spectrometry in combination with gas-phase ion-ion/ion-neutral reactions or ion mobility spectrometry.     

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.     

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.

Protein dynamics revealed by hydrogen/deuterium exchange mass spectrometry: Correlation between experiments and simulation

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.     

Measuring hydrogen–deuterium exchange in protein monolayers

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.     

Comparison of Peptide Ion Conformers Arising from Non-Helical and Helical Peptides Using Ion Mobility Spectrometry and Gas-Phase Hydrogen/Deuterium Exchange

The dominant gas-phase conformer of [M+3H]³⁺ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]³⁺ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system.

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Protein structure and dynamics studied by mass spectrometry: H/D exchange, hydroxyl radical labeling, and related approaches

Mass spectrometry (MS) plays a central role in studies on protein structure and dynamics. This review highlights some of the recent developments in this area, with focus on applications involving the use of electrospray ionization (ESI) MS. Although this technique involves the transformation of analytes into highly nonphysiological species (desolvated gas-phase ions in the vacuum), ESI-MS can provide detailed insights into the solution-phase behavior of proteins. Notably, the ionization process itself occurs in a structurally sensitive manner. An increased degree of solution-phase unfolding is correlated with a higher level of protonation. Also, ESI allows the transfer of intact noncovalent complexes into the gas phase, thereby yielding information on binding partners, stoichiometries, and even affinities. A particular focus of this article is the use of hydrogen/deuterium exchange (HDX) methods and hydroxyl radical (.OH) labeling for monitoring dynamic and structural aspect of solution-phase proteins. Conceptual similarities and differences between the two methods are discussed. We describe a simple method for the computational simulation of protein HDX patterns, a tool that can be helpful for the interpretation of isotope exchange data recorded under mixed EX1/EX2 conditions. Important aspects of .OH labeling include a striking dependence on protein concentration, and the tendency of commonly used solvent additives to act as highly effective radical scavengers. If not properly controlled, both of these factors may lead to experimental artifacts.     

Using Hydrogen/Deuterium Exchange Mass Spectrometry to Study Conformational Changes in Granulocyte Colony Stimulating Factor upon PEGylation

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.     

Hydrogen/deuterium exchange in parallel with acid/base induced protein conformational change in electrospray droplets

The exposure of electrospray droplets to vapors of deuterating reagents during droplet desolvation in the interface of a mass spectrometer results in hydrogen/deuterium exchange (HDX) on the sub‐millisecond time scale. Deuterated water is used to label ubiquitin and cytochrome c with minimal effect on the observed charge state distribution (CSD), suggesting that the protein conformation is not being altered. However, the introduction of deuterated versions of various acids (e.g., CD₃COOD and DCl) and bases (ND₃) induces unfolding or refolding of the protein while also labeling these newly formed conformations. The extent of HDX within a protein CSD associated with a particular conformation is essentially constant, whereas the extent of HDX can differ significantly for CSDs associated with different conformations from the same protein. In some cases, multiple HDX distributions can be observed within a given charge state (as is demonstrated with cytochrome c) suggesting that the extent of HDX and CSDs share a degree of complementarity in their sensitivities for protein conformation. The CSD is established late in the evolution of ions in electrospray whereas the HDX process presumably takes place in the bulk of the droplet throughout the electrospray process. Back exchange is also performed in which proteins are prepared in deuterated solvents prior to ionization and exposed to undeuterated vapors to exchange deuteriums for hydrogens. The degree of deuterium uptake is easily controlled by varying the identity and partial pressure of the reagent introduced into the interface. Since the exchange occurs on the sub‐millisecond time scale, the use of deuterated acids or bases allows for transient species to be generated and labeled for subsequent mass analysis. Copyright © 2014 John Wiley & Sons, Ltd.     

Hydrogen exchange mass spectrometry for studying protein structure and dynamics

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a key technique for monitoring structural and dynamic aspects of proteins in solution. This approach relies on the fact that exposure of a protein to D(2)O induces rapid amide H → D exchange in disordered regions that lack stable hydrogen-bonding. Tightly folded elements are much more protected from HDX, resulting in slow isotope exchange that is mediated by the structural dynamics ("breathing motions") of the protein. MS-based peptide mapping is a well established technique for measuring the mass shifts of individual protein segments. This tutorial review briefly discusses basic fundamentals of HDX/MS, before highlighting a number of recent developments and applications. Gas phase fragmentation strategies represent a promising alternative to the traditional proteolysis-based approach, but experimentalists have to be aware of scrambling phenomena that can be encountered under certain conditions. Electron-based dissociation methods provide a solution to this problem. We also discuss recent advances that facilitate the applicability of HDX/MS to membrane proteins, and to the characterization of short-lived protein folding intermediates. It is hoped that this review will provide a starting point for novices, as well as a useful reference for practitioners, who require an overview of some recent trends in HDX/MS.     

Two tools for applying chromatographic retention data to the mass-based identification of peptides during hydrogen/deuterium exchange experiments by nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry

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.     

Atmospheric Pressure Photo Ionization Hydrogen/Deuterium Exchange Mass Spectrometry—a Method to Differentiate Isomers by Mass Spectrometry

In this report, a method for in-source hydrogen/deuterium (H/D) exchange at atmospheric pressure is reported. The method was named atmospheric pressure photo ionization hydrogen/deuterium exchange mass spectrometry (APPI HDX MS). H/D exchange was performed by mixing samples dissolved in toluene with CH₃OD solvent and analyzing the mixture using atmospheric pressure photo ionization mass spectrometry (APPI-MS). The APPI HDX spectra obtained with contact times between the analyte solution and methanol-OD (CH₃OD) of?<?0.5 s or 1 h showed the same pattern of H/D exchange. Therefore, it was concluded that APPI HDX occurred in the source but not in the solution. The proposed method does not require a specific type of mass spectrometer and can be performed at atmospheric pressure. H/D exchange can be performed in any laboratory with a mass spectrometer and a commercial APPI source. Using this method, multiple H/D exchanges of aromatic hydrogen and/or H/D exchange of active hydrogen were observed. These results demonstrated that H/D exchange can be used to distinguish between isomers containing primary, secondary, and tertiary amines, as well as pyridine and pyrrole functional groups.

Data‐dependent electron transfer dissociation of large peptides and medium size proteins in a QTOF instrument on a liquid chromatography timescale

Liquid chromatography (LC) electron transfer dissociation (ETD) tandem mass spectrometry (MS/MS) of protein digests is demonstrated in a hybrid quadrupole‐hexapole orthogonal time‐of‐flight (OTOF) mass spectrometer. Analyte ions are selected in a mass‐analyzing quadrupole, accumulated in the hexapole linear ETD reaction cell and mutually stored with ETD reagent anions. Product ions are collected in an ion cooler and then analyzed by an OTOF mass analyzer. The hexapole structure of the ETD reaction cell allows for a broad fragment ion mass range distribution and a high ion storage capacity. Analytically useful ETD OTOF‐MS/MS spectra could be obtained at a rate of faster than 2 Hz. When used in conjunction with LC this high speed allows for several MS and MS/MS spectra to be obtained across each LC peak. An MS scan is used to select the precursor ions. With a 1 m flight tube and single reflection, resolutions of about 10 k and a mass accuracy of 5 ppm were achieved. When analyzing a 100 fmol solution of a tryptic digest of bovine serum albumin (BSA) by LC/ETD MS/MS, 27 unique peptides were identified with a summed Mascot score of 1316 using the Swiss Prot database. In addition, we explored the capability for analyzing small proteins with the present hybrid instrument. ETD MS/MS of intact ubiquitin ([M+12H]¹²⁺) leads to the identification of the protein with a Mascot score of 264. Copyright © 2009 John Wiley & Sons, Ltd.     

Inter-molecular migration during collisional activation monitored by hydrogen/deuterium exchange FT-ICR tandem mass spectrometry

The difficulty with integrating solution-phase hydrogen/deuterium exchange (HDX) and tandem mass spectrometry is that the energy added to cause fragmentation might promote gas-phase migration of the added deuterium atoms. Here, we compare the solution-phase HDX profiles generated from a- b- and y-type fragment ion series originating from capillary-skimmer dissociation. The isotopic distributions of fragments from the different fragment ion types were used to determine the isotopic state of the amide hydrogen within a specific residue. Even though the same amide hydrogen was examined, the result was different for different fragment ion types. This observation indicates that different fragment series are not equally subjected to inter-molecular migration during collision-induced dissociation (CID). We also investigated the gas-phase reactivity of originally undeuterated CID fragments of penta-phenylalanine using gas-phase HDX in an external accumulation hexapole. The incorporation of deuterium into the different fragments was studied as a function of hexapole pressure. It was found that different b- and y-ions from the same peptide had different gas-phase reactivity. However, the a-ions did not display significant gas-phase reactivity. The observed behavior has significant impact on any method that involves comparing the isotopic distributions of different fragment ions. Great care has to be taken in the interpretation of the HDX data using CID to increase the spatial resolution. The isotopic state observed after solution-phase exchange might be more preserved for some CID-fragment types.     

Reaction products in mass spectrometry elucidated with infrared spectroscopy

Determining the structure and dynamics of large biologically relevant molecules is one of the key challenges facing biology. Although X-ray crystallography (XRD) and nuclear magnetic resonance (NMR) yield accurate structural information, they are of limited use when sample quantities are low. Mass spectrometry (MS) on the other hand has been very successful in analyzing biological molecules down to atto-mole quantities and has hence begun to challenge XRD and NMR as the key technology in the life sciences. This trend has been further assisted by the development of MS techniques that yield structural information on biomolecules. Of these techniques, collision-induced dissociation (CID) and hydrogen/deuterium exchange (HDX) are among the most popular. Despite advances in applying these techniques, little direct experimental evidence had been available until recently to verify their proposed underlying reaction mechanisms. The possibility to record infrared spectra of mass-selected molecular ions has opened up a novel avenue in the structural characterization of ions and their reaction products. On account of its high pulse energies and wide wavelength tunability, the free electron laser for infrared experiments (FELIX) at FOM Rijnhuizen has been shown to be ideally suited to study trapped molecular ions with infrared photo-dissociation spectroscopy. In this paper, we review recent experiments in our laboratory on the infrared spectroscopic characterization of reaction products from CID and HDX, thereby corroborating some of the reaction mechanisms that have been proposed. In particular, it is shown that CID gives rise to linear fragment ion structures which have been proposed for some time, but also yields fully cyclical ring structures. These latter structures present a possible challenge for using tandem MS in the sequencing of peptides/proteins, as they can lead to a scrambling of the amino acid sequence information. In gas-phase HDX of an amino acid it is shown that the structure can be changed from a charge solvated to a zwitterionic structure, thereby demonstrating that HDX can be an invasive technique, in fact changing the structure of the analyte. These results emphasize that more fundamental work is required in order to understand the underlying mechanisms in two of the most important structural techniques in MS.     

Comparative Analysis of Oxy-Hemoglobin and Aquomet-Hemoglobin by Hydrogen/Deuterium Exchange Mass Spectrometry

The function of hemoglobin (Hb) as oxygen transporter is mediated by reversible O₂ binding to Fe(2+) heme in each of the α and β subunits. X-ray crystallography revealed different subunit arrangements in oxy-Hb and deoxy-Hb. The deoxy state is stabilized by additional contacts, causing a rigidification that results in strong protection against hydrogen/deuterium exchange (HDX). Aquomet-Hb is a dysfunctional degradation product with four water-bound Fe(3+) centers. Heme release from aquomet-Hb is relatively facile, triggering oxidative damage of membrane lipids. Aquomet-Hb crystallizes in virtually the same conformation as oxy-Hb. Hence, it is commonly implied that the solution-phase properties of aquomet-Hb should resemble those of the oxy state. This work compares the structural dynamics of oxy-Hb and aquomet-Hb by HDX mass spectrometry (MS). It is found that the aquomet state exhibits a solution-phase structure that is significantly more dynamic, as manifested by elevated HDX levels. These enhanced dynamics affect the aquomet α and β subunits in a different fashion. The latter undergoes global destabilization, whereas the former shows elevated HDX levels only in the heme binding region. It is proposed that these enhanced dynamics play a role in facilitating heme release from aquomet-Hb. Our findings should be of particular interest to the MS community because oxy-Hb and aquomet-Hb serve as widely used test analytes for probing the relationship between biomolecular structure in solution and in the gas phase. We are not aware of any prior comparative HDX/MS experiments on oxy-Hb and aquomet-Hb.

Solution-phase deuterium/hydrogen exchange at a specific residue using nozzle-skimmer and electron capture dissociation mass spectrometry

Information about protein conformation can be obtained with hydrogen/deuterium exchange (HDX) mass spectrometry. The isotopic solution-phase exchange of specific amide hydrogen atoms can be followed using low-vacuum nozzle-skimmer collision-induced dissociation (CID). In this study, the nozzle-skimmer technique was complemented by electron capture dissociation (ECD) Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). The solution-phase exchange at a specific residue is monitored by comparing isotopic distributions of two consecutive b- or c-type ions. While nozzle-skimmer fragmentation takes place in the low-vacuum region of the mass spectrometer, ECD occurs at ultra-high vacuum within the mass analyzer cell of the FTICR mass spectrometer. The dissociations take place at 10(-4) and 10(-9) mbar, respectively. Low-vacuum nozzle-skimmer fragmentation can result in intramolecular exchange between product ions and solvent molecules in the gas phase. Consequently, the solution-phase information about protein or peptide conformation is lost. It was not possible to monitor isotopic solution-phase exchange at the eighth residue in substance P, (Phe)8, with nozzle-skimmer CID. By using the in-cell ECD fragmentation method, the solution-phase exchange at the (Phe)8 residue was preserved during mass spectrometric analysis. This result shows the complementary aspects of applying fragmentation at low and at high vacuum, when studying isotopic exchange in solution at specific residues using FTICRMS.     

Hydrogen/deuterium exchange of hydrophobic peptides in model membranes by electrospray ionization mass spectrometry

We demonstrate here that the hydrogen/deuterium solvent exchange (HDX) properties of the transmembrane fragment of the M2 protein of Influenza A (M2-TM) incorporated into lipid vesicles or detergent micelles can be studied with straightforward electrospray (ESI) and nanospray mass spectrometry (MS) configurations provided that key factors, including sample preparation techniques, are optimized. Small unilamellar vesicle preparations were obtained by solubilizing dimyristoyl phosphatidylcholine (DMPC) and the M2-TM peptide in aqueous solution with n-octyl-β-D-glycopyranoside, followed by dialysis to remove the detergent. Electron microscopy experiments revealed that subsequent concentration by centrifugation introduced large multilamellar aggregates that were not compatible with ESI-MS. By contrast, a lyophilization-based concentration procedure, followed by thawing above the liquid crystal transition temperature of the lipid component, maintained the liposome size profile and yielded excellent ion fluxes in both ESI-MS and nano-ESI-MS. Using these methods the global HDX profile of M2-TM in aqueous DMPC vesicles was compared with that in methanol, demonstrating that several amide sites were protected from exchange by the lipid membrane. We also show that hydrophobic peptides can be detected by ESI-MS in the presence of a large molar excess of the detergent Triton X-100. The rate of HDX of M2-TM in Triton X-100 micelles was faster than that in DMPC vesicles but slower than when the peptide had been denatured in methanol. These results indicate that the accessibility of backbone amide sites to the solvent can be profoundly affected by membrane protein structure and dynamics, as well as the properties of model bilayer systems.     

Irreversible thermal denaturation of cytochrome c studied by electrospray mass spectrometry

This work uses electrospray ionization mass spectrometry (ESI-MS) in conjunction with hydrogen/deuterium exchange (HDX) and optical spectroscopy for characterizing the solution-phase properties of cytochrome c (cyt c) after heat exposure. Previous work demonstrated that heating results in irreversible denaturation for a subpopulation of proteins in the sample. However, that study did not investigate the physical reasons underlying this interesting effect. Here we report that the formation of oxidative modifications at elevated temperature plays a key role for the observed behavior. Tryptic digestion followed by tandem mass spectrometry is used to identify individual oxidation sites. Trp59 and Met80 are among the modified amino acids. In native cyt c both of these residues are buried deep within the protein structure, such that covalent modifications would be expected to be particularly disruptive. ESI-MS analysis after heat exposure results in a bimodal charge-state distribution. Oxidized protein appears predominantly in charge states around 11+, whereas a considerably lower degree of oxidation is observed for the 7+ and 8+ peaks. This finding confirms that different oxidation levels are associated with different solution-phase conformations. HDX measurements for different charge states are complicated by peak distortions arising from oxygen adduction. Nonetheless, comparison with simulated peak shapes clearly shows that the HDX properties are different for high- and low-charge states, confirming that interconversion between unfolded and folded conformers is blocked in solution. In addition to oxidation, partial aggregation upon heat exposure likely contributes to the formation of irreversibly denatured protein.