Hydrogen/Deuterium Exchange Mass Spectrometry for the Structural Analysis of Detergent-Solubilized Membrane Proteins.

Integral membrane proteins are involved in numerous biological functions and represent important drug targets. Despite their abundance in the human proteome, the number of integral membrane protein structures is largely underrepresented in the Protein Data Bank. The challenges associated with the biophysical characterization of such biological systems are well known. Most structural approaches, including X-ray crystallography, SAXS, or mass spectrometry (MS), require the complete solubilization of membrane proteins in aqueous solutions. Detergents are frequently used for this task, but may interfere with the analysis, as is the case with MS. The use of "MS-friendly" detergents, such as non-ionic alkyl glycoside detergents, has greatly facilitated the analysis of detergent-solubilized membrane proteins. Here, we describe a protocol, which we have successfully implemented in our laboratory to study the structure and dynamics of detergent-solubilized integral membrane proteins by Hydrogen/Deuterium eXchange and Mass Spectrometry (HDX-MS). The procedure does not require detergent removal prior to MS analysis, instead taking advantage of the ultra-high pressure chromatographic system to separate deuterated peptides from "MS-friendly" detergents.

Rapid Solution-Phase Hydrogen/Deuterium Exchange for Metabolite Compound Identification.

Rapid, solution-phase hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (MS) is demonstrated as a means for distinguishing small-molecule metabolites. HDX is achieved using capillary vibrating sharp-edge spray ionization (cVSSI) to allow sufficient time for reagent mixing and exchange in micrometer-sized droplets. Different compounds are observed to incorporate deuterium with varying efficiencies resulting in unique isotopic patterns as revealed in the MS spectra. Using linear regression techniques, parameters representing contribution to exchange by different hydrogen types can be computed. In this proof-of-concept study, the exchange parameters are shown to be useful in the retrodiction of the amount of deuterium incorporated within different compounds. On average, the exchange parameters retrodict the exchange level with ~ 2.2-fold greater accuracy than treating all exchangeable hydrogens equally. The parameters can be used to produce hypothetical isotopic distributions that agree (± 16% RMSD) with experimental measurements. These initial studies are discussed in light of their potential value for identifying challenging metabolite species.

Probing the Dissociation of Protein Complexes by Means of Gas-Phase H/D Exchange Mass Spectrometry.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration. Graphical abstract ᅟ.

Thiol-ene Microfluidic Chip for Performing Hydrogen/Deuterium Exchange of Proteins at Subsecond Time Scales.

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14-1.1 s time frame. The HDX of Hb at 0.14-1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14-1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.

Installation, validation, and application examples of two instrumental setups for gas-phase HDX-MS analysis of peptides and proteins.

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry in a millisecond timeframe after ESI (gas-phase HDX-MS) is a fast and sensitive, yet unharnessed method to analyze the primary- and higher-order structure, intramolecular and intermolecular interactions, surface properties, and charge location of peptides and proteins. During a gas-phase HDX-MS experiment, heteroatom-bound non-amide hydrogens are made to exchange with deuterium during a millisecond timespan after electrospray ionization (ESI) by reaction with the highly basic reagent ND3, enabling conformational analysis of protein states that are pertinent to the native solution-phase. Here, we describe two different instrumental approaches to enable gas-phase HDX-MS for analysis of peptides and proteins on high-resolution Q-TOF mass spectrometers. We include a description of the procedure and equipment required for successful installation as well as suggested procedures for testing, validation, and troubleshooting of a gas-phase HDX-MS setup. In the two described approaches, gas-phase HDX-MS are performed either immediately after ESI in the cone exit region by leading N2-gas over a deuterated ND3/D2O solution, or by leading purified ND3-gas into different traveling wave ion guides (TWIG) of the mass spectrometer. We envision that a detailed description of the two gas-phase HDX-MS setups and their practical implementation and validation can pave the way for gas-phase HDX-MS to become a more routinely used MS technique for structural analysis of peptides and proteins.

Biophysical Interactions of Direct AMPK Activators.

Protein-ligand interactions can be evaluated by a number of different biophysical methods. Here we describe some of the experimental methods that we have used to generate AMPK protein reagents and characterize its interactions with direct synthetic activators. Recombinant heterotrimeric AMPK complexes were generated using standard molecular biology methods by expression either in insect cells via infection with three different viruses or more routinely in Escherichia coli with a tricistronic expression vector. Hydrogen/deuterium exchange (HDX) coupled with mass spectrometry was used to probe protein conformational changes and potential binding sites of activators on AMPK. X-ray crystallographic studies were carried out on crystals of AMPK with bound ligands to reveal detailed molecular interactions formed by AMPK activators at near-atomic resolution. In order to gain insights into the mechanism of enzyme activation and to probe the effects of AMPK activators on kinetic parameters such as Michaelis-Menten constant (K m ) or maximal reaction velocity (V max), we performed classical enzyme kinetic studies using radioactive 33P-ATP-based filter assay. Equilibrium dissociation constants (K D ) and on and off rates of ligand binding were obtained by application of surface plasmon resonance (SPR) technique.

Application of GC/MS Soft Ionization for Isomeric Biological Compound Analysis.

Isomers are compounds with the same molecular formula. Many different types of isomers are ubiquitous and play important roles in living organisms. Despite their early discovery, the actual analysis of isomers has been tricky and has confounded researchers. Using mass spectrometry (MS) to distinguish or identify isomers is an emergent topic and challenge for analytical chemists. We review some techniques for analyzing isomers with emphasis on MS, e.g., the roles of ion reaction, hydrogen-deuterium exchange, ion mobility mass spectrometry, ion spectroscopy, and energy change in producing isomer-specific fragments. In particular, soft ionization for gas chromatography-mass spectrometry (GC-MS) is a focus in this review. Awareness of the advantages and technical problems of these techniques would inspire innovation in future approaches.

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational Dynamics and Interactions of Membrane Proteins by Hydrogen/Deuterium Mass Spectrometry.

Hydrogen/deuterium exchange associated with mass spectrometry has been recently used to characterize the dynamics and the interactions of membrane proteins. Here we describe experimental workflow enabling localization of the regions involved in conformational changes or interactions.

Two-Site Evaluation of the Repeatability and Precision of an Automated Dual-Column Hydrogen/Deuterium Exchange Mass Spectrometry Platform.

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.

Conformational Analysis of Proteins in Highly Concentrated Solutions by Dialysis-Coupled Hydrogen/Deuterium Exchange Mass Spectrometry.

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. Graphical Abstract ᅟ.

Online deuterium hydrogen exchange and protein digestion coupled with ion mobility spectrometry and tandem mass spectrometry.

Online deuterium hydrogen exchange (DHX) and pepsin digestion (PD) is demonstrated using drift tube ion mobility spectrometry (DTIMS) coupled with linear ion trap (LTQ) mass spectrometry (MS) with electron transfer dissociation (ETD) capabilities. DHX of deuterated ubiquitin, followed by subsequent quenching and digestion, is performed within ∼60 s, yielding 100% peptide sequence coverage. The high reproducibility of the IMS separation allows spectral feature matching between two-dimensional IMS-MS datasets (undeuterated and deuterated) without the need for dataset alignment. Extracted ion drift time distributions (XIDTDs) of deuterated peptic peptides are mobility-matched to corresponding XIDTDs of undeuterated peptic peptides that were identified using collision-induced dissociation (CID). Matching XIDTDs allows a straightforward identification and deuterium retention evaluation for labeled peptides. Aside from the mobility separation, the ion trapping capabilities of the LTQ, combined with ETD, are demonstrated to provide single-residue resolution. Deuterium retention for the c- series ions across residues M(1)-L(15) and N(25)-R(42) are in good agreement with the known secondary structural elements within ubiquitin.

In ESI-source H/D exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa: the role of the temperature of the desolvating capillary on H/D exchange.

Transition of proteins from the solution to the gas phase during electrospray ionization remains a challenging problem despite the large amount of attention it has received during the past few decades. One of the major questions relates to the extent to which proteins in the gas phase retain their condensed phase structures. We have used in-electrospray source hydrogen/deuterium exchange to determine the number of deuterium incorporations as a function of protein mass, charge state and temperature of the desolvating capillary where the reaction occurs. All experiments were performed on a Thermo LTQ FT Ultra equipped with a 7-T superconducting magnet. Ions were generated by an IonMax Electrospray ion source operated in the positive ESI mode. Deuterium exchange was performed by introducing a droplet of D2 O beneath the ESI capillary. We systematically investigated gas phase hydrogen/deuterium (H/D) exchange under atmospheric pressure for peptides and proteins of different molecular weights from 1 to 66 kDa. We observed that almost all proteins demonstrate similar exchange rates for all charge states and that these rates increase exponentially with the temperature of the desolvating capillary. We did not observe any clear correlation of the number of H/D exchanges with the value of the cross section for a corresponding charge state. We have demonstrated the possibility of performing in-ESI source H/D exchange of large proteins under atmospheric pressure. The simplicity of the experimental setup makes it a useful experimental technique that can be applied for the investigation of gas phase conformations of proteins.

Characterizing rapid, activity-linked conformational transitions in proteins via sub-second hydrogen deuterium exchange mass spectrometry.

This review outlines the application of time-resolved electrospray ionization mass spectrometry (TRESI-MS) and hydrogen-deuterium exchange (HDX) to study rapid, activity-linked conformational transitions in proteins. The method is implemented on a microfluidic chip which incorporates all sample-handling steps required for a 'bottom-up' HDX workflow: a capillary mixer for sub-second HDX labeling, a static mixer for HDX quenching, a microreactor for rapid protein digestion, and on-chip electrospray. By combining short HDX labeling pulses with rapid digestion, this approach provides a detailed characterization of the structural transitions that occur during protein folding, ligand binding, post-translational modification and catalytic turnover in enzymes. This broad spectrum of applications in areas largely inaccessible to conventional techniques means that microfluidics-enabled TRESI-MS/HDX is a unique and powerful approach for investigating the dynamic basis of protein function.

Analysis of disordered proteins using a simple apparatus for millisecond quench-flow H/D exchange.

Measurement of amide H/D exchange on the ms time scale can provide valuable information about the dynamic behavior of the most flexible regions of proteins. We describe here a simple mixing apparatus, assembled solely from off-the-shelf components, that can be used for H/D exchange mass spectrometry to measure exchange on the 50-5000 ms time scale. Our apparatus utilizes flow-injection to minimize sample consumption. Although the mixer operates at low Reynolds numbers (less than 10(2)) where laminar flow is expected, H/D exchange kinetics were well-approximated using the assumption of plug-flow. We validated this approximation using fluorescence imaging of fluorescein-conjugated bovine serum albumin in the delay line and by demonstrating agreement between measured and calculated H/D exchange kinetics for a mixture of peptides. The performance of the apparatus was further validated by measuring rapid H/D exchange kinetics by an intrinsically disordered protein, murine CBP(2059-2117) (UniProt CBP_MOUSE). H/D exchange data from CBP, both free and in complex with human ACTR(1018-1088) (UniProt NCOA3_HUMAN), were consistent with previous biophysical studies of this protein.

Minimizing carry-over in an online pepsin digestion system used for the H/D exchange mass spectrometric analysis of an IgG1 monoclonal antibody.

Chromatographic carry-over can severely distort measurements of amide H/D exchange in proteins analyzed by LC/MS. In this work, we explored the origin of carry-over in the online digestion of an IgG1 monoclonal antibody using an immobilized pepsin column under quenched H/D exchange conditions (pH 2.5, 0 °C). From a consensus list of 169 different peptides consistently detected during digestion of this large, ~150 kDa protein, approximately 30% of the peptic peptides exhibited carry-over. The majority of carry-over originates from the online digestion. Carry-over can be substantially decreased by washing the online digestion flow-path and pepsin column with two wash cocktails: [acetonitrile (5%)/isopropanol (5%)/acetic acid (20%) in water] and [2 M guanidine hydrochloride in 100 mM phosphate buffer pH 2.5]. Extended use of this two-step washing procedure does not adversely affect the specificity or activity of the immobilized pepsin column. The results suggest that although the mechanism of carry-over appears to be chemical in nature, and not hydrodynamic, carry-over cannot be attributed to a single factor such as mass, abundance, pI, or hydrophobicity of the peptides.

A supplementary coil for ²H decoupling with commercial HCN MAS probes.

Partial deuteration is a powerful tool to increase coherence life times and spectral resolution in proton solid-state NMR. The J coupling to deuterium needs, however, to be decoupled to maintain the good resolution in the (usually indirect) (13)C dimension(s). We present a simple and reversible way to expand a commercial 1.3mm HCN MAS probe with a (2)H channel with sufficient field strength for J-decoupling of deuterium, namely 2-3kHz. The coil is placed at the outside of the stator and requires no significant modifications to the probe. The performance and the realizable gains in sensitivity and resolution are demonstrated using perdeuterated ubiquitin, with selectively CHD(2)-labeled methyl groups.

Ion-water hydrogen-bond switching observed with 2D IR vibrational echo chemical exchange spectroscopy.

The exchange of water hydroxyl hydrogen bonds between anions and water oxygens is observed directly with ultrafast 2D IR vibrational echo chemical exchange spectroscopy (CES). The OD hydroxyl stretch of dilute HOD in H(2)O in concentrated (5.5 M) aqueous solutions of sodium tetrafluoroborate (NaBF(4)) displays a spectrum with a broad water-like band (hydroxyl bound to water oxygen) and a resolved, blue shifted band (hydroxyl bound to BF(4)(-)). At short time (200 fs), the 2D IR vibrational echo spectrum has 4 peaks, 2 on the diagonal and 2 off-diagonal. The 2 diagonal peaks are the 0-1 transitions of the water-like band and the hydroxyl-anion band. Vibrational echo emissions at the 1-2 transition frequencies give rise to 2 off-diagonal peaks. On a picosecond time scale, additional off-diagonal peaks grow in. These new peaks arise from chemical exchange between water hydroxyls bound to anions and hydroxyls bound to water oxygens. The growth of the chemical exchange peaks yields the time dependence of anion-water hydroxyl hydrogen bond switching under thermal equilibrium conditions as T(aw) = 7 +/- 1 ps. Pump-probe measurements of the orientational relaxation rates and vibrational lifetimes are used in the CES data analysis. The pump-probe measurements are shown to have the correct functional form for a system undergoing exchange.

H/D exchange and mass spectrometry-based method for biophysical analysis of multidomain proteins at the domain level.

A protocol was developed to characterize the domain-specific thermodynamic stabilities of multidomain proteins using SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange). The protocol incorporates a protease digestion step into the conventional SUPREX protocol and enables folding free energy (DeltaGf) and cooperativity (m-value) measurements to be made on the individual domains of multidomain proteins in their native context (i.e., in the intact protein). Three multidomain protein systems (calmodulin, a Fyn construct, and transferrin) were used to validate the SUPREX-protease digestion protocol. The DeltaGf and m-value of each domain in the intact test proteins were measured in the absence and presence of ligands using the new protocol. Domain-specific thermodynamic parameters were obtained on each system; and the measured parameters were consistent with known biophysical properties of the test proteins. The known stabilization of the N-terminal domain of CaM in the context of the intact protein and the known binding affinity of a proline-rich peptide to the SH3 domain in the Fyn construct were successfully quantified using the new protocol. Qualitative information about the relative calcium binding affinities of the N- and C-terminal domains of CaM and about the relative iron binding affinities of the N- and C-terminal domains of transferrin was also obtained using the new protocol.

Hydrogen/deuterium exchange mass spectrometry analysis of protein aggregates.

The elucidation of the structure of amyloid fibrils and related aggregates is an important step toward understanding the pathogenesis of diseases like Alzheimer's disease, which feature protein misfolding and/or aggregation. However, the large size, heterogeneous morphology, and poor solubility of amyloid-like fibrils make them resistant to high-resolution structure determination. Using amyloid fibrils and protofibrils of the Alzheimer's plaque peptide amyloid beta as examples, we describe here the use of hydrogen/deuterium exchange methods in conjunction with electrospray ionization mass spectrometry to determine regions of the peptide involved in beta-sheet network when it is incorporated into protein aggregates. The advantages of this method are low sample utilization and high speed. The basic methodology exploits the fact that protons either involved in H-bonded secondary structures or buried in a protein's core structure exchange more slowly with deuterium than do solvent-exposed and non-H-bonded protons. Details of all aspects of this methodology, including sample preparation, data acquisition, and data analysis, are described. These data provide insights into the structures of monomers, protofibrils, and fibrils and to the structural relations among these states.