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Molecular & Cellular Proteomics : MCP Sep 2011A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations....
A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.
Topics: Antibodies; Automation, Laboratory; Deuterium; Deuterium Exchange Measurement; Deuterium Oxide; Hydrogen; Kinetics; Mass Spectrometry; Peptides; Protein Binding; Protein Folding; Protein Structure, Secondary; Protein Unfolding; Proteins; Proteomics
PubMed: 21610101
DOI: 10.1074/mcp.M110.006510 -
Journal of Dairy Science May 1995To develop equations for predicting body composition, mature Holstein cows (n = 21) were slaughtered at three physiological stages (-7, 63, and 269 d postpartum) after...
To develop equations for predicting body composition, mature Holstein cows (n = 21) were slaughtered at three physiological stages (-7, 63, and 269 d postpartum) after consecutive intravenous dosing with urea and D2O. Blood was sampled at 0 and 12 min after dosing with urea for determination of urea space and from 0 to 72 h after dosing with D2O. Empty body water and total body water were estimated by dilution kinetics for D2O using two- and one-compartment models, respectively. At slaughter, body components were ground, sampled, and freeze-dried for chemical analysis. Prediction of empty body water by urea space was not an improvement over the prediction by body weight alone. Prediction by D2O dilution explained 73 and 87% of the variation in empty and total body water, respectively. Estimated body protein, as determined from empty body water, predicted actual body protein with an error of 4.7 kg. Daily DMI explained 84% of the variation in the DM of the gastrointestinal tract contents (DM fill). Estimations of empty body fat (R2 = .85) and empty body energy (R2 = .89) from D2O dilution were capable of detecting significant differences in body fat (42.9 kg) and body energy (375 Mcal) across physiological stages and might be useful for prediction of body composition changes during the lactation cycle.
Topics: Animals; Body Composition; Body Water; Cattle; Deuterium Oxide; Energy Metabolism; Female; Kinetics; Regression Analysis; Urea
PubMed: 7622720
DOI: 10.3168/jds.S0022-0302(95)76725-X -
PloS One 2019Wrist-mounted motion sensors can quantify the volume and intensity of physical activities, but little is known about their long-term validity. Our aim was to validate a... (Observational Study)
Observational Study
INTRODUCTION
Wrist-mounted motion sensors can quantify the volume and intensity of physical activities, but little is known about their long-term validity. Our aim was to validate a wrist motion sensor in estimating daily energy expenditure, including any change induced by long-term participation in endurance and strength training. Supplemental heart rate monitoring during weekly exercise was also investigated.
METHODS
A 13-day doubly labeled water (DLW) measurement of total energy expenditure (TEE) was performed twice in healthy male subjects: during two last weeks of a 12-week Control period (n = 15) and during two last weeks of a 12-week combined strength and aerobic Training period (n = 13). Resting energy expenditure was estimated using two equations: one with body weight and age, and another one with fat-free mass. TEE and activity induced energy expenditure (AEE) were determined from motion sensor alone, and from motions sensor combined with heart rate monitor, the latter being worn during exercise only.
RESULTS
When body weight and age were used in the calculation of resting energy expenditure, the motion sensor data alone explained 78% and 62% of the variation in TEE assessed by DLW at the end of Control and Training periods, respectively, with a bias of +1.75 (p <.001) and +1.19 MJ/day (p = .002). When exercise heart rate data was added to the model, the combined wearable device approach explained 85% and 70% of the variation in TEE assessed by DLW with a bias of +1.89 and +1.75 MJ/day (p <.001 for both). While significant increases in TEE and AEE were detected by all methods as a result of participation in regular training, motion sensor approach underestimated the change measured by DLW: +1.13±0.66 by DLW, +0.59±0.69 (p = .004) by motion sensor, and +0.98±0.70 MJ/day by combination of motion sensor and heart rate. Use of fat-free mass in the estimation of resting energy expenditure removed the biases between the wearable device estimations and the golden standard reference method of TEE and demonstrated a training-induced increase in resting energy expenditure by +0.18±0.13 MJ/day (p <.001).
CONCLUSIONS
Wrist motion sensor combined with a heart rate monitor during exercise sessions, showed high agreement with the golden standard measurement of daily TEE and its change induced by participation in a long-term training protocol. The positive findings concerning the validity, especially the ability to follow-up the change associated with a lifestyle modification, can be considered significant because they partially determine the feasibility of wearable devices as quantifiers of health-related behavior.
Topics: Accelerometry; Adult; Body Weight; Deuterium Oxide; Drinking; Energy Metabolism; Heart Rate; Humans; Life Style; Male; Monitoring, Physiologic; Physical Conditioning, Human; Physical Endurance; Water; Wearable Electronic Devices; Wrist; Young Adult
PubMed: 31291373
DOI: 10.1371/journal.pone.0219563 -
Comparative Biochemistry and... Dec 2016Metabolic costs are central to individual energy budgets, making estimates of metabolic rate vital to understanding how an organism interacts with its environment as... (Review)
Review
Metabolic costs are central to individual energy budgets, making estimates of metabolic rate vital to understanding how an organism interacts with its environment as well as the role of species in their ecosystem. Despite the ecological and commercial importance of fishes, there are currently no widely adopted means of measuring field metabolic rate in fishes. The lack of recognized methods is in part due to the logistical difficulties of measuring metabolic rates in free swimming fishes. However, further development and refinement of techniques applicable for field-based studies on free swimming animals would greatly enhance the capacity to study fish under environmentally relevant conditions. In an effort to foster discussion in this area, from field ecologists to biochemists alike, we review aspects of energy metabolism and give details on approaches that have been used to estimate energetic parameters in fishes. In some cases, the techniques have been applied to field conditions; while in others, the methods have been primarily used on laboratory held fishes but should be applicable, with validation, to fishes in their natural environment. Limitations, experimental considerations and caveats of these measurements and the study of metabolism in wild fishes in general are also discussed. Potential novel approaches to FMR estimates are also presented for consideration. The innovation of methods for measuring field metabolic rate in free-ranging wild fish would revolutionize the study of physiological ecology.
Topics: Animals; Carbon Disulfide; Deuterium Oxide; Ecosystem; Energy Metabolism; Fish Proteins; Fishes; Heart Rate; Otolithic Membrane; Oxygen Consumption; Oxygen Isotopes; Swimming; Telemetry
PubMed: 27139083
DOI: 10.1016/j.cbpa.2016.04.022 -
International Journal of Molecular... Mar 2022Mono- and polysaccharides are an essential part of every biological system. Identifying underivatized carbohydrates using mass spectrometry is still a challenge because...
Mono- and polysaccharides are an essential part of every biological system. Identifying underivatized carbohydrates using mass spectrometry is still a challenge because carbohydrates have a low capacity for ionization. Normally, the intensities of protonated carbohydrates are relatively low, and in order to increase the corresponding peak height, researchers add Na, K, or NHto the solution. However, the fragmentation spectra of the corresponding ions are very poor. Based on this, reliably identifying carbohydrates in complex natural and biological objects can benefit frommeasuring additional molecular descriptors, especially those directly connected to the molecular structure. Previously, we reported that the application of the isotope exchange approach (H/D and O/O) to high-resolution mass spectrometry can increase the reliability of identifying drug-like compounds. Carbohydrates possess many -OH and -COOH groups, making it reasonable to expect that the isotope exchange approach would have considerable potential for detecting carbohydrates. Here, we used a collection of standard carbohydrates to investigate the isotope exchange reaction (H/D and O/O) in carbohydrates and estimate its analytical applications.
Topics: Carbohydrates; Deuterium Oxide; Hexoses; Ions; Polysaccharides; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization
PubMed: 35408942
DOI: 10.3390/ijms23073585 -
PeerJ 2023Premature ovarian failure (POF) is defined as the cessation of ovarian function before the age of 40 years, imposing a significant health burden on patients. However,...
BACKGROUND
Premature ovarian failure (POF) is defined as the cessation of ovarian function before the age of 40 years, imposing a significant health burden on patients. However, effective etiological therapy for POF is scarce. Thus, we aimed to explore the protective role and targets of hydrogen-rich water (HRW) in POF.
METHODS
Based on cyclophosphamide (CTX)-induced POF rat models, the protective role of HRW treatment was mainly determined through serum 17--estradiol (E2), follicle-stimulating hormone (FSH), anti-mullerian hormone (AMH) levels, ovarian histomorphological analysis, and TUNEL assay. Tandem mass tag (TMT)-based quantitative proteomic analysis was then conducted on ovarian tissues, and the targets of HRW in POF were identified integrating differential expression analysis, functional enrichment analysis, and interaction analysis.
RESULTS
In HRW treatment of POF rats, the serum AMH and E2 levels significantly increased, and FSH level significantly reduced, indicating the protective role of HRW. After TMT quantitative proteomic analysis, a total of 16 candidate differentially expressed proteins (DEPs) were identified after the cross analysis of DEPs from POF vs. control and POF+HRW vs. POF groups, which were found to be significantly enriched in 296 GO terms and 36 KEGG pathways. The crucial targets, RT1-Db1 and RT1-Bb, were finally identified based on both protein-protein interaction network and GeneMANIA network.
CONCLUSIONS
The HRW treatment could significantly alleviate the ovarian injury of POF rats; RT1-Db1 and RT1-Bb are identified as two crucial targets of HRW treatment in POF rats.
Topics: Animals; Female; Humans; Rats; Anti-Mullerian Hormone; Follicle Stimulating Hormone; Hydrogen; Menopause, Premature; Primary Ovarian Insufficiency; Proteomics; Deuterium Oxide
PubMed: 37397014
DOI: 10.7717/peerj.15564 -
Scientific Data Sep 2023Metabolic stable isotope labeling with heavy water followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein...
Metabolic stable isotope labeling with heavy water followed by liquid chromatography coupled with mass spectrometry (LC-MS) is a powerful tool for in vivo protein turnover studies. Several algorithms and tools have been developed to determine the turnover rates of peptides and proteins from time-course stable isotope labeling experiments. The availability of benchmark mass spectrometry data is crucial to compare and validate the effectiveness of newly developed techniques and algorithms. In this work, we report a heavy water-labeled LC-MS dataset from the murine liver for protein turnover rate analysis. The dataset contains eighteen mass spectral data with their corresponding database search results from nine different labeling durations and quantification outputs from d2ome+ software. The dataset also contains eight mass spectral data from two-dimensional fractionation experiments on unlabeled samples.
Topics: Animals; Mice; Chromatography, Liquid; Deuterium Oxide; Liver; Proteome; Tandem Mass Spectrometry
PubMed: 37726365
DOI: 10.1038/s41597-023-02537-w -
Pflugers Archiv : European Journal of... Jun 2024Increase in transendothelial water permeability is an essential etiological factor in a variety of diseases like edema and shock. Despite the high clinical relevance,...
Increase in transendothelial water permeability is an essential etiological factor in a variety of diseases like edema and shock. Despite the high clinical relevance, there has been no precise method to detect transendothelial water flow until now. The deuterium oxide (DO) dilution method, already established for measuring transepithelial water transport, was used to precisely determine the transendothelial water permeability. It detected appropriate transendothelial water flow induced by different hydrostatic forces. This was shown in four different endothelial cell types. The general experimental setup was verified by gravimetry and absorbance spectroscopy. Determination of transendothelial electrical resistance (TEER) and immunocytochemical staining for proteins of the cell-cell contacts were performed to ensure that no damage to the endothelium occurred because of the measurements. Furthermore, endothelial barrier function was modulated. Measurement of transendothelial water flux was verified by measuring the TEER, the apparent permeability coefficient and the electrical capacity. The barrier-promoting substances cyclic adenosine monophosphate and iloprost reduced TEER and electrical capacity and increased permeability. This was accompanied by a reduced transendothelial water flux. In contrast, the barrier-damaging substances thrombin, histamine and bradykinin reduced TEER and electrical capacity, but increased permeability. Here, an increased water flow was shown. This newly established in vitro method for direct measurement of transendothelial water permeability was verified as a highly precise technique in various assays. The use of patient-specific endothelial cells enables individualized precision medicine in the context of basic edema research, for example regarding the development of barrier-protective pharmaceuticals.
Topics: Deuterium Oxide; Humans; Electric Impedance; Water; Endothelial Cells; Permeability; Animals; Endothelium, Vascular; Capillary Permeability; Human Umbilical Vein Endothelial Cells
PubMed: 38438679
DOI: 10.1007/s00424-024-02934-z -
PloS One 2013Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date....
Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.
Topics: Aromatase; Deuterium Exchange Measurement; Deuterium Oxide; Enzyme Stability; Humans; Ligands; Mutant Proteins; Protein Structure, Secondary; Recombinant Proteins; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared
PubMed: 24349198
DOI: 10.1371/journal.pone.0082118 -
Experimental Physiology Jan 2013The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a...
The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water ((2)H(2)O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with (2)H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific (2)H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal (2)H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment ((2)H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.
Topics: Animals; Deuterium Oxide; Gluconeogenesis; Hindlimb Suspension; Liver; Male; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Sprague-Dawley
PubMed: 22707505
DOI: 10.1113/expphysiol.2012.067074