Deuterium Exchange Research Articles

Identification of l-Tryptophan by down-field 1 H MRS: A precursor for brain NAD+ and serotonin syntheses.

To explore the presence of new resonances beyond 9.4ppm from the human brain, down-field proton MRS was performed in vivo in the human brain on 6 healthy volunteers at 7 T. To maximize the SNR, a large voxel was placed within the brain to cover the maximal area in such a way that sinus cavities were avoided. A spectrally selective 90° E-BURP pulse with an excitation bandwidth of 2ppm was used to probe the spectral chemical shift range between 9.1 and 10.5ppm. The E-BURP pulse was integrated with PRESS spatial localization to obtain non-water-suppressed proton MR spectra from the desired spectral region. In the down-field proton MRS obtained from all of the volunteers scanned, we identified a new peak consistently resonating at 10.1ppm. Protons associated with this resonance are in cross-relaxation with the bulk water, as demonstrated by the water saturation and deuterium exchange experiments. Based on the chemical shift, this new peak was identified as the indole (-NH) proton of l-tryptophan (l-TRP) and was further confirmed from phantom experiments on l-TRP. These promising preliminary results potentially pave the way to investigate the role of cerebral metabolism of l-TRP in healthy and disease conditions.

A primordial atmospheric origin of hydrospheric deuterium enrichment on Mars

The deuterium-to-hydrogen (D/H or 2H/1H) ratio of Martian atmospheric water (∼6× standard mean ocean water, SMOW) is higher than that of known sources, requiring planetary enrichment. A recent measurement by NASA's Mars Science Laboratory rover Curiosity of Hesperian-era (>3 Ga) clays yields a D/H ratio ∼3×SMOW, demonstrating that most of the enrichment occurs early in Mars's history, reinforcing the conclusions of Martian meteorite studies. As on Venus, Mars's D/H enrichment is widely thought to reflect preferential loss to space of 1H (protium) relative to 2H (deuterium), but both the cause and the global environmental context of large and early hydrogen losses remain to be determined. Here, we apply a recent model of primordial atmosphere evolution to Mars, link the magma ocean of the accretion epoch with a subsequent water-ocean epoch, and calculate the behavior of deuterium for comparison with the observed record. In contrast to earlier works that consider Martian D/H fractionation in atmospheres in which hydrogen reservoirs are present exclusively as H2O or H2, here we consider 2-component (H2O-H2) outgassed atmospheres in which both condensing (H2O) and escaping (H2) components – and their interaction – are explicitly calculated. We find that a ≈2-3× hydrospheric deuterium-enrichment is produced rapidly if the Martian magma ocean is chemically reducing at last equilibration with the primordial atmosphere, making H2 and CO the initially dominant species, with minor abundances of H2O and CO2. Reducing gases – in particular H2 – can cause substantial greenhouse warming and prevent a water ocean from freezing immediately after the magma ocean epoch. We find that greenhouse warming due to plausible H2 inventories (pH=21−102 bars) yields surface temperatures high enough (T=s290−560 K) to stabilize a water ocean and produce an early hydrological cycle through which surface water can be circulated. Moreover, the pressure-temperature conditions are high enough to produce ocean-atmosphere H2O-H2 isotopic equilibrium through gas-phase deuterium exchange such that surface H2O strongly concentrates deuterium relative to H2, which preferentially takes up protium and escapes from the primordial atmosphere. The efficient physical separation of deuterium-rich (H2O) and deuterium-poor (H2) species via condensation permits equilibrium isotopic partitioning and early atmospheric escape to be recorded in modern crustal reservoirs. The proposed scenario of primordial H2-CO-rich outgassing and escape suggests significant durations (>Myr) of chemical conditions on the Martian surface conducive to prebiotic chemistry immediately following magma ocean crystallization.

Ionic liquid-based reverse micelles. Use of hydrogen–deuterium exchange as a tool for surfactant self-assembly characterization

We report the study of the rate for hydrogen–deuterium (H-D) exchange in the cation of the ionic liquid-surfactant 1-butyl-3-methylimidazolium 1,4-bis-2-ethylhexylsulfosuccinate (bmim-AOT) when it forms reverse micelles (RMs). The results were compared with those found for bmim-AOT dissolved in D2O showing an inhibition larger than 500-fold on going from bulk D2O to the reverse micelle. Also, the effect of varying water content and surfactant concentration of the RMs on the deuterium exchange was evaluated by 1H NMR technique. The results demonstrate that the H-D exchange at the C(2) position of bmim+ when it forms RMs significantly reflects the properties of the micellar microenvironment, specifically of the micellar interface due to the localization of bmim+ in the interfacial region, intercalated between the AOT moiety. The rate of deuterium exchange is directly related to the properties of interfacial water, which, in turn, is closely related to the water-surfactant interaction. Thus, the analysis of deuterium exchange in the 2-position of bmim+ is proposed as a useful tool to explore the properties of RMs formed by this cation, with the advantage of being a non-invasive technique since the ionic liquid-surfactant itself acts as the molecular probe.

Schaftoside inhibits 3CLpro and PLpro of SARS-CoV-2 virus and regulates immune response and inflammation of host cells for the treatment of COVID-19.

It is an urgent demand worldwide to control the coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. The 3-chymotrypsin-like protease (3CLpro) and papain-like protease (PLpro) are key targets to discover SARS-CoV-2 inhibitors. After screening 12 Chinese herbal medicines and 125 compounds from licorice, we found that a popular natural product schaftoside inhibited 3CLpro and PLpro with IC50 values of 1.73 ± 0.22 and 3.91 ± 0.19 μmol/L, respectively, and inhibited SARS-CoV-2 virus in Vero E6 cells with EC50 of 11.83 ± 3.23 μmol/L. Hydrogen–deuterium exchange mass spectrometry analysis, quantum mechanics/molecular mechanics calculations, together with site-directed mutagenesis indicated the antiviral activities of schaftoside were related with non-covalent interactions with H41, G143 and R188 of 3CLpro, and K157, E167 and A246 of PLpro. Moreover, proteomics analysis and cytokine assay revealed that schaftoside also regulated immune response and inflammation of the host cells. The anti-inflammatory activities of schaftoside were confirmed on lipopolysaccharide-induced acute lung injury mice. Schaftoside showed good safety and pharmacokinetic property, and could be a promising drug candidate for the prevention and treatment of COVID-19.

Temperature-dependent hydrogen deuterium exchange shows impact of analog binding on adenosine deaminase flexibility but not embedded thermal networks

The analysis of hydrogen deuterium exchange by mass spectrometry as a function of temperature and mutation has emerged as a generic and efficient tool for the spatial resolution of protein networks that are proposed to function in the thermal activation of catalysis. In this work, we extend temperature-dependent hydrogen deuterium exchange from apo-enzyme structures to protein–ligand complexes. Using adenosine deaminase as a prototype, we compared the impacts of a substrate analog (1-deaza-adenosine) and a very tight-binding inhibitor/transition state analog (pentostatin) at single and multiple temperatures. At a single temperature, we observed different hydrogen deuterium exchange-mass spectrometry properties for the two ligands, as expected from their 106-fold differences in strength of binding. By contrast, analogous patterns for temperature-dependent hydrogen deuterium exchange mass spectrometry emerge in the presence of both 1-deaza-adenosine and pentostatin, indicating similar impacts of either ligand on the enthalpic barriers for local protein unfolding. We extended temperature-dependent hydrogen deuterium exchange to a function-altering mutant of adenosine deaminase in the presence of pentostatin and revealed a protein thermal network that is highly similar to that previously reported for the apo-enzyme (Gao et al., 2020, JACS 142, 19936-19949). Finally, we discuss the differential impacts of pentostatin binding on overall protein flexibility versus site-specific thermal transfer pathways in the context of models for substrate-induced changes to a distributed protein conformational landscape that act in synergy with embedded protein thermal networks to achieve efficient catalysis.

Dynamics of integrin α5β1, fibronectin, and their complex reveal sites of interaction and conformational change

Integrin α5β1 mediates cell adhesion to the extracellular matrix by binding fibronectin (Fn). Selectivity for Fn by α5β1 is achieved through recognition of an RGD motif in the 10th type III Fn domain (Fn10) and the synergy site in the ninth type III Fn domain (Fn9). However, details of the interaction dynamics are unknown. Here, we compared synergy-site and Fn-truncation mutations for their α5β1-binding affinities and stabilities. We also interrogated binding of the α5β1 ectodomain headpiece fragment to Fn using hydrogen-deuterium exchange (HDX) mass spectrometry to probe binding sites and sites of integrin conformational change. Our results suggest the synergistic effect of Fn9 requires both specific residues and a folded domain. We found some residues considered important for synergy are required for stability. Additionally, we show decreases in fibronectin HDX are localized to a synergy peptide containing contacting residues in two β-strands, an intervening loop in Fn9, and the RGD-containing loop in Fn10, indicative of binding sites. We also identified binding sites in the α5-subunit β-propeller domain for the Fn9 synergy site and in the β1-subunit βI domain for Fn10 based on decreases in α5β1 HDX. Interestingly, the dominant effect of Fn binding was an increase in α5β1 deuterium exchange distributed over multiple sites that undergo changes in conformation or solvent accessibility and appear to be sites where energy is stored in the higher-energy, open-integrin conformation. Together, our results highlight regions important for α5β1 binding to Fn and dynamics associated with this interaction.

Mechanism of inhibition of bacterial RNA helicases by diazo dyes and implications for antimicrobial drug development

RNA helicases represent attractive drug targets as their activity is linked to several human diseases and impacts microbial infectious processes. While some inhibitors of human RNA helicases demonstrated therapeutic potential as anticancer and antiviral drugs in preclinical trials, chemical inhibition of microbial RNA helicases is less investigated. Here, we address this matter by focusing on the RhlE proteobacterial group of RNA helicases. Having previously shown that the RhlE2 RNA helicase is important for the virulence of the opportunistic pathogen Pseudomonas aeruginosa, we screened a library of 1280 molecules for inhibitors of RhlE2 RNA-dependent ATP hydrolytic activity. The most potent inhibitor is the diazo compound Chicago Sky Blue (CSB). Using hydrogen–deuterium exchange mass spectrometry and biochemical assays, we mapped CSB binding to RhlE2 catalytic core and defined its inhibitory mechanism. Targeting microbial RNA helicases as therapeutic strategy is challenging due to potential side-effects linked to protein conservation across life kingdoms. Interestingly, our structure–activity relationship analysis delineates other diazo dyes closely related to CSB differentially affecting RhlE homologs. Our work could thus be exploited for future drug development studies, which are extremely timely considering the increasing spread of antibiotic resistance among bacterial pathogens.

Novel interaction interfaces mediate the interaction between the NEIL1 DNA glycosylase and mitochondrial transcription factor A.

The maintenance of human mitochondrial DNA (mtDNA) is critical for proper cellular function as damage to mtDNA, if left unrepaired, can lead to a diverse array of pathologies. Of the pathways identified to participate in DNA repair within the mitochondria, base excision repair (BER) is the most extensively studied. Protein-protein interactions drive the step-by-step coordination required for the successful completion of this pathway and are important for crosstalk with other mitochondrial factors involved in genome maintenance. Human NEIL1 is one of seven DNA glycosylases that initiates BER in both the nuclear and mitochondrial compartments. In the current work, we scrutinized the interaction between NEIL1 and mitochondrial transcription factor A (TFAM), a protein that is essential for various aspects of mtDNA metabolism. We note, for the first time, that both the N- and C- terminal domains of NEIL1 interact with TFAM revealing a unique NEIL1 protein-binding interface. The interaction between the two proteins, as observed biochemically, appears to be transient and is most apparent at concentrations of low salt. The presence of DNA (or RNA) also positively influences the interaction between the two proteins, and molar mass estimates indicate that duplex DNA is required for complex formation at higher salt concentrations. Hydrogen deuterium exchange mass spectrometry data reveal that both proteins exchange less deuterium upon DNA binding, indicative of an interaction, and the addition of NEIL1 to the TFAM-DNA complex alters the interaction landscape. The transcriptional activity of TFAM appears to be independent of NEIL1 expression under normal cellular conditions, however, in the presence of DNA damage, we observe a significant reduction in the mRNA expression of TFAM-transcribed mitochondrial genes in the absence of NEIL1. Overall, our data indicate that the interaction between NEIL1 and TFAM can be modulated by local environment such as salt concentrations, protein availability, the presence of nucleic acids, as well as the presence of DNA damage.

Hydrogen-Deuterium Addition and Exchange in N-Ethylmaleimide Reaction with Glutathione Detected by NMR Spectroscopy.

Glutathione (GSH) is an important and ubiquitous thiol compound abundantly present in virtually every living cell. It is a powerful antioxidant critically required to protect cells from oxidative damage and free radical injury. Its quantification in ex vivo analysis remains a major challenge because it spontaneously oxidizes to form glutathione disulfide. N-Ethylmaleimide (NEM) is a well-known Michael acceptor, which reacts rapidly and irreversibly with thiol and prevents disulfide bond formation. Based on thiol conjugation to NEM, recently, the concentration of GSH was determined in human blood using NMR spectroscopy [Anal. Chem, 2021, 93(44): 14844–14850]. It was found that hydrogen–deuterium addition and exchange occur during the thiol–maleimide reaction as well as NMR analysis, generating a series of poorly explored diastereomers/isotopomers. Here, we establish a general NMR approach to identify the thiosuccinimide diastereomers/isotopomers derived from the thiol–maleimide reaction. The thiol-Michael addition reaction was conducted for GSH and another thiol compound, cysteine, separately, using D2O and H2O. The conjugates were characterized by 1H/13C 1D/2D NMR under different solvent, buffer, and pH conditions. The Michael addition combined with the H/D exchange formed twelve unique diastereomers/isotopomers. NMR measurements allowed the distinct assignment of all structures in solutions and quantification of H/D addition and exchange. Interestingly, the deuterium exchange rate was dependent on structure, pH, and buffer. The elucidation of the thiol–maleimide reaction and H/D exchange mechanism can potentially impact areas including metabolomics, small molecule synthesis, and bioconjugation chemistry.

Simple and Fast Maximally Deuterated Control (maxD) Preparation for Hydrogen-Deuterium Exchange Mass Spectrometry Experiments.

During the analysis steps of hydrogen-deuterium exchange (HDX) mass spectrometry (MS), there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during liquid chromatography (LC)-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD), in which the protein is deuterated at positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here, we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ∼40 min for nonmembrane proteins. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with earlier protocols that either require much more time or require isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed for maximum deuteration in a short time (∼10 min) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy-to-perform protocol should enhance routine preparation of maxD controls.

Impact of Bioconjugation on Structure and Function of Antibodies for Use in Immunoassay by Hydrogen-Deuterium Exchange Mass Spectrometry

Monoclonal antibodies (mAbs) are widely used as analytical components in immunoassays to detect target molecules in applications such as clinical diagnostics, food analysis and drug discovery. Functional groups are often conjugated to lysine or cysteine residues to aid immobilization of mAbs or to enable their detection in an antibody antigen complex. Good assay performance depends on the affinity and specificity of the mAbs for the antigen. The conjugation reaction however can cause higher order structural (HOS) changes and ultimately affect the assay performance. In this study, four differently conjugated mAbs were selected as model systems and characterized by mass spectrometry. Particularly, intact protein analysis by liquid-chromatography mass-spectrometry (LC-MS) was performed to determine the amount and distribution of conjugation. Hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments were carried out for the structural characterization of the conjugated mAbs. Immunoassay experiments were performed to monitor the effects of conjugation on the binding properties of the antibodies selected. Good agreement between the mass spectrometry and binding experiment results was found. Particularly, it was noted that the overall structural flexibility of the antibodies increases upon cysteine conjugation and decreases for lysine conjugation. The conjugation of mAbs with bulky functional groups tends to decrease the deuterium uptake kinetics due to induced steric effects. Overall, this study shows correlations between conjugation, structure and function of immunoassay antibodies and the benefits of mass spectrometry to improve understanding of the conjugation reaction and provide insights that can predict immunoassay performance.

Tracking global invasion pathways of the spongy moth (Lepidoptera: Erebidae) to the United States using stable isotopes as endogenous biomarkers.

The spread of invasive insect species causes enormous ecological damage and economic losses worldwide. A reliable method that tracks back an invaded insect's origin would be of great use to entomologists, phytopathologists, and pest managers. The spongy moth (Lymantria dispar, Linnaeus 1758) is a persistent invasive pest in the Northeastern United States and periodically causes major defoliations in temperate forests. We analyzed field‐captured (Europe, Asia, United States) and laboratory‐reared L. dispar specimens for their natal isotopic hydrogen and nitrogen signatures imprinted in their biological tissues (δ2H and δ15N) and compared these values to the long‐term mean δ2H of regional precipitation (Global Network of Isotopes in Precipitation) and δ15N of regional plants at the capture site. We established the percentage of hydrogen–deuterium exchange for L. dispar tissue (Pex = 8.2%) using the comparative equilibration method and two‐source mixing models, which allowed the extraction of the moth's natal δ2H value. We confirmed that the natal δ2H and δ15N values of our specimens are related to the environmental signatures at their geographic origins. With our regression models, we were able to isolate potentially invasive individuals and give estimations of their geographic origin. To enable the application of these methods on eggs, we established an egg‐to‐adult fraction factor for L. dispar (Δegg‐adult = 16.3 ± 4.3‰). Our models suggested that around 25% of the field‐captured spongy moths worldwide were not native in the investigated capture sites. East Asia was the most frequently identified location of probable origin. Furthermore, our data suggested that eggs found on cargo ships in the United States harbors in Alaska, California, and Louisiana most probably originated from Asian L. dispar in East Russia. These findings show that stable isotope biomarkers give a unique insight into invasive insect species pathways, and thus, can be an effective tool to monitor the spread of insect pest epidemics.

Allosteric inhibition of PPM1D serine/threonine phosphatase via an altered conformational state

PPM1D encodes a serine/threonine phosphatase that regulates numerous pathways including the DNA damage response and p53. Activating mutations and amplification of PPM1D are found across numerous cancer types. GSK2830371 is a potent and selective allosteric inhibitor of PPM1D, but its mechanism of binding and inhibition of catalytic activity are unknown. Here we use computational, biochemical and functional genetic studies to elucidate the molecular basis of GSK2830371 activity. These data confirm that GSK2830371 binds an allosteric site of PPM1D with high affinity. By further incorporating data from hydrogen deuterium exchange mass spectrometry and sedimentation velocity analytical ultracentrifugation, we demonstrate that PPM1D exists in an equilibrium between two conformations that are defined by the movement of the flap domain, which is required for substrate recognition. A hinge region was identified that is critical for switching between the two conformations and was directly implicated in the high-affinity binding of GSK2830371 to PPM1D. We propose that the two conformations represent active and inactive forms of the protein reflected by the position of the flap, and that binding of GSK2830371 shifts the equilibrium to the inactive form. Finally, we found that C-terminal truncating mutations proximal to residue 400 result in destabilization of the protein via loss of a stabilizing N- and C-terminal interaction, consistent with the observation from human genetic data that nearly all PPM1D mutations in cancer are truncating and occur distal to residue 400. Taken together, our findings elucidate the mechanism by which binding of a small molecule to an allosteric site of PPM1D inhibits its activity and provides insights into the biology of PPM1D.

NMR-monitoring of H/D exchange reaction of ketones in solutions of imidazolium ionic liquids

NMR spectroscopy was used to study hydrogen–deuterium exchange in CH3, CH2 and CH moieties of ketones dissolved in mixtures of solvents containing exchangeable deuteriums and imidazolium ionic liquids (IL) acting as catalysts. Factors affecting the efficiency of the exchange, as well as the role of the deuterated solvent, temperature and concentration, were investigated. Depending on the sample composition and temperature, the degree of deuteration can reach different values at equilibrium state, and the exchange rate can vary from several minutes to several months. ILs with OAc anions and with ethyl chain cations exhibit significant catalytic properties and high degrees of deuteration (up to 98%), which can be achieved by consecutive deuteration cycles. A convenient practical protocol for monitoring the exchange process by NMR spectroscopy and calculating the degree of deuteration was developed and can be used for various molecular systems.

A Novel PNGase Rc for Improved Protein N-Deglycosylation in Bioanalytics and Hydrogen-Deuterium Exchange Coupled With Mass Spectrometry Epitope Mapping under Challenging Conditions.

N-linked glycosylation is a ubiquitous posttranslational modification of proteins. While it plays an important role in the biological function of proteins, it often poses a major challenge for their analytical characterization. Currently available peptide N-glycanases (PNGases) are often inefficient at deglycosylating proteins due to sterically inaccessible N-glycosylation sites. This usually leads to poor sequence coverage in bottom-up analysis using liquid chromatography with tandem mass spectrometry and makes it impossible to obtain an intact mass signal in top-down MS analysis. In addition, most PNGases operate optimally only in the neutral to slightly acidic pH range and are severely compromised in the presence of reducing and denaturing substances, which limits their use for advanced bioanalysis based on hydrogen-deuterium exchange in combination with mass spectrometry (HDX-MS). Here, we present a novel peptide N-glycanase from Rudaea cellulosilytica (PNGase Rc) for which we demonstrate broad substrate specificity for N-glycan hydrolysis from multiply occupied and natively folded proteins. Our results show that PNGase Rc is functional even under challenging, HDX quenching conditions (pH 2.5, 0 °C) and in the presence of 0.4 M tris(2-carboxyethyl)phosphine, 4 M urea, and 1 M guanidinium chloride. Most importantly, we successfully applied the PNGase Rc in an HDX-MS workflow to determine the epitope of a nanobody targeting the extracellular domain of human signal-regulating protein alpha (SIRPα).

Preparation of Deuterium-Labeled Armodafinil by Hydrogen-Deuterium Exchange and Its Application in Quantitative Analysis by LC-MS.

Armodafinil, the R enantiomer of modafinil, was approved in 2007 by the US Food and Drug Administration as a wake-promoting agent for excessive sleepiness treatment. Due to its abuse by students and athletes, there is a need of its quantification. Quantitative analysis by liquid chromatography-mass spectrometry, however, though very common and sensitive, frequently cannot be performed without isotopically labeled standards which usually have to be specially synthesized. Here we reported our investigation on the preparation of deuterated standard of armodafinil based on the simple and inexpensive hydrogen–deuterium exchange reaction at the carbon centers. The obtained results clearly indicate the possibility of introduction of three deuterons into the armodafinil molecule. The introduced deuterons do not undergo back exchange under neutral and acidic conditions. Moreover, the deuterated and non-deuterated armodafinil isotopologues revealed co-elution during the chromatographic analysis. The ability to control the degree of deuteration using different reaction conditions was determined. The proposed method of deuterated armodafinil standard preparation is rapid, cost-efficient and may be successfully used in its quantitative analysis by LC-MS.

Chromatography at -30 °C for Reduced Back-Exchange, Reduced Carryover, and Improved Dynamic Range for Hydrogen-Deuterium Exchange Mass Spectrometry.

For hydrogen–deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during protein analyses should be minimized to promote greater reproducibility. Standard HDX-MS analysis systems that digest proteins and separate peptides at pH 2.7 and 0 °C can lose >30% of the deuterium marker within 15 min of sample injection. This report describes the architecture and performance of a dual-enzyme, HDX-MS instrument that conducts liquid chromatography (LC) separations at subzero temperature, thereby reducing back-exchange and supporting longer LC separations with improved chromatographic resolution. LC separations of perdeuterated, fully reduced, iodoacetamide-treated BSA protein digest standard peptides were performed at 0, −10, −20, and −30 °C in ethylene glycol (EG)/H2O mixtures. Analyses conducted at −20 and −30 °C produced similar results. After subtracting for deuterium retained in arginine side chains, the average peptide eluted during a 40 min gradient contained ≈16% more deuterium than peptides eluted with a conventional 8 min gradient at 0 °C. A subset of peptides exhibited ≈26% more deuterium. Although chromatographic peaks shift with EG concentration and temperature, the apparatus elutes unbroadened LC peaks. Electrospray ion intensity does not decline with increasing EG fraction. To minimize bias from sample carryover, the fluidic circuits allow flush and backflush cleaning of all enzyme and LC columns. The system can perform LC separations and clean enzyme columns simultaneously. Temperature zones are controlled ±0.058 °C. The potential of increased sensitivity by mixing acetonitrile with the analytical column effluent was also examined.

Multiplexed Conformationally Selective, Localized Gas-Phase Hydrogen Deuterium Exchange of Protein Ions Enabled by Transmission-Mode Electron Capture Dissociation.

In this article, we present an approach for conformationally multiplexed, localized hydrogen deuterium exchange (HDX) of gas-phase protein ions facilitated by ion mobility (IM) followed by electron capture dissociation (ECD). A quadrupole-IM-time of flight instrument previously modified to enable ECD in transmission mode (without ion trapping) immediately following a mobility separation was further modified to allow for deuterated ammonia (ND3) to be leaked in after m/z selection. Collisional activation was minimized to prevent deuterium scrambling from giving structurally irrelevant results. Gas-phase HDX with ECD fragmentation for exchange site localization was demonstrated with the extensively studied protein folding models ubiquitin and cytochrome c. Ubiquitin was ionized from conditions that stabilize the native state and conditions that stabilize the partially folded A-state. IM of deuterated ubiquitin 6+ ions allowed the separation of more compact conformers from more extended conformers. ECD of the separated subpopulations revealed that the more extended (later arriving) conformers had significant, localized differences in the amount of HDX observed. The 5+ charge state showed many regions with protection from HDX, and the 11+ charge state, ionized from conditions that stabilize the A-state, showed high levels of deuterium incorporation throughout most of the protein sequence. The 7+ ions of cytochrome c ionized from aqueous conditions showed greater HDX with unstructured regions of the protein relative to interior, structured regions, especially those involved in heme binding. With careful tuning and attention to deuterium scrambling, our approach holds promise for determining region-specific information on a conformer-selected basis for gas-phase protein structures, including localized characterizations of ligand, epitope, and protein-protein binding.

Empirical Bayes functional models for hydrogen deuterium exchange mass spectrometry

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a technique to explore differential protein structure by examining the rate of deuterium incorporation for specific peptides. This rate will be altered upon structural perturbation and detecting significant changes to this rate requires a statistical test. To determine rates of incorporation, HDX-MS measurements are frequently made over a time course. However, current statistical testing procedures ignore the correlations in the temporal dimension of the data. Using tools from functional data analysis, we develop a testing procedure that explicitly incorporates a model of hydrogen deuterium exchange. To further improve statistical power, we develop an empirical Bayes version of our method, allowing us to borrow information across peptides and stabilise variance estimates for low sample sizes. Our approach has increased power, reduces false positives and improves interpretation over linear model-based approaches. Due to the improved flexibility of our method, we can apply it to a multi-antibody epitope-mapping experiment where current approaches are inapplicable due insufficient flexibility. Hence, our approach allows HDX-MS to be applied in more experimental scenarios and reduces the burden on experimentalists to produce excessive replicates. Our approach is implemented in the R-package “hdxstats”: https://github.com/ococrook/hdxstats.

Exploring Performance Parameters of Artificial Allosteric Protein Switches

Biological information processing networks rely on allosteric protein switches that dynamically interconvert biological signals. Construction of their artificial analogues is a central goal of synthetic biology and bioengineering. Receptor domain insertion is one of the leading methods for constructing chimeric protein switches. Here we present an in vitro expression-based platform for the analysis of chimeric protein libraries for which traditional cell survival or cytometric high throughput assays are not applicable. We utilise this platform to screen a focused library of chimeras between PQQ-glucose dehydrogenase and calmodulin. Using this approach, we identified 50 chimeras (approximately 23% of the library) that were activated by calmodulin-binding peptides. We analysed performance parameters of the active chimeras and demonstrated that their dynamic range and response times are anticorrelated, pointing to the existence of an inherent thermodynamic trade-off. We show that the structure of the ligand peptide affects both the response and activation kinetics of the biosensors suggesting that the structure of a ligand:receptor complex can influence the chimera’s activation pathway. In order to understand the extent of structural changes in the reporter protein induced by the receptor domains, we have analysed one of the chimeric molecules by CD spectroscopy and hydrogen–deuterium exchange mass spectrometry. We concluded that subtle ligand-induced changes in the receptor domain propagated into the GDH domain and affected residues important for substrate and cofactor binding. Finally, we used one of the identified chimeras to construct a two-component rapamycin biosensor and demonstrated that core switch optimisation translated into improved biosensor performance.