Probing High-order Protein Complexes Using Native Mass Spectrometry and Hydrogen/Deuterium Exchange Mass Spectrometry: A Case Study Using Fresh and Commercial Hemoglobin Samples.

Native mass spectrometry (MS) analysis of protein complexes is highly susceptible to matrix effect, and addressing this predicament using buffer exchange is a common approach. Nevertheless, optimization of the buffer exchange protocol is not trivial. With the use of hemoglobin (Hb) as the model entity, it was discovered that the native mass spectrum of protein assembly is highly dependent on the buffer-exchange protocol. Given the dependence of native MS on the purification protocol, this work attempts to use hydrogen/deuterium exchange mass spectrometry (HDX-MS) for comparative studies of hemoglobin complexes in untreated fresh and commercial samples. The information obtained from the HDX study was found to correlate well with the native mass spectrometry analysis of the properly buffer-exchanged Hb samples. Both native MS and HDX-MS showed that the fresh Hb sample has retained the expected tetrameric structure, whereas the commercial Hb has largely been denatured to the dimeric form. These findings prove the complementarity of native MS and HDX-MS in the analysis of high-order protein complexes and stress the necessity to validate the integrity of the high-order structures of the proteins prior to the use of the protein samples for other biomedical studies.

Quantitative Hydrogen-Deuterium Exchange Mass Spectrometry for Simultaneous Structural Characterization and Affinity Indexing of Single Target Drug Candidate Libraries.

Hydrogen-deuterium eXchange mass spectrometry (HDX-MS) is increasingly used in drug development to locate binding sites and to identify allosteric effects in drug/target interactions. However, the potential of this technique to quantitatively analyze drug candidate libraries remains largely unexplored. Here, a collection of 13 WDR5-targeting small molecules with surface plasmon resonance (SPR) dissociation coefficients (KD) ranging from 20 nM to ∼116 µM were characterized using differential HDX-MS (ΔHDX-MS). Conventional qualitative analysis of the ΔHDX-MS data set revealed the binding interfaces for all compounds and allosteric effects where present. We then demonstrated that ΔHDX-MS signal-to-noise (S/N) not only can rank library-relative affinity but also can accurately predict KD from a calibration curve constructed from high-quality SPR data. Three methods for S/N calculation are explored, each suitable for libraries with different characteristics. Our results demonstrate the potential for ΔHDX-MS use in drug candidate library affinity validation and/or determination while simultaneously characterizing structure.

HDXBoxeR: an R package for statistical analysis and visualization of multiple Hydrogen-Deuterium Exchange Mass-Spectrometry datasets of different protein states.

SUMMARY: Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) is a powerful protein characterization technique that provides insights into protein dynamics and flexibility at the peptide level. However, analyzing HDX-MS data presents a significant challenge due to the wealth of information it generates. Each experiment produces data for hundreds of peptides, often measured in triplicate across multiple time points. Comparisons between different protein states create distinct datasets containing thousands of peptides that require matching, rigorous statistical evaluation, and visualization. Our open-source R package, HDXBoxeR, is a comprehensive tool designed to facilitate statistical analysis and comparison of multiple sets among samples and time points for different protein states, along with data visualization. AVAILABILITY AND IMPLEMENTATION: HDXBoxeR is accessible as the R package (https://cran.r-project.org/web//packages/HDXBoxeR) and GitHub: mkajano/HDXBoxeR.

Elucidation of the Reversible Self-Association Interface of a Diabody-Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling.

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

MSe Collision Energy Optimization for the Analysis of Membrane Proteins Using HDX-cIMS.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has evolved as an essential technique in structural proteomics. The use of ion mobility separation (IMS) coupled to HDX-MS has increased the applicability of the technique to more complex systems and has been shown to improve data quality and robustness. The first step when running any HDX-MS workflow is to confirm the sequence and retention time of the peptides resulting from the proteolytic digestion of the nondeuterated protein. Here, we optimized the collision energy ramp of HDMSE experiments for membrane proteins using a Waters SELECT SERIES cIMS-QTOF system following an HDX workflow using Phosphorylase B, XylE transporter, and Smoothened receptor (SMO) as model systems. Although collision energy (CE) ramp 10-50 eV gave the highest amount of positive identified peptides when using Phosphorylase B, XylE, and SMO, results suggest optimal CE ramps are protein specific, and different ramps can produce a unique set of peptides. We recommend cIMS users use different CE ramps in their HDMSE experiments and pool the results to ensure maximum peptide identifications. The results show how selecting an appropriate CE ramp can change the sequence coverage of proteins ranging from 4 to 94%.

Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange-Mass Spectrometry Experiments Using the Digestible Peptide Score.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS).

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the Fab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the CH2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.

Higher-Order Structure of Adeno-Associated Virus Serotype 8 by Hydrogen/Deuterium Exchange Mass Spectrometry.

The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.

Interpretation of Hydrogen/Deuterium Exchange Mass Spectrometry.

This paper sheds light on the meaning of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) data. HDX-MS data provide not structural information but dynamic information on an analyte protein. First, the reaction mechanism of backbone amide HDX reaction is considered and the correlation between the parameters from an X-ray crystal structure and the protection factors of HDX reactions of cytochrome c is evaluated. The presence of H-bonds in a protein structure has a strong influence on HDX rates which represent protein dynamics, while the solvent accessibility only weakly affects the HDX rates. Second, the energy diagrams of the HDX reaction at each residue in the presence and absence of perturbation are described. Whereas the free energy change upon mutation can be directly measured by the HDX rates, the free energy change upon ligand binding may be complicated due to the presence of unbound analyte protein in the protein-ligand mixture. Third, the meanings of HDX and other biophysical techniques are explained using a hypothetical protein folding well. The shape of the protein folding well describes the protein dynamics and provides Boltzmann distribution of open and closed states which yield HDX protection factors, while a protein's crystal structure represents a snapshot near the bottom of the well. All biophysical data should be consistent yet provide different information because they monitor different parts of the same protein folding well.

Cyclic Ion Mobility for Hydrogen/Deuterium Exchange-Mass Spectrometry Applications.

Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a powerful tool to probe protein dynamics. As a bottom-up technique, HDX-MS provides information at peptide-level resolution, allowing structural localization of dynamic changes. Consequently, the HDX-MS data quality is largely determined by the number of peptides that are identified and monitored after deuteration. Integration of ion mobility (IM) into HDX-MS workflows has been shown to increase the data quality by providing an orthogonal mode of peptide ion separation in the gas phase. This is of critical importance for challenging targets such as integral membrane proteins (IMPs), which often suffer from low sequence coverage or redundancy in HDX-MS analyses. The increasing complexity of samples being investigated by HDX-MS, such as membrane mimetic reconstituted and in vivo IMPs, has generated need for instrumentation with greater resolving power. Recently, Giles et al. developed cyclic ion mobility (cIM), an IM device with racetrack geometry that enables scalable, multipass IM separations. Using one-pass and multipass cIM routines, we use the recently commercialized SELECT SERIES Cyclic IM spectrometer for HDX-MS analyses of four detergent solubilized IMP samples and report its enhanced performance. Furthermore, we develop a novel processing strategy capable of better handling multipass cIM data. Interestingly, use of one-pass and multipass cIM routines produced unique peptide populations, with their combined peptide output being 31 to 222% higher than previous generation SYNAPT G2-Si instrumentation. Thus, we propose a novel HDX-MS workflow with integrated cIM that has the potential to enable the analysis of more complex systems with greater accuracy and speed.

Dynamics, allostery, and stabilities of whole virus particles by amide hydrogen/deuterium exchange mass spectrometry (HDXMS).

X-ray crystallography and cryo-electron microscopy have enabled the determination of structures of numerous viruses at high resolution and have greatly advanced the field of structural virology. These structures represent only a subset of snapshot end-state conformations, without describing all conformational transitions that virus particles undergo. Allostery plays a critical role in relaying the effects of varied perturbations both on the surface through environmental changes and protein (receptor/antibody) interactions into the genomic core of the virus. Correspondingly, allostery carries implications for communicating changes in genome packaging to the overall stability of the virus particle. Amide hydrogen/deuterium exchange mass spectrometry (HDXMS) of whole viruses is a powerful probe for uncovering virus allostery. Here we critically discuss advancements in understanding virus dynamics by HDXMS with single particle cryo-EM and computational approaches.

OligoR: A Native HDX/MS Data Processing Application Dedicated to Oligonucleotides.

Hydrogen-deuterium exchange mass spectrometry (HDX/MS) is increasingly used to study the dynamics of protein conformation. Coupled to native MS, HDX can also characterize the conformations of oligonucleotides and their binding to cations, small molecules, and proteins. Data processing and visualization of native HDX/MS of oligonucleotides requires dedicated software solutions. OligoR is a web-browser-based application that addresses the specific needs of DNA HDX/MS and native MS experiments from raw data in an open format to visualization and export of results. Whole experiments spanning many time points can be processed in minutes for several mass-separated species. To access valuable folding dynamics information, we have developed a simple and robust approach to deconvolute bimodal isotope distributions, even when they are highly overlapping. This approach is based on modeling physically possible isotope distributions determined from chemical formulae and could be extended to any type of analyte (proteins, peptides, sugars, and small molecules). All results are presented in interactive data tables, and publication-quality figures can be generated, customized, and exported.

Hydrogen/deuterium exchange-mass spectrometry of integral membrane proteins in native-like environments: current scenario and the way forward.

Integral membrane proteins (IMPs) perform a range of diverse functions and their dysfunction underlies numerous pathological conditions. Consequently, IMPs constitute most drug targets, and the elucidation of their mechanism of action has become an intense field of research. Historically, IMP studies have relied on their extraction from membranes using detergents, which have the potential to perturbate their structure and dynamics. To circumnavigate this issue, an array of membrane mimetics has been developed that aim to reconstitute IMPs into native-like lipid environments that more accurately represent the biological membrane. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has emerged as a versatile tool for probing protein dynamics in solution. The continued development of HDX-MS methodology has allowed practitioners to investigate IMPs using increasingly native-like membrane mimetics, and even pushing the study of IMPs into the in vivo cellular environment. Consequently, HDX-MS has come of age and is playing an ever-increasingly important role in the IMP structural biologist toolkit. In the present mini-review, we discuss the evolution of membrane mimetics in the HDX-MS context, focusing on seminal publications and recent innovations that have led to this point. We also discuss state-of-the-art methodological and instrumental advancements that are likely to play a significant role in the generation of high-quality HDX-MS data of IMPs in the future.

HDX-MS and MD Simulations Provide Evidence for Stabilization of the IgG1-FcγRIa (CD64a) Immune Complex Through Intermolecular Glycoprotein Bonds.

Previous reports present different models for the stabilization of the Fc-FcγRI immune complex. Although accord exists on the importance of L235 in IgG1 and some hydrophobic contacts for complex stabilization, discord exists regarding the existence of stabilizing glycoprotein contacts between glycans of IgG1 and a conserved FG-loop (171MGKHRY176) of FcγRIa. Complexes formed from the FcγRIa receptor and IgG1s containing biantennary glycans with N-acetylglucosamine, galactose, and α2,6-N-acetylneuraminic terminations were measured by hydrogen-deuterium exchange mass spectrometry (HDX-MS), classified for dissimilarity with Welch's ANOVA and Games-Howell post hoc procedures, and modeled with molecular dynamics (MD) simulations. For each glycoform of the IgG1-FcγRIa complex peptic peptides of Fab, Fc and FcγRIa report distinct H/D exchange rates. MD simulations corroborate the differences in the peptide deuterium content through calculation of the percent of time that transient glycan-peptide bonds exist. These results indicate that stability of IgG1-FcγRIa complexes correlate with the presence of intermolecular glycoprotein interactions between the IgG1 glycans and the 173KHR175 motif within the FG-loop of FcγRIa. The results also indicate that intramolecular glycan-protein bonds stabilize the Fc region in isolated and complexed IgG1. Moreover, HDX-MS data evince that the Fab domain has glycan-protein binding contacts within the IgG1-FcγRI complex.

Rapid Assessment of Pepsin Column Activity for Reliable HDX-MS Studies.

Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) is a widely used technique to probe protein structural dynamics, track conformational changes, and map protein-protein interactions. Most HDX-MS studies employ a bottom-up approach utilizing the acid active protease pepsin to digest the protein of interest, often utilizing immobilized protease in a column format. The extent of proteolytic cleavage will greatly influence data quality and presents a major source of variation in HDX-MS studies. Here, we present a simple cocktail of commonly available peptides that are substrates of pepsin and can serve as a rapid check of pepsin column activity. The peptide-based assay requires no system modifications and provides an immediate readout to check and benchmark pepsin activity across different HDX-MS platforms.

Complementary Structural Information for Stressed Antibodies from Hydrogen-Deuterium Exchange and Covalent Labeling Mass Spectrometry.

Identifying changes in the higher-order structure (HOS) of therapeutic monoclonal antibodies upon storage, stress, or mishandling is important for ensuring efficacy and avoiding adverse effects. Here, we demonstrate diethylpyrocarbonate (DEPC)-based covalent labeling (CL) mass spectrometry (MS) and hydrogen-deuterium exchange (HDX)/MS can be used together to provide site-specific information about subtle conformational changes that are undetectable by traditional techniques. Using heat-stressed rituximab as a model protein, we demonstrate that CL/MS is more sensitive than HDX/MS to subtle HOS structural changes under low stress conditions (e.g., 45 and 55 °C for 4 h). At higher heat stress (65 °C for 4 h), we find CL/MS and HDX/MS provide complementary information, as CL/MS reports on changes in side chain orientation while HDX/MS reveals changes in backbone dynamics. More interestingly, we demonstrate that the two techniques work synergistically to identify likely aggregation sites in the heat-stressed protein. In particular, the CH3 and CL domains experience decreases in deuterium uptake after heat stress, while only the CH3 domain experiences decreases in DEPC labeling extent as well, suggesting the CH3 domain is a likely site of aggregation and the CL domain only undergoes a decrease in backbone dynamics. The combination of DEPC-CL/MS and HDX/MS provides valuable structural information, and the two techniques should be employed together when investigating the HOS of protein therapeutics.

Combining Small-Molecule Bioconjugation and Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) to Expose Allostery: the Case of Human Cytochrome P450 3A4.

We report a novel approach to study allostery which combines the use of carefully selected bioconjugates and hydrogen-deuterium exchange mass spectrometry (HDX-MS). This strategy avoids issues related to weak substrate binding and ligand relocalization. The utility of our method is demonstrated using human cytochrome P450 3A4 (CYP3A4), the most important drug-metabolizing enzyme. Allosteric activation and inhibition of CYP3A4 by pharmaceuticals is an important mechanism of drug interactions. We performed HDX-MS analysis on several CYP3A4-effector bioconjugates, some of which mimic the allosteric effect of positive effectors, while others show activity enhancement even though the label does not occupy the allosteric pocket (agonistic) or do not show activation while still blocking the allosteric site (antagonistic). This allowed us to better define the position of the allosteric site, the protein structural dynamics associated with allosteric activation, and the presence of coexisting conformers.

Conformational Dynamics of α-Synuclein during the Interaction with Phospholipid Nanodiscs by Millisecond Hydrogen-Deuterium Exchange Mass Spectrometry.

Both normal and pathological functions of α-synuclein (αSN), an abundant protein in the central and peripheral nervous system, have been linked to its interaction with membrane lipid bilayers. The ability to characterize structural transitions of αSN upon membrane complexation will clarify molecular mechanisms associated with αSN-linked pathologies, including Parkinson's disease (PD), multiple systems atrophy, and other synucleinopathies. In this work, time-resolved electrospray ionization hydrogen/deuterium exchange mass spectrometry (TRESI-HDX-MS) was employed to acquire a detailed picture of αSN's conformational transitions as it undergoes complexation with nanodisc membrane mimics with different headgroup charges (zwitterionic DMPC and negative POPG). Using this approach, αSN interactions with DMPC nanodiscs were shown to be rapid exchanging and to have little impact on the αSN conformational ensemble. Interactions with nanodiscs containing lipids known to promote amyloidogenesis (e.g., POPG), on the other hand, were observed to induce substantial and specific changes in the αSN conformational ensemble. Ultimately, we identify a region corresponding residues 19-28 and 45-57 of the αSN sequence that is uniquely impacted by interactions with "amyloidogenic" lipid membranes, supporting the existing "broken-helix" model for α-synuclein/membrane interactions, but do not detect a "helical extension" that is also thought to play a role in αSN aggregation.

Epitope and Paratope Mapping by HDX-MS Combined with SPR Elucidates the Difference in Bactericidal Activity of Two Anti-NadA Monoclonal Antibodies.

Characterization of antigen-antibody interactions is crucial for understanding antibody-mediated protection against pathogens, biopharmaceutical development, as well as evaluation of the immune response post vaccination. Bexsero is a multicomponent vaccine against Neisseria meningitidis serogroup B in which one of the key vaccine antigens is Neisserial adhesin A (NadA), a trimeric coiled-coil protein. Two NadA-specific monoclonal antibodies (mAbs) isolated from Bexsero-vaccinated individuals have been shown to have similar binding affinity and appear to recognize a similar antigen region, yet only one of the mAbs is bactericidal. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to perform an in-depth study of the interaction of the two mAbs with NadA antigen using a combined epitope and paratope mapping strategy. In addition, we use surface plasmon resonance (SPR) to investigate the stoichiometry of the binding of the two mAbs to NadA. While epitope mapping only identifies a clear binding impact of one of the mAbs on NadA, the paratope mapping analyses shows that both mAbs are binding to NadA through several complementarity determining regions spanning both heavy and light chains. Our results highlight the advantage of combined epitope and paratope mapping HDX-MS experiments and supporting biochemical experiments to characterize antigen-antibody interactions. Through this combined approach, we provide a rationale for how the binding stoichiometry of the two mAbs to the trimeric NadA antigen can explain the difference in bactericidal activity of the two mAbs.

Investigation of D76N ß2-Microglobulin Using Protein Footprinting and Structural Mass Spectrometry.

NMR studies and X-ray crystallography have shown that the structures of the 99-residue amyloidogenic protein ß2-microglobulin (ß2m) and its more aggregation-prone variant, D76N, are indistinguishable, and hence, the reason for the striking difference in their aggregation propensities remains elusive. Here, we have employed two protein footprinting methods, hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP), in conjunction with ion mobility-mass spectrometry, to probe the differences in conformational dynamics of the two proteins. Using HDX-MS, a clear difference in HDX protection is observed between these two proteins in the E-F loop (residues 70-77) which contains the D76N substitution, with a significantly higher deuterium uptake being observed in the variant protein. Conversely, following FPOP-MS only minimal differences in the level of oxidation between the two proteins are observed in the E-F loop region, suggesting only modest side-chain movements in that area. Together the HDX-MS and FPOP-MS data suggest that a tangible perturbation to the hydrogen-bonding network in the E-F loop has taken place in the D76N variant and furthermore illustrate the benefit of using multiple complementary footprinting methods to address subtle, but possibly biologically important, differences between highly similar proteins.