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

Japanese encephalitis virus (JEV) remains a global public health concern due to its epidemiological distribution and the existence of multiple strains. Neutralizing antibodies against this infection have shown efficacy in in vivo studies. Thus, elucidation of the epitopes of neutralizing antibodies can aid in the design and development of effective vaccines against different strains of JEV. Here, we describe a combination of native mass spectrometry (native-MS) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to complete screening of eight mouse monoclonal antibodies (MAbs) against JEV E-DIII to identify epitope regions. Native-MS was used as a first pass to identify the antibodies that formed a complex with the target antigen, and it revealed that seven of the eight monoclonal antibodies underwent binding. Native mass spectra of a MAb (JEV-27) known to be non-binding showed broad native-MS peaks and poor signal, suggesting the protein is a mixture or that there are impurities in the sample. We followed native-MS with HDX-MS to locate the binding sites for several of the complex-forming antibodies. This combination of two mass spectrometry-based approaches should be generally applicable and particularly suitable for screening of antigen–antibody and other protein–protein interactions when other traditional approaches give unclear results or are difficult, unavailable, or need to be validated. [ABSTRACT FROM AUTHOR]

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as ‘fuzzy’. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation – a heterotrimer of INCENP, Survivin, and Borealin – we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

Several platinum (II) complexes bearing heteroatom‐substituted phosphine oxides ligand H(O)P(OCMe2CMe2O) were obtained. The H‐phosphonate was synthesized in an alternative, one‐pot procedure with the use of water from hexamethylphosphorus triamide and pinacol. In the presence of H(O)P (OCMe2CMe2O) ligand, [PtCl2(cod)] was converted into three new complexes: [PtCl2{P(OH)(OCMe2CMe2O)}2] [Pt1], [PtCl{P(O)(OCMe2CMe2O)}{P(OH)(OCMe2CMe2O)}2] [Pt2], and [Pt{P(OH)(OCMe2CMe2O)}4]Cl2 [Pt3]. When [PtCl2(PPh3)2] was utilized as the substrate, [PtCl(PPh3)2{P(OH)(OCMe2CMe2O)}]Cl [Pt4] and [PtCl(PPh3)2{P(O)(OCMe2CMe2O)}] [Pt5] complexes were obtained, as the results of reaction with H(O)P(OCMe2CMe2O) and HP(OCMe2CMe2O)(OCH2CMe2NH) ligands. The structural features of all complexes were characterized by physicochemical and spectroscopic methods as well as single X‐ray diffraction studies. Complex [Pt1] was found to promote cascade reaction, the addition of water to triple bond, and hydrogen/deuterium exchange reaction. The hydration reaction of substituted terminal aryl alkynes gave exclusively Markovnikov products and tolerated different functional groups. Various aryl ketones were synthesized in good to excellent yield. H/D exchange reaction was exemplified by the model reaction of acetophenone in deuterium containing methanol. Factors influencing the effectiveness of the hydrogen/deuterium exchange reaction of the methyl group were determined. The cascade procedure bears the potential to overcome the limitations of conventional synthesis and unlocks practical use in the synthesis of deuterium‐labeled pharmaceuticals. [ABSTRACT FROM AUTHOR]

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity.

Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation in vitro, we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.

High resolution and tandem mass spectrometry are popular techniques for the structure elucidation of small molecule impurities and metabolites. However, proposing definitive structures for isomers solely based on the mass spectrometry data can be challenging. Hydrogen/Deuterium exchange mass spectrometry can be used as an effective tool for the structure elucidation of isomers by determining the number of exchangeable hydrogen atoms on heteroatomic functional groups. The study demonstrates a simple and effective online hydrogen/deuterium exchange technique using two-dimensional liquid chromatography mass spectrometry for unknown impurity and metabolite structure elucidation. A mixture of isomeric compounds were separated in the first dimension of the two-dimensional liquid chromatography and transferred to the second dimension using heart-cutting valve. The hydrogen/deuterium exchange was carried out in the second dimension using deuterium oxide as one of the mobile phase solvents. The eluents were analyzed after hydrogen/deuterium exchange with mass spectrometry. The application of two-dimensional liquid chromatography mass spectrometry for hydrogen/deuterium exchange experiments provides unique advantages of simplicity along with minimized time and resources. [ABSTRACT FROM AUTHOR]

The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6 M guanidinium chloride. BDPA is a small three‐helix‐bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two‐state process between the native and the fully unfolded states. We employed a dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange 2D NMR techniques with the use of spin desalting columns, which allowed us to investigate the H/D‐exchange behavior of individually identified peptide amide (NH) protons. We obtained H/D‐exchange protection factors of the 21 NH protons that form an α‐helical hydrogen bond in the native structure, and the majority of these NH protons were significantly protected with a protection factor of 2.0–5.2 in 6 M guanidinium chloride, strongly suggesting that these weakly protected NH protons form much stronger hydrogen bonds under native folding conditions. The results can be used to deduce the structure of an early folding intermediate, when such an intermediate is shown by other methods. Among three native helical regions, the third helix in the C‐terminal side was highly protected and stabilized by side‐chain salt bridges, probably acting as the folding initiation site of BDPA. The present results are discussed in relation to previous experimental and computational findings on the folding mechanisms of BDPA. [ABSTRACT FROM AUTHOR]

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. [ABSTRACT FROM AUTHOR]

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab. [ABSTRACT FROM AUTHOR]

This note describes theoretical and experimental considerations to observe perturbation of a protein upon binding to a ligand with weak affinity by hydrogen/deuterium exchange mass spectrometry (HDX-MS). The most popular application of HDX-MS is to determine the binding site of a drug or drug lead in a protein target. However, when the affinity of a ligand is weak, driving the equilibrium to the formation of a complex is difficult, and thus, observing the perturbation upon binding is also challenging. Theoretical consideration indicates that the original concentration of a ligand over the dissociation constant ([L0]/K D) is roughly equal to the maximum protection factor expected for the experiment when the original concentration of a ligand is significantly larger than the original concentration of a protein and the dissociation constant ([L0] ⇌ [P0] and [L0] ⇌ K D). When HDX-MS analysis of a protein with a ligand of low affinity and low solubility is carried out, it may be challenging to achieve high enough ligand concentration to drive the equilibrium in favor of the complex due to the low solubility. There are two methods to alleviate this issue: (i) spiking a low affinity/low solubility ligand to exchange buffer to lower the required ligand concentration in aqueous protein stock solution and (ii) mixing a 1:1 ratio of aqueous protein-ligand stock solution and deuterated buffer to initiate the exchange reaction instead of the commonly used 1:9 ratio. [ABSTRACT FROM AUTHOR]

This Account describes considerations for the data generation, data analysis, and data interpretation of a hydrogen/deuterium exchange-mass spectrometry (HDX-MS) experiment to have a quantitative argument. Although HDX-MS has gained its popularity as a biophysical tool, the argument from its data often remains qualitative. To generate HDX-MS data that are more suitable for a quantitative argument, the sequence coverage and sequence resolution should be optimized during the feasibility stage, and the time window coverage and time window resolution should be improved during the HDX stage. To extract biophysically meaningful values for a certain perturbation from medium-resolution HDX-MS data, there are two major ways: (i) estimating the area between the two deuterium buildup curves using centroid values with and without the perturbation when plotted against log time scale and (ii) dissecting into multiple single-exponential curves using the isotope envelopes. To have more accurate arguments for an HDX-MS perturbation study, (i) false negatives due to sequence coverage, (ii) false negatives due to time window coverage, (iii) false positives due to sequence resolution, and (iv) false positives due to allosteric effects should be carefully examined. [ABSTRACT FROM AUTHOR]

[Display omitted] • Amphiphilic ionic liquid-based solutions indicated hydrogen/deuterium exchange. • Kinetic H/D exchange was observed. • H/D exchange was controlled by the ion-pairing strength. The hydrogen/deuterium (H/D) exchange of ionic liquid (IL) occurred in the deuterated solutions. Kinetic H/D exchange was investigated via Raman spectroscopy and pH measurements. The ILs employed were amphiphilic 1-alkyl-3-methylimidazolium triflate ([C n mim][OTf] (n = 2, 4, 6, and 8)) and hydrophilic [C 2 mim][X] (X = Cl, Br, I, and BF 4). The H/D exchange occurred in the [C 2 mim][OTf]-deuterated methanol (MeOD-d 4) solutions and was dependent on the additive concentration. For [C 2 mim][X]-D 2 O (X = Cl, Br, and I), which did not possess nano-heterogeneity and the anionic anisotropic shape factor, significant relationships were observed among the H/D exchange rate, pD, and ion-pairing strength. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) has made enormous contributions to comprehensive protein identification and quantification in proteomics. MS is also gaining momentum for structural biology in a variety of ways, complementing conventional structural biology techniques. Here, we will review how MS-based techniques, such as hydrogen/deuterium exchange, covalent labeling, and chemical cross-linking, enable the characterization of protein structure, dynamics, and interactions, especially from a perspective of their data analyses. Structural information encoded by chemical probes in intact proteins is decoded by interpreting MS data at a peptide level, i.e., revealing conformational and dynamic changes in local regions of proteins. The structural MS data are not amenable to data analyses in traditional proteomics workflow, requiring dedicated software for each type of data. We first provide basic principles of data interpretation, including isotopic distribution and peptide sequencing. We then focus particularly on computational methods for structural MS data analyses and discuss outstanding challenges in a proteome-wide large scale analysis.

The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX‐MS), differential scanning calorimetry (DSC), and nano‐differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX‐MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the CH2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry‐Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI. [ABSTRACT FROM AUTHOR]

The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia coli by transiently encapsulating non-native substrate in a nano-cage formed by the GroEL ring cavity and the lid-shaped GroES. Mechanistic studies of GroEL/ES with heterologous protein substrates suggested that the chaperonin is inefficient, typically requiring multiple ATP-dependent encapsulation cycles with only a few percent of protein folded per cycle. Here we analyzed the spontaneous and chaperonin-assisted folding of the essential enzyme 5,10-methylenetetrahydrofolate reductase (MetF) of E. coli , an obligate GroEL/ES substrate. We found that MetF, a homotetramer of 33-kDa subunits with (β/α) 8 TIM-barrel fold, populates a kinetically trapped folding intermediate(s) (MetF-I) upon dilution from denaturant that fails to convert to the native state, even in the absence of aggregation. GroEL/ES recognizes MetF-I and catalyzes rapid folding, with ~ 50% of protein folded in a single round of encapsulation. Analysis by hydrogen/deuterium exchange at peptide resolution showed that the MetF subunit folds to completion in the GroEL/ES nano-cage and binds its cofactor flavin adenine dinucleotide. Rapid folding required the net negative charge character of the wall of the chaperonin cavity. These findings reveal a remarkable capacity of GroEL/ES to catalyze folding of an endogenous substrate protein that would have coevolved with the chaperonin system. Unlabelled Image • MetF fails to fold spontaneously, even in the absence of aggregation. • MetF forms a kinetically trapped folding intermediate, MetF-I. • The GroEL/ES chaperonin catalyzes MetF-I folding. • MetF folds rapidly and with high efficiency upon encapsulation in the GroEL/ES cage. [ABSTRACT FROM AUTHOR]

Recent developments in neutron scattering instrumentation and sample handling have enabled studies of more complex biological samples and measurements at shorter exposure times. The experiments are typically conducted in D2O‐based buffers to emphasize or diminish scattering from a particular component or to minimize background noise in the experiment. To extract most information from such experiments it is thus desirable to determine accurate estimates of how and when closely bound hydrogen atoms from the biomolecule exchange with the deuterium in the solvent. This article introduces and documents software, PSX, for exploring the effect of hydrogen–deuterium exchange for proteins solubilized in D2O as well as the underlying bioinformatical models. The software aims to be generally applicable for any atomistic structure of a protein and its surrounding environment, and thus captures effects of both heterogenous exchange rates throughout the protein structure and varying the experimental conditions such as pH and temperature. The paper concludes with examples of applications and estimates of the effect in typical scenarios emerging in small‐angle neutron scattering on biological macromolecules in solution. The analysis presented here suggests that the common assumption of 90% exchange is in many cases an overestimate with the rapid sample handling systems currently available, which leads to fitting and calibration issues when analysing the data. Source code for the presented software is available from an online repository in which it is published under version 3 of the GNU publishing licence. [ABSTRACT FROM AUTHOR]

In-electrospray (ESI) hydrogen/deuterium exchange–mass spectrometry (HDX-MS) has been used to characterize solvated carbohydrate structures. However, the rapid exchange rate of hydroxyls, as well as variations in source conditions and ambient humidity, alter the extent of forward and back exchange, resulting in poor repeatability when quantifying D-uptake on different days. Herein, we compare two internal standards, a peptide and derivatized carbohydrate, to improve the repeatability of in-ESI HDX of carbohydrate-metal adducts. Our results show that maltoheptaose, derivatized with Girard's T reagent, is a suitable internal standard for improving the repeatability of in-ESI HDX analyses of carbohydrates of varying size. [ABSTRACT FROM AUTHOR]

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a powerful method to characterize protein conformational dynamics. Workflows typically utilize pepsin digestion prior to MS analysis to yield peptide level structural resolution. Tandem mass spectrometry (MS/MS) can potentially facilitate determination of site-specific deuteration to single-residue resolution. However, to be effective, MS/MS activation must minimize the occurrence of gas-phase intramolecular randomization of solution-generated deuterium labels. While significant work has focused on understanding this process in positive-ion mode, little is known about hydrogen/deuterium (H/D) scrambling processes in negative-ion mode. Here, we utilize selectively deuterated model peptides to investigate the extent of intramolecular H/D scrambling upon several negative-ion mode MS/MS techniques, including negative-ion collision-induced dissociation (nCID), electron detachment dissociation (EDD), negative-ion free radical–initiated peptide sequencing (nFRIPS), and negative-ion electron capture dissociation (niECD). H/D scrambling was extensive in deprotonated peptides upon nCID and nFRIPS. In fact, the energetics required to induce dissociation in nCID are sufficient to allow histidine C-2 and Cβ hydrogen atoms to participate in the scrambling process. EDD and niECD demonstrated moderate H/D scrambling with niECD being superior in terms of minimizing hydrogen migration, achieving ~ 30% scrambling levels for small c-type fragment ions. We believe the observed scrambling is likely due to activation during ionization and ion transport rather than during the niECD event itself. [ABSTRACT FROM AUTHOR]

Abstract The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural model for human PrP23-144 amyloid. [ABSTRACT FROM AUTHOR]

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

Kidneys play a crucial role in maintaining metabolic homeostasis in a body. Serum creatinine concentration is a simple test used as an indicator of renal function. One of the known ways of quantifying creatinine concentration is the liquid chromatography-mass spectrometry (LC-MS) method, using an isotopically labeled analog of creatinine as an internal standard. Unfortunately, such isotope-labeled analogs are expensive and their synthesis is complex. Here we demonstrate a facile preparation of deuterated analogues of creatinine, via the H/D exchange of hydrogens located at the α-carbon (α-C) of the N-methylated amino acid part, under basic conditions. The stability of retrieved isotopologues was analyzed under both neutral or acidic conditions, and the results revealed that the introduced deuterons do not undergo back-exchange. In addition, the coelution of deuterated and non-deuterated forms under acidic and neutral conditions was observed. The prepared isotopologues were successfully applied in the quantitative LC-MS analysis of urine samples, and the results demonstrated that the presented strategy is novel and inexpensive, and that the quantification correlates with the commonly used Jaffe test method.

The emergence of multi-drug resistant Enterococcus faecalis raises a serious threat to global public health. E. faecalis is a gram-positive intestinal commensal bacterium found in humans. E. faecalis can endure extreme environments such as high temperature, pressure, and high salt, which facilitates them to cause infection in hospitals. E. faecalis has two acyl carrier proteins, AcpA (EfAcpA) in de novo fatty acid synthesis (FAS) and AcpB (EfAcpB) which utilizes exogenous fatty acids. Previously, we determined the tertiary structures of these two ACPs and investigated their structure-function relationships. Solution structures revealed that overall folding of these two ACPs is similar to those of other bacterial ACPs. However, circular dichroism (CD) experiments showed that the melting temperature of EfAcpA is 76.3°C and that of EfAcpB is 79.2°C, which are much higher than those of other bacterial ACPs. In this study, to understand the origin of their structural stabilities, we verified the important residues for stable folding of these two ACPs by monitoring thermal and chemical denaturation. Hydrogen/deuterium exchange and chemical denaturation experiments on wild-type and mutant proteins revealed that Ile10 of EfAcpA and Ile14 of EfAcpB mediate compact intramolecular packing and promote high thermostability and stable folding. E. faecalis may maximize efficiency of FAS and increase adaptability to the environmental stress by having two thermostable ACPs. This study may provide insight into bacterial adaptability and development of antibiotics against multi-drug-resistant E. faecalis .

Highlights • Temperature programmed desorption shows that the exchange of hydrogen and deuterium occurs below 200 K on Au(111). • The exchange of hydrogen and deuterium between ethanol and water molecules occurs without the presence of a reactive surface species or reactive substrate. • Isotopic studies show that the exchange occurs between both ethanol/deuterated water and ethanol–OD/water adsorbates on Au(111). • The formation of ethanol/water hydrogen–bonded networks on Au(111) is expected to facilitate the concerted motion of hydrogen/deuterium atoms at low temperatures. Abstract Proton transfer and hydrogen-bonding networks are critical in biochemical processes including protein folding and protein–protein interactions. Surface science can simplify these complex systems by studying the exchange of hydrogen and deuterium atoms between molecules supported on surfaces. Temperature programmed desorption (TPD) was used to show that the exchange of hydrogen and deuterium between co–adsorbed isotopologues of ethanol and water occurs readily at temperatures below 200 K on Au(111). Since Au(111) is an inert substrate that does not cause dissociation of water or ethanol at any temperature, the results herein suggest that Au(111) acts as a support to facilitate H/D exchange via hydrogen-bonded networks of molecular ethanol and water at low temperatures. Graphical abstract Image, graphical abstract [ABSTRACT FROM AUTHOR]

A pepsin/FPXIII (protease from Aspergillus saitoi, type XIII) mixed bed column significantly improved the resolution of bottom-up hydrogen/deuterium exchange mass spectrometry (HDX-MS) data compared with a pepsin-only column. The HDX-MS method using the mixed bed column determined 65 amide hydrogen exchange rates out of one hundred cytochrome c backbone amide hydrogens. Different cleavage specificities of the two enzymes generated 138 unique high-quality peptic fragments, which allows fine sub-localization of deuterium. The exchange rates determined in this method are consistent within the current study as well as with the previous HDX-NMR study. High-resolution HDX-MS data can determine the exchange rate of each residue not the deuterium buildup curve of a peptic fragment. The exchange rates provide more precise and quantitative measurements of protein dynamics in a more reproducible manner.ᅟ [ABSTRACT FROM AUTHOR]

The conformations of glycans are crucial for their biological functions. In-electrospray ionization (ESI) hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a promising technique for studying carbohydrate conformations since rapidly exchanging functional groups, e.g., hydroxyls, can be labeled on the timeframe of ESI. However, regular application of in-ESI HDX to characterize carbohydrates requires further analysis of the in-ESI HDX methodology. For instance, in this method, HDX occurs concurrently to the analyte transitioning from solution to gas-phase ions. Therefore, there is a possibility of sampling both gas-phase and solution-phase conformations of the analyte. Herein, we differentiate in-ESI HDX of metal-adducted carbohydrates from gas-phase HDX and illustrate that this method analyzes solvated species. We also systematically examine the effects of ESI parameters, including spray solvent composition, auxiliary gas flow rate, sheath gas flow rate, sample infusion rate, sample concentration, and spray voltage, and discuss their effects on in-ESI HDX. Further, we model the structural changes of a trisaccharide, melezitose, and its intramolecular and intermolecular hydrogen bonding in solvents with different compositions of methanol and water. These molecular dynamic simulations support our experimental results and illustrate how an individual ESI parameter can alter the conformations we sample by in-ESI HDX. In total, this work illustrates how the fundamental processes of ESI alter the magnitude of HDX for carbohydrates and suggest parameters that should be considered and/or optimized prior to performing experiments with this in-ESI HDX technique.ᅟ [ABSTRACT FROM AUTHOR]

Gas-phase hydrogen/deuterium exchange measured by mass spectrometry (gas-phase HDX-MS) is a fast method to probe the conformation of protein ions. The use of gas-phase HDX-MS to investigate the structure and interactions of protein complexes is however mostly unharnessed. Ionizing proteins under conditions that maximize preservation of their native structure (native MS) enables the study of solution-like conformation for milliseconds after electrospray ionization (ESI), which enables the use of ND3-gas inside the mass spectrometer to rapidly deuterate heteroatom-bound non-amide hydrogens. Here, we explored the utility of gas-phase HDX-MS to examine protein-protein complexes and inform on their binding surface and the structural consequences of gas-phase dissociation. Protein complexes ranging from 24 kDa dimers to 395 kDa 24mers were analyzed by gas-phase HDX-MS with subsequent collision-induced dissociation (CID). The number of exchangeable sites involved in complex formation could, therefore, be estimated. For instance, dimers of cytochrome c or α-lactalbumin incorporated less deuterium/subunit than their unbound monomer counterparts, providing a measure of the number of heteroatom-bound side-chain hydrogens involved in complex formation. We furthermore studied if asymmetric charge-partitioning upon dissociation of protein complexes caused intermolecular H/D migration. In larger multimeric protein complexes, the dissociated monomer showed a significant increase in deuterium. This indicates that intermolecular H/D migration occurs as part of the asymmetric partitioning of charge during CID. We discuss several models that may explain this increase deuterium content and find that a model where only deuterium involved in migrating charge can account for most of the deuterium enrichment observed on the ejected monomer. In summary, the deuterium content of the ejected subunit can be used to estimate that of the intact complex with deviations observed for large complexes accounted for by charge migration.ᅟ [ABSTRACT FROM AUTHOR]

Characterizing glycans is analytically challenging since glycans are heterogeneous, branched polymers with different three-dimensional conformations. Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been used to analyze native conformations and dynamics of biomolecules by measuring the mass increase of analytes as labile protons are replaced with deuterium following exposure to deuterated solvents. The rate of exchange is dependent on the chemical functional group, the presence of hydrogen bonds, pH, temperature, charge, and solvent accessibility. HDX-MS of carbohydrates is challenging due to the rapid exchange rate of hydroxyls. Here, we describe an observed HDX reaction associated with residual solvent vapors saturating electrospray sources. When undeuterated melezitose was infused after infusing D2O, samples with up to 73% deuterium exchange were detected. This residual solvent HDX was observed for both carbohydrates and peptides in multiple instruments and dependent on sample infusion rate, infusion time, and deuterium content of the solvent. This residual solvent HDX was observed over several minutes of sample analysis and persisted long enough to alter the measured deuterium labeling and possibly change the interpretation of the results. This work illustrates that residual solvent HDX competes with in-solution HDX for rapidly exchanging functional groups. Thus, we propose conditions to minimize this effect, specifically for top-down, in-electrospray ionization, and quench-flow HDX experiments.ᅟ [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Mass spectrometry (MS) is an integral component of the structural biology toolkit. • Native MS and footprinting MS methods can be used to study membrane proteins. • Native MS can inform on stoichiometry and ligand binding. • Ion mobility provides structural information. • Chemical crosslinking can be used to inform on the structure of proteins/complexes. Abstract The last ∼25 years has seen mass spectrometry (MS) emerge as an integral method in the structural biology toolkit. In particular, MS has enabled the structural characterization of proteins and protein assemblies that have been intractable by other methods, especially those that are large, heterogeneous or transient, providing experimental evidence for their structural organization in support of, and in advance of, high resolution methods. The most recent frontier conquered in the field of MS-based structural biology has been the application of established methods for studying water soluble proteins to the more challenging targets of integral membrane proteins. The power of MS in obtaining structural information has been enabled by advances in instrumentation and the development of hyphenated mass spectrometry-based methods, such as ion mobility spectrometry-MS, chemical crosslinking-MS and other chemical labelling/footprinting-MS methods. In this review we detail the insights garnered into the structural biology of membrane proteins by applying such techniques. Application and refinement of these methods has yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligand binding, which can be challenging to study using other methods. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Naphthoquine (NQ) is one of important partner drugs of artemisinin‐based combination therapy (ACT), which is recommended for the treatment of uncomplicated Plasmodium falciparum. NQ shows a high cure rate after a single oral administration. It is absorbed quickly (time to peak concentration 2–4 h) and has a long elimination half‐life (255 h). However, the metabolism of NQ has not been clarified. In this work, the metabolite profiling of NQ was studied in six liver microsomal incubates (human, cynomolgus monkey, beagle dog, mini pig, rat and CD1 mouse), seven recombinant CYP enzymes (1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and rat (plasma, urine, bile and feces) using liquid chromatography tandem high‐resolution LTQ‐Orbitrap mass spectrometry (HRMSn) in conjunction with online hydrogen/deuterium exchange. The biological samples were pretreated by protein precipitation and solid‐phase extraction. For data processing, multiple data‐mining tools were applied in tandem, i.e. background subtraction and followed by mass defect filter. NQ metabolites were characterized by accurate MS/MS fragmentation characteristics, the hydrogen/deuterium exchange data and cLogP simulation. As a result, five phase I metabolites (M1–M5) of NQ were characterized for the first time. Two metabolic pathways were involved: hydroxylation and N‐oxidation. This study demonstrates that LC‐HRMSn in combination with multiple data‐mining tools in tandem can be a valuable analytical strategy for rapid metabolite profiling of drugs. [ABSTRACT FROM AUTHOR]

The technological goal of hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is to determine backbone amide hydrogen exchange rates. The most critical challenge to achieve this goal is obtaining the deuterium incorporation in single-amide resolution, and gas-phase fragmentation may provide a universal solution. The gas-phase fragmentation may generate the daughter ions which differ by a single amino acid and the difference in deuterium incorporations in the two analogous ions can yield the deuterium incorporation at the sub-localized site. Following the pioneering works by Jørgensen and Rand, several papers utilized the electron transfer dissociation (ETD) to determine the location of deuterium in single-amide resolution. This paper demonstrates further advancement of the strategy by determining backbone amide hydrogen exchange rates, instead of just determining deuterium incorporation at a single time point, in combination with a wide time window monitoring. A method to evaluate the effects of scrambling and to determine the exchange rates from partially scrambled HDX-ETD-MS data is described. All parent ions for ETD fragmentation were regio-selectively scrambled: The deuterium in some regions of a peptide ion was scrambled while that in the other regions was not scrambled. The method determined 31 backbone amide hydrogen exchange rates of cytochrome c in the non-scrambled regions. Good fragmentation of a parent ion, a low degree of scrambling, and a low number of exchangeable hydrogens in the preceding side chain are the important factors to determine the exchange rate. The exchange rates determined by the HDX-MS are in good agreement with those determined by NMR.ᅟ [ABSTRACT FROM AUTHOR]

Cyclic B-type proanthocyanidins in red wines and grapes have been discovered recently. However, proanthocyanidins of a different chemical structure (non-cyclic A-type proanthocyanidins) already known to be present in cranberries and wine possess an identical theoretical mass. As a matter of fact, the retention times and the MS/MS fragmentations found for the proposed novel cyclic B-type tetrameric proanthocyanidin in red wine and the known tetrameric proanthocyanidin in a cranberry extract are herein shown to be identical. Thus, hydrogen/deuterium (H/D) exchange was applied to HPLC-HRMS/MS to confirm the actual chemical structure of the new oligomeric proanthocyanidins. The comparison of the results in water and deuterium oxide and between wine and cranberry extract indicates that the cyclic B-type tetrameric proanthocyanidin is the actual constituent of the recently proposed novel tetrameric species ([C60H49O24]+, m/z 1153.2608). Surprisingly, the same compound was also identified as the main tetrameric proanthocyanidin in cranberries. Finally, a totally new cyclic B-type hexameric proanthocyanidin ([C90H73O36]+, m/z 1729.3876) belonging to this novel class was identified for the first time in red wine.ᅟ [ABSTRACT FROM AUTHOR]

The usefulness of higher-order structural information provided by hydrogen/deuterium exchange-mass spectrometry (H/DX-MS) for the structural impact analyses of chemical and post-translational antibody modifications has been demonstrated in various studies. However, the structure−function assessment for protein drugs in biopharmaceutical research and development is often impeded by the relatively low-abundance (below 5%) of critical quality attributes or by overlapping effects of modifications, such as glycosylation, with chemical amino acid modifications; e.g., oxidation or deamidation. We present results demonstrating the applicability of the H/DX-MS technique to monitor conformational changes of specific Fc glycosylation variants produced by in vitro glyco-engineering technology. A trend towards less H/DX in Fc Cγ2 domain segments correlating with larger glycan structures could be confirmed. Furthermore, significant deuterium uptake differences and corresponding binding properties to Fc receptors (as monitored by SPR) between α-2,3- and α-2,6-sialylated Fc glycosylation variants were verified at sensitive levels.

In this study, continuous in-source hydrogen/deuterium exchange (HDX) atmospheric pressure photoionization (APPI) mass spectrometry (MS) with continuous feeding of DO was developed and validated. DO was continuously fed using a capillary line placed on the center of a metal plate positioned between the UV lamp and nebulizer. The proposed system overcomes the limitations of previously reported APPI HDX-MS approaches where deuterated solvents were premixed with sample solutions before ionization. This is particularly important for APPI because solvent composition can greatly influence ionization efficiency as well as the solubility of analytes. The experimental parameters for APPI HDX-MS with continuous feeding of DO were optimized, and the optimized conditions were applied for the analysis of nitrogen-, oxygen-, and sulfur-containing compounds. The developed method was also applied for the analysis of the polar fraction of a petroleum sample. Thus, the data presented in this study clearly show that the proposed HDX approach can serve as an effective analytical tool for the structural analysis of complex mixtures. [ABSTRACT FROM AUTHOR]

Fibrillization of the microtubule-associated protein tau has been recognized as one of the signature pathologies of the nervous system in Alzheimer's disease, progressive supranuclear palsy, and other tauopathies. The conformational transition of tau in the fibrillization process, tau monomer to soluble aggregates to fibrils in particular, remains unclear. Here we report on the use of hydrogen/deuterium exchange mass spectrometry (HDX-MS) in combination with other biochemical approaches, including Thioflavin S fluorescence measurements, enzyme-linked immunosorbent assay (ELISA), and Western blotting to understand the heparin-induced tau's fibrillization. HDX-MS studies including anti-tau antibody epitope mapping experiments provided molecular level details of the full-length tau's conformational dynamics and its regional solvent accessibility upon soluble aggregates formation. The results demonstrate that R3 region in the full-length tau's microtubule binding repeat region (MTBR) is stabilized in the aggregation process, leaving both N and C terminal regions to be solvent exposed in the soluble aggregates and fibrils. The findings also illustrate the practical utility of orthogonal analytical methodologies for the characterization of protein higher order structure. [ABSTRACT FROM AUTHOR]

Hydrogen/Deuterium exchange (HDX) has been applied, since the 1930s, as an analytical tool to study the structure and dynamics of (small) biomolecules. The popularity of using HDX to study proteins increased drastically in the last two decades due to the successful combination with mass spectrometry (MS). Together with this growth in popularity, several technological advances have been made, such as improved quenching and fragmentation. As a consequence of these experimental improvements and the increased use of protein-HDXMS, large amounts of complex data are generated, which require appropriate analysis. Computational analysis of HDXMS requires several steps. A typical workflow for proteins consists of identification of (non-)deuterated peptides or fragments of the protein under study (local analysis), or identification of the deuterated protein as a whole (global analysis); determination of the deuteration level; estimation of the protection extent or exchange rates of the labile backbone amide hydrogen atoms; and a statistically sound interpretation of the estimated protection extent or exchange rates. Several algorithms, specifically designed for HDX analysis, have been proposed. They range from procedures that focus on one specific step in the analysis of HDX data to complete HDX workflow analysis tools. In this review, we provide an overview of the computational methods and discuss outstanding challenges. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 36:649-667, 2017 [ABSTRACT FROM AUTHOR]

Herein we report the observation of atmospheric pressure in-source hydrogen-deuterium exchange (HDX) of thiol group for the first time. The HDX for thiol group was optimized for positive atmospheric pressure photoionization (APPI) mass spectrometry (MS). The optimized HDX-MS was applied for 31 model compounds (thiols, thiophenes, and sulfides) to demonstrate that exchanged peaks were observed only for thiols. The optimized method has been successfully applied to the isolated fractions of sulfur-rich oil samples. The exchange of one and two thiol hydrogens with deuterium was observed in the thiol fraction; no HDX was observed in the other fractions. Thus, the results presented in this study demonstrate that the HDX-MS method using APPI ionization source can be effective for speciation of sulfur compounds. This method has the potential to be used to access corrosion problems caused by thiol-containing compounds. [ABSTRACT FROM AUTHOR]

Sickle hemoglobin (HbS) polymerization initiates in the deoxy state with the binding of hydrophobic patch formed by the isopropyl group of βVal6 residue of a hemoglobin tetramer with the hydrophobic pocket of another tetramer, whose hydrophobic patch binds to the hydrophobic groove of a third molecule. Subsequent elongation of a single stranded polymer followed by the formation of a double strand and finally combination of seven such pairs of double strands results in a fourteen stranded fibrous polymer. Precipitation of this fiber inside the erythrocytes results in sickling of red blood cells. Surprisingly, the polymerization does not occur in the oxy state of HbS. Due to the unavailability of crystal structure of oxy form of HbS, the molecular basis of inhibition of polymerization in the oxy state is unknown to date. In the present study, we have attempted to understand the molecular mechanism of inhibition of polymerization by exploiting the exchange of backbone amide hydrogens of HbS with deuterated solvent. Hydrogen/deuterium exchange kinetics of peptide amide hydrogens of both oxy and deoxy form of HbS were monitored through ESI mass spectrometry. Upon oxygenation changes in the conformational flexibility across different regions of α and β globin chains in the tetrameric HbS molecule were investigated. It was observed that oxygenation led to perturbation in the conformation of several residues around the hydrophobic patch, groove of a tetramer and axial, lateral contacts across the double strands that are involved in HbS polymerization. [ABSTRACT FROM AUTHOR]

The computational analysis of the isomer- and conformer-dependent photochemistry of 1- and 2-naphthols and their microsolvated water clusters is motivated by their very different excited state reactivities. We present evidence that 1- and 2-naphthol follow distinct excited state deactivation pathways. The deactivation of 2-naphthols, 2-naphthol water clusters, as well as of the anti conformer of 1-naphthol is mediated by the optically dark 1πσ* state. The dynamics of the 1πσ* surface leads to the homolytic cleavage of the OH bond. On the contrary, the excited state deactivation of syn 1-naphthol and 1-naphthol water clusters follows an uncommon reaction pathway. Upon excitation to the bright 1ππ*(La) state, a highly specific excited state hydrogen transfer (ESHT) to carbon atoms C8 and C5 takes place, yielding 1,8- and 1,5-naphthoquinone methides. The ESHT pathway arises from the intrinsic electronic properties of the 1ππ*(La) state of 1-naphthols. [ABSTRACT FROM AUTHOR]

Cytochrome c (cyt c ) is a mitochondrial protein responsible for transferring electrons between electron transport chain complexes III and IV. The release of cyt c from the mitochondria has been considered as a commitment step in intrinsic apoptosis. Transfer RNA (tRNA) has recently been found to interact with the released cyt c to prevent the formation of the apoptosome complex, thus preventing cell apoptosis. To understand the molecular basis of tRNA–cyt c interactions, we applied hydrogen/deuterium exchange mass spectrometry (HDXMS) to analyze the interactions between tRNA and cyt c . tRNA Phe binding to cyt c reduced the deuteration level of cyt c in all analyzed regions, indicating that tRNA binding blocks the solvent-accessible regions and results in the formation of a more compact conformation. Substitution of the tRNA Phe with the total tRNA from brewer's yeast in the HDXMS experiment significantly reduced deuteration in the N-terminus and the region 18–32 residue of cyt c , where all tRNAs are bound. To clarify the cause of binding, we used synthesized single-stranded oligonucleotides of 12-mer dA and dT to form complexes with cyt c . The exchange of the nucleotide bases between adenine and thymine did not affect the deuteration level of cyt c . However, the regions 1–10 and 65–82 showed minor decreases after unstructured dA or dT DNA binding. Collectively, these results reveal that cyt c maintains its globular structure to interact with tRNA. The region 18–32 selectively interacts with tRNA, and N-terminal 1–10 interacts with oligonucleotides electrostatically. [ABSTRACT FROM AUTHOR]