RESUMO
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 â¼116 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.
Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério , Bibliotecas de Moléculas Pequenas , Ressonância de Plasmônio de Superfície , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Ressonância de Plasmônio de Superfície/métodos , HumanosRESUMO
Low molecular weight heparin and synthetic mimetics such as fondaparinux show different binding kinetics, protease specificity, and clinical effects. A combination of allosteric and template-mediated bridging mechanisms have been proposed to explain the differences in rate acceleration and specificity. The difficulty in working with heterogeneous heparin species has rendered a crystallographic interpretation of the differences in antithrombin activation between mimetics and natural heparin inaccessible. In this study, we examine the allosteric changes in antithrombin caused by binding fondaparinux, enoxaparin and depolymerized natural heparins using millisecond hydrogen deuterium exchange mass spectrometry (TRESI-HDX MS) and relate these conformational changes to complex stability in the gas phase using collision induced unfolding (CIU). This exploration reveals that in addition to the dynamic changes caused by fondaparinux, long chain heparins reduce structural flexibility proximal to Arg393, the cleavable residue in the reactive centre loop of the protein. These local changes in protein dynamics are associated with an increase in overall complex stability that increases with heparin chain length. Ultimately, these results shed light on the molecular mechanisms underlying differences in activity and specificity between heparin mimetics and natural heparins.
Assuntos
Antitrombinas , Fondaparinux , Heparina , Fondaparinux/química , Heparina/química , Antitrombinas/química , Antitrombinas/farmacologia , Desdobramento de Proteína/efeitos dos fármacos , Medição da Troca de Deutério , Humanos , Cinética , Ligação Proteica , Polissacarídeos/química , Polissacarídeos/farmacologia , Modelos MolecularesRESUMO
The structurally conserved B-cell lymphoma 2 (Bcl-2) family of protein function to promote or inhibit apoptosis through an exceedingly complex web of specific, intrafamilial protein-protein interactions. The critical role of these proteins in lymphomas and other cancers has motivated a widespread interest in understanding the molecular mechanisms that drive specificity in Bcl-2 family interactions. However, the high degree of structural similarity among Bcl-2 homologues has made it difficult to rationalize the highly specific (and often divergent) binding behavior exhibited by these proteins using conventional structural arguments. In this work, we use time-resolved hydrogen deuterium exchange mass spectrometry to explore shifts in conformational dynamics associated with binding partner engagement in the Bcl-2 family proteins Bcl-2 and Mcl-1. Using this approach combined with homology modeling, we reveal that Mcl-1 binding is driven by a large-scale shift in conformational dynamics, while Bcl-2 complexation occurs primarily through a classical charge compensation mechanism. This work has implications for understanding the evolution of internally regulated biological systems composed of structurally similar proteins and for the development of drugs targeting Bcl-2 family proteins for promotion of apoptosis in cancer.
Assuntos
Proteínas Reguladoras de Apoptose , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Ligação Proteica , ApoptoseRESUMO
Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a rapidly growing technique for protein characterization in industry and academia, complementing the "static" picture provided by classical structural biology with information about the dynamic structural changes that accompany biological function. Conventional hydrogen deuterium exchange experiments, carried out on commercially available systems, typically collect 4-5 exchange timepoints on a timescale ranging from tens of seconds to hours using a workflow that can require 24 h or more of continuous data collection for triplicate measurements. A small number of groups have developed setups for millisecond timescale HDX, allowing for the characterization of dynamic shifts in weakly structured or disordered regions of proteins. This capability is particularly important given the central role that weakly ordered protein regions often play in protein function and pathogenesis. In this work, we introduce a new continuous flow injection setup for time-resolved HDX-MS (CFI-TRESI-HDX) that allows automated, continuous or discrete labeling time measurements from milliseconds to hours. The device is composed almost entirely of "off-the-shelf" LC components and can acquire an essentially unlimited number of timepoints with substantially reduced runtimes compared to conventional systems.
Assuntos
Medição da Troca de Deutério , Tetranitrato de Pentaeritritol , Espectrometria de Massa com Troca Hidrogênio-Deutério , Coleta de Dados , HidrogênioRESUMO
The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.
Assuntos
Metalochaperonas , Proteína Supressora de Tumor p53 , Metalochaperonas/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ligantes , Linhagem Celular Tumoral , Domínios ProteicosRESUMO
Biological macromolecules, such as proteins, nucleic acids, and carbohydrates, contain heteroatom-bonded hydrogens that undergo exchange with solvent hydrogens on timescales ranging from microseconds to hours. In hydrogen-deuterium exchange mass spectrometry (HDX-MS), this exchange process is used to extract information about biomolecular structure and dynamics. This minireview focuses on millisecond timescale HDX-MS measurements, which, while less common than 'conventional' timescale (seconds to hours) HDX-MS, provide a unique window into weakly structured species, weak (or fast cycling) binding interactions, and subtle shifts in conformational dynamics. This includes intrinsically disordered proteins and regions (IDPs/IDRs) that are associated with cancer and amyloidotic neurodegenerative disease. For nucleic acids and carbohydrates, structures such as isomers, stems, and loops, can be elucidated and overall structural rigidity can be assessed. We will provide a brief overview of technical developments in rapid HDX followed by highlights of various applications, emphasising the importance of broadening the HDX timescale to improve throughput and to capture a wider range of function-relevant dynamic and structural shifts.
Assuntos
Proteínas Intrinsicamente Desordenadas , Doenças Neurodegenerativas , Humanos , Deutério , Medição da Troca de Deutério/métodos , Hidrogênio/química , Proteínas Intrinsicamente Desordenadas/química , Conformação ProteicaRESUMO
The properties of milk proteins differ between mammalian species. ß-Lactoglobulin (ßlg) proteins from caprine and bovine milk are sequentially and structurally highly similar, yet their physicochemical properties differ, particularly in response to pH. To resolve this conundrum, we compared the dynamics of both the monomeric and dimeric states for each homologue at pH 6.9 and 7.5 using hydrogen/deuterium exchange experiments. At pH 7.5, the rate of exchange is similar across both homologues, but at pH 6.9 the dimeric states of the bovine ßlg B variant homologue have significantly more conformational flexibility compared with caprine ßlg. Molecular dynamics simulations provide a mechanistic rationale for the experimental observations, revealing that variant-specific substitutions encode different conformational ensembles with different dynamic properties consistent with the hydrogen/deuterium exchange experiments. Understanding the dynamic differences across ßlg homologues is essential to understand the different responses of these milks to processing, human digestion, and differences in immunogenicity.
Assuntos
Cabras , Lactoglobulinas , Humanos , Animais , Lactoglobulinas/genética , Lactoglobulinas/química , Deutério , Cabras/genética , Hidrogênio , Concentração de Íons de HidrogênioRESUMO
Life at the molecular level is a dynamic world, where the key playersâproteins, oligonucleotides, lipids, and carbohydratesâare in a perpetual state of structural flux, shifting rapidly between local minima on their conformational free energy landscapes. The techniques of classical structural biology, X-ray crystallography, structural NMR, and cryo-electron microscopy (cryo-EM), while capable of extraordinary structural resolution, are innately ill-suited to characterize biomolecules in their dynamically active states. Subsecond time-resolved mass spectrometry (MS) provides a unique window into the dynamic world of biological macromolecules, offering the capacity to directly monitor biochemical processes and conformational shifts with a structural dimension provided by the electrospray charge-state distribution, ion mobility, covalent labeling, or hydrogen-deuterium exchange. Over the past two decades, this suite of techniques has provided important insights into the inherently dynamic processes that drive function and pathogenesis in biological macromolecules, including (mis)folding, complexation, aggregation, ligand binding, and enzyme catalysis, among others. This Review provides a comprehensive account of subsecond time-resolved MS and the advances it has enabled in dynamic structural biology, with an emphasis on insights into the dynamic drivers of protein function.
Assuntos
Biologia , Proteínas , Microscopia Crioeletrônica/métodos , Espectrometria de Massas/métodos , Conformação Proteica , Proteínas/químicaRESUMO
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.
Assuntos
Espectrometria de Massa com Troca Hidrogênio-Deutério/métodos , Fosfolipídeos/química , alfa-Sinucleína/química , Cromatografia em Gel , Dimiristoilfosfatidilcolina/química , Humanos , Bicamadas Lipídicas/química , Espectroscopia de Ressonância Magnética , Lipídeos de Membrana/química , Modelos Químicos , Nanoestruturas/química , Fosfatidilgliceróis/química , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray/métodos , alfa-Sinucleína/isolamento & purificaçãoRESUMO
Acetaminophen (APAP)-related toxicity is caused by the formation of N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite able to covalently bind to protein thiols. A targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, using multiple reaction monitoring (MRM), was developed to measure APAP binding on selected target proteins, including glutathione S-transferases (GSTs). In vitro incubations with CYP3A4 were performed to form APAP in the presence of different proteins, including four purified GST isozymes. A custom alkylation agent was used to prepare heavy labeled modified protein containing a structural isomer of APAP on all cysteine residues for isotope dilution. APAP incubations were spiked with heavy labeled protein, digested with either trypsin or pepsin, followed by peptide fractionation by HPLC prior to LC-MRM analysis. Relative site occupancy on the protein-level was used for comparing levels of modification of different sites in target proteins, after validation of protein and peptide-level relative quantitation using human serum albumin as a model system. In total, seven modification sites were quantified, namely Cys115 and 174 in GSTM2, Cys15, 48 and 170 in GSTP1, and Cys50 in human MGST1 and rat MGST1. In addition, APAP site occupancies of three proteins from liver microsomes were also quantified by using heavily labeled microsomes spiked into APAP microsomal incubations. A novel approach employing an isotope-labeled alkylation reagent was used to determine site occupancies on multiple protein thiols.
RESUMO
The success of bevacizumab (Avastin), a monoclonal antibody (mAb) anticancer drug targeting vascular endothelial growth factor A (VEGF-A), has motivated the development of biosimilars. Establishing target epitope similarity using epitope mapping is a critical step in preclinical mAb biosimilar development. Here we use time-resolved electrospray ionization hydrogen-deuterium exchange (HDX) mass spectrometry to rapidly compare the epitopes of commercial Avastin and a biosimilar in preclinical development (ApoBev) on an extended construct of VEGF-A. The Avastin and ApoBev epitopes determined in our experiments agree with each other and with the known epitope derived from the Avastin Fab domain/truncated VEGF co-crystal structure. However, subtly different allosteric effects observed exclusively at short (millisecond) HDX labeling times may reflect a slightly different binding mode for ApoBev.
Assuntos
Bevacizumab/imunologia , Medicamentos Biossimilares/farmacologia , Mapeamento de Epitopos , Espectrometria de Massa com Troca Hidrogênio-Deutério , Fator A de Crescimento do Endotélio Vascular/química , Humanos , Cinética , Microfluídica , Modelos MolecularesRESUMO
Virtually all protein functions in the cell, including pathogenic processes, require coordinated motion of atoms or domains, i.e., conformational dynamics. Understanding protein dynamics is therefore critical both for drug development and to learn about the underlying molecular causes of many diseases. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) provides valuable information about protein dynamics, which is highly complementary to the static picture provided by conventional high-resolution structural tools (i.e., X-ray crystallography and structural NMR). The amount of protein required to carry out HDX-MS experiments is a fraction of the amount required by alternative biophysical techniques, which are also usually lower resolution. Use of HDX-MS is growing quickly both in industry and academia, and it has been successfully used in numerous drug and vaccine development efforts, with important roles in understanding allosteric effects and mapping binding sites.
RESUMO
Collision induced unfolding (CIU) is increasingly used to characterize protein complexes in the gas phase and is often employed to detect ligand binding-induced conformational stabilization. However, the extent to which gas-phase conformational stabilities measured by CIU reflect analogous parameters in solution is not yet clear, particularly for systems where conformational and protein complex stability are modulated by point mutation. Here, we compare CIU-derived relative stabilities of four point mutants of the homotetramer pyruvate kinase to solution stabilities measured by differential scanning fluorimetry (DSF) and solution conformational dynamics measured by time-resolved electrospray ionization hydrogen-deuterium exchange (TRESI-HDX). Our results demonstrate that both destabilization of the tetrameric state and generally reduced conformational stability of the monomer in solution are well correlated to lower onset energies for specific unfolding transitions observed in CIU. However, this correlation not fully retained when comparing CIU to HDX data, where the latter measurement is strongly impacted by conformational dynamics within the tetramer.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Piruvato Quinase/química , Medição da Troca de Deutério , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fluorometria , Gases/química , Modelos Moleculares , Mutação Puntual , Conformação Proteica , Multimerização Proteica , Desdobramento de Proteína , Piruvato Quinase/genética , Soluções , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Lcn2 is a host defense protein induced via the innate immune response to sequester iron-loaded bacterial siderophores. However, excess or prolonged elevation of Lcn2 levels can induce adverse cellular effects, including oxidative stress and inflammation. In this work, we use Hydrogen-Deuterium eXchange (HDX) and Isothermal Titration Calorimetry (ITC) to characterize the binding interaction between Lcn2 and siderophores enterobactin and 2,3-DHBA, in the presence and absence of iron. Our results indicate a rare "Type II" interaction in which binding of siderophores drives the protein conformational equilibrium toward an unfolded state. Linking our molecular model to cellular assays, we demonstrate that this "distorted binding mode" facilitates a deleterious cellular accumulation of reactive oxygen species that could represent the molecular origin of Lcn2 pathology. These results add important insights into mechanisms of Lcn2 action and have implications in Lcn2-mediated effects including inflammation.
Assuntos
Anti-Infecciosos/química , Proteínas de Bactérias/química , Deutério/química , Lipocalina-2/química , Sideróforos/química , Anti-Infecciosos/metabolismo , Proteínas de Bactérias/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Descoberta de Drogas , Enterobactina/química , Humanos , Hidroxibenzoatos/química , Imunidade Inata/efeitos dos fármacos , Ferro/química , Cinética , Lipocalina-2/metabolismo , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo , Sideróforos/metabolismo , Coloração e Rotulagem , Relação Estrutura-AtividadeRESUMO
Current research has identified S-nitrosoglutathione reductase (GSNOR) as the central enzyme for regulating protein S-nitrosylation. In addition, the dysregulation of GSNOR expression is implicated in several organ system pathologies including respiratory, cardiovascular, hematologic, and neurologic, making GSNOR a primary target for pharmacological intervention. This study demonstrates the kinetic activation of GSNOR by its substrate S-nitrosoglutathione (GSNO). GSNOR kinetic analysis data resulted in nonhyperbolic behavior that was successfully accommodated by the Hill-Langmuir equation with a Hill coefficient of +1.75, indicating that the substrate, GSNO, was acting as a positive allosteric affector. Docking and molecular dynamics simulations were used to predict the location of the GSNO allosteric domain comprising the residues Asn185, Lys188, Gly321, and Lys323 in the vicinity of the structural Zn2+-binding site. GSNO binding to Lys188, Gly321, and Lys323 was further supported by hydrogen-deuterium exchange mass spectroscopy (HDXMS), as deuterium exchange significantly decreased at these residues in the presence of GSNO. The site-directed mutagenesis of Lys188Ala and Lys323Ala resulted in the loss of allosteric behavior. Ultimately, this work unambiguously demonstrates that GSNO at large concentrations activates GSNOR by binding to an allosteric site comprised of the residues Asn185, Lys188, Gly321, and Lys323. The identification of an allosteric GSNO-binding domain on GSNOR is significant, as it provides a platform for pharmacological intervention to modulate the activity of this essential enzyme.
RESUMO
Acetaminophen (APAP)-induced hepatotoxicity is the most common cause of acute liver failure in the Western world. APAP is bioactivated to N-acetyl p-benzoquinone imine (NAPQI), a reactive metabolite, which can subsequently covalently bind to glutathione and protein thiols. In this study, we have used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize NAPQI binding to human glutathione S-transferases (GSTs) in vitro. GSTs play a crucial role in the detoxification of reactive metabolites and therefore are interesting target proteins to study in the context of APAP covalent binding. Recombinantly-expressed and purified GSTs were used to assess NAPQI binding in vitro. APAP biotransformation to NAPQI was achieved using rat liver microsomes or human cytochrome P450 Supersomes in the presence of GSTA1, M1, M2, or P1. Resulting adducts were analyzed using bottom-up proteomics, with or without LC fractionation prior to LC-MS/MS analysis on a quadrupole-time-of-flight instrument with data-dependent acquisition (DDA). Targeted methods using multiple reaction monitoring (MRM) on a triple quadrupole platform were also developed by quantitatively labeling all available cysteine residues with a labeling reagent yielding isomerically-modified peptides following enzymatic digestion. Seven modified cysteine sites were confirmed, including Cys112 in GSTA1, Cys78 in GSTM1, Cys115 and 174 in GSTM2, as well as Cys15, 48, and 170 in GSTP1. Most modified peptides could be detected using both untargeted (DDA) and targeted (MRM) approaches, however the latter yielded better detection sensitivity with higher signal-to-noise and two sites were uniquely found by MRM.
RESUMO
Primarily known for its function in the electron transport chain, cytochrome c (Cyt c) also plays a critical role in the initiation of mitochondrially induced apoptosis through specific interactions with cardiolipin (CL), a negatively charged phospholipid found in the inner mitochondrial membrane. In this work, we study the conformational dynamics of Cyt c in the presence of CL and phosphatidylcholine (PC) phospholipids also present in the mitochondrial membrane to better understand how these interactions might drive transformation to the peroxidase-active protein. Using ion mobility mass spectrometry and millisecond hydrogen-deuterium exchange mass spectrometry, we demonstrate heterogeneity in the lipid-bound ensemble, with zwitterionic (PC) phospholipids inducing destabilization of residues necessary for peroxidase coordination, and increased dynamics on the proximal face of the heme binding pocket. In contrast to what might be expected from classical models for CL-driven Cyt c peroxidase activation, interactions with CL are shown to rigidify heme coordination. To reconcile this observation with the well-supported view that CL is linked to peroxidase activation, we propose a mechanism in which CL stabilizes the conformational transition between the peroxidase-active and inactive forms.
Assuntos
Cardiolipinas/metabolismo , Citocromos c/metabolismo , Fosfatidilcolinas/metabolismo , Animais , Cavalos/metabolismo , Conformação ProteicaRESUMO
Conformational dynamics are increasingly recognized as being essential for enzyme function. However, there is virtually no direct experimental evidence to support the notion that individual dynamic modes are required for specific catalytic processes, apart from the initial step of substrate binding. In this work, we use a unique approach based on millisecond hydrogen-deuterium exchange mass spectrometry to identify dynamic modes linked to individual catalytic processes in the antibiotic resistance enzyme TEM-1 ß-lactamase. Using a "good" substrate (ampicillin), a poorly hydrolyzed substrate (cephalexin) and a covalent inhibitor (clavulanate), we are able to isolate dynamic modes that are specifically linked to substrate binding, productive lactam ring hydrolysis and deacylation. These discoveries are ultimately translated into specific targets for allosteric TEM-1 inhibitor development.
Assuntos
Simulação de Dinâmica Molecular , Conformação Proteica , beta-Lactamases/química , Acilação , Catálise , Ligação de Hidrogênio , Cinética , beta-Lactamases/metabolismoRESUMO
Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) emerged as a tool for biochemistry and structural biology around 25â¯years ago. It has since become a key approach for studying protein dynamics, protein-ligand interactions, membrane proteins and intrinsically disordered proteins (IDPs). In HDX labeling, proteins are exposed to deuterated solvent (usually D2O) for a variable 'labeling time', resulting in isotope exchange of unprotected labile protons on the amide backbone and amino acid side chains. By comparing the levels of deuterium uptake in different regions of a protein, information on conformational and dynamic changes in the system can be acquired. When coupled with MS, HDX is suitable for probing allosteric effects in catalysis and ligand binding, epitope mapping, validation of biosimilars, drug candidate screening and mapping membrane-protein interactions among many other bioanalytical applications. This review introduces HDX-MS via a brief description of HDX-MS development, followed by an overview of HDX theory and ultimately an outline of methods and procedures involved in performing HDX-MS experiments.