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Journal of Mass Spectrometry : JMS Feb 2022Stable isotope tracing can be safely used for metabolic studies in animals and humans. The endogenous biosynthesis of lipids (lipogenesis) is a key process throughout...
Stable isotope tracing can be safely used for metabolic studies in animals and humans. The endogenous biosynthesis of lipids (lipogenesis) is a key process throughout the entire life but especially during brain and lung growth. Adequate synthesis of pulmonary surfactant lipids is indispensable for life. With this study, we report the use of deuterium-depleted water (DDW), suitable for human consumption, as metabolic precursor for lipogenesis. We studied 13 adult rabbits for 5 days. Four rabbits drank tap water (TW) and served as controls; in four animals, DDW was substituted to drinking water, whereas five drank deuterium-enriched water (DEW). After 5 days, a blood sample and a bronchoalveolar lavage (BAL) sample were collected. The H/ H (δ H) of BAL palmitic acid (PA) desaturated phosphatidylcholine (DSPC), the major phospholipid of pulmonary surfactant, and of plasma water was determined by high-resolution mass spectrometry. We found that the δ H values of DDW, DEW and TW were -984 ± 2‰, +757 ± 2‰ and -58 ± 1‰, respectively. After 5 days, plasma water values were -467 ± 87‰, +377 ± 56‰ and -53 ± 6‰, and BAL DSPC-PA was -401 ± 27‰, -96 ± 38‰ and -249 ± 9‰ in the DDW, DEW and TW, respectively. With this preliminary study, we demonstrated the feasibility of using DDW to label pulmonary surfactant lipids. This novel approach can be used in animals and in humans, and we speculate that it could be associated with more favourable study compliance than DEW in human studies.
Topics: Animals; Deuterium; Drinking Water; Phosphatidylcholines; Phospholipids; Pulmonary Surfactants; Rabbits
PubMed: 35060656
DOI: 10.1002/jms.4808 -
Molecular & Cellular Proteomics : MCP Jan 2004Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled... (Review)
Review
Mass spectrometric approaches using electrospray ionization charge states and hydrogen-deuterium exchange for determining protein structures and their conformational changes.
Electrospray ionization (ESI) mass spectrometry (MS) is a powerful analytical tool for elucidating structural details of proteins in solution especially when coupled with amide hydrogen/deuterium (H/D) exchange analysis. ESI charge-state distributions and the envelopes of charges they form from proteins can provide an abundance of information on solution conformations that is not readily available through other biophysical techniques such as near ultraviolet circular dichroism (CD) and tryptophan fluorescence. The most compelling reason for the use of ESI-MS over nuclear magnetic resonance (NMR) for measuring H/D after exchange is that larger proteins and lesser amounts of samples can be studied. In addition, MS can provide structural details on transient or folding intermediates that may not be accessible by CD, fluorescence, and NMR because these techniques measure the average properties of large populations of proteins in solution. Correlations between measured H/D and calculated parameters that are often available from crystallographic data can be used to extend the range of structural details obtained on proteins. Molecular dynamics and energy minimization by simulation techniques such as assisted model building with energy refinement (AMBER) force field can be very useful in providing structural models of proteins that rationalize the experimental H/D exchange results. Charge-state envelopes and H/D exchange information from ESI-MS data used complementarily with NMR and CD data provides the most powerful approach available to understanding the structures and dynamics of proteins in solution.
Topics: Deuterium; Humans; Hydrogen; Models, Molecular; Molecular Structure; Protein Conformation; Protein Denaturation; Proteins; Spectrometry, Mass, Electrospray Ionization; Statistics as Topic
PubMed: 14623985
DOI: 10.1074/mcp.R300010-MCP200 -
BMC Bioinformatics Dec 2014Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is...
BACKGROUND
Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry permits analysis of structure, dynamics, and molecular interactions of proteins. HDX mass spectrometry is confounded by deuterium exchange-associated peaks overlapping with peaks of heavy, natural abundance isotopes, such as carbon-13. Recent studies demonstrated that high-performance mass spectrometers could resolve isotopic fine structure and eliminate this peak overlap, allowing direct detection and quantification of deuterium incorporation.
RESULTS
Here, we present a graphical tool that allows for a rapid and automated estimation of deuterium incorporation from a spectrum with isotopic fine structure. Given a peptide sequence (or elemental formula) and charge state, the mass-to-charge ratios of deuterium-associated peaks of the specified ion is determined. Intensities of peaks in an experimental mass spectrum within bins corresponding to these values are used to determine the distribution of deuterium incorporated. A theoretical spectrum can then be calculated based on the estimated distribution of deuterium exchange to confirm interpretation of the spectrum. Deuterium incorporation can also be detected for ion signals without a priori specification of an elemental formula, permitting detection of exchange in complex samples of unidentified material such as natural organic matter. A tool is also incorporated into QUDeX-MS to help in assigning ion signals from peptides arising from enzymatic digestion of proteins. MATLAB-deployable and standalone versions are available for academic use at qudex-ms.sourceforge.net and agarlabs.com .
CONCLUSION
Isotopic fine structure HDX-MS offers the potential to increase sequence coverage of proteins being analyzed through mass accuracy and deconvolution of overlapping ion signals. As previously demonstrated, however, the data analysis workflow for HDX-MS data with resolved isotopic fine structure is distinct. QUDeX-MS we hope will aid in the adoption of isotopic fine structure HDX-MS by providing an intuitive workflow and interface for data analysis.
Topics: Carbon Isotopes; Deuterium; Deuterium Exchange Measurement; Humans; Hydrogen; Isotope Labeling; Mass Spectrometry; Peptide Fragments; Proteins; Software
PubMed: 25495703
DOI: 10.1186/s12859-014-0403-1 -
Journal of the American Society For... Jul 2022For hydrogen-deuterium exchange mass spectrometry (HDX-MS) to have an increased role in quality control of biopharmaceuticals, H for D back-exchange occurring during...
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)/HO 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.
Topics: Chromatography, Liquid; Deuterium; Deuterium Exchange Measurement; Hydrogen Deuterium Exchange-Mass Spectrometry; Peptides; Proteins; Reproducibility of Results
PubMed: 35732031
DOI: 10.1021/jasms.2c00096 -
Molecules (Basel, Switzerland) Nov 2019This article presents the original descriptions of some recent physics mechanisms (based on the thermodynamic, kinetic, and quantum tunnel effects) providing stable H/H... (Review)
Review
This article presents the original descriptions of some recent physics mechanisms (based on the thermodynamic, kinetic, and quantum tunnel effects) providing stable H/H isotope fractionation, leading to the accumulation of particular isotopic forms in intra- or intercellular space, including the molecular effects of deuterium interaction with O/O/O, N/N, C/C, and other stable biogenic isotopes. These effects were observed mainly at the organelle (mitochondria) and cell levels. A new hypothesis for heavy nonradioactive isotope fractionation in living systems via neutron effect realization is discussed. The comparative analysis of some experimental studies results revealed the following observation: "Isotopic shock" is highly probable and is observed mostly when chemical bonds form between atoms with a summary odd number of neutrons (i.e., bonds with a non-compensated neutron, which correspond to the following equation: Nn - Np = 2k + 1, where k ϵ Z, k is the integer, Z is the set of non-negative integers, Nn is number of neutrons, and Np is number of protons of each individual atom, or in pair of isotopes with a chemical bond). Data on the efficacy and metabolic pathways of the therapy also considered H-modified drinking and diet for some diseases, such as Alzheimer's disease, Friedreich's ataxia, mitochondrial disorders, diabetes, cerebral hypoxia, Parkinson's disease, and brain cancer.
Topics: Animals; Chemical Fractionation; Deuterium; Humans; Isotopes; Models, Chemical; Neutrons; Organelles; Proton Therapy; Protons
PubMed: 31766268
DOI: 10.3390/molecules24224101 -
Methods (San Diego, Calif.) Sep 2018The focus of this review is the basic methodology for applications of static deuteron NMR for studies of dynamics in the side chains of proteins. We review experimental... (Review)
Review
The focus of this review is the basic methodology for applications of static deuteron NMR for studies of dynamics in the side chains of proteins. We review experimental approaches for the measurements of static line shapes and relaxation rates as well as signal enhancement strategies using the multiple echo acquisition scheme. Further, we describe computational strategies for modeling jump and diffusive motions underlying experimental data. Applications are chosen from studies of amyloid fibrils comprising the amyloid-β protein.
Topics: Amyloid beta-Peptides; Deuterium; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular
PubMed: 29705208
DOI: 10.1016/j.ymeth.2018.04.030 -
Organic Letters Jan 2019A conceptually novel sulfoxonium metathesis reaction between TMSOI and cost-effective DMSO- d is developed for the efficient generation of a new trideuteromethylation...
A conceptually novel sulfoxonium metathesis reaction between TMSOI and cost-effective DMSO- d is developed for the efficient generation of a new trideuteromethylation reagent TDMSOI. The new reagent TDMSOI is produced highly efficiently by simply heating a mixture of TMSOI and DMSO- d and directly used for subsequent trideuteromethylation in a "one-pot" operation. The preparative power of the new versatile reagent and the "one-pot" protocol is demonstrated by its use to install the -CD moiety into broad functionalities including phenols, thiophenols, acidic amines, and enolizable methylene units in high yield and at a useful level of deuteration (>87% D).
Topics: Deuterium; Indicators and Reagents; Phenols; Sulfhydryl Compounds
PubMed: 30615462
DOI: 10.1021/acs.orglett.8b03641 -
Journal of Chromatography. A Nov 2017Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen...
Hydrogen deuterium exchange mass spectrometry (HDX MS) reports on the conformational landscape of proteins by monitoring the exchange between backbone amide hydrogen atoms and deuterium in the solvent. To maintain the label for analysis, quench conditions of low temperature and pH are required during the chromatography step performed after protease digestion but before mass spectrometry. Separation at 0°C is often chosen as this is the temperature where the most deuterium can be recovered without freezing of the typical water and acetonitrile mobile phases. Several recent reports of separations at subzero Celsius emphasize the promise for retaining more deuterium and using a much longer chromatographic gradient or direct infusion time. Here we present the construction and validation of a modified Waters nanoACQUITY HDX manager with a third temperature-controlled zone for peptide separations at subzero temperatures. A new Peltier-cooled door replaces the door of a traditional main cooling chamber and the separations and trapping column are routed through the door housing. To prevent freezing, 35% methanol is introduced post online digestion. No new pumps are required and online digestion is performed as in the past. Subzero separations, using conventional HPLC column geometry of 3μ m particles in a 1×50mm column, did not result in major changes to chromatographic efficiency when lowering the temperature from 0 to -20°C. There were significant increases in deuterium recovery for both model peptides and biologically relevant protein systems. Given the higher levels of deuterium recovery, expanded gradient programs can be used to allow for higher chromatographic peak capacity and therefore the analysis of larger and more complex proteins and systems.
Topics: Amides; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid; Cold Temperature; Deuterium; Deuterium Exchange Measurement; Hydrogen; Mass Spectrometry; Peptides; Proteins; Solvents
PubMed: 28596009
DOI: 10.1016/j.chroma.2017.05.067 -
European Journal of Pharmaceutical... Aug 2023The discovery of cephalosporin and demonstration of its improved stability in aqueous solution, as well as enhanced in vitro activity against penicillin-resistant...
The discovery of cephalosporin and demonstration of its improved stability in aqueous solution, as well as enhanced in vitro activity against penicillin-resistant organisms, were major breakthroughs in the development of β-lactam antibiotics. Although cephalosporins are more stable with respect to hydrolytic degradation than penicillins, they still experience a variety of chemical transformations. The present study offers an insight into the rates and mechanisms of ceftriaxone degradation at the therapeutic concentration in water, a mixture of water and deuterium oxide, and deuterium oxide itself at the neutral pH. Specific ceftriaxone degradation products were observed in aged samples (including a previously unreported dimer-type species), and by comparing the degradation rates in HO and DO, the observation of a kinetic isotope effect provided some valuable insight as to the nature of the initial ceftriaxone degradation. The effect of protium to deuterium isotope change on the degradation kinetics of ceftriaxone was evaluated using the method of initial rates based on HPLC analysis as well as by quantitative H NMR spectroscopy. Moreover, computational analysis was utilized to get a molecular insight into chemical processes governing the ceftriaxone degradation and to rationalize the stabilizing effect of replacing HO with DO.
Topics: Deuterium Oxide; Deuterium; Ceftriaxone; Kinetics; Water; Cephalosporins
PubMed: 37160178
DOI: 10.1016/j.ejps.2023.106461 -
Scientific Reports Jan 2021Filamentous fungi grow exclusively at their tips, where many growth-related fungal processes, such as enzyme secretion and invasion into host cells, take place. Hyphal...
Filamentous fungi grow exclusively at their tips, where many growth-related fungal processes, such as enzyme secretion and invasion into host cells, take place. Hyphal tips are also a site of active metabolism. Understanding metabolic dynamics within the tip region is therefore important for biotechnology and medicine as well as for microbiology and ecology. However, methods that can track metabolic dynamics with sufficient spatial resolution and in a nondestructive manner are highly limited. Here we present time-lapse Raman imaging using a deuterium (D) tracer to study spatiotemporally varying metabolic activity within the hyphal tip of Aspergillus nidulans. By analyzing the carbon-deuterium (C-D) stretching Raman band with spectral deconvolution, we visualize glucose accumulation along the inner edge of the hyphal tip and synthesis of new proteins from the taken-up D-labeled glucose specifically at the central part of the apical region. Our results show that deuterium-labeled Raman imaging offers a broadly applicable platform for the study of metabolic dynamics in filamentous fungi and other relevant microorganisms in vivo.
Topics: Aspergillus nidulans; Deuterium; Fungal Proteins; Glucose; Hyphae; Spectrum Analysis, Raman
PubMed: 33446770
DOI: 10.1038/s41598-020-80270-9