Use Of Isotope Labeling Research Articles

Assessing microbial plastic degradation requires robust methods.

Plastics are versatile materials that have the potential to propel humanity towards circularity and ultimate societal sustainability. However, the escalating concern surrounding plastic pollution has garnered significant attention, leading to widespread negative perceptions of these materials. Here, we question the role microbes may play in plastic pollution bioremediation by (i) defining polymer biodegradability (i.e., recalcitrant, hydrolysable and biodegradable polymers) and (ii) reviewing best practices for evaluating microbial biodegradation of plastics. We establish recommendations to facilitate the implementation of rigorous methodologies in future studies on plastic biodegradation, aiming to push this field towards the use of isotopic labelling to confirm plastic biodegradation and further determine the molecular mechanisms involved.

Estradiol, Estrone and Ethinyl Estradiol Metabolism Studied by High Resolution LC-MS/MS Using Stable Isotope Labeling and Trapping of Reactive Metabolites.

Biotransformation reactions that xenobiotics undergo during their metabolism are crucial for their proper excretion from the body, but can also be a source of toxicity, especially in the case of reactive metabolite formation. Unstable, reactive metabolites are capable of covalent binding to proteins, and have often been linked to liver damage and other undesired side effects. A common technique to assess the formation of reactive metabolites employs trapping them in vitro with glutathione and characterizing the resulting adducts by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Some endogenous compounds, however, can interfere with xenobiotic metabolites of interest, making the analysis more difficult. This study demonstrates the usefulness of isotope-labeled compounds to detect and elucidate the structures of both stable metabolites and trapped adducts of three estrogen analogs using an untargeted LC-MS/MS workflow. The metabolism of estradiol, estrone and ethinyl estradiol was investigated. Unlabeled and deuterated versions of these three compounds were incubated with human or rat liver microsomes in the presence of two different trapping agents, namely glutathione and N-acetylcysteine. The detection of closely eluting deuterated peaks allowed us to confirm the formation of several known metabolites, as well as many previously uncharacterized ones. The structure of each adduct was elucidated by the detailed analysis of high-resolution MS/MS spectra for elucidating fragmentation pathways with accurate mass measurements. The use of isotopic labeling was crucial in helping confirm many metabolites and adduct structures, as well as removing endogenous interferences.

A rich catalog of C\u2013C bonded species formed in CO2 reduction on a plasmonic photocatalyst

The understanding and rational design of heterogeneous catalysts for complex reactions, such as CO2 reduction, requires knowledge of elementary steps and chemical species prevalent on the catalyst surface under operating conditions. Using in situ nanoscale surface-enhanced Raman scattering, we probe the surface of a Ag nanoparticle during plasmon-excitation-driven CO2 reduction in water. Enabled by the high spatiotemporal resolution and surface sensitivity of our method, we detect a rich array of C1–C4 species formed on the photocatalytically active surface. The abundance of multi-carbon compounds, such as butanol, suggests the favorability of kinetically challenging C–C coupling on the photoexcited Ag surface. Another advance of this work is the use of isotope labeling in nanoscale probing, which allows confirmation that detected species are the intermediates and products of the catalytic reaction rather than spurious contaminants. The surface chemical knowledge made accessible by our approach will inform the modeling and engineering of catalysts.

Novel Secondary Ion Mass Spectrometry Methods for the Examination of Metabolic Effects at the Cellular and Subcellular Levels.

The behavior of an animal has substantial effects on its metabolism. Such effects, including changes in the lipid composition of different organs, or changes in the turnover of the proteins, have typically been observed using liquid mass spectrometry methods, averaging the effect of animal behavior across tissue samples containing multiple cells. These methods have provided the scientific community with valuable information, but have limited resolution, making it difficult if not impossible to examine metabolic effects at the cellular and subcellular levels. Recent advances in the field of secondary ion mass spectrometry (SIMS) have made it possible to examine the metabolic effects of animal behavior with high resolution at the nanoscale, enabling the analysis of the metabolic effects of behavior on individual cells. In this review we summarize and present these emerging methods, beginning with an overview of the SIMS technique. We then discuss the specific application of nanoscale SIMS (NanoSIMS) to examine cell behavior. This often requires the use of isotope labeling to highlight specific sections of the cell for analysis, an approach that is presented at length in this review article. We also present SIMS applications concerning animal and cell behavior, from development and aging to changes in the cellular activity programs. We conclude that the emerging group of SIMS technologies represents an exciting set of tools for the study of animal behavior and of its effects on internal metabolism at the smallest possible scales.

A high sensitivity, high resolution tandem mass spectrometer to research low-energy, reactive ion-surface interactions.

The device described is the combination of two mass spectrometers, with a surface sample placed between them. Its aim is to allow for detailed research on low-energy ion-surface interactions, involving and triggering surface chemistry. This task is fulfilled by a carefully chosen geometry: Projectile ions from an electron impact source are mass-per-charge selected using a quadrupole. Such continuous bombardment allows for good control of the surface condition. Species emerging from the collisions are focused onto a beam and analyzed using a purpose-built orthogonal pulsing time-of-flight mass spectrometer. Neutral species can be post-ionized using a second electron impact source. Neutral gases can be adsorbed to the surface from the gas phase in a controlled manner, using a feedback-controlled pressure regulator. In order to minimize the discrimination of secondary ions, the distance from the surface to the analyzing mass spectrometer system was kept as short as possible and the acceptance angle of the lens system as large as possible. This increased the sensitivity five orders of magnitude compared to its predecessor. The rigorous use of computer aided design software is responsible for the successful commissioning of the new device. This article describes first which parameters can be measured or controlled. Then, these are linked to the physical processes that occur in reactive ion-surface interactions. Next, the design goal and the design implementation are presented. In the end, a performance comparison, measurements of hydrogen surface chemistry with extensive use of isotope labeling, and measurements of post-ionized beryllium are presented.

Isotope Labelling for Reaction Mechanism Analysis in DBD Plasma Processes

Dielectric barrier discharge (DBD) plasmas and plasma catalysis are becoming an alternative procedure to activate various gas phase reactions. A low-temperature and normal operating pressure are the main advantages of these processes, but a limited energy efficiency and little selectivity control hinder their practical implementation. In this work, we propose the use of isotope labelling to retrieve information about the intermediate reactions that may intervene during the DBD processes contributing to a decrease in their energy efficiency. The results are shown for the wet reforming reaction of methane, using D2O instead of H2O as reactant, and for the ammonia synthesis, using NH3/D2/N2 mixtures. In the two cases, it was found that a significant amount of outlet gas molecules, either reactants or products, have deuterium in their structure (e.g., HD for hydrogen, CDxHy for methane, or NDxHy for ammonia). From the analysis of the evolution of the labelled molecules as a function of power, useful information has been obtained about the exchange events of H by D atoms (or vice versa) between the plasma intermediate species. An evaluation of the number of these events revealed a significant progression with the plasma power, a tendency that is recognized to be detrimental for the energy efficiency of reactant to product transformation. The labelling technique is proposed as a useful approach for the analysis of plasma reaction mechanisms.

Historical perspective: phosphatidylserine and phosphatidylethanolamine from the 1800s to the present

This article provides a historical account of the discovery, chemistry, and biochemistry of two ubiquitous phosphoglycerolipids, phosphatidylserine (PS) and phosphatidylethanolamine (PE), including the ether lipids. In addition, the article describes the biosynthetic pathways for these phospholipids and how these pathways were elucidated. Several unique functions of PS and PE in mammalian cells in addition to their ability to define physical properties of membranes are discussed. For example, the translocation of PS from the inner to the outer leaflet of the plasma membrane of cells occurs during apoptosis and during some other specific physiological processes, and this translocation is responsible for profound life-or-death events. Moreover, mitochondrial function is severely impaired when the PE content of mitochondria is reduced below a threshold level. The discovery and implications of the existence of membrane contact sites between the endoplasmic reticulum and mitochondria and their relevance for PS and PE metabolism, as well as for mitochondrial function, are also discussed. Many of the recent advances in these fields are due to the use of isotope labeling for tracing biochemical pathways. In addition, techniques for disruption of specific genes in mice are now widely used and have provided major breakthroughs in understanding the roles and metabolism of PS and PE in vivo.

Sensing site-specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides.

Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site-specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini-review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra.

Effect of cholesterol on the molecular structure and transitions in a clinical-grade lung surfactant extract

The lipid-protein film covering the interface of the lung alveolar in mammals is vital for proper lung function and its deficiency is related to a range of diseases. Here we present a molecular-level characterization of a clinical-grade porcine lung surfactant extract using a multitechnique approach consisting of [Formula: see text]-[Formula: see text] solid-state nuclear magnetic spectroscopy, small- and wide-angle X-ray scattering, and mass spectrometry. The detailed characterization presented for reconstituted membranes of a lung extract demonstrates that the molecular structure of lung surfactant strongly depends on the concentration of cholesterol. If cholesterol makes up about 11% of the total dry weight of lung surfactant, the surfactant extract adopts a single liquid-ordered lamellar phase, [Formula: see text], at physiological temperatures. This [Formula: see text] phase gradually changes into a liquid-disordered lamellar phase, [Formula: see text], when the temperature is increased by a few degrees. In the absence of cholesterol the system segregates into one lamellar gel phase and one [Formula: see text] phase. Remarkably, it was possible to measure a large set of order parameter magnitudes [Formula: see text] from the liquid-disordered and -ordered lamellar phases and assign them to specific C-H bonds of the phospholipids in the biological extract with no use of isotopic labeling. These findings with molecular details on lung surfactant mixtures together with the presented NMR methodology may guide further development of pulmonary surfactant pharmaceuticals that better mimic the physiological self-assembly compositions for treatment of pathological states such as respiratory distress syndrome.

Elemental Analysis of Complex Organic Aerosol Using Isotopic Labeling and Unit-Resolution Mass Spectrometry

Elemental analysis of unit-mass resolution (UMR) mass spectra is limited by the amount of information available to definitively elucidate the molecular formula of a molecule ionized by electron impact. The problem is compounded when a mixture of organic molecules (such as those found in organic aerosols) is analyzed without the benefit of prior separation. For this reason, quadrupole mass spectrometry is not usually suited to the elemental analysis of organic mixtures. Here, we present a mathematical method for the elemental analysis of UMR mass spectra of a complex organic aerosol through the use of isotopic labeling. Quadrupole aerosol mass spectrometry was used to obtain UMR data of (13)C-labeled and unlabeled aerosol generated by far ultraviolet (FUV) photochemistry of gas mixtures containing 0.1% of either CH4 or (13)CH4 in N2. In this method, the differences in the positions of ion groups in the resulting spectra are used to estimate the mass fraction of carbon in the aerosol, and estimation of the remaining elements follows. Analysis of the UMR data yields an elemental composition of 63 ± 7% C, 8 ± 1% H, and 29 ± 7% N by mass. Unlabeled aerosols formed under the same conditions are found by high-resolution time-of-flight aerosol mass spectrometry to have an elemental composition of 63 ± 3% C, 8 ± 1% H, 20 ± 4% N, and 9 ± 3% O by mass, in good agreement with the UMR method. This favorable comparison verifies the method, which expands the UMR mass spectrometry toolkit.

FTIR study of CPD photolyase with substrate in single strand DNA.

Photolyases (PHRs) utilize near UV/blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer (CPD)-PHR binds flavin adenine dinucleotide (FAD) as a cofactor and repairs CPD lesions in double-stranded DNA. To understand the activation and repair mechanism of CPD-PHR, we applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to CPD-PHR, whose signals were identified by use of isotope-labeling. To further investigate the enzymatic function, here we study the activation and repair mechanism of CPD-PHR with the substrate in single strand DNA, and the obtained FTIR spectra are compared with those in double-stranded DNA, the natural substrate. The difference spectra of photoactivation, the fully-reduced (FADH−) minus semiquinone (FADH•) spectra, are almost identical in the presence of single strand and double-stranded DNA, except for slight spectral modification in the amide-I region. On the other hand, the difference spectra of photorepair were highly substrate dependent. Strong bands of the C=O stretch (1,720–1,690 cm−1) and phosphate vibrations (1,090–1,060 cm−1) of double-stranded DNA may have disappeared in the case of single strand DNA. However, an isotope-labeled enzyme study revealed that spectral features upon DNA repair are similar between both substrates, and the main reason for the apparent spectral difference originates from structural flexibility of DNA after repair.

Chylomicrons produced by Caco-2 cells contained ApoB-48 with diameter of 80-200 nm.

The small intestine generally transports dietary fats to circulation in triglyceride (TG)‐rich lipoproteins. The two main intestinal lipoproteins are chylomicron (CM) and very low‐density lipoprotein (VLDL). Unfortunately, studies on the CM biogenesis and intestinal transport of dietary fats have been hampered by the lack of an adequate in vitro model. In this study, we investigated the possible factors that might increase the efficiency of CM production by Caco‐2 cells. We utilized sequential NaCl gradient ultracentrifugation to isolate the CMs that were secreted by the Caco‐2 cells. To confirm the successful isolation of the CMs, we performed Fat Red 7B staining, TG reading, apolipoprotein B (ApoB) measurement, and transmission electron microcopy (TEM) analysis. We then tested the effects of cell differentiation, oleic acid, mono‐olein, egg lecithin, incubation time, and collagen matrix on CM secretion. We found that cell differentiation, oleic acid, and lecithin were critical for CM secretion. Using the Transwell system, we further confirmed that the CMs produced by our Caco‐2 cells contained significant amount of TGs and ApoB‐48 such that they could be detected without the use of isotope labeling. In conclusion, when fully differentiated Caco‐2 were challenged with oleic acid, lecithin, and sodium taurocholate, 21% of their total number of lipoproteins were CMs with the diameter of 80–200 nm.

Efficient polarization transfer between spin-1/2 and [formula omitted]N nuclei in solid-state MAS NMR spectroscopy

Polarization transfer between spin-1/2 nuclei and quadrupolar spin-1 nuclei such as 14N in solid-state NMR is severely challenged by the typical presence of large quadrupole coupling interactions. This has effectively prevented the use of the abundant 14N spin as a probe to structural information and its use as an element in multi-dimensional solid-state NMR correlation experiments for assignment and structural characterization. In turn, this has been a contributing factor to the extensive use of isotope labeling in biological solid-state NMR, where 14N is replaced with 15N. The alternative strategy of using the abundant 14N spins calls for methods enabling efficient polarization transfer between 14N and its binding partners. This work demonstrates that the recently introduced RESPIRATIONCP transfer method can be optimized to achieve efficient 1H ↔14N polarization transfer under magic angle spinning conditions. The method is demonstrated numerically and experimentally on powder samples of NH4NO3 and l-alanine.

Place du kinésithérapeute au sein des réseaux des maladies neuromusculaires

Polarization transfer between spin-1/2 nuclei and quadrupolar spin-1 nuclei such as 14N in solid-state NMR is severely challenged by the typical presence of large quadrupole coupling interactions. This has effectively prevented the use of the abundant 14N spin as a probe to structural information and its use as an element in multi-dimensional solid-state NMR correlation experiments for assignment and structural characterization. In turn, this has been a contributing factor to the extensive use of isotope labeling in biological solid-state NMR, where 14N is replaced with 15N. The alternative strategy of using the abundant 14N spins calls for methods enabling efficient polarization transfer between 14N and its binding partners. This work demonstrates that the recently introduced RESPIRATIONCP transfer method can be optimized to achieve efficient 1H ↔ 14N polarization transfer under magic angle spinning conditions. The method is demonstrated numerically and experimentally on powder samples of NH4NO3 and l-alanine.

Mechanism of IAPP amyloid fibril formation involves an intermediate with a transient β-sheet

Amyloid formation is implicated in more than 20 human diseases, yet the mechanism by which fibrils form is not well understood. We use 2D infrared spectroscopy and isotope labeling to monitor the kinetics of fibril formation by human islet amyloid polypeptide (hIAPP or amylin) that is associated with type 2 diabetes. We find that an oligomeric intermediate forms during the lag phase with parallel β-sheet structure in a region that is ultimately a partially disordered loop in the fibril. We confirm the presence of this intermediate, using a set of homologous macrocyclic peptides designed to recognize β-sheets. Mutations and molecular dynamics simulations indicate that the intermediate is on pathway. Disrupting the oligomeric β-sheet to form the partially disordered loop of the fibrils creates a free energy barrier that is the origin of the lag phase during aggregation. These results help rationalize a wide range of previous fragment and mutation studies including mutations in other species that prevent the formation of amyloid plaques.

Hydrodeoxygenation by deuterium gas – a powerful way to provide insight into the reaction mechanisms

This study demonstrates the use of isotopic labelling and NMR to study the HDO process. As far as we know, this is the first reported effort to trace the incorporation of hydrogen in the HDO process of lignin pyrolysis oil thereby providing key fundamental insight into its reaction mechanism.

Comparison of ryegrass and red clover on the fermentation pattern, microbial community and efficiency of diet utilisation in the rumen simulation technique (Rusitec)

An in vitro experiment was designed to investigate the effects of incubating two forages with a different energy/nitrogen (N) ratio [perennial ryegrass (GR) vs red clover (RC)] on the efficiency of N utilisation by rumen microbes. Second-cut forages were incubated in artificial rumen fermenters (n = 8). Ryegrass represented a supply of quickly available N and energy for the rumen microorganism which led to a rapid fermentation and bacterial growth 2–4 h after feeding. Ryegrass also promoted greater numbers of anaerobic fungi, methanogens and cellulolytic bacteria, which tended to increase neutral detergent fibre disappearance, gas production, volatile fatty acid and methane production than observed using RC diets. On the contrary, RC provided slowly degradable N and energy, which led to a relatively slow bacterial growth (4–8 h after feeding). In terms of diet utilisation, RC diets promoted a higher N outflow (mainly as undegraded-N) and efficiency of microbial protein synthesis per organic matter disappeared. Even so, microbial protein yield was similar on both diets indicating a better N capture by microorganisms fed GR than in those fed RC diets. The use of 15N-labelled forages demonstrated that this high ammonia incorporation by bacteria-fed GR occurred mainly during the early fermentation coinciding with the highest bacterial growth. In conclusion, this experiment demonstrated that the use of isotopic labelling combined with molecular techniques provided an insight into forage utilisation by the rumen microbes; GR diets led to a better efficiency of N utilisation compared with RC; moreover the lower N outflow on GR diets may be partially compensated for a higher proportion of microbial protein leaving the system and the greater volatile fatty acid production. These findings seem to indicate that RC grazing may increase the N pollution compared with GR without substantial improvements on the rumen function, however this must be confirmed in vivo.

Matrix-free dynamic nuclear polarization enables solid-state NMR 13C–13C correlation spectroscopy of proteins at natural isotopic abundance

We introduce a general approach for dynamic nuclear polarization (DNP) enhanced solid-state NMR that overcomes the current problems in DNP experiments caused by the use of frozen solutions. Notably, we report for the first time a 2D (13)C-(13)C correlation spectrum of a protein without the use of isotopic labeling.

Use of a novel proteomic method to determine potential markers of preterm labour

IntroductionProteomic quantification methods have developed significantly, due to the use of isotope labelling. More recently label-free methods have been developed to allow faster and simpler analysis. We have conducted a...

Medium energy ion scattering of Gr on SiC(0001) and Si(100)

Depth profiling of graphene with high-resolution ion beam analysis is a practical method for analysis of monolayer thicknesses of graphene. Not only is the energy resolution sufficient to resolve graphene from underlying SiC but by use of isotope labeling it is possible to tag graphene generated from reacted ethylene. Furthermore, we are able to analyze graphene supported by oxidized Si(100) substrates, allowing the study of graphene films grown by chemical vapor deposition on metal and transferred to silicon. This introduces a powerful method to explore the fundamentals of graphene formation.