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Rationale: Heavy-labelled internal standards increasingly represent the gold standard for absolute quantitation in mass spectrometry (MS)-based bottom-up proteomics. The biggest drawbacks of using these standards are that they have high costs and lengthy lead times., Methods: We describe an efficient, low-cost optimised method to enable 'in-house' heavy labelling of synthetic tryptic peptides for absolute quantification using tandem LC-MS/MS mass spectrometry. Our methodology uses 18O water in a trypsin-catalysed oxygen exchange reaction at the carboxyl terminus with the overall aim of reducing the costs and lead time associated with sourcing heavy standards from commercial vendors., Results: Step-by-step instructions are provided on how to execute this protocol with high-throughput adaptations utilising a 96-well plate and a liquid-handling robot. Detailed notes on experimental setup, tips for troubleshooting and suggested improvements to maximise labelling efficiencies are highlighted to achieve the best results. Under optimum conditions, labelling efficiencies of peptides can reach from 95% to 100%., Conclusions: The application of the 'in-house' labelled standards in generating calibration curves to quantify endogenous peptide concentrations is just as effective as using the synthetically sourced standards while also having great cost reduction implications as well as saving time spent waiting for peptides to arrive. The protocol is highly adaptable and can be customized to fit the specific setup of any laboratory, maximizing achievable labelling efficiencies., (© 2024 The Author(s). Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)

Isobaric chemical labeling is a widely used strategy for high-throughput quantitative proteomics based on mass spectrometry. However, commercially available reagents have high costs in applications as well as the sensitivity limitations for detection of the trace protein samples. Previously, we developed a 2-plex isobaric labeling strategy based on phosphorus chemistry for ultrasensitive proteome quantification with high accuracy. In this work, 6-plex tandem phosphorus tags (TPT) were developed with 3-fold increase in the multiplexing quantitative capacity compared to the 2-plex isobaric phosphorus reagents introduced previously. High isotope enrichment of 18 O labeling was incorporated into the phosphoryl group with three exchangeable oxygen atoms by using commercially available H 2 18 O. The combinational incorporations of 18 O atom in reporter ions and balance group set up the low-cost foundation for development of multiplex TPT reagents. The novel 6-plex TPT reagents could produce phosphoramidate as unique reporter ions with approximately 1 Da mass difference and thus enable 6-plex quantitative analysis in high-resolution ESI-MS/MS analysis. Using HeLa cell tryptic peptides, we concluded that 6-plex TPT reagents could facilitate large-scale accurate quantitative proteomics with very high labeling efficiency.

ProTides are nucleotide analogues used for the treatment of specific viral infections. These compounds consist of a masked nucleotide that undergoes in vivo enzymatic and spontaneous chemical transformations to generate a free mononucleotide that is ultimately transformed to the pharmaceutically active triphosphorylated drug. The three FDA approved ProTides are composed of a phosphoramidate (P-N) core coupled with a nucleoside analogue, phenol, and an l-alanyl carboxylate ester. The previously proposed mechanism of activation postulates the existence of an unstable 5-membered mixed anhydride cyclic intermediate formed from the direct attack of the carboxylate group of the l-alanyl moiety with expulsion of phenol. The mixed anhydride cyclic intermediate is further postulated to undergo spontaneous hydrolysis to form a linear l-alanyl phosphoramidate product. In the proposed mechanism of activation, the 5-membered mixed anhydride intermediate has been detected previously using mass spectrometry, but the specific site of nucleophilic attack by water (P-O versus C-O) has not been determined. To further interrogate the mechanism for hydrolysis of the putative 5-membered cyclic intermediate formed during ProTide activation, the reaction was conducted in 18 O-labeled water using a ProTide analogue that could be activated by carboxypeptidase Y. Mass spectrometry and 31 P NMR spectroscopy were used to demonstrate that the hydrolysis of the mixed anhydride 5-membered intermediate occurs with exclusive attack at the phosphorus center.

Whether rapid oxygen isotopic exchange between bicarbonate and water occurs in photosynthesis is the key to determine the source of oxygen by classic 18 O-labeled photosynthetic oxygen evolution experiments. Here we show that both Microcystis aeruginosa and Chlamydomonas reinhardtii utilize a significant proportion (>16%) of added bicarbonate as a carbon source for photosynthesis. However, oxygen isotopic signal in added bicarbonate cannot be traced in the oxygen in organic matter synthesized by these photosynthetic organisms. This contradicts the current photosynthesis theory, which states that photosynthetic oxygen evolution comes only from water, and oxygen in photosynthetic organic matter comes only from carbon dioxide. We conclude that the photosynthetic organisms undergo rapid exchange of oxygen isotope between bicarbonate and water during photosynthesis. At the same time, this study also provides isotopic evidence for a new mechanism that half of the oxygen in photosynthetic oxygen evolution comes from bicarbonate photolysis and half comes from water photolysis, which provides a new explanation for the bicarbonate effect, and suggests that the Kok-Joliot cycle of photosynthetic oxygen evolution, must be modified to include a molecule of bicarbonate in addition to one molecule of water which in turn must be incorporated into the cycle instead of two water molecules. Furthermore, this study provides a theoretical basis for constructing a newer artificial photosynthetic reactor coupling light reactions with the dark reactions., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Traditionally, nuclear spin is not considered to affect biological processes. Recently, this has changed as isotopic fractionation that deviates from classical mass dependence was reported both in vitro and in vivo. In these cases, the isotopic effect correlates with the nuclear magnetic spin. Here, we show nuclear spin effects using stable oxygen isotopes ( 16 O, 17 O, and 18 O) in two separate setups: an artificial dioxygen production system and biological aquaporin channels in cells. We observe that oxygen dynamics in chiral environments (in particular its transport) depend on nuclear spin, suggesting future applications for controlled isotope separation to be used, for instance, in NMR. To demonstrate the mechanism behind our findings, we formulate theoretical models based on a nuclear-spin-enhanced switch between electronic spin states. Accounting for the role of nuclear spin in biology can provide insights into the role of quantum effects in living systems and help inspire the development of future biotechnology solutions.

Although dissolved inorganic phosphate (DIP) is an important nutrient in the hydrosphere, it is difficult to quantitatively clarify the dynamics of DIP in the hydrosphere using the δ 18 O value of DIP as a tracer. In this study, we quantified the triple oxygen isotopic compositions (Δ' 17 O) of DIP relative to VSMOW with the reference line with a slope of 0.528 as an additional tracer to clarify the sources and dynamics of DIP in the hydrosphere. We found significant variation in the Δ' 17 O values of riverine DIP in urban areas, ranging from -107 × 10 -6 to +3 × 10 -6 , while those of DIP in the effluents from wastewater treatment plants (WWTP) and DIP extracted from the chemical fertilizers showed -56 ± 5 × 10 -6 (1SD) and -98 ± 5 × 10 -6 , respectively. We conclude that both the DIP supplied directly from the artificial loads (the WWTP effluent and chemical fertilizers) showing 17 O-depleted Δ' 17 O values and the DIP turned over via the aquatic biosphere showing 17 O-enriched Δ' 17 O values similar to ambient H 2 O were the major sources of riverine DIP. High-precision determination of the Δ' 17 O value of DIP can contribute to quantitative clarification of the dynamics of DIP in the hydrosphere.

Precision measurements of the stable isotope ratios of oxygen ( 18 O/ 16 O and 17 O/ 16 O) in CO 2 are critical to atmospheric monitoring and terrestrial climate research. High-precision 17 O measurements by isotope ratio mass spectrometry (IRMS) are challenging because they require complicated sample preparation procedures, long measurement times, and relatively large samples sizes. Recently, tunable infrared laser direct absorption spectroscopy (TILDAS) has shown significant potential as an alternative technique for triple oxygen isotope analysis of CO 2 , although the ultimate level of reproducibility is unknown, partly because it is unclear how to relate TILDAS measurements to an internationally accepted isotope abundance scale (e.g., VSMOW2-SLAP2). Here, we present a method for high-precision triple oxygen isotope analysis of CO 2 by TILDAS, requiring ∼8-9 μmol of CO 2 (or 0.9 mg carbonate) in 50 min, plus ∼1.5 h for sample preparation and dilution of CO 2 in N 2 to a nominal 400 μmol mol -1 . Overall reproducibility of Δ' 17 O (CO 2 ) was 0.004‰ (4 per meg) for IAEA603 (SE, n = 6) and 10 per meg for NBS18 (SE, n = 4). Values corrected to the VSMOW2-SLAP2 scale are in good agreement with established techniques of high-precision IRMS, with the exception of Δ' 17 O measured by platinum-catalyzed exchange of CO 2 with O 2 . Compared to high-precision IRMS, TILDAS offers the advantage of ∼ 10 times less sample, and greater throughput, without loss of reproducibility. The flexibility of the technique should allow for many important applications to global biogeochemical monitoring and investigation of 17 O anomalies in a range of geological materials.

Contact electrification between water and a solid surface is crucial for physicochemical processes at water-solid interfaces. However, the nature of the involved processes remains poorly understood, especially in the initial stage of the interface formation. Here we report that H 2 O 2 is spontaneously produced from the hydroxyl groups on the solid surface when contact occurred. The density of hydroxyl groups affects the H 2 O 2 yield. The participation of hydroxyl groups in H 2 O 2 generation is confirmed by mass spectrometric detection of 18 O in the product of the reaction between 4-carboxyphenylboronic acid and 18 O-labeled H 2 O 2 resulting from 18 O 2 plasma treatment of the surface. We propose a model for H 2 O 2 generation based on recombination of the hydroxyl radicals produced from the surface hydroxyl groups in the water-solid contact process. Our observations show that the spontaneous generation of H 2 O 2 is universal on the surfaces of soil and atmospheric fine particles in a humid environment.

High-quality oxygen isotope analysis of composition-variable minerals (e.g., ubiquitous carbonates) using secondary ion mass spectrometry (SIMS) is extremely challenging. The classical off-line procedure, which requires additional electron probe microanalyzer (EPMA) chemical compositions for calibrating instrumental mass fractionation (IMF), is inherently inaccurate and analytically inefficient. In this study, the first accurate and paired SIMS analysis of δ 18 O and Fe# [molar Fe/(Mg + Fe)] in dolomite is reported. Based on five newly developed dolomite O-isotopic standards with an Fe# range of 0.01-0.35 obtained by SIMS, a novel accurate and rapid online matrix effect calibration method for dolomite O-isotope analysis was developed using concurrent SIMS 18 O- 16 O- 56 Fe 16 O- 24 Mg 16 O measurements without additional chemical electron probe microanalysis. A logistic equation was proposed as the best-fit curve to represent the δ 18 O matrix effect based on the 56 Fe 16 O/ 24 Mg 16 O ratios. For CTD-4 carbonatitic dolomite with variable Fe# but homogeneous oxygen isotopes, the off-line method exhibited highly variable apparent δ 18 O values in the range of 5.74-10.11‰. The online method yielded a homogeneous δ 18 O value of 7.94 ± 0.34‰ (2SD, n = 40), which is comparable with that of bulk analysis (7.94 ± 0.20‰; 2SD). Comprehensive analyses validated the online method as the best strategy for performing accurate δ 18 O analysis of samples with highly heterogeneous compositions. Based on its accuracy, simplicity, and economic feasibility, this method has potential applications in the analysis of composition-complex dolomites, detrital dolomites, and other precious terrestrial and extraterrestrial materials.

Nuclear magnetic resonance (NMR) studies involving 17 O are increasingly important in molecular biology, material science, and other disciplines. A large number of these studies employ H 2 17 O as a source of 17 O, and this reliance can be limiting because the high cost of H 2 17 O. To overcome this constraint, a recent study proposed a distillation scheme capable of producing significant quantities of H 2 17 O at a low cost. Although this method is reported to be effective, the reactions proposed to quantify percent of 17 O enrichment are either time intensive or have a risk of errors due to the isotope effect. Here, an alternative reaction scheme is described to measure 17 O water that ultimately creates methyl benzoate as the sole 17 O-containing product. The proposed reaction is completed in a matter of minutes at room temperature, produces only one 17 O product, and requires no clean-up step. The large isotope shift observed in solution NMR between the 13 C═ 16 O and 13 C═ 17 O resonances allows for integration of the individual peaks. This 13 C NMR analysis is found to be highly accurate over a wide enrichment range and is accessible to most NMR spectroscopists.

Stable isotope labelling is state-of-the-art in quantitative mass spectrometry, yet often accessing the required standards is cumbersome and very expensive. Here, a unifying synthetic concept for 18 O-labelled phosphates is presented, based on a family of modified 18 O 2 -phosphoramidite reagents. This toolbox offers access to major classes of biologically highly relevant phosphorylated metabolites as their isotopologues including nucleotides, inositol phosphates, -pyrophosphates, and inorganic polyphosphates. 18 O-enrichment ratios >95 % and good yields are obtained consistently in gram-scale reactions, while enabling late-stage labelling. We demonstrate the utility of the 18 O-labelled inositol phosphates and pyrophosphates by assignment of these metabolites from different biological matrices. We demonstrate that phosphate neutral loss is negligible in an analytical setup employing capillary electrophoresis electrospray ionisation triple quadrupole mass spectrometry., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Oxygen isotope compositions of fossil foraminifera tests are commonly used proxies for ocean paleotemperatures, with reconstructions spanning the last 112 million years. However, the isotopic composition of these calcitic tests can be substantially altered during diagenesis without discernible textural changes. Here, we investigate fluid-mediated isotopic exchange in pristine tests of three modern benthic foraminifera species (Ammonia sp., Haynesina germanica, and Amphistegina lessonii) following immersion into an 18 O-enriched artificial seawater at 90 °C for hours to days. Reacted tests remain texturally pristine but their bulk oxygen isotope compositions reveal rapid and species-dependent isotopic exchange with the water. NanoSIMS imaging reveals the 3-dimensional intra-test distributions of 18 O-enrichment that correlates with test ultra-structure and associated organic matter. Image analysis is used to quantify species level differences in test ultrastructure, which explains the observed species-dependent rates of isotopic exchange. Consequently, even tests considered texturally pristine for paleo-climatic reconstruction purposes may have experienced substantial isotopic exchange; critical paleo-temperature record re-examination is warranted., (© 2022. The Author(s).)

Rationale: This study determines if saliva collection procedures for the doubly labeled water (DLW) method, used for measuring total energy expenditure (TEE), are comparable to urine and plasma collection. Both the cavity ring-down spectroscopy (CRDS) and isotope ratio mass spectrometry (IRMS) analyses techniques are compared., Methods: Saliva specimens were collected from participants for the DLW method. The specimens were collected under different conditions: after consumption of tap water, after chewing gum, and during exposure to conditions of high and low relative humidity. The isotopes in saliva were compared with simultaneous plasma and urine collection. TEE calculated from saliva and analyzed using CRDS was compared to that of plasma analyzed using IRMS., Results: The within-individual variances were not significantly different between the saliva specimens (0.4‰) and plasma (0.3‰). After the oral dose of DLW, the saliva specimens displayed a shorter equilibration time to urine. When participants consumed 500 mL of tap water, the enrichment of saliva specimens reached a new plateau value faster than urine. Saliva collection exposed to high ambient humidity conditions was slightly less enriched as compared to low-humidity conditions while urine enrichment was unaffected. In contrast, whereas the within-individual effects of gum chewing during saliva collection on 18 O were unaffected, the abundance of 2 H in saliva was slightly lower after chewing the gum. The within-individual difference between TEE calculated from saliva and that calculated from plasma analyzed using IRMS did not differ from zero, and the standard deviation was not different from that predicted by a propagation of error analysis based on analytical error alone., Conclusions: Our findings support using saliva specimens for the DLW method. The analysis of plasma and urine, however, requires reducing the memory effect resulting from contaminants. Also, it should be performed in a manner that minimizes exposure to air where specimens may be exposed to evaporation or contamination from water vapor during sampling., (© 2021 John Wiley & Sons Ltd.)

We report two- and three-dimensional (2D and 3D) 13 C- 17 O heteronuclear correlation solid-state NMR experiments under magic-angle spinning (MAS) conditions. These experiments utilize the D-RINEPT (Dipolar-mediated Refocused Insensitive Nuclei Enhanced by Polarization Transfer) scheme with symmetry-based SR4 1 2 recoupling blocks for coherence transfer between 13 C and 17 O nuclei. First, a 2D 17 O → 13 C correlation experiment was performed for the [1- 13 C, 17 O]-Gly/Gly·HCl cocrystal and [U- 13 C, 1- 17 O]-α/β-d-glucose samples. Second, a 2D 17 O → 13 C MQ-D-RINEPT correlation experiment where the indirect dimension incorporates the multiple-quantum MAS (MQMAS) scheme was tested for obtaining isotropic 17 O resolution with [U- 13 C, 1- 17 O]-α/β-d-glucose. Third, a new 3D 17 O → 13 C → 13 C correlation experiment was demonstrated where 17 O → 13 C and 13 C → 13 C correlations are achieved by D-RINEPT and DARR (Dipolar Assisted Rotational Resonance) sequences, respectively (thus termed as a 3D D-RINEPT/DARR OCC experiment). This new 3D 17 O NMR experiment is implemented with the aim for site-resolved solid-state 17 O NMR studies.

The demand for more efficient methods of establishing the undetermined stereochemistries of peptidic natural products continues unabated. A new method for microscale stereochemical determination was devised by integrating solid-phase synthesis, split-and-mix randomization, 18 O/ 16 O-encoding of d/l-configurations, tandem mass spectrometry, and biological evaluation. Here we applied gramicidin A as the molecule for a blind test. Gramicidin A and its 31 diastereomers were randomly prepared in microgram scale with 18 O/ 16 O-stereochemical encoding and subjected to MS/MS-structural determination and cytotoxicity assay. Only the parent gramicidin A was selected from among the 32 stereoisomers, validating the high reliability of the present strategy., (© 2021 Wiley-VCH GmbH.)

The removal of plant pigments such as β-carotene is an aspect of vegetable oil processing often desired by the food and pharmaceutical industries. Adsorption of β-carotene to acid-activated clay (AAC) is a well-established method for purification. Despite this, the removal mechanism of β-carotene is not well understood. UPLC-MS/MS analysis of surface compounds extracted from β-carotene-AAC (BC-AAC) complexes show that AAC acts as an oxidiser. Oxidation products detected included canthaxanthin and 3',4'-didehydro-β-caroten-4-one. AAC had surface water exchanged with an 18 O labelled water and was then exposed to β-carotene. Carotenoids labelled with 18 O were produced from this reaction, suggesting surface water is necessary for β-carotene removal., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The international wine market has been repeatedly hit by cases of fraud in recent decades. While several studies attested a special vulnerability of the fast growing wine business in China, reports on chemical analyses of commercial wine samples are rare. We examined 50 predominantly red wines with European labelling, which were purchased on the Chinese market, for fraud-relevant parameters. More than 20% of the tested samples revealed anomalies in relation to the stable isotope ratios of D/H, 18 O/ 16 O and 13 C/ 12 C, contents of technical glycerol by-products or anthocyanin composition. These results strongly suggested watering of the wines, chaptalisation, glycerol addition or the use of non- Vitis anthocyanin sources, respectively. Some of these samples also showed suspicious spelling errors or other irregularities in the labelling, but the majority appeared genuine to the eye. Hence, this spot check demonstrates the importance of chemical authenticity analysis of market samples in order to detect fraudulent products. Moreover, we used the same sample set for an evaluation of the Chinese standard method for carbon stable isotope determination of wine ethanol in comparison to the current OIV (International Organisation of Vine and Wine) standard method. The results of a Bland-Altman analysis indicated that the methods can be applied interchangeably. As the two methods differ in their workflow and in the requested equipment, this might eventually enable more laboratories to perform 13 C/ 12 C analysis of wine and spirits.

The electrons that nature uses to reduce CO 2 during photosynthesis come from water oxidation at the oxygen-evolving complex of photosystem II. Molecular catalysts have served as models to understand its mechanism, in particular the O-O bond-forming reaction, which is still not fully understood. Here we report a Ru(IV) side-on peroxo complex that serves as a 'missing link' for the species that form after the rate-determining O-O bond-forming step. The Ru(IV) side-on peroxo complex (η 2 -1 iv -OO) is generated from the isolated Ru(IV) oxo complex (1 iv =O) in the presence of an excess of oxidant. The oxidation (IV) and spin state (singlet) of η 2 -1 iv -OO were determined by a combination of experimental and theoretical studies. 18 O- and 2 H-labelling studies evidence the direct evolution of O 2 through the nucleophilic attack of a H 2 O molecule on the highly electrophilic metal-oxo species via the formation of η 2 -1 iv -OO. These studies demonstrate water nucleophilic attack as a viable mechanism for O-O bond formation, as previously proposed based on indirect evidence., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Two mechanochemical procedures for 17O/18O-isotope labeling of fatty acids are reported: a carboxylic acid activation/hydrolysis approach and a saponification approach. The latter route allowed first-time enrichment of important polyunsaturated fatty acids (PUFAs) including docosahexaenoic acid (DHA). Overall, a total of 9 pure labeled products were isolated in high yields (≥80%) and with high enrichment levels (≥37% average labeling of C=O and C-OH carboxylic oxygen atoms), under mild conditions, and in short time (

The recently proposed glymphatic pathway for solute transport and waste clearance from the brain has been the focus of intense debate. By exploiting an isotopically enriched MRI tracer, H217O, we directly imaged glymphatic water transport in the rat brain in vivo. Our results reveal glymphatic transport that is dramatically faster and more extensive than previously thought and unlikely to be explained by diffusion alone. Moreover, we confirm the critical role of aquaporin-4 channels in glymphatic transport.

In the present study we investigated the isotope effects associated with water loss from closed low-density polyethylene (LDPE) bottles via diffusion at temperatures between 4 and 60 °C. While at low temperatures (4 and 10 °C) no substantial diffusional loss of water was observed within storage time, a pronounced loss was found for the experiments at room temperature and 60 °C. The latter was associated with a substantial increase in δ 18 O, δ 17 O, and δ 2 Η values, and a decrease in the deuterium excess. The magnitude of the isotope effects essentially depended on the extent of water evaporation from the closed bottles through the LDPE membrane.

In mammals, carotenoids are converted by two carotenoid cleavage oxygenases into apocarotenoids, including vitamin A. Although knowledge about β-carotene oxygenase-1 (BCO1) and vitamin A metabolism has tremendously increased, the function of β-carotene oxygenase-2 (BCO2) remains less well-defined. We here studied the role of BCO2 in the metabolism of long chain β-apocarotenoids, which recently emerged as putative regulatory molecules in mammalian biology. We showed that recombinant murine BCO2 converted the alcohol, aldehyde, and carboxylic acid of a β-apocarotenoid substrate by oxidative cleavage at position C9,C10 into a β-ionone and a diapocarotenoid product. Chain length variation (C20 to C40) and ionone ring site modifications of the apocarotenoid substrate did not impede catalytic activity or alter the regioselectivity of the double bond cleavage by BCO2. Isotope labeling experiments revealed that the double bond cleavage of an apocarotenoid followed a dioxygenase reaction mechanism. Structural modeling and site directed mutagenesis identified amino acid residues in the substrate tunnel of BCO2 that are critical for apocarotenoid binding and catalytic processing. Mice deficient for BCO2 accumulated apocarotenoids in their livers, indicating that the enzyme engages in apocarotenoid metabolism. Together, our study provides novel structural and functional insights into BCO2 catalysis and establishes the enzyme as a key component of apocarotenoid homeostasis in mice.

Magnetic resonance imaging (MRI) has a leading place in medicine as an imaging tool of high resolution for anatomical studies and diagnosis of diseases, in particular for soft tissues that cannot be accessible by other modalities. Many research works are thus focused on improving the images obtained with MRI. This technique has indeed poor sensitivity, which can be compensated by using a contrast agent (CA). Today, the clinically approved CAs on market are solely based on gadolinium complexes that may induce nephrogenic systemic fibrosis for patients with kidney failure, whereas more recent studies on healthy rats also showed Gd retention in the brain. Consequently, researchers try to elaborate other types of safer MRI CAs like manganese-based complexes. In this context, the synthesis of Mn 2+ complexes of four 12-membered pyridine-containing macrocyclic ligands based on the pyclen core was accomplished and described herein. Then, the properties of these Mn(II) complexes were studied by two relaxometric methods, 17 O NMR spectroscopy and 1 H NMR dispersion profiles. The time of residence (τ M ) and the number of water molecules ( q ) present in the inner sphere of coordination were determined by these two experiments. The efficacy of the pyclen-based Mn(II) complexes as MRI CAs was evaluated by proton relaxometry at a magnetic field intensity of 1.41 T near those of most medical MRI scanners (1.5 T). Both the 17 O NMR and the nuclear magnetic relaxation dispersion profiles indicated that the four hexadentate ligands prepared herein left one vacant coordination site to accommodate one water molecule, rapidly exchanging, in around 6 ns. Furthermore, it has been shown that the presence of an additional amide bond formed when the paramagnetic complex is conjugated to a molecule of interest does not alter the inner sphere of coordination of Mn, which remains monohydrated. These complexes exhibit r 1 relaxivities, large enough to be used as clinical MRI CAs (1.7-3.4 mM -1 ·s -1 , at 1.41 T and 37 °C).

A theoretical approach is developed for the description of all possible recombination pathways in the ozone forming reaction, without neglecting any process a priori , and without decoupling the individual pathways one from another. These pathways become physically distinct when a rare isotope of oxygen is introduced, such as 18 O, which represents a sensitive probe of the ozone forming reaction. Each isotopologue of O 3 contains two types of physically distinct entrance channels and two types of physically distinct product wells, creating four recombination pathways. Calculations are done for singly and doubly substituted isotopologues of ozone, eight rate coefficients total. Two pathways for the formation of asymmetric ozone isotopomer exhibit rather different rate coefficients, indicating large isotope effect driven by ΔZPE-difference. Rate coefficient for the formation of symmetric isotopomer of ozone (third pathway) is found to be in between of those two, while the rate of insertion pathway is smaller by two orders of magnitude. These trends are in good agreement with experiments, for both singly and doubly substituted ozone. The total formation rates for asymmetric isotopomers are found to be somewhat larger than those for symmetric isotopomers, but not as much as in the experiment. Overall, the distribution of lifetimes is found to be very similar for the metastable states in symmetric and asymmetric ozone isotopomers.

In erythrocytes, nitric oxide (NO) and its autoxidation product nitrite undergo multiple reactions with hemoglobin species to form nitrate, Fe-nitrosyl hemoglobin (HbFe (II) NO), S-nitrosohemoglobin (HbCys β93 SNO), and other intermediates including nitrito-methemoblobin HbFe (III) ONO, nitro-methemoblobin HbFe (III) NO 2 and nitrous anhydride (N 2 O 3 ). Here, we report a stable-isotope dilution GC-MS assay that allows studying reactions of nitrite in hemolysate. The method is based on the use of 18 O-labelled nitrite in combination with l-cysteine or N-acetyl-l-cysteine ethyl ester and GC-MS measurement of unlabelled and labelled nitrite and nitrate species. This approach reveals reactions that are hidden at physiological nitrite concentrations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Sheep remains constitute the main archaeozoological evidence for the presence of Early Neolithic human groups in the highlands of the Southern Pyrenees but understanding the role of herding activities in the Neolithisation process of this mountain ecosystem calls for the analysis of large and well-dated faunal assemblages. Cova de Els Trocs (Bisaurri, Huesca, Spain), a cave located at 1564 m a.s.l on the southern slopes of the Central Pyrenees, is an excellent case study since it was seasonally occupied throughout the Neolithic (ca. 5312-2913 cal. BC) and more than 4000 caprine remains were recovered inside. The multi-proxy analytical approach here presented has allowed us to offer new data elaborating on vertical mobility practices and herd management dynamics as has not been attempted up until now within Neolithic high-mountain sites in the Iberian Peninsula. For the first time, δ18O and δ13C stable isotope analyses offer direct evidence on both the regular practice of altitudinal movements of sheep flocks and the extended breeding season of sheep. Autumn births are recorded from the second half of the fifth millennium cal. BC onwards. Age-at-death distributions illustrate the progressive decline in caprine perinatal mortality together with the rising survival rate of individuals older than six months of age and the larger frequency of adults. This trend alongside the 'off-season' lambing signal at the implementation of husbandry techniques over time, probably aiming to increase the size of the flocks and their productivity. Palaeoparasitological analyses of sediment samples document also the growing reliance on herding activities of the human groups visiting the Els Trocs cave throughout the Neolithic sequence. In sum, our work provides substantial arguments to conclude that the advanced herding management skills of the Early Neolithic communities arriving in Iberia facilitated the anthropisation process of the subalpine areas of the Central Pyrenees., Competing Interests: The authors have declared that no competing interests exist

Fatty acids are ubiquitous in biological systems and widely used in materials science, including for the formulation of drugs and the surface-functionalization of nanoparticles. However, important questions regarding the structure and reactivity of these molecules are still to be elucidated, including their mode of binding to certain metal cations or materials surfaces. In this context, we have developed novel, efficient, user-friendly, and cost-effective synthetic protocols based on ball-milling, for the 17 O and 18 O isotopic labeling of two key fatty acids which are widely used in (nano)materials science, namely stearic and oleic acid. Labeled molecules were analyzed by 1 H and 13 C solution NMR, IR spectroscopy, and mass spectrometry (ESI-TOF and LC-MS), as well as 17 O solid state NMR (for the 17 O labeled species). In both cases, the labeling procedures were scaled-up to produce up to gram quantities of 17 O- or 18 O-enriched molecules in just half-a-day, with very good synthetic yields (all ≥84%) and enrichment levels (up to an average of 46% per carboxylic oxygen). The 17 O-labeled oleic acid was then used for the synthesis of a metal soap (Zn-oleate) and the surface-functionalization of ZnO nanoparticles (NPs), which were characterized for the first time by high-resolution 17 O NMR (at 14.1 and 35.2 T). This allowed very detailed insight into (i) the coordination mode of the oleate ligand in Zn-oleate to be achieved (including information on Zn···O distances) and (ii) the mode of attachment of oleic-acid at the surface of ZnO (including novel information on its photoreactivity upon UV-irradiation). Overall, this work demonstrates the high interest of these fatty acid-enrichment protocols for understanding the structure and reactivity of a variety of functional (nano)materials systems using high resolution analyses like 17 O NMR.

Cholesterol is a crucial component of biological membranes and can interact with other membrane components through hydrogen bonding. NMR spectroscopy has been used previously to investigate this bonding, however this study represents the first 17O NMR spectroscopy study of isotopically enriched cholesterol. We demonstrate the 17O chemical shift is dependent on hydrogen bonding, providing a novel method for the study of cholesterol in bilayers.

Background: The high molecular complexity of variably O-glycosylated and degraded pro B-type natriuretic peptide (proBNP) derived molecular forms challenges current immunoassays. Antibodies used show pronounced differences in cross-reactivities with these circulating fragments, which still need to be better characterized on a molecular level. To pave the way for advanced quantitative assays in the future, it is critical to fully understand these circulating forms., Methods: Plasma samples were collected from 8 heart failure (HF) patients and 2 healthy controls. NT-proBNP and proBNP were purified by immunoprecipitation and analyzed by nano-flow liquid chromatography coupled to high-resolution mass spectrometry. Fragments formed during proteolysis in solution digestion were distinguished from naturally occurring peptides by using an 18O stable isotope labeling strategy., Results: We detected 16 previously unknown circulating fragments of proBNP peptides (9 of which are located in the N-terminal and 7 in the C-terminal region), revealing a more advanced state of degradation than previously known. Two of these fragments are indicative of either unidentified processing modes or a far-reaching C-terminal degradation (or a combination thereof) of the precursor proBNP., Conclusions: Our results further restrict ideal target epitopes for immunoassay antibodies and expand the current thinking of diversity, degradation, and processing of proBNP, as well as the distribution of circulating forms., (© American Association for Clinical Chemistry 2020. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Natural-abundance 17O NMR experiments are used to investigate the hydrated water in magnetically aligned synthetic polymer based lipid-nanodiscs. Residual quadrupole couplings (RQCs) measured from the observed five 17O (central and satellite) transitions, and molecular dynamics simulations, are used to probe the ordering of water molecules across the lipid bilayer.

In this study we attempted to assess whether seasonal upwelling or a steady thermocline persisted at the western margin of the Tethys Ocean during the late Turonian-early Coniacian interval. For this scope, we employed novel and published stable oxygen isotope (δ18O) data of various organisms (bivalves, bivalves, brachiopods, fish and belemnites). New seasonally resolved temperature estimates were based on the δ18O record of sequentially sampled inoceramid (Inoceramus sp.) and rudist (Hippurites resectus) shells from the Scaglia Rossa and Gosau deposits of northern Italy and western Austria, respectively. Diagenetic screening was performed using reflected light, cathodoluminescence (CL), scanning electron microscopy (SEM) and stable isotope analysis. Originally preserved δ13C and δ18O values were used to characterize the lifestyle of the bivalves and detect vital effects that could have biased oxygen isotope-based temperature reconstructions. Inoceramid δ18O values provide-for the first time-information on temperatures of Tethyan benthic waters, which were, on average, 14.4 ± 0.6 °C and fluctuated seasonally within a range of less than 2 °C. Such a thermal regime is in line with the temperatures postulated for late Turonian boreal water masses and support the existence of a cold water supply from the North Atlantic to the Tethyan bottom. Bottom cooling, however, did not affect the shallow water environment. In fact, the rudist-based temperature estimates for shallow water environment revealed a mean annual range of 11 °C, between 24 and 35 °C (assuming a seasonally constant δ18Ow = 1.0 ‰), which are among the warmest temperatures recorded over the entire Late Cretaceous. Our findings, thus, suggest a strong thermal and food web decoupling between the two environments. The absence of a seasonal vertical homogenization of different water bodies suggests the existence of a steady thermocline and, therefore, contrasts with the presence of an active coastal upwelling in the region as hypothesized by previous authors., Competing Interests: The authors have declared that no competing interests exist.

Dynamic nuclear polarization (DNP) significantly enhances the sensitivity of nuclear magnetic resonance (NMR), increasing its applications and the quality of NMR spectroscopy as a characterization tool for materials. Efficient spin diffusion among the nuclear spins is considered to be essential for spreading the hyperpolarization throughout the sample, enabling large DNP enhancements. This scenario mostly limits the polarization enhancement of low-sensitivity nuclei in inorganic materials to the surface sites when the polarization source is an exogenous radical. In metal-ion-based DNP, the polarization agents are distributed in the bulk sample and act as a source of both relaxation and polarization enhancement. We have found that as long as the polarization agent is the main source of relaxation, the enhancement does not depend on the distance between the nucleus and dopant. As a consequence, the requirement of efficient spin diffusion is lifted, and the entire sample can be directly polarized. We exploit this finding to measure high-quality NMR spectra of 17 O in the electrode material Li 4 Ti 5 O 12 doped with Fe(III) despite its low abundance and long relaxation time.

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18 O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18 O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

Most developments in synthetic biology try to depart from life as we know it, attempting to create orthogonal constructions. Here, following a variational principle, I try to explore how slight changes in the buildup of cells reveal critical features of life's physics. In a first section, I suggest that we use stable isotopes of the atoms of life to see how living cells fare, beginning with life in heavy water. Subsequently, isotopes of the other main biogenic atoms are suggested as an extension of the variational principle, despite their likely very small influence on the course of biological activity. Finally, two atoms of the second row of Mendeleev's table, boron and fluorine are explored as a further extension of the principle. The use of the former is still in its infancy, whereas the latter, based on existing fluorinases, could open the door to a more general use of halogens in synthetic biology., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Reactive oxygen and nitrogen species have been implicated in many biological processes and diseases, including immune responses, cardiovascular dysfunction, neurodegeneration, and cancer. These chemical species are short-lived in biological settings, and detecting them in these conditions and diseases requires the use of molecular probes that form stable, easily detectable, products. The chemical mechanisms and limitations of many of the currently used probes are not well-understood, hampering their effective applications. Boronates have emerged as a class of probes for the detection of nucleophilic two-electron oxidants. Here, we report the results of an oxygen-18-labeling MS study to identify the origin of oxygen atoms in the oxidation products of phenylboronate targeted to mitochondria. We demonstrate that boronate oxidation by hydrogen peroxide, peroxymonocarbonate, hypochlorite, or peroxynitrite involves the incorporation of oxygen atoms from these oxidants. We therefore conclude that boronates can be used as probes to track isotopically labeled oxidants. This suggests that the detection of specific products formed from these redox probes could enable precise identification of oxidants formed in biological systems. We discuss the implications of these results for understanding the mechanism of conversion of the boronate-based redox probes to oxidant-specific products., (© 2020 Rios et al.)

Earth's ocean-atmosphere system has undergone a dramatic but protracted increase in oxygen (O 2 ) abundance. This environmental transition ultimately paved the way for the rise of multicellular life and provides a blueprint for how a biosphere can transform a planetary surface. However, estimates of atmospheric oxygen levels for large intervals of Earth's history still vary by orders of magnitude-foremost for Earth's middle history. Historically, estimates of mid-Proterozoic (1.9-0.8 Ga) atmospheric oxygen levels are inferred based on the kinetics of reactions occurring in soils or in the oceans, rather than being directly tracked by atmospheric signatures. Rare oxygen isotope systematics-based on quantifying the rare oxygen isotope 17 O in addition to the conventionally determined 16 O and 18 O-provide a means to track atmospheric isotopic signatures and thus potentially provide more direct estimates of atmospheric oxygen levels through time. Oxygen isotope signatures that deviate strongly from the expected mass-dependent relationship between 16 O, 17 O, and 18 O develop during ozone formation, and these "mass-independent" signals can be transferred to the rock record during oxidation reactions in surface environments that involve atmospheric O 2 . The magnitude of these signals is dependent upon p O 2 , p CO 2 , and the overall extent of biospheric productivity. Here, we use a stochastic approach to invert the mid-Proterozoic Δ 17 O record for a new estimate of atmospheric p O 2 , leveraging explicit coupling of p O 2 and biospheric productivity in a biogeochemical Earth system model to refine the range of atmospheric p O 2 values that is consistent with a given observed Δ 17 O. Using this approach, we find new evidence that atmospheric oxygen levels were less than ∼1% of the present atmospheric level (PAL) for at least some intervals of the Proterozoic Eon.

With the rapid development of immunometabolism, 2-hydroxyglutarate (2-HG) is being promoted as a key immunometabolite to regulate the immune system. Based on the well-established crosstalk between 2-HG and other immunometabolites, here we firstly constructed a 2-HG metabolic panel by mapping the related metabolic pathways. Quantitative methods to globally monitor 2-HG metabolic panel are of great importance for immunometabolism study. However, the existence of enantiomer hampers the accurate measurement of these immunometabolites. This study addressed an original isotopically-paired chiral derivatization approach for UPLC-MS/MS quantification of 2-HG metabolic panel. To achieve better chromatographic separation, N-(p-toluenesulfonyl)-L-phenylalanyl chloride (TSPC) was utilized as an optical resolving reagent to form diastereomers. For accurate quantitation, an 18 O 2 -labeled-TSPC reagent was designed and readily synthesized to produce one-to-one internal standards. The developed approach enabled an accurate quantification of 13 immunometabolites in 2-HG metabolic panel with good linearity (R 2  > 0.99) and high sensitivity (0.5-120 fmol for LLOQ). With this method, we were able to simultaneously monitor the specific alterations of 2-HG metabolic panel in collagen-induced rheumatoid arthritis (CIA) rats. The measured levels of this panel ranged from 0.02 to 85.14 μg g -1 for synovium tissue and 0.012 to 87.75 μmol L -1 for serum samples. We envisage that the present isotopically-paired chiral derivatization approach will be practicable for different bio-samples to quantitatively profile the amino- and hydroxyl acids submetabolome, especially for the endogenous enantiomers. By virtue of the low cost of reagents and the simple procedure used in the assay, this method could be readily implemented., (Copyright © 2019 Elsevier B.V. All rights reserved.)

18 O-labeled water (Water- 18 O) is a widely used starting material of 18 F-labeled diagnostic agents in positron emission tomography (PET). Conventionally, Water- 18 O has been separated from other stable oxygen isotope species ( 16 O, 17 O) by water distillation or nitric oxide distillation. However, conventional methods are costly and may have safety issues. In 2004, we developed the first unit of our novel oxygen isotope separation process by cryogenic oxygen distillation to overcome these issues. To meet the needs of the market, we built a second unit in 2013 and a third in 2016. We are now operating three commercially viable separation units with a total capacity of 600 kg of Water- 18 O per year., (© 2019 John Wiley & Sons, Ltd.)

Stable isotopes have been widely used in the literature both to discuss current ocean circulation processes, as well as to reconstitute paleoceanographic parameters. The distribution of oxygen and deuterium stable isotopes in seawater (δ18Osw and δDsw) at the Western Tropical South Atlantic border was investigated to better understand the main fractionation processes of these isotopes and establish a regional salinity and δ18Osw relation to improve the paleoceanographic knowledge in the region. This study was conducted during a quasi-synoptic oceanographic cruise in which 98 discrete seawater samples were collected in the core of the main water masses for stable isotope analysis. A strong correlation between δ18Osw and δD was found, which made it possible to extrapolate the results for δ18Osw to δD. Although it was not possible to distinguish the water masses based only on their isotopic signatures, the water masses had a strong salinity and δ18Osw relation, and compared with previous studies, a seasonal pattern was observed. Paleosalinity differences of up to 0.2 psu between Summer and Winter are reported. Considering the limitations of the current techniques to seasonally separate the samples for the paleoceanographic studies, an intermediate Mixing Line for the Tropical South Atlantic (SSS = 1.942* δ18Osw + 34.56) was proposed to reduce the estimated errors associated with these seasonal variations.

Analytical methods for authenticity testing of organically grown vegetables are urgently needed. Here we present a novel method for organic authentication based on stable isotope ratio analysis of oxygen in plant-derived sulphate. We combined this method with stable isotope ratio analysis of bulk plant tissue and plant-derived nitrate to discriminate organic and conventional potato, carrot, and cabbage from rigidly controlled long-term field trials and from a case study using retail potatoes. It was shown that oxygen isotope ratios of sulphate from organic vegetables were significantly lower compared to their conventional counterparts and the values were directly linked to the fertilisation strategy. The classification power of sulphate isotope analysis was superior compared to known bulk tissue isotope markers and nitrate isotope values. In conclusion, oxygen isotope analysis of plant-derived sulphate represents a promising new method for authentication of organic vegetables., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

To test the potential of different analytical tools to determine the geographical origin of Ethiopian coffee, 103 green arabica coffee samples from four coffee regions in Ethiopia were subjected to multi-elements and δ 13 C, δ 15 N and δ 18 O determinations. Multi-elements were determined by using inductively coupled plasma (ICP)- and X-ray fluorescence spectrometry (XRF)-based techniques, and δ 13 C, δ 15 N and δ 18 O were determined by using elemental analyzer-isotope ratio mass spectrometry. Using linear discriminant analysis, XRF-based multi-elements with and without δ 13 C appeared to be most effective in discriminating the geographical origin of coffee, giving higher classification accuracy (89 and 86%, respectively) than ICP-based multi-elements with and without stable isotopes (80%, each). These results demonstrate the potential of XRF-based multi-element profiling as a relatively fast and low-cost tool to trace the geographical origin of Ethiopian coffee. All together this study offers the proof of concept for a promising method that, upon standardization, could be used for coffee provenance authentication and fraud detection., (Copyright © 2019. Published by Elsevier Ltd.)

Fast photochemical oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical ( • OH) generated by photolysis of hydrogen peroxide (H 2 O 2 ) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of • OH oxidative modification of 13 amino acid residues by using 18 O isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H 2 O 2 . Methionine residues competitively take oxygen from H 2 O 2 and dissolved oxygen (O 2 ), whereas the remaining residues take oxygen exclusively from O 2 . Results reported in this work deepen the understanding of • OH labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.

Tropospheric ozone (O 3 ) is a key component of air pollution and an important anthropogenic greenhouse gas 1 . During the twentieth century, the proliferation of the internal combustion engine, rapid industrialization and land-use change led to a global-scale increase in O 3 concentrations 2,3 ; however, the magnitude of this increase is uncertain. Atmospheric chemistry models typically predict 4-7 an increase in the tropospheric O 3 burden of between 25 and 50 per cent since 1900, whereas direct measurements made in the late nineteenth century indicate that surface O 3 mixing ratios increased by up to 300 per cent 8-10 over that time period. However, the accuracy and diagnostic power of these measurements remains controversial 2 . Here we use a record of the clumped-isotope composition of molecular oxygen ( 18 O 18 O in O 2 ) trapped in polar firn and ice from 1590 to 2016 AD, as well as atmospheric chemistry model simulations, to constrain changes in tropospheric O 3 concentrations. We find that during the second half of the twentieth century, the proportion of 18 O 18 O in O 2 decreased by 0.03 ± 0.02 parts per thousand (95 per cent confidence interval) below its 1590-1958 AD mean, which implies that tropospheric O 3 increased by less than 40 per cent during that time. These results corroborate model predictions of global-scale increases in surface pollution and vegetative stress caused by increasing anthropogenic emissions of O 3 precursors 4,5,11 . We also estimate that the radiative forcing of tropospheric O 3 since 1850 AD is probably less than +0.4 watts per square metre, consistent with results from recent climate modelling studies 12 .

The importance of studying site-specific interactions of structurally similar water molecules in complex systems is well known. We demonstrate the ability to resolve four distinct bound water environments within the crystal structure of lanthanum magnesium nitrate hydrate via 17 O solid state nuclear magnetic resonance (NMR) spectroscopy. Using high-resolution multidimensional experiments at high magnetic fields (18.8-35.2 T), each individual water environment was resolved. The quadrupole coupling constants and asymmetry parameters of the 17 O of each water were determined to be between 6.6 and 7.1 MHz, 0.83 and 0.90, respectively. The resolution of the four unique, yet similar, structural waters within a hydrated crystal via 17 O NMR spectroscopy demonstrates the ability to decipher the unique electronic environment of structural water within a single hydrated crystal structure.

The enzymatic breakdown of recalcitrant polysaccharides is achieved by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive, meaning that they stay bound to the substrate between subsequent catalytic interactions. Cellulases are GHs that catalyze the hydrolysis of cellulose [β-1,4-linked glucose (Glc)]. Here, we have determined the relative subsite binding affinity for a glucose moiety as well as the thermodynamic signatures for (Glc) 6 binding to three of the seven cellulases produced by the bacterium Thermobifida fusca. TfCel48A is exo-processive, TfCel9A endo-processive, and TfCel5A endo-nonprocessive. Initial hydrolysis of (Glc) 5 and (Glc) 6 was performed in H 2 18 O enabling the incorporation of an 18 O atom at the new reducing end anomeric carbon. A matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the products reveals the intensity ratios of otherwise identical 18 O- and 16 O-containing products to provide insight into how the substrate is placed during productive binding. The two processive cellulases have significant binding affinity in subsites where products dissociate during processive hydrolysis, aligned with a need to have a pushing potential to remove obstacles on the substrate. Moreover, we observed a correlation between processive ability and favorable binding free energy, as previously postulated. Upon ligand binding, the largest contribution to the binding free energy is desolvation for all three cellulases as determined by isothermal titration calorimetry. The two endo-active cellulases show a more favorable solvation entropy change compared to the exo-active cellulase, while the two processive cellulases have less favorable changes in binding enthalpy compared to the nonprocessive TfCel5A.

The Indian Ocean Dipole (IOD) is an interannual mode of climate variability in the Indian Ocean that has intensified with 20 th century global-warming. However, instrumental data shows a global-warming hiatus between the late-1990s and 2015. It is presently not clear how the global-warming hiatus affects modes of climate variability such as the IOD, and their basin-wide ocean-atmosphere teleconnections. Here, we present a 26-year long, biweekly record of Sr/Ca and δ 18 O from a Porites coral drilled in the Gulf of Oman. Sea surface temperature (SST anom ) is calculated from Sr/Ca ratios, and seawater δ 18 O (δ 18 O sw-anom ) is estimated by subtracting the temperature component from coral δ 18 O. Our δ 18 O sw-anom record reveals a significant regime shift in 1999, towards lower mean δ 18 O sw values, reflecting intensified upwelling in the western Indian Ocean. Prior to the 1999 regime shift, our SST anom and δ 18 O sw-anom show a clear IOD signature, with higher values in the summer of positive-IOD years due to weakened upwelling. The IOD signature in SST anom and δ 18 O sw-anom disappears with the overall intensification of upwelling after the 1999 regime shift. The inferred increase in upwelling is likely driven by an intensified Walker circulation during the global-warming hiatus. Upwelling in the Western Indian Ocean uncouples from the IOD.

Enzymatic 18 O exchange, the well-established approach in comparative proteomics, has some disadvantages such as back exchange of labeled oxygen and overlapping the peak of a labeled peptide with isotopic peaks of an unlabeled one. Herein we demonstrated a simple procedure in which samples digested with a trypsin (with and without H 2 18 O) were reacted with unlabeled and quadrupled 13 C-labeled pyrylium salt respectively which results in formation of pyridinium cations. Thus, each isobarically labeled peptide containing zero or four 13 C atoms in the mass reporter group, during tandem MS/MS forms an unique reporter ion useful for a relative quantitation. Such a sample treatment improves the signal to noise ratio, reduces overlapping of the isotopic peaks and completely eliminates the back exchange problem., (Copyright © 2018 Elsevier B.V. All rights reserved.)

A method is proposed in this paper for the determination of oxygen-18 labeled phosphate so that positional isotope experiments using sensitive and rapid liquid chromatography-QTOF-mass spectrometry (LC-QTOF-MS) experiments can be carried out. The positional isotope exchange technique is a useful tool in understanding the mechanisms and kinetics of many enzyme-catalyzed reactions. Detection of the positions and concentration of these exchanged isotopes is the key. Gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance imaging are commonly used analytical techniques for measurement of 18 O/ 16 O, 31 P and 15 N isotope enrichment. Since these techniques either require a time-consuming derivatization step or have a limited sensitivity, an LC and accurate mass-based method for monitoring 18 O/ 16 O exchange was developed and compared with a standard GC-MS method. Our results showed that the LC-QTOF-MS method developed was not only as accurate as the standard GC-MS method, but also a sensitive and robust analytical platform for the simultaneous determination of isotope enrichment and the analysis of positional isotopes without chemical derivation. The LC-QTOF-MS method developed was successfully applied to the measurement of 18 O/ 16 O in the reversibility study of ATP hydrolysis by Lon proteases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pyridine nucleotides which include NAD + , NADH, NADP, and NADPH play vital roles in many different biological processes. These metabolites can be accurately quantified in a wide variety of biological samples using LC-MS/MS. The quality and precision of these measurements was enhanced using heavy isotope-labeled internal standards and carefully crafted protocols for sample processing.

Quantitative cross-linking/mass spectrometry (QCLMS/QXL-MS) probes structural changes of proteins in solution. This method has revealed induced conformational changes, composition shifts in conformational ensembles and changes in protein interactions. It illuminates different structural states of proteins or protein complexes by comparing which residue pairs can be cross-linked in these states. Cross-links provide information about structural changes that may be inaccessible by alternative technologies. Small local conformational changes affect relative abundances of nearby cross-links, whereas larger conformational changes cause new cross-links to be formed. Distinguishing between minor and major changes requires a robust analysis based on carefully selected replicates and, when using isotope-labeled cross-linkers, replicated analysis with a permutated isotope-labeling scheme. A label-free workflow allows for application of a wide range of cross-linking chemistries and enables parallel comparison of multiple conformations. In this protocol, we demonstrate both label-free and isotope-labeled cross-linker-based workflows using the cross-linker bis(sulfosuccinimidyl)suberate (BS3). The software XiSearch, developed by our group, is used to identify cross-linked residue pairs, although the workflow is not limited to this search software. The open-access software Skyline is used for automated quantitation. Note that additional manual correction greatly enhances quantitation accuracy. The protocol has been applied to purified multi-protein complexes but is not necessarily limited to that level of sample complexity. Optimizing the cross-linker/protein ratio and fractionating peptides increase the data density of quantified cross-links, and thus the resolution of QCLMS. The entire procedure takes ~1-3 weeks.