Hydrogen Exchange Mass Spectrometry Research Articles

Single-domain antibodies reveal unique borrelicidal epitopes on the Lyme disease vaccine antigen, outer surface protein A (OspA).

Camelid-derived, single-domain antibodies (VHHs) have proven to be extremely powerful tools in defining the antigenic landscape of immunologically heterogeneous surface proteins. In this report, we generated a phage-displayed VHH library directed against the candidate Lyme disease vaccine antigen, outer surface protein A (OspA). Two alpacas were immunized with recombinant OspA serotype 1 from Borrelia burgdorferi sensu stricto strain B31, in combination with the canine vaccine RECOMBITEK Lyme containing lipidated OspA. The phage library was subjected to two rounds of affinity enrichment ("panning") against recombinant OspA, yielding 21 unique VHHs within two epitope bins, as determined through competition enzyme linked immunosorbent assays (ELISAs) with a panel of OspA-specific human monoclonal antibodies. Epitope refinement was conducted by hydrogen exchange-mass spectrometry. Six of the monovalent VHHs were expressed as human IgG1-Fc fusion proteins and shown to have functional properties associated with protective human monoclonal antibodies, including B. burgdorferi agglutination, outer membrane damage, and complement-dependent borreliacidal activity. The VHHs displayed unique reactivity profiles with the seven OspA serotypes associated with B. burgdorferi genospecies in the United States and Europe consistent with there being unique epitopes across OspA serotypes that should be considered when designing and evaluating multivalent Lyme disease vaccines.

Optimization of a Hydrogen Exchange-Mass Spectrometry Robotic Liquid Handler Using Tracers.

Hydrogen exchange-mass spectrometry (HX-MS) is a valuable analytical technique that can provide insight into protein interactions and structure. The deuterium labeling necessary to gain this insight is affected by many physical and chemical factors, making it challenging to achieve high reproducibility. Poor precision during dispensing, transfer, and mixing of solutions during the experiment contributes substantially to the overall variability. While the use of a robotic liquid handler can potentially improve precision, its operation must be optimized. We observed poor precision in data collected using a robotic liquid handler to perform HX-MS. In this work, we describe how we were able to improve that system's precision considerably based on tracking performance using caffeine, caffeine-d3, and caffeine-d9 as tracers for the sample, label, and quench to report on each operation of the liquid handling workflow. The insights gained about liquid handler performance and the three-tracer approach can aid in optimizing HX-MS workflow operations, whether performed manually or when using a liquid handling system. Additionally, these tracers can be incorporated as internal tracers during an experiment to report on the labeling and quench operations of each sample throughout the run and, if desired, be used to implement an uptake correction described previously.

Interaction between preservatives and a monoclonal antibody in support of multidose formulation development

Multidose formulations have patient-centric advantages over single-dose formats. A major challenge in developing multidose formulations is the prevention of microbial growth that can potentially be introduced during multiple drawings. The incorporation of antimicrobial preservatives (APs) is a common approach to inhibit this microbial growth. Selection of the right preservative while maintaining drug product stability is often challenging. We explored the effects of three APs, 1.1 % (w/v) benzyl alcohol, 0.62 % (w/v) phenol, and 0.42 % (w/v) m-cresol, on a model immunoglobulin G1 monoclonal antibody, termed the “NIST mAb.” As measured by hydrogen exchange-mass spectrometry (HX-MS) and differential scanning calorimetry, conformational stability was decreased in the presence of APs. Specifically, flexibility (faster HX) was significantly increased in the CH2 domain (HC 238–255) across all APs. The addition of phenol caused the greatest conformational destabilization, followed by m-cresol and benzyl alcohol. Storage stability studies conducted by subvisible particle (SVP) analysis at 40 °C over 4 weeks further revealed an increase in SVPs in the presence of phenol and m-cresol but not in the presence of benzyl alcohol. However, as monitored by size exclusion chromatography, there was neither a significant change in the monomeric content nor an accumulation of soluble aggregate in the presence of APs.

The Deuterium Calculator: An Open-Source Tool for Hydrogen-Deuterium Exchange Mass Spectrometry Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time-consuming. Moreover, the HDX community is expanding to investigate multiprotein and highly complex protein systems which further complicates data analysis. Thus, a simple, open-source software package designed to analyze large and highly complex protein systems is needed. In this vein, we have developed "The Deuterium Calculator", a Python-based software package for HDX-MS data analysis. The Deuterium Calculator is capable of differential and nondifferential HDX-MS analysis, produces standardized data files according to recommendations from the International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX) to increase transparency in data analysis, and generates Woods' plots for statistical analysis and data visualization. This standard output can be used to perform time dependent deuteration studies and for the study of protein folding kinetics or differential uptake. Moreover, The Deuterium Calculator is capable of performing these analyses on large HDX-MS data sets (e.g., LC-HDX-MS from cell lysate digest). The Deuterium Calculator is freely available for download at https://github.com/OUWuLab/TheDeuteriumCalculator.git. Data are available via ProteomeXchange with identifier PXD036813.

High-Throughput Epitope Mapping by Hydrogen Exchange-Mass Spectrometry.

In this paper, we introduce a screening protocol for epitope mapping by hydrogen exchange mass spectrometry (HX-MS) that has higher throughput than a traditional HX-MS epitope mapping. In the screening protocol, three HX labeling times (20, 1000, and 86400 s) are each measured without replicates. The experimental protocol is anchored on a single epitope mapping experiment conducted using the traditional complete protocol (five HX times measured in triplicate) that is used to define HX times and define significance limits. Previously, we reported traditional epitope mapping results on the Borrelia burgdorferi outer surface protein A (OspA) antigen that are in excellent agreement with the X-ray crystallography results. Here, we show that the screening protocol and complete HX-MS identify identical epitopes of OspA but that the screening protocol has a 5-fold higher throughput.

Human B Cell Epitope Map of the Lyme Disease Vaccine Antigen, OspA.

The Lyme disease (LD) vaccine formerly approved for use in the United States consisted of recombinant outer surface protein A (OspA) from Borrelia burgdorferi sensu stricto (ss), the bacterial genospecies responsible for the vast majority of LD in North America. OspA is an ∼30 kDa lipoprotein made up of 21 antiparallel β-strands and a C-terminal α-helix. In clinical trials, protection against LD following vaccination correlated with serum antibody titers against a single epitope near the C-terminus of OspA, as defined by the mouse monoclonal antibody (MAb), LA-2. However, the breadth of the human antibody response to OspA following vaccination remains undefined even as next-generation multivalent OspA-based vaccines are under development. In this report, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes recognized by a unique panel of OspA human MAbs, including four shown to passively protect mice against experimental B. burgdorferi infection and one isolated from a patient with antibiotic refractory Lyme arthritis. The epitopes grouped into three spatially distinct bins that, together, encompass more than half the surface-exposed area of OspA. The bins corresponded to OspA β-strands 8-10 (bin 1), 11-13 (bin 2), and 16-20 plus the C-terminal α-helix (bin 3). Bin 3 was further divided into sub-bins relative to LA-2's epitope. MAbs with complement-dependent borreliacidal activity, as well as B. burgdorferi transmission-blocking activity in the mouse model were found within each bin. Therefore, the resulting B cell epitope map encompasses functionally important targets on OspA that likely contribute to immunity to B. burgdorferi.

Epitope mapping of a blood-brain barrier crossing antibody targeting the cysteine-rich region of IGF1R using hydrogen-exchange mass spectrometry enabled by electrochemical reduction.

Pathologies of the central nervous system impact a significant portion of our population, and the delivery of therapeutics for effective treatment is challenging. The insulin-like growth factor-1 receptor (IGF1R) has emerged as a target for receptor-mediated transcytosis, a process by which antibodies are shuttled across the blood-brain barrier (BBB). Here, we describe the biophysical characterization of VHH-IR4, a BBB-crossing single-domain antibody (sdAb). Binding was confirmed by isothermal titration calorimetry and an epitope was highlighted by surface plasmon resonance that does not overlap with the IGF-1 binding site or other known BBB-crossing sdAbs. The epitope was mapped with a combination of linear peptide scanning and hydrogen-deuterium exchange mass spectrometry (HDX-MS). IGF1R is large and heavily disulphide bonded, and comprehensive HDX analysis was achieved only through the use of online electrochemical reduction coupled with a multiprotease approach, which identified an epitope for VHH-IR4 within the cysteine-rich region (CRR) of IGF1R spanning residues W244-G265. This is the first report of an sdAb binding the CRR. We show that VHH-IR4 inhibits ligand induced auto-phosphorylation of IGF1R and that this effect is mediated by downstream conformational effects. Our results will guide the selection of antibodies with improved trafficking and optimized IGF1R binding characteristics.

Identification of Agitation-Induced Unfolding Events Causing Aggregation of Monoclonal Antibodies Using Hydrogen Exchange-Mass Spectrometry

Due to significant safety tolerances on maximum levels of visible and sub-visible particles in parenterally dosed drug products like monoclonal antibodies (mAbs), particle formation rates must be determined during development and minimized. Agitation stress, encountered during transportation and manufacturing, increases particle formation rates in a protein and formulation dependent fashion in a phenomenon thought to be partially mediated by mAb adsorption to the continuously regenerating air-water interface that results from agitation. The goal of this study was to explore the structural dynamics of three mAbs with variable sensitivity to agitation to develop a mechanistic understanding of exactly what occurs at the air-water interface that leads to aggregation and particle formation. We observed preferential orientation at the interface and subsequent cooperative unfolding for the molecule which aggregates most extensively under agitation, and also that the magnitude of destabilization appears to scale with particle formation rates. We also show that polysorbate, a widely-used excipient in parenteral formulations to protect against particle formation, eliminates interface-induced destabilization. This study provides direct evidence that local unfolding events resulting from interface exposure precede particle formation and may play a causal role in the process.

Potassium channel folding assessed by hydrogen exchange mass spectrometry and tryptophan fluorescence identifies pore helix and selectivity filter folding events

While the function of ion channels is critical for numerous physiological processes, how they achieve their three-dimensional fold and assemble remains an open topic. Outstanding questions concern the identification of the rate-limiting step, when the selectivity filter folds, and the role of a protein-dense phase in folding. We have made significant progress in characterizing the in vitro folding of the KcsA transmembrane pore domain, a robust model system for the pore domain of human potassium channels such as hERG and Kv1.2.

Virus breathing, metastability and epitope dynamics

RNA viruses are metastable macromolecular assemblies that sense their environment through dynamic breathing motions. Amide hydrogen/exchange mass spectrometry (HDXMS) is a powerful method to uncover dynamics of whole viral particles in solution. I will describe dynamics of viral surface regions and core ribonucleoprotein in two model viruses: Dengue and Turnip Crinkle Virus (TCV). We have identified a broad neutralizing antibody, C10 that induces altered ‘breathing’ dynamics in dengue viral particles and shows unique stoichiometry-specific responses.

Native State Hydrogen Exchange-Mass Spectrometry Methods to Probe Protein Folding and Unfolding.

Native state hydrogen exchange (HX) methods provide high-resolution structural data on the rare and transient opening motions in proteins under native conditions. Mass spectrometry-based HX methods (HX-MS) have gained popularity because of their ability to delineate population distributions, which allow a direct determination of the mechanism of inter conversion of the partially folded states under native conditions. Various technological advancements have provided further impetus to the development of HX-MS-based experiments to study protein folding. Classical HX-MS studies use proteolytic digestion to produce fragments of the protein subsequent to HX in solution, in order to obtain structural data. New chemical fragmentation methods, which achieve the same result as proteolysis and cause minimal change to the HX pattern in the protein, provide an attractive alternative to proteolysis. Moreover, when used in conjunction with proteolysis, chemical fragmentation methods have significantly increased the structural resolution afforded by HX-MS studies, even bringing them at par with the single amino acid resolution observed in NMR-based measurements. Experiments based on one such chemical fragmentation method, electron transfer dissociation (ETD), are described in this chapter. The ETD HX-MS method is introduced using data from a protein which is inherently resistant to proteolytic digestion as example of how such an experiment can provide high-resolution structural data on the folding-unfolding transitions of the protein under native conditions.

Interaction of Aluminum-adjuvanted Recombinant P[4] Protein Antigen With Preservatives: Storage Stability and Backbone Flexibility Studies

Eight antimicrobial preservatives used in parenteral multidose formulations (thimerosal, 2-phenoxy ethanol, phenol, benzyl alcohol, m-cresol, chlorobutanol, methyl paraben, propyl paraben) were examined for their effects on the storage stability (4 °C, 25 °C) of an Alhydrogel® (AH) adjuvanted formulation of the non-replicating rotavirus vaccine (NRRV) recombinant P[4] protein antigen. The stability of AH-adsorbed P[4] was monitored for antigen-antibody binding, conformational stability, and antigen-adjuvant interaction via competitive ELISA, DSC, and SDS-PAGE, respectively. There was an unexpected correlation between increasing storage stability of the AH-adsorbed P[4] and preservative hydrophobicity (log P) (e.g., the parabens and chlorobutanol were least destabilizing). We used hydrogen exchange-mass spectrometry (HX-MS) to better understand the destabilizing effects of temperature and preservative on backbone flexibility of AH-adsorbed P[4]. Thimerosal addition immediately increased the backbone flexibility across much of the AH-adsorbed P[4] protein backbone (except the N-terminal P2 region and residues G17-Y38), and further increase in P[4] backbone flexibility was observed after storage (4 °C, 4 weeks). HX-MS analysis of AH-adsorbed P[4] stored for 4 weeks at 25 °C revealed structural alterations in some regions of the epitope involved in P[4] specific mAb binding. These combined results are discussed in terms of a generalized workflow for multi-dose vaccine formulation development for recombinant protein antigens.

Structural Characterization of the Cooperativity of Unfolding of a Heterodimeric Protein using Hydrogen Exchange-Mass Spectrometry

Little is known about how the sequence of structural changes in one chain of a heterodimeric protein is coupled to those in the other chain during protein folding and unfolding reactions, and whether individual secondary structural changes in the two chains occur in one or many coordinated steps. Here, the unfolding mechanism of a small heterodimeric protein, double chain monellin, has been characterized using hydrogen exchange-mass spectrometry. Transient structure opening, which enables HX, was found to be describable by a five state N ↔ I1 ↔ I2 ↔ I3 ↔ U mechanism. Structural changes occur gradually in the first three steps, and cooperatively in the last step. β strands 2, 4 and 5, as well as the α-helix undergo transient unfolding during all three non-cooperative steps, while β1 and the two loops on both sides of the helix undergo transient unfolding during the first two steps. In the absence of GdnHCl, only β3 in chain A of the protein unfolds during the last cooperative step, while in the presence of 1 M GdnHCl, not only β3, but also β2 in chain B unfolds cooperatively. Hence, the extent of cooperative structural change and size of the cooperative unfolding unit increase when the protein is destabilized by denaturant. The naturally evolved two-chain variant of monellin folds and unfolds in a more cooperative manner than does a single chain variant created artificially, suggesting that increasing folding cooperativity, even at the cost of decreasing stability, may be a driving force in the evolution of proteins.

Statistical Equivalence Testing of Higher-Order Protein Structures with Differential Hydrogen Exchange-Mass Spectrometry (HX-MS).

Hydrogen exchange-mass spectrometry (HX-MS) is widely recognized for its potential utility for establishing the equivalence of the higher-order structures of proteins, particularly in comparability and similarity contexts. However, recent progress in the statistical analysis of HX-MS data has instead placed an emphasis on significance testing to identify regions of proteins where there are significant differences in HX between two or more protein states. In the cases involving assessment of similarity or equivalence of the higher-order structure of different protein samples (e.g., biosimilars), significance testing of HX-MS data is unsuitable. To meet this need, we have adapted the univariate two one-sided test (TOST) equivalence testing method for HX-MS data. Equivalence acceptance criteria were determined using maximum deviations from randomized resampling of truly equivalent samples to define hybrid equivalence criteria (maximum deviation of true equivalents, MDTE). Application of the TOST-MDTE test on differential HX-MS measurements of wild-type and mutated maltose-binding proteins demonstrates that the equivalence testing method was fit-for-purpose. Three infliximab biosimilars (Remsima, Renflexis, and Inflectra) were found to be equivalent to their Remicade reference product based on differential HX-MS measurements, while 5% deglycosylated NIST mAb was not statistically equivalent to the unmodified NIST mAb reference.

Recommendations for the Propagation of Uncertainty in Hydrogen Exchange-Mass Spectrometric Measurements.

Hydrogen exchange-mass spectrometry (HX-MS) is used widely to characterize higher-order protein structure and to locate changes in protein structure and dynamics that accompany, for example, ligand binding and protein-protein interactions. Quantitative differences in the amount of hydrogen exchange between two states (i.e., differential HX) are taken as evidence of significant differences in higher-order structure or dynamics. The quantitative measures range from simple mass differences at one HX labeling time to differences averaged across an HX time course with correction for deuterium recovery. This work applies the principles of uncertainty propagation to differential HX measurements to facilitate the identification of significant differences. Furthermore, it is shown that pooled estimates of experimental uncertainty result in a lower false positive rate than estimates of uncertainty based on individual standard deviations.

Protein A does not induce allosteric structural changes in an IgG1 antibody during binding.

Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix. HX-MS measurements of NIST mAb bound to in-solution and resin forms of ProA revealed regions of the CH2 and CH3 domains with increased protection from HX upon ProA binding, consistent with the known ProA binding region. In-solution ProA experiments revealed regions in the Fab with increased HX uptake when the ProA:mAb molar ratio was increased to 2:1, suggesting an allosterically induced increase in backbone flexibility. Such effects were not observed with lower ProA concentration (1:1 molar ratio) or when ProA resin was used, suggesting some kind of change in binding mode. Since all pharmaceutical processes use ProA bound to resin, our results rule out reversible allosteric effects on the NIST mAb during interaction with resin ProA. However, irreversible effects cannot be ruled out since the NIST mAb was previously exposed to ProA during its original purification.

Adenylate control in cAMP signaling: implications for adaptation in signalosomes.

In cAMP-Protein Kinase A (PKA) signaling, A-kinase anchoring protein scaffolds assemble PKA in close proximity to phosphodiesterases (PDE), kinase-substrates to form signaling islands or 'signalosomes'. In its basal state, inactive PKA holoenzyme (R2:C2) is activated by binding of cAMP to regulatory (R)-subunits leading to dissociation of active catalytic (C)-subunits. PDEs hydrolyze cAMP-bound to the R-subunits to generate 5'-AMP for termination and resetting the cAMP signaling. Mechanistic basis for cAMP signaling has been derived primarily by focusing on the proteins in isolation. Here, we set out to simulate cAMP signaling activation-termination cycles in a signalosome-like environment with PDEs and PKA subunits in close proximity to each other. Using a combination of fluorescence polarization and amide hydrogen exchange mass spectrometry with regulatory (RIα), C-subunit (Cα) and PDE8 catalytic domain, we have tracked movement of cAMP through activation-termination cycles. cAMP signaling operates as a continuum of four phases: (1) Activation and dissociation of PKA into R- and C-subunits by cAMP and facilitated by substrate (2) PDE recruitment to R-subunits (3) Hydrolysis of cAMP to 5'-AMP (4) Reassociation of C-subunit to 5'-AMP-bound-RIα in the presence of excess ATP to reset cAMP signaling to form the inactive PKA holoenzyme. Our results demonstrate that 5'-AMP is not merely a passive hydrolysis end-product of PDE action. A 'ligand-free' state R subunit does not exist in signalosomes as previously assumed. Instead the R-subunit toggles between cAMP- or 5'-AMP bound forms. This highlights, for the first time, the importance of 5'-AMP in promoting adaptation and uncovers adenylate control in cAMP signaling.

Structural characterization of an Arf dimer interface: molecular mechanism of Arf-dependent membrane scission.

Dimerization of the small GTPase Arf is prerequisite for the scission of COPI-coated transport vesicles. Here, we quantify the monomer/dimer equilibrium of Arf within the membrane and show that after membrane scission, Arf dimers are restricted to donor membranes. By hydrogen exchange mass spectrometry, we define the interface of activated dimeric Arf within its switch II region. Single amino acid exchanges in this region reduce the propensity of Arf to dimerize. We suggest a mechanism of membrane scission by which the dimeric form of Arf is segregated to the donor membrane. Our data are consistent with the previously reported absence of dimerized Arf in COPI vesicles and could explain the presence of one single scar-like noncoated region in each COPI vesicle.

Structural and kinetic basis for the regulation and potentiation of Hsp104 function

Hsp104 provides a valuable model for the many essential proteostatic functions performed by the AAA+ superfamily of protein molecular machines. We developed and used a powerful hydrogen exchange mass spectrometry (HX MS) analysis that can provide positionally resolved information on structure, dynamics, and energetics of the Hsp104 molecular machinery, even during functional cycling. HX MS reveals that the ATPase cycle is rate-limited by ADP release from nucleotide-binding domain 1 (NBD1). The middle domain (MD) serves to regulate Hsp104 activity by slowing ADP release. Mutational potentiation accelerates ADP release, thereby increasing ATPase activity. It reduces time in the open state, thereby decreasing substrate protein loss. During active cycling, Hsp104 transits repeatedly between whole hexamer closed and open states. Under diverse conditions, the shift of open/closed balance can lead to premature substrate loss, normal processing, or the generation of a strong pulling force. HX MS exposes the mechanisms of these functions at near-residue resolution.

Unfolding Nature's Origami Acetonitrile's Role as a Protein Denaturant

Protein function is determined not only by the structure of the folded, or native, conformation, but also by the stability and dynamics of the native state. To study folding, stability, and dynamics, denaturants such as Urea and GdmCl are used to perturb the native state, enabling detection of non-native conformations through spectroscopic methods. Linear extrapolation of multiple measurements as a function of denaturant concentration allows for extrapolation of ΔGNU to conditions in the absence of denaturant. The volatile solvent acetonitrile (AcN) is known to denature proteins but has not been systematically investigated. Here we validate the ability of acetonitrile to cooperatively denature two model proteins, myoglobin and trypsinogen, yielding ΔGNU values that closely match those obtained from traditional denaturants. In addition, we establish a method for accurately determining AcN concentration in denaturation samples, which is of critical importance given AcN's volatility. Together these results lay the foundation for use of AcN as a complementary denaturant that is compatible with hydrogen exchange mass spectrometry.