Probe capsid structure stability and dynamics of adeno-associated virus as an important viral vector for gene therapy by hydrogen-deuterium exchange-mass spectrometry.

Adeno-associated virus (AAV), a widely used gene therapy vector, is a small, nonenveloped virus that contains a single-stranded DNA genome with a maximum length of 4.7 kb. Despite extensive biophysical and structural characterization, many aspects of AAV functions remain elusive. This knowledge gap is primarily due to a lack of structurally resolved dynamic information and the absence of structural coverage of functionally critical segments on the AAV capsid. Here, we developed a protocol to study AAV structural dynamics by hydrogen-deuterium exchange mass spectrometry (HDX-MS), a powerful method for monitoring protein structure stability and dynamics in solution. We performed HDX-MS measurements on AAVs without or with different DNA payloads of different sizes, and obtained detailed dynamic information on the entire AAV sequence including the two functionally important segments not previously structurally characterized. The unique N terminus of the capsid protein VP1 (VP1u) was found to adopt a highly dynamic and unstable conformation with low HDX protection across the entire region, whereas the presence of a DNA payload increased its protection. The VP1 and VP2 shared region (VP1/2) showed no measurable protection, with or without DNA. Differential HDX between empty and full capsid samples allowed us to identify potential new DNA-capsid interaction sites located primarily around the five-fold channel, which differ from the three-fold pocket binding site previously identified. Our HDX-MS method for characterizing AAV structural dynamics opens a new way for future efforts to understand AAV structure-function relationships and engineer next-generation AAV vectors with improved gene delivery properties.

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.

Allosteric Regulation and Inhibition of Coronavirus 3CLpro Revealed by HDX-MS.

Coronavirus (CoV) infections have caused contagious and fatal respiratory diseases in humans worldwide. CoV 3-chymotrypsin-like proteases (3CLpro or Mpro) play an important role in viral maturation, and maintenance of their dimeric conformation is crucial for viral activity. Therefore, allosterically regulated dimerization of 3CLpro can be employed as a drug development target. Here, we investigated the allosteric regulatory mechanism of 3CLpro dimerization by using hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) technology. We found that the FLAG tag directly coupled to the N-finger of 3CLpro significantly increased HDX kinetics at the dimer interface, and 3CLpro transformed from a dimer to a monomer. The 3CLpro mutants of SARS-CoV-2, which are monomeric, also exhibited increased deuterium exchange. Binding of the allosteric inhibitor Gastrodenol to most betacoronavirus 3CLpros led to increased allosteric deuterium exchange, resulting in the monomeric conformation of the CoV 3CLpro upon binding. Molecular dynamics (MD) simulation analysis further indicated the molecular mechanism of action of Gastrodenol on CoV 3CLpro: binding of Gastrodenol to SARS-CoV-2 3CLpro destroyed the hydrogen bond in the dimer interface. These results suggest that Gastrodenol may be a potential broad-spectrum anti-betacoronavirus drug.

The Light Chain Allosterically Enhances the Protease Activity of Murine Urokinase-Type Plasminogen Activator.

The active form of the murine urokinase-type plasminogen activator (muPA) is formed by a 27-residue disordered light chain connecting the amino-terminal fragment (ATF) with the serine protease domain. The two chains are tethered by a disulfide bond between C1CT in the disordered light chain and C122CT in the protease domain. Previous work showed that the presence of the disordered light chain affected the inhibition of the protease domain by antibodies. Here we show that the disordered light chain induced a 3.7-fold increase in kcat of the protease domain of muPA. In addition, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and accelerated molecular dynamics (AMD) were performed to identify the interactions between the disordered light chain and the protease domain. HDX-MS revealed that the light chain is contacting the 110s, the turn between the ß10- and ß11-strand, and the ß7-strand. A reduction in deuterium uptake was also observed in the activation loop, the 140s loop and the 220s loop, which forms the S1-specificty pocket where the substrate binds. These loops are further away from where the light chain seems to be interacting with the protease domain. Our results suggest that the light chain most likely increases the activity of muPA by allosterically favoring conformations in which the specificity pocket is formed. We propose a model by which the allostery would be transmitted through the ß-strands of the ß-barrels to the loops on the other side of the protease domain.

Native dynamics and allosteric responses in PTP1B probed by high-resolution HDX-MS.

Protein tyrosine phosphatase 1B (PTP1B) is a validated therapeutic target for obesity, diabetes, and certain types of cancer. In particular, allosteric inhibitors hold potential for therapeutic use, but an incomplete understanding of conformational dynamics and allostery in this protein has hindered their development. Here, we interrogate solution dynamics and allosteric responses in PTP1B using high-resolution hydrogen-deuterium exchange mass spectrometry (HDX-MS), an emerging and powerful biophysical technique. Using HDX-MS, we obtain a detailed map of backbone amide exchange that serves as a proxy for the solution dynamics of apo PTP1B, revealing several flexible loops interspersed among more constrained and rigid regions within the protein structure, as well as local regions that exchange faster than expected from their secondary structure and solvent accessibility. We demonstrate that our HDX rate data obtained in solution adds value to estimates of conformational heterogeneity derived from a pseudo-ensemble constructed from ~200 crystal structures of PTP1B. Furthermore, we report HDX-MS maps for PTP1B with active-site versus allosteric small-molecule inhibitors. These maps suggest distinct and widespread effects on protein dynamics relative to the apo form, including changes in locations distal (>35 Å) from the respective ligand binding sites. These results illuminate that allosteric inhibitors of PTP1B can induce unexpected changes in dynamics that extend beyond the previously understood allosteric network. Together, our data suggest a model of BB3 allostery in PTP1B that combines conformational restriction of active-site residues with compensatory liberation of distal residues that aid in entropic balancing. Overall, our work showcases the potential of HDX-MS for elucidating aspects of protein conformational dynamics and allosteric effects of small-molecule ligands and highlights the potential of integrating HDX-MS alongside other complementary methods, such as room-temperature X-ray crystallography, NMR spectroscopy, and molecular dynamics simulations, to guide the development of new therapeutics.

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.

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.

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.

Hydrophilic Interaction Liquid Chromatography-Hydrogen/Deuterium Exchange-Mass Spectrometry (HILIC-HDX-MS) for Untargeted Metabolomics.

Liquid chromatography with mass spectrometry (LC-MS)-based metabolomics detects thousands of molecular features (retention time-m/z pairs) in biological samples per analysis, yet the metabolite annotation rate remains low, with 90% of signals classified as unknowns. To enhance the metabolite annotation rates, researchers employ tandem mass spectral libraries and challenging in silico fragmentation software. Hydrogen/deuterium exchange mass spectrometry (HDX-MS) may offer an additional layer of structural information in untargeted metabolomics, especially for identifying specific unidentified metabolites that are revealed to be statistically significant. Here, we investigate the potential of hydrophilic interaction liquid chromatography (HILIC)-HDX-MS in untargeted metabolomics. Specifically, we evaluate the effectiveness of two approaches using hypothetical targets: the post-column addition of deuterium oxide (D2O) and the on-column HILIC-HDX-MS method. To illustrate the practical application of HILIC-HDX-MS, we apply this methodology using the in silico fragmentation software MS-FINDER to an unknown compound detected in various biological samples, including plasma, serum, tissues, and feces during HILIC-MS profiling, subsequently identified as N1-acetylspermidine.

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.

Epitope Mapping of Human Polyclonal Antibodies to the fHbp Antigen of a Neisseria Meningitidis Vaccine by Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Antigen-antibody interactions play a key role in the immune response post vaccination and the mechanism of action of antibody-based biopharmaceuticals. 4CMenB is a multicomponent vaccine against Neisseria meningitidis serogroup B in which factor H binding protein (fHbp) is one of the key antigens. In this study, we use hydrogen/deuterium exchange mass spectrometry (HDX-MS) to identify epitopes in fHbp recognized by polyclonal antibodies (pAb) from two human donors (HDs) vaccinated with 4CMenB. Our HDX-MS data reveal several epitopes recognized by the complex mixture of human pAb. Furthermore, we show that the pAb from the two HDs recognize the same epitope regions. Epitope mapping of total pAb and purified fHbp-specific pAb from the same HD reveals that the two antibody samples recognize the same main epitopes, showing that HDX-MS based epitope mapping can, in this case at least, be performed directly using total IgG pAb samples that have not undergone Ab-selective purification. Two monoclonal antibodies (mAb) were previously produced from B-cell repertoire sequences from one of the HDs and used for epitope mapping of fHbp with HDX-MS. The epitopes identified for the pAb from the same HD in this study, overlap with the epitopes recognized by the two individual mAbs. Overall, HDX-MS epitope mapping appears highly suitable for simultaneous identification of epitopes recognized by pAb from human donors and to thus both guide vaccine development and study basic human immunity to pathogens, including viruses.

Toxoplasma gondii mitochondrial association factor 1b interactome reveals novel binding partners including Ral GTPase accelerating protein α1.

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a Kd of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness.

Characterization of Higher Order Structural Changes of a Thermally Stressed Monoclonal Antibody via Mass Spectrometry Footprinting and Other Biophysical Approaches.

Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress. The biophysical techniques report a nuanced characterization of the HOS in which CD detects no changes to the secondary or tertiary structure, yet DLS measurements show an increase in the hydrodynamic radius. DSC indicates that the stability decreases, and chemical or conformational changes accumulate with incubation time according to NMR. Furthermore, whereas HDX-MS does not indicate HOS changes, FPOP-MS footprinting reveals conformational changes at residue resolution for some amino acids. The local phenomena observed with FPOP-MS indicate that several residues show various patterns of degradation during thermal stress: no change, an increase in solvent exposure, and a biphasic response to solvent exposure. All evidences show that FPOP-MS efficiently resolves subtle structural changes and novel degradation pathways upon thermal stress treatment at residue-level resolution.

Structure of phosphorylated-like RssB, the adaptor delivering σs to the ClpXP proteolytic machinery, reveals an interface switch for activation.

In enterobacteria such as Escherichia coli, the general stress response is mediated by σs, the stationary phase dissociable promoter specificity subunit of RNA polymerase. σs is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-ß5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σs-binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σs engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation.

Recent advances in structural mass spectrometry methods in the context of biosimilarity assessment: from sequence heterogeneities to higher order structures.

Biotherapeutics and their biosimilar versions have been flourishing in the biopharmaceutical market for several years. Structural and functional characterization is needed to achieve analytical biosimilarity through the assessment of critical quality attributes as required by regulatory authorities. The role of analytical strategies, particularly mass spectrometry-based methods, is pivotal to gathering valuable information for the in-depth characterization of biotherapeutics and biosimilarity assessment. Structural mass spectrometry methods (native MS, HDX-MS, top-down MS, etc.) provide information ranging from primary sequence assessment to higher order structure evaluation. This review focuses on recent developments and applications in structural mass spectrometry for biotherapeutic and biosimilar characterization.

Probing Antibody Structures by Hydrogen/Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange (HDX) followed by mass spectrometry detection (MS) provides a fast, reliable, and detailed solution for the assessment of a protein structure. It has been widely recognized as an indispensable tool and already approved by several regulatory agencies as a structural technique for the validation of protein biopharmaceuticals, including antibody-based drugs. Antibodies are of a key importance in life and medical sciences but considered to be challenging analytical targets because of their compact structure stabilized by disulfide bonds and due to the presence of glycosylation. Despite these difficulties, there are already numerous excellent studies describing MS-based antibody structure characterization. In this chapter, we describe a universal HDX-MS workflow. Deeper attention is paid to sample handling, optimization procedures, and feasibility stages, as these elements of the HDX experiment are crucial for obtaining reliable detailed and spatially well-resolved information.

HDX-MS Reveals Substrate-Dependent, Localized EX1 Conformational Dynamics in the Retaining GT-B Glycosyltransferase, MshA.

Glycosyltransferases (GTs) are well-characterized with respect to static 3D structures and molecular dynamics simulations, but there is a lack of reports on in-solution dynamics on time scales relevant to turnover. Here, backbone amide hydrogen/deuterium exchange followed by mass spectrometry (HDX-MS) was used to investigate the in-solution dynamics of the model retaining GT MshA from Corynebacterium glutamicum (CgMshA). CgMshA has a GT-B fold and catalyzes the transfer of N-acetyl-glucosamine (GlcNAc) from UDP-GlcNAc to l-myo-inositol-1-phosphate in the first step in mycothiol biosynthesis. HDX-MS results identify several key regions of conformational changes in response to UDP-GlcNAc binding, including residues 159-198 in the N-terminal domain and residues 323-354 in the C-terminal domain. These regions also exhibited substrate-dependent EX1 exchange kinetics consistent with conformational tension on the milliseconds to seconds time scale. A potential source of this conformational change is the flexible ß4/α5 loop in the C-terminal domain, which sits at the interface of the two domains and likely interacts with the GlcNAc ring of UDP-GlcNAc. In contrast to UDP-GlcNAc, the UDP-CgMshA product complex exhibited severe decreases in deuterium incorporation, suggesting a less dynamic conformation. The HDX-MS results are complemented by solvent viscosity effects of 1.8-2.3 on the CgMshA kcat value, which are consistent with product release as a rate-determining step and possibly a direct role for protein dynamics in catalysis. The identification of in-solution dynamics that are sensitive to substrate binding allows for the proposal of a more detailed mechanism in CgMshA including conformation tension between the donor sugar and the flexible C-terminal domain ß4/α5 loop providing sufficient conformational sampling for substrate-assisted catalysis to occur.

Mass Spectrometry-Based Approaches for the Analysis of Protein Higher Order Structure.

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A Functional Bayesian Model for Hydrogen-Deuterium Exchange Mass Spectrometry.

Proteins often undergo structural perturbations upon binding to other proteins or ligands or when they are subjected to environmental changes. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can be used to explore conformational changes in proteins by examining differences in the rate of deuterium incorporation in different contexts. To determine deuterium incorporation rates, HDX-MS measurements are typically made over a time course. Recently introduced methods show that incorporating the temporal dimension into the statistical analysis improves power and interpretation. However, these approaches have technical assumptions that hinder their flexibility. Here, we propose a more flexible methodology by reframing these methods in a Bayesian framework. Our proposed framework has improved algorithmic stability, allows us to perform uncertainty quantification, and can calculate statistical quantities that are inaccessible to other approaches. We demonstrate the general applicability of the method by showing it can perform rigorous model selection on a spike-in HDX-MS experiment, improved interpretation in an epitope mapping experiment, and increased sensitivity in a small molecule case-study. Bayesian analysis of an HDX experiment with an antibody dimer bound to an E3 ubiquitin ligase identifies at least two interaction interfaces where previous methods obtained confounding results due to the complexities of conformational changes on binding. Our findings are consistent with the cocrystal structure of these proteins, demonstrating a bayesian approach can identify important binding epitopes from HDX data. We also generate HDX-MS data of the bromodomain-containing protein BRD4 in complex with GSK1210151A to demonstrate the increased sensitivity of adopting a Bayesian approach.