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.

Differentiation of isomeric chalcone and dihydroflavone using liquid chromatography coupled with hydrogen-deuterium exchange tandem mass spectrometry (HDX-MS/MS): An application for flavonoids-focused characterization of Snow chrysanthemum.

Although the co-occurrences of isomeric chalcones and dihydroflavones widely appear in medicinal plants, the differentiation of such isomerism seldom succeeds using MS/MS, attributing to totally identical MS/MS spectra. Here, efforts were paid to pursue an eligible tool allowing to address the technical challenge. Being inspired by that one more proton signal is observed in 1H NMR spectrum of isoliquiritigenin than liquiritigenin when employing DMSO­d6 as solvent, hydrogen-deuterium exchange (HDX)-MS/MS was evaluated towards differentiating isomeric chalcones and dihydroflavones through replacing H2O with D2O to prepare the mobile phase. As a result, differences were observed for either MS1 or MS2 spectrum when comparing two pairs of isomers, such as liquiritigenin vs. isoliquiritigenin and liquiritin vs. isoliquiritin, because the isomeric precursor and fragment ion species owned different amounts of hydroxyl protons and those reactive protons could be partially or completely substituted by deuterium protons at the exposure in D2O to result in n × 1.006 mass increments. Moreover, utmost four hydrogen/deuterium exchanges occurred for a single glucosyl moiety. Thereafter, HDX-MS/MS was applied to characterize the flavonoids of Snow chrysanthemum, a precious edible herbal medicine that is rich in isomeric chalcones and dihydroflavones. Through paying special attention to the deuterium labeling styles of (de)protonated molecules as well as those featured fragment ions, five pairs of isomeric chalcones and dihydroflavones were confirmatively differentiated, in addition to that 28 flavonoids were structurally annotated by applying those well-defined mass fragmentation rules. Hence, this study offered an in-depth insight towards the flavonoids-focused characterization of Snow chrysanthemum, and more importantly, HDX-MS/MS is a superior tool to differentiate, but not limited to, isomeric chalcones and dihydroflavones.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

HDX-MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker.

Eukaryotic cells form condensates to sense and adapt to their environment [S. F. Banani, H. O. Lee, A. A. Hyman, M. K. Rosen, Nat. Rev. Mol. Cell Biol. 18, 285-298 (2017), H. Yoo, C. Triandafillou, D. A. Drummond, J. Biol. Chem. 294, 7151-7159 (2019)]. Poly(A)-binding protein (Pab1), a canonical stress granule marker, condenses upon heat shock or starvation, promoting adaptation [J. A. Riback et al., Cell 168, 1028-1040.e19 (2017)]. The molecular basis of condensation has remained elusive due to a dearth of techniques to probe structure directly in condensates. We apply hydrogen-deuterium exchange/mass spectrometry to investigate the mechanism of Pab1's condensation. Pab1's four RNA recognition motifs (RRMs) undergo different levels of partial unfolding upon condensation, and the changes are similar for thermal and pH stresses. Although structural heterogeneity is observed, the ability of MS to describe populations allows us to identify which regions contribute to the condensate's interaction network. Our data yield a picture of Pab1's stress-triggered condensation, which we term sequential activation (Fig. 1A), wherein each RRM becomes activated at a temperature where it partially unfolds and associates with other likewise activated RRMs to form the condensate. Subsequent association is dictated more by the underlying free energy surface than specific interactions, an effect we refer to as thermodynamic specificity. Our study represents an advance for elucidating the interactions that drive condensation. Furthermore, our findings demonstrate how condensation can use thermodynamic specificity to perform an acute response to multiple stresses, a potentially general mechanism for stress-responsive proteins.

Automating data analysis for hydrogen/deuterium exchange mass spectrometry using data-independent acquisition methodology.

We present a hydrogen/deuterium exchange workflow coupled to tandem mass spectrometry (HX-MS2) that supports the acquisition of peptide fragment ions alongside their peptide precursors. The approach enables true auto-curation of HX data by mining a rich set of deuterated fragments, generated by collisional-induced dissociation (CID), to simultaneously confirm the peptide ID and authenticate MS1-based deuteration calculations. The high redundancy provided by the fragments supports a confidence assessment of deuterium calculations using a combinatorial strategy. The approach requires data-independent acquisition (DIA) methods that are available on most MS platforms, making the switch to HX-MS2 straightforward. Importantly, we find that HX-DIA enables a proteomics-grade approach and wide-spread applications. Considerable time is saved through auto-curation and complex samples can now be characterized and at higher throughput. We illustrate these advantages in a drug binding analysis of the ultra-large protein kinase DNA-PKcs, isolated directly from mammalian cells.

Investigation of the Solid-State Interactions in Lyophilized Human G-CSF Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) previously elucidated the interactions between excipients and proteins for liquid granulocyte colony stimulating factor (G-CSF) formulations, confirming predictions made using computational structure docking. More recently, solid-state HDX mass spectrometry (ssHDX-MS) was developed for proteins in the lyophilized state. Deuterium uptake in ssHDX-MS has been shown for various proteins, including monoclonal antibodies, to be highly correlated with storage stability, as measured by protein aggregation and chemical degradation. As G-CSF is known to lose activity through aggregation upon lyophilization, we applied the ssHDX-MS method with peptide mapping to four different lyophilized formulations of G-CSF to compare the impact of three excipients on local structure and exchange dynamics. HDX at 22 °C was confirmed to correlate well with the monomer content remaining after lyophilization and storage at -20 °C, with sucrose providing the greatest protection, and then phenylalanine, mannitol, and no excipient leading to progressively less protection. Storage at 45 °C led to little difference in final monomer content among the formulations, and so there was no discernible relationship with total deuterium uptake on ssHDX. Incubation at 45 °C may have led to a structural conformation and/or aggregation mechanism no longer probed by HDX at 22 °C. Such a conformational change was observed previously at 37 °C for liquid-formulated G-CSF using NMR. Peptide mapping revealed that tolerance to lyophilization and -20 °C storage was linked to increased stability in the small helix, loop AB, helix C, and loop CD. LC-MS HDX and NMR had previously linked loop AB and loop CD to the formation of a native-like state (N*) prior to aggregation in liquid formulations, suggesting a similar structural basis for G-CSF aggregation in the liquid and solid states.

Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides.

Carbohydrates are critical for cellular functions as well as an important class of metabolites. Characterizing carbohydrate structures is a difficult analytical challenge due to the presence of isomers. In-electrospray hydrogen/deuterium exchange mass spectrometry (in-ESI HDX-MS) is a method of HDX that samples the solvated structure of carbohydrates during the ESI process and requires little to no instrument modification. Traditionally, solution-phase HDX is utilized with proteins to sample conformational differences, and pH is a critical parameter to monitor and control due to the presence of both acid- and base-catalyzed mechanisms of exchange. For In-ESI HDX, the pH surrounding the analyte changes before and during labeling, which has the potential to affect the rate of labeling for analytes. Herein, we alter the pH of spray solutions containing model carbohydrates and peptides, perform in-ESI HDX-MS, and characterize the deuterium uptake trends. Varying pH results in altered D uptake, though the overall trends differ from the expected bulk-solution trends due to the electrospray process. These findings show the utility of varying pH prior to in-ESI HDX-MS for establishing different extents of HDX as well as distinguishing labile functional groups that are present in different analytes.

Deep Learning Enables Automatic Correction of Experimental HDX-MS Data with Applications in Protein Modeling.

Observed mass shifts associated with deuterium incorporation in hydrogen-deuterium exchange mass spectrometry (HDX-MS) frequently deviate from the initial signals due to back and forward exchange. In typical HDX-MS experiments, the impact of these disparities on data interpretation is generally low because relative and not absolute mass changes are investigated. However, for more advanced data processing including optimization, experimental error correction is imperative for accurate results. Here the potential for automatic HDX-MS data correction using models generated by deep neural networks is demonstrated. A multilayer perceptron (MLP) is used to learn a mapping between uncorrected HDX-MS data and data with mass shifts corrected for back and forward exchange. The model is rigorously tested at various levels including peptide level mass changes, residue level protection factors following optimization, and ability to correctly identify native protein folds using HDX-MS guided protein modeling. AI is shown to demonstrate considerable potential for amending HDX-MS data and improving fidelity across all levels. With access to big data, online tools may eventually be able to predict corrected mass shifts in HDX-MS profiles. This should improve throughput in workflows that require the reporting of real mass changes as well as allow retrospective correction of historic profiles to facilitate new discoveries with these data.

Structural flexibility and heterogeneity of recombinant human glial fibrillary acidic protein (GFAP).

Glial fibrillary acidic protein (GFAP) is a promising biomarker for brain and spinal cord disorders. Recent studies have highlighted the differences in the reliability of GFAP measurements in different biological matrices. The reason for these discrepancies is poorly understood as our knowledge of the protein's 3-dimensional conformation, proteoforms, and aggregation remains limited. Here, we investigate the structural properties of GFAP under different conditions. For this, we characterized recombinant GFAP proteins from various suppliers and applied hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a snapshot of the conformational dynamics of GFAP in artificial cerebrospinal fluid (aCSF) compared to the phosphate buffer. Our findings indicate that recombinant GFAP exists in various conformational species. Furthermore, we show that GFAP dimers remained intact under denaturing conditions. HDX-MS experiments show an overall decrease in H-bonding and an increase in solvent accessibility of GFAP in aCSF compared to the phosphate buffer, with clear indications of mixed EX2 and EX1 kinetics. To understand possible structural interface regions and the evolutionary conservation profiles, we combined HDX-MS results with the predicted GFAP-dimer structure by AlphaFold-Multimer. We found that deprotected regions with high structural flexibility in aCSF overlap with predicted conserved dimeric 1B and 2B domain interfaces. Structural property predictions combined with the HDX data show an overall deprotection and signatures of aggregation in aCSF. We anticipate that the outcomes of this research will contribute to a deeper understanding of the structural flexibility of GFAP and ultimately shed light on its behavior in different biological matrices.

Transient Structural Dynamics of Glycogen Phosphorylase from Nonequilibrium Hydrogen/Deuterium-Exchange Mass Spectrometry.

It remains a major challenge to ascertain the specific structurally dynamic changes that underpin protein functional switching. There is a growing need in molecular biology and drug discovery to complement structural models with the ability to determine the dynamic structural changes that occur as these proteins are regulated and function. The archetypal allosteric enzyme glycogen phosphorylase is a clinical target of great interest to treat type II diabetes and metastatic cancers. Here, we developed a time-resolved nonequilibrium millisecond hydrogen/deuterium-exchange mass spectrometry (HDX-MS) approach capable of precisely locating dynamic structural changes during allosteric activation and inhibition of glycogen phosphorylase. We resolved obligate transient changes in the localized structure that are absent when directly comparing active/inactive states of the enzyme and show that they are common to allosteric activation by AMP and inhibition by caffeine, operating at different sites. This indicates that opposing allosteric regulation by inhibitor and activator ligands is mediated by pathways that intersect with a common structurally dynamic motif. This mass spectrometry approach uniquely stands to discover local transient structural dynamics and could be used broadly to identify features that influence the structural transitions of proteins.

Probing the Protein-Excipient Interaction in the Orally Delivered Protein by Solid-State Hydrogen-Deuterium Exchange Mass Spectrometry and Molecular Dynamics.

The oral administration of protein therapeutics in solid dosage form is gaining popularity due to its benefits, such as improved medication adherence, convenience, and ease of use for patients compared to traditional parental delivery. However, formulating oral biologics presents challenges related to pH barriers, enzymatic breakdown, and poor bioavailability. Therefore, understanding the interaction between excipients and protein therapeutics in the solid state is crucial for formulation development. In this Letter, we present a case study focused on investigating the role of excipients in protein aggregation during the production of a solid dosage form of a single variable domain on a heavy chain (VHH) protein. We employed solid-state hydrogen-deuterium exchange coupled with mass spectrometry (ssHDX-MS) at both intact protein and peptide levels to assess differences in protein-excipient interactions between two formulations. ssHDX-MS analysis revealed that one formulation effectively prevents protein aggregation during compaction by blocking ß-sheets across the VHH protein, thereby preventing ß-sheet-ß-sheet interactions. Spatial aggregation propensity (SAP) mapping and cosolvent simulation from molecular dynamics (MD) simulation further validated the protein-excipient interaction sites identified through ssHDX-MS. Additionally, the MD simulation demonstrated that the interaction between the VHH protein and excipients involves hydrophilic interactions and/or hydrogen bonding. This novel approach holds significant potential for understanding protein-excipient interactions in the solid state and can guide the formulation and process development of orally delivered protein dosage forms, ultimately enhancing their efficacy and stability.

A General Framework to Interpret Hydrogen-Deuterium Exchange Native Mass Spectrometry of G-Quadruplex DNA.

G-quadruplexes (G4s) are secondary structures formed by guanine-rich oligonucleotides involved in various biological processes. However, characterizing G4s is challenging, because of their structural polymorphism. Here, we establish how hydrogen-deuterium exchange native mass spectrometry (HDX/MS) can help to characterize the G4 structures and dynamics in solution. We correlated the time range of G4 exchange to the number of guanines involved in the inner and outer tetrads. We also established relationships among exchange rates, numbers of tetrads and bound cations, and stability. The use of HDX/native MS allows for the determination of tetrads formed and assessment of G4 stability at a constant temperature. A key finding is that stable G4s exchange through local fluctuations (EX2 exchange), whereas less stable G4s also undergo exchange through partial or complete unfolding (EX1 exchange). Deconvolution of the bimodal isotope distributions resulting from EX1 exchange provides valuable insight into the kinetics of folding and unfolding processes and allows one to detect and characterize transiently unfolded intermediates, even if scarcely populated. HDX/native MS thus represents a powerful tool for a more comprehensive exploration of the folding landscapes of G4s.

Antibody Binding Captures High Energy State of an Antigen: The Case of Nsp1 SARS-CoV-2 as Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry.

We describe an investigation using structural mass spectrometry (MS) of the impact of two antibodies, 15497 and 15498, binding the highly flexible SARS-CoV-2 Nsp1 protein. We determined the epitopes and paratopes involved in the antibody-protein interactions by using hydrogen-deuterium exchange MS (HDX-MS). Notably, the Fab (Fragment antigen binding) for antibody 15498 captured a high energy form of the antigen exhibiting significant conformational changes that added flexibility over most of the Nsp1 protein. The Fab for antibody 15497, however, showed usual antigen binding behavior, revealing local changes presumably including the binding site. These findings illustrate an unusual antibody effect on an antigen and are consistent with the dynamic nature of the Nsp1 protein. Our studies suggest that this interaction capitalizes on the high flexibility of Nsp1 to undergo conformational change and be trapped in a higher energy state by binding with a specific antibody.

Hydrogen-Deuterium Exchange Dynamics of NISTmAb Measured by Small Angle Neutron Scattering.

Understanding protein dynamics and conformational stability holds great significance in biopharmaceutical research. Hydrogen-deuterium exchange (HDX) is a quantitative methodology used to examine these fundamental properties of proteins. HDX involves measuring the exchange of solvent-accessible hydrogens with deuterium, which yields valuable insights into conformational fluctuations and conformational stability. While mass spectrometry is commonly used to measure HDX on the peptide level, we explore a different approach using small-angle neutron scattering (SANS). In this work, SANS is demonstrated as a complementary and noninvasive HDX method (HDX-SANS). By assessing subtle changes in the tertiary and quaternary structure during the exchange process in deuterated buffer, along with the influence of added electrolytes on protein stability, SANS is validated as a complementary HDX technique. The HDX of a model therapeutic antibody, NISTmAb, an IgG1κ, is monitored by HDX-SANS over many hours using several different formulations, including salts from the Hofmeister series of anions, such as sodium perchlorate, sodium thiocyanate, and sodium sulfate. The impact of these formulation conditions on the thermal stability of NISTmAb is probed by differential scanning calorimetry. The more destabilizing salts led to heightened conformational dynamics in mAb solutions even at temperatures significantly below the denaturation point. HDX-SANS is demonstrated as a sensitive and noninvasive technique for quantifying HDX kinetics directly in mAb solution, providing novel information about mAb conformational fluctuations. Therefore, HDX-SANS holds promise as a potential tool for assessing protein stability in formulation.

Evaluation of Hydrogen-Deuterium Exchange during Transient Vapor Binding of MeOD with Model Peptide Systems Angiotensin II and Bradykinin.

The advancement of hybrid mass spectrometric tools as an indirect probe of molecular structure and dynamics relies heavily upon a clear understanding between gas-phase ion reactivity and ion structural characteristics. This work provides new insights into gas-phase ion-neutral reactions of the model peptides (i.e., angiotensin II and bradykinin) on a per-residue basis by integrating hydrogen/deuterium exchange, ion mobility, tandem mass spectrometry, selective vapor binding, and molecular dynamics simulations. By comparing fragmentation patterns with simulated probabilities of vapor uptake, a clear link between gas-phase hydrogen/deuterium exchange and the probabilities of localized vapor association is established. The observed molecular dynamics trends related to the sites and duration of vapor binding track closely with experimental observation. Additionally, the influence of additional charges and structural characteristics on exchange kinetics and ion-neutral cluster formation is examined. These data provide a foundation for the analysis of solvation dynamics of larger, native-like conformations of proteins in the gas phase.

Evidence of H/D Exchange within Metal-Adducted Carbohydrates after Ion/Ion-Dissociation Reactions.

Tandem mass spectrometry (MS/MS) using fragmentation has become one of the most effective methods for gaining sequence and structural information on biomolecules. Ion/ion reactions are competitive reactions, where either proton transfer (PT) or electron transfer (ET) can occur from interactions between multiply charged cations and singly charged anions. Utilizing ion/ion reactions with fluoranthene has offered a unique method of fragment formation for the structural elucidation of biomolecules. Fluoranthene is considered an ideal anion reagent because it selectively causes electron-transfer dissociation (ETD) and minimizes PT when interacting with peptides. However, limited investigations have sought to understand how fluoranthene─the primary, commercially available anion reagent─interacts with other biomolecules. Here, we apply deuterium labeling to investigate ion/ion reaction mechanisms between fluoranthene and divalent, metal-adducted carbohydrates (Ca2+, Mg2+, Co2+, and Ni2+). Deuterium labeling of carbohydrates allowed us to observe evidence of hydrogen/deuterium exchange (HDX) occurring after ion/ion dissociation reactions. The extent of deuterium loss is dependent on several factors, including the physical properties of the metal ion and the fragment structure. Based on the deuterium labeling data, we have proposed ETD, PTD, and intermolecular PT─also described as HDX─mechanisms. This research provides a fundamental perspective of ion/ion and ion/molecule reaction mechanisms and illustrates properties that impact ion/ion and ion/molecule reactions for carbohydrates. Together, this could improve the capability to distinguish complex and heterogeneous biomolecules, such as carbohydrates.

H/D Exchange Coupled with 2H-labeled Stable Isotope-Assisted Metabolomics Discover Transformation Products of Contaminants of Emerging Concern.

Stable isotope-assisted metabolomics (SIAM) is a powerful tool for discovering transformation products (TPs) of contaminants. Nevertheless, the high cost or lack of isotope-labeled analytes limits its application. In-house H/D (hydrogen/deuterium) exchange reactions enable direct 2H labeling to target analytes with favorable reaction conditions, providing intuitive and easy-to-handle approaches for environmentally relevant laboratories to obtain cost-effective 2H-labeled contaminants of emerging concern (CECs). We first combined the use of in-house H/D exchange and 2H-SIAM to discover potential TPs of 6PPD (N-1,3-dimethylbutyl-N'-phenyl-p-phenylenediamine), providing a new strategy for finding TPs of CECs. 6PPD-d9 was obtained by in-house H/D exchange with favorable reaction conditions, and the impurities were carefully studied. Incomplete deuteride, for instance, 6PPD-d8 in this study, constitutes a major part of the impurities. Nevertheless, it has few adverse effects on the 2H-SIAM pipeline in discovering TPs of 6PPD. The 2H-SIAM pipeline annotated 9 TPs of 6PPD, and commercial standards further confirmed the annotated 6PPDQ (2-anilino-5-(4-methylpentan-2-ylamino)cyclohexa-2,5-diene-1,4-dione) and PPPD (N-phenyl-p-phenylenediamine). Additionally, a possible new formation mechanism for 6PPDQ was proposed, highlighting the performance of the strategy. In summary, this study highlighted a new strategy for discovering the TPs of CECs and broadening the application of SIAM in environmental studies.

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.

Mapping HDX-MS Data to Protein Conformations through Training Ensemble-Based Models.

An original approach that adopts machine learning inference to predict protein structural information using hydrogen-deuterium exchange mass spectrometry (HDX-MS) is described. The method exploits an in-house optimization program that increases the resolution of HDX-MS data from peptides to amino acids. A system is trained using Gradient Tree Boosting as a type of machine learning ensemble technique to assign a protein secondary structure. Using limited training data we generate a discriminative model that uses optimized HDX-MS data to predict protein secondary structure with an accuracy of 75%. This research could form the basis for new methods exploiting artificial intelligence to model protein conformations by HDX-MS.