Sample records for efficient isotopic labeling

  1. Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture

    PubMed Central

    Snyder, Nathaniel W.; Tombline, Gregory; Worth, Andrew J.; Parry, Robert C.; Silvers, Jacob A.; Gillespie, Kevin P.; Basu, Sankha S.; Millen, Jonathan; Goldfarb, David S.; Blair, Ian A.

    2015-01-01

    Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and β-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the gold standard for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope labeled metabolites such as acyl-coenzyme A thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell media with commercially available [13C3 15N1]-pantothenic acid, mammalian cells exclusively incorporated [13C3 15N1]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope labeled CoA and acyl-CoAs from [13C3 15N1]-pantothenate using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC) in Pan6 deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof-of-concept for generating other labeled metabolites in yeast mutants. PMID:25572876

  2. A novel design for a dual stable isotope continuous labeling chamber: results on labeling efficiency and C and N allocation in Andropogon gerardii

    NASA Astrophysics Data System (ADS)

    Soong, J.; Stewart, C.; Reuss, D.; Pinney, C.; Cotrufo, F. M.

    2010-12-01

    The use of stable isotope enriched plant material can provide an unobstructed method of studying ecosystem nutrient dynamics between plants, soil, and atmosphere. However, the production of uniformly labeled perennial plant material is challenging due to plant physiological constraints and the mechanics of building and operating an isotope labeling system. In this study we present the design of a novel dual 13C and 15N continuous isotope labeling chamber located at Colorado State University. The chamber is equipped with automatic controls for CO2 concentration, temperature, and humidity, and has successfully been used to grow and label the tallgrass perennial Andropogon gerardii in pots from rhizomes. Three different nitrogen fertilization levels were applied to assess how substrate availability may alter growth and overall performance in the system. The efficiency of the 13C and 15N labeling chamber, its design and overall performance, as well as a full C, N, 13C, and 15N budget of the aboveground biomass, belowground biomass, and soil will be presented. Solid samples were analyzed on an EA-IRMS, while air samples from the chamber were analyzed using a precon-GC-IRMS system. The dual stable isotope labeled A. gerardii produced from this chamber will be used in a decomposition experiment to quantify the relative contribution of aboveground litter derived C to soil respiration, dissolved organic carbon, and various soil organic matter pools. Based on the results of our A. gerardii 13C and 15N labeling experiment we believe that this chamber design can be used to successfully produce dual stable isotope labeled plants for a wide variety of terrestrial nutrient flux experiments.

  3. Simple, rapid method for the preparation of isotopically labeled formaldehyde

    DOEpatents

    Hooker, Jacob Matthew [Port Jefferson, NY; Schonberger, Matthias [Mains, DE; Schieferstein, Hanno [Aabergen, DE; Fowler, Joanna S [Bellport, NY

    2011-10-04

    Isotopically labeled formaldehyde (*C.sup..sctn.H.sub.2O) is prepared from labeled methyl iodide (*C.sup..sctn.H.sub.3I) by reaction with an oxygen nucleophile having a pendant leaving group. The mild and efficient reaction conditions result in good yields of *C.sup..sctn.H.sub.2O with little or no *C isotopic dilution. The simple, efficient production of .sup.11CH.sub.2O is described. The use of the .sup.11CH.sub.2O for the formation of positron emission tomography tracer compounds is described. The reaction can be incorporated into automated equipment available to radiochemistry laboratories. The isotopically labeled formaldehyde can be used in a variety of reactions to provide radiotracer compounds for imaging studies as well as for scintillation counting and autoradiography.

  4. Stable isotope labeling by essential nutrients in cell culture for preparation of labeled coenzyme A and its thioesters.

    PubMed

    Basu, Sankha S; Mesaros, Clementina; Gelhaus, Stacy L; Blair, Ian A

    2011-02-15

    Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

  5. IsoCor: correcting MS data in isotope labeling experiments.

    PubMed

    Millard, Pierre; Letisse, Fabien; Sokol, Serguei; Portais, Jean-Charles

    2012-05-01

    Mass spectrometry (MS) is widely used for isotopic labeling studies of metabolism and other biological processes. Quantitative applications-e.g. metabolic flux analysis-require tools to correct the raw MS data for the contribution of all naturally abundant isotopes. IsoCor is a software that allows such correction to be applied to any chemical species. Hence it can be used to exploit any isotopic tracer, from well-known ((13)C, (15)N, (18)O, etc) to unusual ((57)Fe, (77)Se, etc) isotopes. It also provides new features-e.g. correction for the isotopic purity of the tracer-to improve the accuracy of quantitative isotopic studies, and implements an efficient algorithm to process large datasets. Its user-friendly interface makes isotope labeling experiments more accessible to a wider biological community. IsoCor is distributed under OpenSource license at http://metasys.insa-toulouse.fr/software/isocor/

  6. SAIL--stereo-array isotope labeling.

    PubMed

    Kainosho, Masatsune; Güntert, Peter

    2009-11-01

    Optimal stereospecific and regiospecific labeling of proteins with stable isotopes enhances the nuclear magnetic resonance (NMR) method for the determination of the three-dimensional protein structures in solution. Stereo-array isotope labeling (SAIL) offers sharpened lines, spectral simplification without loss of information and the ability to rapidly collect and automatically evaluate the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as before. This review gives an overview of stable isotope labeling methods for NMR spectroscopy with proteins and provides an in-depth treatment of the SAIL technology.

  7. Chemo-enzymatic synthesis of isotopically labeled nicotinamide riboside.

    PubMed

    Tran, Ai; Yokose, Ryota; Cen, Yana

    2018-05-15

    As a cofactor for numerous reactions, NAD+ is found widely dispersed across many maps of cellular metabolism. This core redox role alone makes the biosynthesis of NAD+ of great interest. Recent studies have revealed new biological roles for NAD+ as a substrate for diverse enzymes that regulate a broad spectrum of key cellular tasks. These NAD+-consuming enzymes further highlight the importance of understanding NAD+ biosynthetic pathways. In this study, we developed a chemo-enzymatic synthesis of isotopically labeled NAD+ precursor, nicotinamide riboside (NR). The synthesis of NR isotopomers allowed us to unambiguously determine that NR is efficiently converted to NAD+ in the cellular environment independent of degradation to nicotinamide, and it is incorporated into NAD+ in its intact form. The versatile synthetic method along with the isotopically labeled NRs will provide powerful tools to further decipher the important yet complicated NAD+ metabolism.

  8. Optimal design of isotope labeling experiments.

    PubMed

    Yang, Hong; Mandy, Dominic E; Libourel, Igor G L

    2014-01-01

    Stable isotope labeling experiments (ILE) constitute a powerful methodology for estimating metabolic fluxes. An optimal label design for such an experiment is necessary to maximize the precision with which fluxes can be determined. But often, precision gained in the determination of one flux comes at the expense of the precision of other fluxes, and an appropriate label design therefore foremost depends on the question the investigator wants to address. One could liken ILE to shadows that metabolism casts on products. Optimal label design is the placement of the lamp; creating clear shadows for some parts of metabolism and obscuring others.An optimal isotope label design is influenced by: (1) the network structure; (2) the true flux values; (3) the available label measurements; and, (4) commercially available substrates. The first two aspects are dictated by nature and constrain any optimal design. The second two aspects are suitable design parameters. To create an optimal label design, an explicit optimization criterion needs to be formulated. This usually is a property of the flux covariance matrix, which can be augmented by weighting label substrate cost. An optimal design is found by using such a criterion as an objective function for an optimizer. This chapter uses a simple elementary metabolite units (EMU) representation of the TCA cycle to illustrate the process of experimental design of isotope labeled substrates.

  9. Highly efficient preparation of selectively isotope cluster-labeled long chain fatty acids via two consecutive C(sp3)-C(sp3) cross-coupling reactions.

    PubMed

    Lethu, Sébastien; Matsuoka, Shigeru; Murata, Michio

    2014-02-07

    An efficient synthesis involving two copper-catalyzed alkyl-alkyl coupling reactions has been designed to easily access doubly isotope-labeled fatty acids. Such NMR- and IR-active compounds were obtained in excellent overall yields and will be further used for determining the conformation of an alkyl chain of lipidic biomolecules upon interaction with proteins.

  10. Isotope-Labeled Composition B for Tracing Detonation Signatures

    NASA Astrophysics Data System (ADS)

    Manner, Virginia; Podlesak, David; Huber, Rachel; Amato, Ronald; Giambra, Anna; Bowden, Patrick; Hartline, Ernest; Dattelbaum, Dana

    2017-06-01

    To better understand how solid carbon forms and evolves during detonation, we have prepared Composition B with 13 C and 15 N-labeled 1,3,5-trinitro-1,3,5-triazacyclohexane (RDX) and 2,4,6-trinitrotoluene (TNT) in order to trace the formation of soot from the carbon and nitrogen atoms in these explosives. Isotope-labeling of explosives has been performed in the recent past for a variety of reasons, including environmental remediation and reaction mechanism studies. Because it is expensive and time consuming to prepare these materials, and our detection equipment only requires trace amounts of isotopes, we have prepared fully-labeled materials and substituted them into unlabeled RDX and TNT at less than the 1% level. We will discuss the preparation and full characterization of this labeled Composition B, the detonation tests performed, along with the results of the post-detonation soot analysis. Various detonation models predict differing amounts and forms of carbon and nitrogen; these isotopically-labeled precursors have allowed these models to be tested.

  11. [Progress in stable isotope labeled quantitative proteomics methods].

    PubMed

    Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

    2013-06-01

    Quantitative proteomics is an important research field in post-genomics era. There are two strategies for proteome quantification: label-free methods and stable isotope labeling methods which have become the most important strategy for quantitative proteomics at present. In the past few years, a number of quantitative methods have been developed, which support the fast development in biology research. In this work, we discuss the progress in the stable isotope labeling methods for quantitative proteomics including relative and absolute quantitative proteomics, and then give our opinions on the outlook of proteome quantification methods.

  12. Stable isotope dimethyl labelling for quantitative proteomics and beyond

    PubMed Central

    Hsu, Jue-Liang; Chen, Shu-Hui

    2016-01-01

    Stable-isotope reductive dimethylation, a cost-effective, simple, robust, reliable and easy-to- multiplex labelling method, is widely applied to quantitative proteomics using liquid chromatography-mass spectrometry. This review focuses on biological applications of stable-isotope dimethyl labelling for a large-scale comparative analysis of protein expression and post-translational modifications based on its unique properties of the labelling chemistry. Some other applications of the labelling method for sample preparation and mass spectrometry-based protein identification and characterization are also summarized. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644970

  13. 13C metabolic flux analysis: optimal design of isotopic labeling experiments.

    PubMed

    Antoniewicz, Maciek R

    2013-12-01

    Measuring fluxes by 13C metabolic flux analysis (13C-MFA) has become a key activity in chemical and pharmaceutical biotechnology. Optimal design of isotopic labeling experiments is of central importance to 13C-MFA as it determines the precision with which fluxes can be estimated. Traditional methods for selecting isotopic tracers and labeling measurements did not fully utilize the power of 13C-MFA. Recently, new approaches were developed for optimal design of isotopic labeling experiments based on parallel labeling experiments and algorithms for rational selection of tracers. In addition, advanced isotopic labeling measurements were developed based on tandem mass spectrometry. Combined, these approaches can dramatically improve the quality of 13C-MFA results with important applications in metabolic engineering and biotechnology. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Measuring the labeling efficiency of pseudocontinuous arterial spin labeling.

    PubMed

    Chen, Zhensen; Zhang, Xingxing; Yuan, Chun; Zhao, Xihai; van Osch, Matthias J P

    2017-05-01

    Optimization and validation of a sequence for measuring the labeling efficiency of pseudocontinuous arterial spin labeling (pCASL) perfusion MRI. The proposed sequence consists of a labeling module and a single slice Look-Locker echo planar imaging readout. A model-based algorithm was used to calculate labeling efficiency from the signal acquired from the main brain-feeding arteries. Stability of the labeling efficiency measurement was evaluated with regard to the use of cardiac triggering, flow compensation and vein signal suppression. Accuracy of the measurement was assessed by comparing the measured labeling efficiency to mean brain pCASL signal intensity over a wide range of flip angles as applied in the pCASL labeling. Simulations show that the proposed algorithm can effectively calculate labeling efficiency when correcting for T1 relaxation of the blood spins. Use of cardiac triggering and vein signal suppression improved stability of the labeling efficiency measurement, while flow compensation resulted in little improvement. The measured labeling efficiency was found to be linearly (R = 0.973; P < 0.001) related to brain pCASL signal intensity over a wide range of pCASL flip angles. The optimized labeling efficiency sequence provides robust artery-specific labeling efficiency measurement within a short acquisition time (∼30 s), thereby enabling improved accuracy of pCASL CBF quantification. Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine Magn Reson Med 77:1841-1852, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

  15. Highly Efficient Quantum Sieving in Porous Graphene-like Carbon Nitride for Light Isotopes Separation

    NASA Astrophysics Data System (ADS)

    Qu, Yuanyuan; Li, Feng; Zhou, Hongcai; Zhao, Mingwen

    2016-01-01

    Light isotopes separation, such as 3He/4He, H2/D2, H2/T2, etc., is crucial for various advanced technologies including isotope labeling, nuclear weapons, cryogenics and power generation. However, their nearly identical chemical properties made the separation challenging. The low productivity of the present isotopes separation approaches hinders the relevant applications. An efficient membrane with high performance for isotopes separation is quite appealing. Based on first-principles calculations, we theoretically demonstrated that highly efficient light isotopes separation, such as 3He/4He, can be reached in a porous graphene-like carbon nitride material via quantum sieving effect. Under moderate tensile strain, the quantum sieving of the carbon nitride membrane can be effectively tuned in a continuous way, leading to a temperature window with high 3He/4He selectivity and permeance acceptable for efficient isotopes harvest in industrial application. This mechanism also holds for separation of other light isotopes, such as H2/D2, H2/T2. Such tunable quantum sieving opens a promising avenue for light isotopes separation for industrial application.

  16. Existing and emerging technologies for measuring stable isotope labelled retinol in biological samples: isotope dilution analysis of body retinol stores.

    PubMed

    Preston, Tom

    2014-01-01

    This paper discusses some of the recent improvements in instrumentation used for stable isotope tracer measurements in the context of measuring retinol stores, in vivo. Tracer costs, together with concerns that larger tracer doses may perturb the parameter under study, demand that ever more sensitive mass spectrometric techniques are developed. GCMS is the most widely used technique. It has high sensitivity in terms of sample amount and uses high resolution GC, yet its ability to detect low isotope ratios is limited by background noise. LCMSMS may become more accessible for tracer studies. Its ability to measure low level stable isotope tracers may prove superior to GCMS, but it is isotope ratio MS (IRMS) that has been designed specifically for low level stable isotope analysis through accurate analysis of tracer:tracee ratios (the tracee being the unlabelled species). Compound-specific isotope analysis, where GC is interfaced to IRMS, is gaining popularity. Here, individual 13C-labelled compounds are separated by GC, combusted to CO2 and transferred on-line for ratiometric analysis by IRMS at the ppm level. However, commercially-available 13C-labelled retinol tracers are 2 - 4 times more expensive than deuterated tracers. For 2H-labelled compounds, GC-pyrolysis-IRMS has now become more generally available as an operating mode on the same IRMS instrument. Here, individual compounds are separated by GC and pyrolysed to H2 at high temperature for analysis by IRMS. It is predicted that GC-pyrolysis-IRMS will facilitate low level tracer procedures to measure body retinol stores, as has been accomplished in the case of fatty acids and amino acids. Sample size requirements for GC-P-IRMS may exceed those of GCMS, but this paper discusses sample preparation procedures and predicts improvements, particularly in the efficiency of sample introduction.

  17. Glycan reductive isotope labeling for quantitative glycomics.

    PubMed

    Xia, Baoyun; Feasley, Christa L; Sachdev, Goverdhan P; Smith, David F; Cummings, Richard D

    2009-04-15

    Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed glycan reductive isotope labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [(12)C(6)]aniline and [(13)C(6)]aniline. These dual-labeled aniline-tagged glycans can be recovered by reverse-phase chromatography and can be quantified based on ultraviolet (UV) absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins, using this method. This technique allows linear relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of glycomics.

  18. Gluconeogenesis from labeled carbon: estimating isotope dilution

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kelleher, J.K.

    1986-03-01

    To estimate the rate of gluconeogenesis from steady-state incorporation of labeled 3-carbon precursors into glucose, isotope dilution must be considered so that the rate of labeling of glucose can be quantitatively converted to the rate of gluconeogenesis. An expression for the value of this isotope dilution can be derived using mathematical techniques and a model of the tricarboxylic acid (TCA) cycle. The present investigation employs a more complex model than that used in previous studies. This model includes the following pathways that may affect the correction for isotope dilution: 1) flux of 3-carbon precursor to the oxaloacetate pool via acetyl-CoAmore » and the TCA cycle; 2) flux of 4- or 5-carbon compounds into the TCA cycle; 3) reversible flux between oxaloacetate (OAA) and pyruvate and between OAA and fumarate; 4) incomplete equilibrium between OAA pools; and 5) isotope dilution of 3-carbon tracers between the experimentally measured pool and the precursor for the TCA-cycle OAA pool. Experimental tests are outlined which investigators can use to determine whether these pathways are significant in a specific steady-state system. The study indicated that flux through these five pathways can significantly affect the correction for isotope dilution. To correct for the effects of these pathways an alternative method for calculating isotope dilution is proposed using citrate to relate the specific activities of acetyl-CoA and OAA.« less

  19. Isotope labeling for studying RNA by solid-state NMR spectroscopy.

    PubMed

    Marchanka, Alexander; Kreutz, Christoph; Carlomagno, Teresa

    2018-04-12

    Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.

  20. Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling[S

    PubMed Central

    Cai, Tanxi; Shu, Qingbo; Liu, Peibin; Niu, Lili; Guo, Xiaojing; Ding, Xiang; Xue, Peng; Xie, Zhensheng; Wang, Jifeng; Zhu, Nali; Wu, Peng; Niu, Lili; Yang, Fuquan

    2016-01-01

    Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling. PMID:26733148

  1. Simplified Synthesis of Isotopically Labeled 5,5-Dimethyl-pyrroline N-Oxide

    PubMed Central

    Leinisch, Fabian; Jiang, JinJie; Deterding, Leesa J.; Mason, Ronald P.

    2011-01-01

    5,5-Dimethylpyrroline N-oxide (15N) and 5,5-di(trideuteromethyl)pyrroline N-oxide were synthesized from the respective isotopically labeled 2-nitropropane analogs obtained from the reaction of sodium nitrate with 2-halopropanes. This facile, straightforward process allows synthesizing isotopically labeled DMPO analogs in a 4-step reaction without special equipment. PMID:21986521

  2. The synthesis of a tritium, carbon-14, and stable isotope-labeled cathepsin C inhibitors.

    PubMed

    Allen, Paul; Bragg, Ryan A; Caffrey, Moya; Ericsson, Cecilia; Hickey, Michael J; Kingston, Lee P; Elmore, Charles S

    2017-02-01

    As part of a medicinal chemistry program aimed at developing a highly potent and selective cathepsin C inhibitor, tritium, carbon-14, and stable isotope-labeled materials were required. The synthesis of tritium-labeled methanesulfonate 5 was achieved via catalytic tritiolysis of a chloro precursor, albeit at a low radiochemical purity of 67%. Tritium-labeled AZD5248 was prepared via a 3-stage synthesis, utilizing amide-directed hydrogen isotope exchange. Carbon-14 and stable isotope-labeled AZD5248 were successfully prepared through modifications of the medicinal chemistry synthetic route, enabling the use of available labeled intermediates. Copyright © 2016 John Wiley & Sons, Ltd.

  3. GLYCAN REDUCTIVE ISOTOPE LABELING (GRIL) FOR QUANTITATIVE GLYCOMICS

    PubMed Central

    Xia, Baoyun; Feasley, Christa L.; Sachdev, Goverdhan P.; Smith, David F.; Cummings, Richard D.

    2009-01-01

    Many diseases and disorders are characterized by quantitative and/or qualitative changes in complex carbohydrates. Mass spectrometry methods show promise in monitoring and detecting these important biological changes. Here we report a new glycomics method, termed Glycan Reductive Isotope Labeling (GRIL), where free glycans are derivatized by reductive amination with the differentially coded stable isotope tags [12C6]-aniline and [13C6]-aniline. These dual-labeled aniline-tagged glycans can be recovered by reversed-phase chromatography and quantified based on UV-absorbance and relative ion abundances. Unlike previously reported isotopically coded reagents for glycans, GRIL does not contain deuterium, which can be chromatographically resolved. Our method shows no chromatographic resolution of differentially labeled glycans. Mixtures of differentially tagged glycans can be directly compared and quantified using mass spectrometric techniques. We demonstrate the use of GRIL to determine relative differences in glycan amount and composition. We analyze free glycans and glycans enzymatically or chemically released from a variety of standard glycoproteins, as well as human and mouse serum glycoproteins using this method. This technique allows for linear, relative quantitation of glycans over a 10-fold concentration range and can accurately quantify sub-picomole levels of released glycans, providing a needed advancement in the field of Glycomics. PMID:19454239

  4. Isotopic labeling of milk disialogangliosides (GD3).

    PubMed

    Reis, Mariza Gomes; Bibiloni, Rodrigo; McJarrow, Paul; MacGibbon, Alastair; Fong, Bertram; Bassett, Shalome; Roy, Nicole; Dos Reis, Marlon Martins

    2016-10-01

    The most abundant ganglioside group in both human milk and bovine milk during the first postnatal week is ganglioside GD3. This group of disialogangliosides forms up to 80% of the total ganglioside content of colostrum. Although dietary gangliosides have shown biological activity such as improvement of cognitive development, gastrointestinal health, and immune function, there is still a gap in our understanding of the molecular mechanisms governing its uptake and the metabolic processes affecting its bioavailability. The use of isotopically labeled ganglioside to track the bioavailability, absorption, distribution, and metabolism of gangliosides may provide key information to bridge this gap. However, isotope labeled GD3 is not commercially available and its preparation has not been described. We report for the first time the preparation of labeled GD3 with stable isotopes. Using alkaline hydrolysis, we were able to selectively remove both acetyl groups from the tetrasaccharide portion of GD3 without promoting significant hydrolysis of the ceramide portion of the molecule to generate N-deacetyl-GD3 (Neu5α2-8Neu5-GD3). The N-deacetyl-GD3 was then chemoselectively re-acetylated in aqueous medium using deuterated acetic anhydride in the presence of Triton X 100 to produce 2 H 6 -GD3 {GD3[(Neu5Ac-11- 2 H 3 )-(Neu5Ac-11- 2 H 3 )]}. This method provided 2 H 6 -GD3 with approximately 60% yield. This compound was characterized by proton nuclear magnetic resonance ( 1 H NMR) and liquid chromatography mass spectrometry (LC-MS). The oral absorption of the 2 H 6 -GD3 was demonstrated using a Sprague-Dawley weaning rats. Our results indicate that some ingested labeled milk gangliosides are absorbed and transported into the bloodstream without modification. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  5. Advances in stable isotope assisted labeling strategies with information science.

    PubMed

    Kigawa, Takanori

    2017-08-15

    Stable-isotope (SI) labeling of proteins is an essential technique to investigate their structures, interactions or dynamics by nuclear magnetic resonance (NMR) spectroscopy. The assignment of the main-chain signals, which is the fundamental first step in these analyses, is usually achieved by a sequential assignment method based on triple resonance experiments. Independently of the triple resonance experiment-based sequential assignment, amino acid-selective SI labeling is beneficial for discriminating the amino acid type of each signal; therefore, it is especially useful for the signal assignment of difficult targets. Various combinatorial selective labeling schemes have been developed as more sophisticated labeling strategies. In these strategies, amino acids are represented by combinations of SI labeled samples, rather than simply assigning one amino acid to one SI labeled sample as in the case of conventional amino acid-selective labeling. These strategies have proven to be useful for NMR analyses of difficult proteins, such as those in large complex systems, in living cells, attached or integrated into membranes, or with poor solubility. In this review, recent advances in stable isotope assisted labeling strategies will be discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Segmental Isotopic Labeling of Proteins for Nuclear Magnetic Resonance

    PubMed Central

    Dongsheng, Liu; Xu, Rong; Cowburn, David

    2009-01-01

    Nuclear Magnetic Resonance (NMR) spectroscopy has emerged as one of the principle techniques of structural biology. It is not only a powerful method for elucidating the 3D structures under near physiological conditions, but also a convenient method for studying protein-ligand interactions and protein dynamics. A major drawback of macromolecular NMR is its size limitation caused by slower tumbling rates and greater complexity of the spectra as size increases. Segmental isotopic labeling allows specific segment(s) within a protein to be selectively examined by NMR thus significantly reducing the spectral complexity for large proteins and allowing a variety of solution-based NMR strategies to be applied. Two related approaches are generally used in the segmental isotopic labeling of proteins: expressed protein ligation and protein trans-splicing. Here we describe the methodology and recent application of expressed protein ligation and protein trans-splicing for NMR structural studies of proteins and protein complexes. We also describe the protocol used in our lab for the segmental isotopic labeling of a 50 kDa protein Csk (C-terminal Src Kinase) using expressed protein ligation methods. PMID:19632474

  7. Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

    PubMed

    Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

    2015-01-01

    Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

  8. A Novel Method for Relative Quantitation of N-Glycans by Isotopic Labeling Using 18O-Water

    PubMed Central

    Tao, Shujuan; Orlando, Ron

    2014-01-01

    Quantitation is an essential aspect of comprehensive glycomics study. Here, a novel isotopic-labeling method is described for N-glycan quantitation using 18O-water. The incorporation of the 18O-labeling into the reducing end of N-glycans is simply and efficiently achieved during peptide-N4-(N-acetyl-β-glucosaminyl) asparagine amidase F release. This process provides a 2-Da mass difference compared with the N-glycans released in 16O-water. A mathematical calculation method was also developed to determine the 18O/16O ratios from isotopic peaks. Application of this method to several standard glycoprotein mixtures and human serum demonstrated that this method can facilitate the relative quantitation of N-glycans over a linear dynamic range of two orders, with high accuracy and reproducibility. PMID:25365792

  9. Doubly labeled water method: in vivo oxygen and hydrogen isotope fractionation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schoeller, D.A.; Leitch, C.A.; Brown, C.

    The accuracy and precision of the doubly labeled water method for measuring energy expenditure are influenced by isotope fractionation during evaporative water loss and CO/sub 2/ excretion. To characterize in vivo isotope fractionation, we collected and isotopically analyzed physiological fluids and gases. Breath and transcutaneous water vapor were isotopically fractionated. The degree of fractionation indicated that the former was fractionated under equilibrium control at 37/sup 0/C, and the latter was kinetically fractionated. Sweat and urine were unfractionated. By use of isotopic balance models, the fraction of water lost via fractionating routes was estimated from the isotopic abundances of body water,more » local drinking water, and dietary solids. Fractionated water loss averaged 23% (SD = 10%) of water turnover, which agreed with our previous estimates based on metabolic rate, but there was a systematic difference between the results based on O/sub 2/ and hydrogen. Corrections for isotopic fractionation of water lost in breath and (nonsweat) transcutaneous loss should be made when using labeled water to measure water turnover or CO/sub 2/ production.« less

  10. Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA).

    PubMed

    Guo, Guangyu; Li, Ning

    2011-07-01

    In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. (15)N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of (15)N or (14)N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the (15)N supply, which led to a uniform incorporation of (15)N into the whole plant protein complement. The incorporation rate (97.43±0.11%) of (15)N into (15)N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of (15)N/(14)N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified (15)N/(14)N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed (15)N-coded and (14)N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

  11. Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

    PubMed

    Terada, Takaho; Yokoyama, Shigeyuki

    2015-01-01

    This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

  12. Stable isotope-labelled feed nutrients to assess nutrient-specific feed passage kinetics in ruminants.

    PubMed

    Warner, Daniel; Dijkstra, Jan; Hendriks, Wouter H; Pellikaan, Wilbert F

    2014-03-30

    Knowledge of digesta passage kinetics in ruminants is essential to predict nutrient supply to the animal in relation to optimal animal performance, environmental pollution and animal health. Fractional passage rates (FPR) of feed are widely used in modern feed evaluation systems and mechanistic rumen models, but data on nutrient-specific FPR are scarce. Such models generally rely on conventional external marker techniques, which do not always describe digesta passage kinetics in a satisfactory manner. Here the use of stable isotope-labelled dietary nutrients as a promising novel tool to assess nutrient-specific passage kinetics is discussed. Some major limitations of this technique include a potential marker migration, a poor isotope distribution in the labelled feed and a differential disappearance rate of isotopes upon microbial fermentation in non-steady state conditions. Such limitations can often be circumvented by using intrinsically stable isotope-labelled plant material. Data are limited but indicate that external particulate markers overestimate rumen FPR of plant fibre compared with the internal stable isotope markers. Stable isotopes undergo the same digestive mechanism as the labelled feed components and are thus of particular interest to specifically measure passage kinetics of digestible dietary nutrients. © 2013 Society of Chemical Industry.

  13. Relative quantification of biomarkers using mixed-isotope labeling coupled with MS

    PubMed Central

    Chapman, Heidi M; Schutt, Katherine L; Dieter, Emily M; Lamos, Shane M

    2013-01-01

    The identification and quantification of important biomarkers is a critical first step in the elucidation of biological systems. Biomarkers take many forms as cellular responses to stimuli and can be manifested during transcription, translation, and/or metabolic processing. Increasingly, researchers have relied upon mixed-isotope labeling (MIL) coupled with MS to perform relative quantification of biomarkers between two or more biological samples. MIL effectively tags biomarkers of interest for ease of identification and quantification within the mass spectrometer by using isotopic labels that introduce a heavy and light form of the tag. In addition to MIL coupled with MS, a number of other approaches have been used to quantify biomarkers including protein gel staining, enzymatic labeling, metabolic labeling, and several label-free approaches that generate quantitative data from the MS signal response. This review focuses on MIL techniques coupled with MS for the quantification of protein and small-molecule biomarkers. PMID:23157360

  14. Highly enriched multiply-labeled stable isotopic compounds as atmospheric tracers

    DOEpatents

    Goldblatt, M.; McInteer, B.B.

    1974-01-29

    Compounds multiply-labeled with stable isotopes and highly enriched in these isotopes are readily capable of detection in tracer experiments involving high dilutions. Thus, for example, /sup 13/C/sup 18/O/sub 2/ provides a useful tracer for following atmospheric pol lution produced as a result of fossil fuel burning. (Official Gazette)

  15. Stable isotope labelling methods in mass spectrometry-based quantitative proteomics.

    PubMed

    Chahrour, Osama; Cobice, Diego; Malone, John

    2015-09-10

    Mass-spectrometry based proteomics has evolved as a promising technology over the last decade and is undergoing a dramatic development in a number of different areas, such as; mass spectrometric instrumentation, peptide identification algorithms and bioinformatic computational data analysis. The improved methodology allows quantitative measurement of relative or absolute protein amounts, which is essential for gaining insights into their functions and dynamics in biological systems. Several different strategies involving stable isotopes label (ICAT, ICPL, IDBEST, iTRAQ, TMT, IPTL, SILAC), label-free statistical assessment approaches (MRM, SWATH) and absolute quantification methods (AQUA) are possible, each having specific strengths and weaknesses. Inductively coupled plasma mass spectrometry (ICP-MS), which is still widely recognised as elemental detector, has recently emerged as a complementary technique to the previous methods. The new application area for ICP-MS is targeting the fast growing field of proteomics related research, allowing absolute protein quantification using suitable elemental based tags. This document describes the different stable isotope labelling methods which incorporate metabolic labelling in live cells, ICP-MS based detection and post-harvest chemical label tagging for protein quantification, in addition to summarising their pros and cons. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Production of isotopically labeled standards from a uniformly labeled precursor for quantitative volatile metabolomic studies.

    PubMed

    Gómez-Cortés, Pilar; Brenna, J Thomas; Sacks, Gavin L

    2012-06-19

    Optimal accuracy and precision in small-molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time-consuming, and many labeled compounds are not available in pure form. We used a single-prototype uniformly labeled [U-(13)C]compound to generate [U-(13)C]-labeled volatile standards for use in subsequent experimental profiling studies. [U-(13)C]-α-Linolenic acid (18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-(13)C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography/time-of-flight mass spectrometry (HS-SPME-GC/TOF-MS) by comparison of spectra with unlabeled volatiles. Labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-(13)C]-labeled standards, limits of detection comparable to or better than those of previous HS-SPME reports were achieved, 0.010-1.04 ng/g. The performance of the [U-(13)C]-labeled volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60 °C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-(13)C]-labeled oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-(13)C]-labeled standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-(13)C]-labeled sugars and amino acids, for profiling studies should be feasible and can dramatically improve

  17. An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes.

    PubMed

    Rinkel, Jan; Litzenburger, Martin; Bernhardt, Rita; Dickschat, Jeroen Sidney

    2018-04-26

    The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for enzymatic oxidation of the sesquiterpene alcohols T-muurolol and isodauc-8-en-11-ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro-scale reactions, for determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450-catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Subcellular SIMS imaging of isotopically labeled amino acids in cryogenically prepared cells

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2004-06-01

    Ion microscopy is a potentially powerful technique for localization of isotopically labeled molecules. In this study, L-arginine and phenylalanine amino acids labeled with stable isotopes 13C and 15N were localized in cultured cells with the ion microscope at 500 nm spatial resolution. Cells were exposed to the labeled amino acids and cryogenically prepared. SIMS analyses were made in fractured freeze-dried cells. A dynamic distribution was observed from labeled arginine-treated LLC-PK 1 kidney cells at mass 28 ( 13C15N) in negative secondaries, revealing cell-to-cell heterogeneity and preferential accumulation of the amino acid (or its metabolite) in the nucleus and nucleolus of some cells. The smaller nucleolus inside the nucleus was clearly resolved in SIMS images and confirmed by correlative light microscopy. The distribution of labeled phenylalanine contrasted with arginine as it was rather homogeneously distributed in T98G human glioblastoma cells. Images of 39K, 23Na and 40Ca were also recorded to confirm the reliability of sample preparation and authenticity of the observed amino acid distributions. These observations indicate that SIMS techniques can provide a valuable technology for subcellular localization of nitrogen-containing molecules in proteomics since nitrogen does not have a radionuclide tracer isotope. Amino acids labeled with stable isotopes can be used as tracers for studying their transport and metabolism in distinct subcellular compartments with SIMS. Further studies of phenylalanine uptake in human glioblastoma cells may have special significance in boron neutron capture therapy (BNCT) as a boron analogue of phenylalanine, boronophenylalanine is a clinically approved compound for the treatment of brain tumors.

  19. Isotope Labeling for Solution and Solid-State NMR Spectroscopy of Membrane Proteins

    PubMed Central

    Verardi, Raffaello; Traaseth, Nathaniel J.; Masterson, Larry R.; Vostrikov, Vitaly V.; Veglia, Gianluigi

    2013-01-01

    In this chapter, we summarize the isotopic labeling strategies used to obtain high-quality solution and solid-state NMR spectra of biological samples, with emphasis on integral membrane proteins (IMPs). While solution NMR is used to study IMPs under fast tumbling conditions, such as in the presence of detergent micelles or isotropic bicelles, solid-state NMR is used to study the structure and orientation of IMPs in lipid vesicles and bilayers. In spite of the tremendous progress in biomolecular NMR spectroscopy, the homogeneity and overall quality of the sample is still a substantial obstacle to overcome. Isotopic labeling is a major avenue to simplify overlapped spectra by either diluting the NMR active nuclei or allowing the resonances to be separated in multiple dimensions. In the following we will discuss isotopic labeling approaches that have been successfully used in the study of IMPs by solution and solid-state NMR spectroscopy. PMID:23076578

  20. Microwave-assisted deuterium exchange: the convenient preparation of isotopically labelled analogues for stable isotope dilution analysis of volatile wine phenols.

    PubMed

    Crump, Anna M; Sefton, Mark A; Wilkinson, Kerry L

    2014-11-01

    This study reports the convenient, low cost, one-step synthesis of labelled analogues of six volatile phenols, guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, eugenol and vanillin, using microwave-assisted deuterium exchange, for use as internal standards for stable isotope dilution analysis. The current method improves on previous strategies in that it enables incorporation of deuterium atoms on the aromatic ring, thereby ensuring retention of the isotope label during mass spectrometry fragmentation. When used as standards for SIDA, these labelled volatile phenols will improve the accuracy and reproducibility of quantitative food and beverage analysis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. A new method for the labelling of proteins with radioactive arsenic isotopes

    NASA Astrophysics Data System (ADS)

    Jennewein, M.; Hermanne, A.; Mason, R. P.; Thorpe, P. E.; Rösch, F.

    2006-12-01

    Radioarsenic labelled radiopharmaceuticals could be a valuable asset to positron emission tomography. In particular, the long half-lives of 72As ( T=26 h) and 74As ( T=17.8 d) allow to investigate slow physiological or metabolical processes, like the enrichment and distribution of monoclonal antibodies (mab) in tumour tissue. In this work, a new method for the labelling of proteins with various radioactive arsenic isotopes was developed. For this purpose, two proteins, namely a chimeric IgG 3 monoclonal antibody, ch3G4, directed against anionic phospholipids, and Rituxan (Rituximab), were labelled as a proof of principle with no-carrier-added radioarsenic isotopes ( 74As and 77As). The developed labelling chemistry gives high yields (>99.9%), is reliable and could easily be transferred to automated labelling systems in a clinical environment. At least for the mab used in this work, this route of radioarsenic labelling does not affect the immunoreactivity of the product. The arsenic label stays stable for up to 72 h at the molecular mass of the monoclonal antibody, which is in particular relevant to follow the pharmacology and pharmacokinetics of the labelled mab for several days.

  2. Metabolomics relative quantitation with mass spectrometry using chemical derivatization and isotope labeling

    DOE PAGES

    O'Maille, Grace; Go, Eden P.; Hoang, Linh; ...

    2008-01-01

    Comprehensive detection and quantitation of metabolites from a biological source constitute the major challenges of current metabolomics research. Two chemical derivatization methodologies, butylation and amination, were applied to human serum for ionization enhancement of a broad spectrum of metabolite classes, including steroids and amino acids. LC-ESI-MS analysis of the derivatized serum samples provided a significant signal elevation across the total ion chromatogram to over a 100-fold increase in ionization efficiency. It was also demonstrated that derivatization combined with isotopically labeled reagents facilitated the relative quantitation of derivatized metabolites from individual as well as pooled samples.

  3. A free-air system for long-term stable carbon isotope labeling of adult forest trees

    EPA Science Inventory

    Stable carbon (C) isotopes, in particular employed in labeling experiments, are an ideal tool to broaden our understanding of C dynamics in trees and forest ecosystems. Here, we present a free-air exposure system, named isoFACE, designed for long-term stable C isotope labeling in...

  4. Determining metal assimilation efficiency in aquatic invertebrates using enriched stable metal isotope tracers

    USGS Publications Warehouse

    Croteau, M.-N.; Luoma, S.N.; Pellet, B.

    2007-01-01

    We employ a novel approach that combines pulse-chase feeding and multi-labelled stable isotopes to determine gut passage time (GPT), gut retention time (GRT), food ingestion rate (IR) and assimilation efficiency (AE) of three trace elements for a freshwater gastropod. Lettuce isotopically enriched in 53Cr, 65Cu and 106Cd was fed for 2 h to Lymnaea stagnalis. The release of tracers in feces and water was monitored for 48 h, during which unlabelled lettuce was provided ad libidum. The first defecation of 53Cr occurred after 5 h of depuration (GPT), whereas 90% of the ingested 53Cr was recovered in the feces after 22.5 h of depuration (GRT). 53Chromium was not significantly accumulated in the soft tissues upon exposure. In contrast, 65Cu and 106Cd assimilation was detectable for most experimental snails, i.e., 65/63Cu and 106/114Cd ratios in exposed snails were higher than those for controls. Food IR during the labelled feeding phase was 0.16 ?? 0.07 g g-1 d-1. IR was inferred from the amount of 53Cr egested in the feces during depuration and the concentration of 53Cr in the labelled lettuce. Assimilation efficiencies (??95% CI) determined using mass balance calculations were 84 ?? 4% for Cu and 85 ?? 3% for Cd. The ratio method yields similar AE estimates. Expanding the application of this novel stable isotope tracer technique to other metals in a wide variety of species will provide unique opportunities to evaluate the interplay between digestive processes and dietary influx of metals. Understanding the biological processes that modulate dietborne metal uptake is crucial to assess the toxicity of dietborne metals. ?? 2007 Elsevier B.V. All rights reserved.

  5. The topology of metabolic isotope labeling networks.

    PubMed

    Weitzel, Michael; Wiechert, Wolfgang; Nöh, Katharina

    2007-08-29

    Metabolic Flux Analysis (MFA) based on isotope labeling experiments (ILEs) is a widely established tool for determining fluxes in metabolic pathways. Isotope labeling networks (ILNs) contain all essential information required to describe the flow of labeled material in an ILE. Whereas recent experimental progress paves the way for high-throughput MFA, large network investigations and exact statistical methods, these developments are still limited by the poor performance of computational routines used for the evaluation and design of ILEs. In this context, the global analysis of ILN topology turns out to be a clue for realizing large speedup factors in all required computational procedures. With a strong focus on the speedup of algorithms the topology of ILNs is investigated using graph theoretic concepts and algorithms. A rigorous determination of all cyclic and isomorphic subnetworks, accompanied by the global analysis of ILN connectivity is performed. Particularly, it is proven that ILNs always brake up into a large number of small strongly connected components (SCCs) and, moreover, there are natural isomorphisms between many of these SCCs. All presented techniques are universal, i.e. they do not require special assumptions on the network structure, bidirectionality of fluxes, measurement configuration, or label input. The general results are exemplified with a practically relevant metabolic network which describes the central metabolism of E. coli comprising 10390 isotopomer pools. Exploiting the topological features of ILNs leads to a significant speedup of all universal algorithms for ILE evaluation. It is proven in theory and exemplified with the E. coli example that a speedup factor of about 1000 compared to standard algorithms is achieved. This widely opens the door for new high performance algorithms suitable for high throughput applications and large ILNs. Moreover, for the first time the global topological analysis of ILNs allows to comprehensively

  6. In-Gel Stable-Isotope Labeling (ISIL): a strategy for mass spectrometry-based relative quantification.

    PubMed

    Asara, John M; Zhang, Xiang; Zheng, Bin; Christofk, Heather H; Wu, Ning; Cantley, Lewis C

    2006-01-01

    Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification.

  7. Reliable LC-MS quantitative glycomics using iGlycoMab stable isotope labeled glycans as internal standards.

    PubMed

    Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia

    2016-06-01

    Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Using phylogenetic probes for quantification of stable isotope labeling and microbial community analysis

    DOEpatents

    Brodie, Eoin L; DeSantis, Todd Z; Karaoz, Ulas; Andersen, Gary L

    2014-12-09

    Herein is described methods for a high-sensitivity means to measure the incorporation of stable isotope labeled substrates into RNA following stable isotope probing experiments (SIP). RNA is hybridized to a set of probes such as phylogenetic microarrays and isotope incorporation is quantified such as by secondary ion mass spectrometer imaging (NanoSIMS).

  9. Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities

    PubMed Central

    Verastegui, Y.; Cheng, J.; Engel, K.; Kolczynski, D.; Mortimer, S.; Lavigne, J.; Montalibet, J.; Romantsov, T.; Hall, M.; McConkey, B. J.; Rose, D. R.; Tomashek, J. J.; Scott, B. R.

    2014-01-01

    ABSTRACT Soil microbial diversity represents the largest global reservoir of novel microorganisms and enzymes. In this study, we coupled functional metagenomics and DNA stable-isotope probing (DNA-SIP) using multiple plant-derived carbon substrates and diverse soils to characterize active soil bacterial communities and their glycoside hydrolase genes, which have value for industrial applications. We incubated samples from three disparate Canadian soils (tundra, temperate rainforest, and agricultural) with five native carbon (12C) or stable-isotope-labeled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose, and cellulose). Indicator species analysis revealed high specificity and fidelity for many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Actinomycetales (Salinibacterium), Rhizobiales (Devosia), Rhodospirillales (Telmatospirillum), and Caulobacterales (Phenylobacterium and Asticcacaulis) were bacterial indicator species for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (Phenylobacterium) were associated with metabolism of cellulose, and Alphaproteobacteria were associated with the metabolism of arabinose; members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycoside hydrolase gene representation within the pooled heavy DNA. By screening 2,876 cloned fragments derived from the 13C-labeled DNA isolated from soils incubated with cellulose, we demonstrate the power of combining DNA-SIP, multiple-displacement amplification (MDA), and functional metagenomics by efficiently isolating multiple clones with activity on carboxymethyl cellulose and fluorogenic proxy substrates for carbohydrate-active enzymes. PMID:25028422

  10. Copper absorption from foods labelled intrinsically and extrinsically with Cu-65 stable isotope.

    PubMed

    Harvey, L J; Dainty, J R; Beattie, J H; Majsak-Newman, G; Wharf, S G; Reid, M D; Fairweather-Tait, S J

    2005-03-01

    To determine copper absorption from copper containing foods labelled either intrinsically or extrinsically with a highly enriched Cu-65 stable isotope label. A longitudinal cross-over study. The study was conducted at the Institute of Food Research, Human Nutrition Unit, Norwich, UK. Subjects were recruited locally via advertisements placed around the Norwich Research Park. A total of 10 volunteers (nine female, one male) took part in the study, but not all volunteers completed each of the test meals. A highly enriched Cu-65 stable isotope label was administered to volunteers in the form of a reference dose or in breakfast test meals consisting of red wine, soya beans, mushrooms or sunflower seeds. Faecal monitoring and mass spectrometry techniques were used to estimate the relative quantities of copper absorbed from the different test meals. True copper absorption from the reference dose (54%) was similar to extrinsically labelled red wine (49%) and intrinsically labelled sunflower seeds (52%), but significantly higher than extrinsically labelled mushrooms (35%), intrinsically (29%) and extrinsically (15%) labelled soya beans and extrinsically labelled sunflower seed (32%) test meals. The use of Cu-65 extrinsic labels in copper absorption studies requires validation according to the food being examined; intrinsic and extrinsic labelling produced significantly different results for sunflower seeds.

  11. Evaluation of stereo-array isotope labeling (SAIL) patterns for automated structural analysis of proteins with CYANA.

    PubMed

    Ikeya, Teppei; Terauchi, Tsutomu; Güntert, Peter; Kainosho, Masatsune

    2006-07-01

    Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems.

  12. UNiquant, a program for quantitative proteomics analysis using stable isotope labeling.

    PubMed

    Huang, Xin; Tolmachev, Aleksey V; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A; Smith, Richard D; Chan, Wing C; Hinrichs, Steven H; Fu, Kai; Ding, Shi-Jian

    2011-03-04

    Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for postmeasurement normalization of peptide ratios, which is required by the other programs.

  13. UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

    PubMed Central

    Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven H.; Fu, Kai; Ding, Shi-Jian

    2011-01-01

    Stable isotope labeling (SIL) methods coupled with nanoscale liquid chromatography and high resolution tandem mass spectrometry are increasingly useful for elucidation of the proteome-wide differences between multiple biological samples. Development of more effective programs for the sensitive identification of peptide pairs and accurate measurement of the relative peptide/protein abundance are essential for quantitative proteomic analysis. We developed and evaluated the performance of a new program, termed UNiquant, for analyzing quantitative proteomics data using stable isotope labeling. UNiquant was compared with two other programs, MaxQuant and Mascot Distiller, using SILAC-labeled complex proteome mixtures having either known or unknown heavy/light ratios. For the SILAC-labeled Jeko-1 cell proteome digests with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10), UNiquant quantified a similar number of peptide pairs as MaxQuant for the H/L = 1:1 and 1:5 mixtures. In addition, UNiquant quantified significantly more peptides than MaxQuant and Mascot Distiller in the H/L = 1:10 mixtures. UNiquant accurately measured relative peptide/protein abundance without the need for post-measurement normalization of peptide ratios, which is required by the other programs. PMID:21158445

  14. Two efficient label-equivalence-based connected-component labeling algorithms for 3-D binary images.

    PubMed

    He, Lifeng; Chao, Yuyan; Suzuki, Kenji

    2011-08-01

    Whenever one wants to distinguish, recognize, and/or measure objects (connected components) in binary images, labeling is required. This paper presents two efficient label-equivalence-based connected-component labeling algorithms for 3-D binary images. One is voxel based and the other is run based. For the voxel-based one, we present an efficient method of deciding the order for checking voxels in the mask. For the run-based one, instead of assigning each foreground voxel, we assign each run a provisional label. Moreover, we use run data to label foreground voxels without scanning any background voxel in the second scan. Experimental results have demonstrated that our voxel-based algorithm is efficient for 3-D binary images with complicated connected components, that our run-based one is efficient for those with simple connected components, and that both are much more efficient than conventional 3-D labeling algorithms.

  15. Isotope labeling of proteins in insect cells.

    PubMed

    Skora, Lukasz; Shrestha, Binesh; Gossert, Alvar D

    2015-01-01

    Protein targets of contemporary research are often membrane proteins, multiprotein complexes, secreted proteins, or other proteins of human origin. These are difficult to express in the standard expression host used for most nuclear magnetic resonance (NMR) studies, Escherichia coli. Insect cells represent an attractive alternative, since they have become a well-established expression system and simple solutions have been developed for generation of viruses to efficiently introduce the target protein DNA into cells. Insect cells enable production of a larger fraction of the human proteome in a properly folded way than bacteria, as insect cells have a very similar set of cytosolic chaperones and a closely related secretory pathway. Here, the limited and defined glycosylation pattern that insect cells produce is an advantage for structural biology studies. For these reasons, insect cells have been established as the most widely used eukaryotic expression host for crystallographic studies. In the past decade, significant advancements have enabled amino acid type-specific as well as uniform isotope labeling of proteins in insect cells, turning them into an attractive expression host for NMR studies. © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

    Keiderling, Timothy A

    2017-12-01

    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. © 2017 Wiley Periodicals, Inc.

  17. High-coverage quantitative proteomics using amine-specific isotopic labeling.

    PubMed

    Melanson, Jeremy E; Avery, Steven L; Pinto, Devanand M

    2006-08-01

    Peptide dimethylation with isotopically coded formaldehydes was evaluated as a potential alternative to techniques such as the iTRAQ method for comparative proteomics. The isotopic labeling strategy and custom-designed protein quantitation software were tested using protein standards and then applied to measure proteins levels associated with Alzheimer's disease (AD). The method provided high accuracy (10% error), precision (14% RSD) and coverage (70%) when applied to the analysis of a standard solution of BSA by LC-MS/MS. The technique was then applied to measure protein abundance levels in brain tissue afflicted with AD relative to normal brain tissue. 2-D LC-MS analysis identified 548 unique proteins (p<0.05). Of these, 349 were quantified with two or more peptides that met the statistical criteria used in this study. Several classes of proteins exhibited significant changes in abundance. For example, elevated levels of antioxidant proteins and decreased levels of mitochondrial electron transport proteins were observed. The results demonstrate the utility of the labeling method for high-throughput quantitative analysis.

  18. Stable Isotope Labeling Strategy for Curcumin Metabolite Study in Human Liver Microsomes by Liquid Chromatography-Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Gao, Dan; Chen, Xiaowu; Yang, Xiaomei; Wu, Qin; Jin, Feng; Wen, Hongliang; Jiang, Yuyang; Liu, Hongxia

    2015-04-01

    The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an 18O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the 18O labeled curcumin was successfully synthesized. The non-labeled and 18O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp., Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites.

  19. Recent advances in stable isotope labeling based techniques for proteome relative quantification.

    PubMed

    Zhou, Yuan; Shan, Yichu; Zhang, Lihua; Zhang, Yukui

    2014-10-24

    The large scale relative quantification of all proteins expressed in biological samples under different states is of great importance for discovering proteins with important biological functions, as well as screening disease related biomarkers and drug targets. Therefore, the accurate quantification of proteins at proteome level has become one of the key issues in protein science. Herein, the recent advances in stable isotope labeling based techniques for proteome relative quantification were reviewed, from the aspects of metabolic labeling, chemical labeling and enzyme-catalyzed labeling. Furthermore, the future research direction in this field was prospected. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Electron linac for medical isotope production with improved energy efficiency and isotope recovery

    DOEpatents

    Noonan, John; Walters, Dean; Virgo, Matt; Lewellen, John

    2015-09-08

    A method and isotope linac system are provided for producing radio-isotopes and for recovering isotopes. The isotope linac is an energy recovery linac (ERL) with an electron beam being transmitted through an isotope-producing target. The electron beam energy is recollected and re-injected into an accelerating structure. The ERL provides improved efficiency with reduced power requirements and provides improved thermal management of an isotope target and an electron-to-x-ray converter.

  1. Segmental isotopic labeling of HIV-1 capsid protein assemblies for solid state NMR.

    PubMed

    Gupta, Sebanti; Tycko, Robert

    2018-02-01

    Recent studies of noncrystalline HIV-1 capsid protein (CA) assemblies by our laboratory and by Polenova and coworkers (Protein Sci 19:716-730, 2010; J Mol Biol 426:1109-1127, 2014; J Biol Chem 291:13098-13112, 2016; J Am Chem Soc 138:8538-8546, 2016; J Am Chem Soc 138:12029-12032, 2016; J Am Chem Soc 134:6455-6466, 2012; J Am Chem Soc 132:1976-1987, 2010; J Am Chem Soc 135:17793-17803, 2013; Proc Natl Acad Sci USA 112:14617-14622, 2015; J Am Chem Soc 138:14066-14075, 2016) have established the capability of solid state nuclear magnetic resonance (NMR) measurements to provide site-specific structural and dynamical information that is not available from other types of measurements. Nonetheless, the relatively high molecular weight of HIV-1 CA leads to congestion of solid state NMR spectra of fully isotopically labeled assemblies that has been an impediment to further progress. Here we describe an efficient protocol for production of segmentally labeled HIV-1 CA samples in which either the N-terminal domain (NTD) or the C-terminal domain (CTD) is uniformly 15 N, 13 C-labeled. Segmental labeling is achieved by trans-splicing, using the DnaE split intein. Comparisons of two-dimensional solid state NMR spectra of fully labeled and segmentally labeled tubular CA assemblies show substantial improvements in spectral resolution. The molecular structure of HIV-1 assemblies is not significantly perturbed by the single Ser-to-Cys substitution that we introduce between NTD and CTD segments, as required for trans-splicing.

  2. Efficient Thread Labeling for Monitoring Programs with Nested Parallelism

    NASA Astrophysics Data System (ADS)

    Ha, Ok-Kyoon; Kim, Sun-Sook; Jun, Yong-Kee

    It is difficult and cumbersome to detect data races occurred in an execution of parallel programs. Any on-the-fly race detection techniques using Lamport's happened-before relation needs a thread labeling scheme for generating unique identifiers which maintain logical concurrency information for the parallel threads. NR labeling is an efficient thread labeling scheme for the fork-join program model with nested parallelism, because its efficiency depends only on the nesting depth for every fork and join operation. This paper presents an improved NR labeling, called e-NR labeling, in which every thread generates its label by inheriting the pointer to its ancestor list from the parent threads or by updating the pointer in a constant amount of time and space. This labeling is more efficient than the NR labeling, because its efficiency does not depend on the nesting depth for every fork and join operation. Some experiments were performed with OpenMP programs having nesting depths of three or four and maximum parallelisms varying from 10,000 to 1,000,000. The results show that e-NR is 5 times faster than NR labeling and 4.3 times faster than OS labeling in the average time for creating and maintaining the thread labels. In average space required for labeling, it is 3.5 times smaller than NR labeling and 3 times smaller than OS labeling.

  3. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

    PubMed

    Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

    2011-09-22

    Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

  4. Development of high-performance chemical isotope labeling LC-MS for profiling the human fecal metabolome.

    PubMed

    Xu, Wei; Chen, Deying; Wang, Nan; Zhang, Ting; Zhou, Ruokun; Huan, Tao; Lu, Yingfeng; Su, Xiaoling; Xie, Qing; Li, Liang; Li, Lanjuan

    2015-01-20

    Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively

  5. Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies

    PubMed Central

    Zhu, Jinge; Rao, Hongyu; Tonelli, Marco; Westler, Milo; Singarapu, Kiran K.; Markley, John L.; DeLuca, Hector F.; Assadi-Porter, Fariba M.

    2012-01-01

    Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from E. coli inclusion bodies. The heterologously expressed protein constructs retain full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with, 13C, and 15N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed. PMID:22750673

  6. Analysis of Hydrogen Isotopic Exchange: Lava Creek Tuff Ash and Isotopically Labeled Water

    NASA Astrophysics Data System (ADS)

    Ross, A. M.; Seligman, A. N.; Bindeman, I. N.; Nolan, G. S.

    2015-12-01

    Nolan and Bindeman (2013) placed secondarily hydrated ash from the 7.7 ka eruption of Mt. Mazama (δD=-149‰, 2.3wt% H2Ot) in isotopically labeled water (+650 ‰ δD, +56 ‰ δ18O) and observed that the H2Ot and δ18O values remained constant, but the δD values of ash increased with the surrounding water at 20, 40 and 70 °C. We expand on this work by conducting a similar experiment with ash from the 640 ka Lava Creek Tuff (LCT, δD of -128 ‰; 2.1 wt.% H2Ot) eruption of Yellowstone to see if significantly older glass (with a hypothesized gel layer on the surface shielding the interior from alteration) produces the same results. We have experiments running at 70, 24, and 5 °C, and periodically remove ~1.5 mg of glass to measure the δD (‰) and H2Ot (wt.%) of water extracted from the glass on a TC/EA MAT 253 continuous flow system. After 600 hours, the δD of the samples left at 5 and 24 °C remains at -128 ‰, but increased 8‰ for the 70 °C run series. However, there is no measurable change in wt.% of H2Ot, indicating that hydrogen exchange is not dictated by the addition of water. We are measuring and will report further progress of isotope exchange. We also plan to analyze the water in the LCT glass for δ18O (‰) to see if, as is the case for the Mt. Mazama glass, the δ18O (‰) remains constant. We also analyzed Mt. Mazama glass from the Nolan and Bindeman (2013) experiments that have now been sitting in isotopically labeled water at room temperature for ~5 years. The water concentration is still unchanged (2.3 wt.% H2Ot), and the δD of the water in the glass is now -111 ‰, causing an increase of 38 ‰. Our preliminary results show that exchange of hydrogen isotopes of hydrated glass is not limited by the age of the glass, and that the testing of hydrogen isotopes of secondarily hydrated glass, regardless of age, may not be a reliable paleoclimate indicator.

  7. Metabolomics of Early Stage Plant Cell–Microbe Interaction Using Stable Isotope Labeling

    PubMed Central

    Pang, Qiuying; Zhang, Tong; Wang, Yang; Kong, Wenwen; Guan, Qijie; Yan, Xiufeng; Chen, Sixue

    2018-01-01

    Metabolomics has been used in unraveling metabolites that play essential roles in plant–microbe (including pathogen) interactions. However, the problem of profiling a plant metabolome with potential contaminating metabolites from the coexisting microbes has been largely ignored. To address this problem, we implemented an effective stable isotope labeling approach, where the metabolome of a plant bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was labeled with heavy isotopes. The labeled bacterial cells were incubated with Arabidopsis thaliana epidermal peels (EPs) with guard cells, and excessive bacterial cells were subsequently removed from the plant tissues by washing. The plant metabolites were characterized by liquid chromatography mass spectrometry using multiple reactions monitoring, which can differentiate plant and bacterial metabolites. Targeted metabolomic analysis suggested that Pst DC3000 infection may modulate stomatal movement by reprograming plant signaling and primary metabolic pathways. This proof-of-concept study demonstrates the utility of this strategy in differentiation of the plant and microbe metabolomes, and it has broad applications in studying metabolic interactions between microbes and other organisms. PMID:29922325

  8. An optimized method for measuring fatty acids and cholesterol in stable isotope-labeled cells

    PubMed Central

    Argus, Joseph P.; Yu, Amy K.; Wang, Eric S.; Williams, Kevin J.; Bensinger, Steven J.

    2017-01-01

    Stable isotope labeling has become an important methodology for determining lipid metabolic parameters of normal and neoplastic cells. Conventional methods for fatty acid and cholesterol analysis have one or more issues that limit their utility for in vitro stable isotope-labeling studies. To address this, we developed a method optimized for measuring both fatty acids and cholesterol from small numbers of stable isotope-labeled cultured cells. We demonstrate quantitative derivatization and extraction of fatty acids from a wide range of lipid classes using this approach. Importantly, cholesterol is also recovered, albeit at a modestly lower yield, affording the opportunity to quantitate both cholesterol and fatty acids from the same sample. Although we find that background contamination can interfere with quantitation of certain fatty acids in low amounts of starting material, our data indicate that this optimized method can be used to accurately measure mass isotopomer distributions for cholesterol and many fatty acids isolated from small numbers of cultured cells. Application of this method will facilitate acquisition of lipid parameters required for quantifying flux and provide a better understanding of how lipid metabolism influences cellular function. PMID:27974366

  9. Preparation of stable isotope-labeled peripheral cannabinoid receptor CB2 by bacterial fermentation

    PubMed Central

    Berger, Christian; Ho, Jenny T.C.; Kimura, Tomohiro; Hess, Sonja; Gawrisch, Klaus; Yeliseev, Alexei

    2010-01-01

    We developed a bacterial fermentation protocol for production of a stable isotope-labeled cannabinoid receptor CB2 for subsequent structural studies of this protein by nuclear magnetic resonance spectroscopy. The human peripheral cannabinoid receptor was expressed in Escherichia coli as a fusion with maltose binding protein and two affinity tags. The fermentation was performed in defined media comprised of mineral salts, glucose and 15N2-L-tryptophan to afford incorporation of the labeled amino acid into the protein. Medium, growth and expression conditions were optimized so that the fermentation process produced about 2 mg of purified, labeled CB2 per liter of culture medium. By performing a mass spectroscopic characterization of the purified CB2, we determined that one of the two 15N atoms in tryptophan was incorporated into the recombinant protein. NMR analysis of 15N chemical shifts strongly suggests that the 15N atoms are located in Trp-indole rings. Importantly, analysis of the peptides derived from the CNBr cleavage of the purified protein confirmed a minimum of 95% incorporation of the labeled tryptophan into the CB2 sequence. The labeled CB2, purified and reconstituted into liposomes at a protein-to-lipid molar ratio of 1:500, was functional as confirmed by activation of cognate G proteins in an in vitro coupled assay. To our knowledge, this is the first reported production of a biologically active, stable isotope-labeled G protein-coupled receptor by bacterial fermentation. PMID:20044006

  10. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

    PubMed

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Asquith, Becca; Macallan, Derek

    2016-06-30

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. © 2016 by The American Society of Hematology.

  11. The effects of isotope-labeled analogs on the LC-IDMS measurement by comparison of ESI responses and matrix effect of melamine, 13C3-melamine, 13C3+15N3-melamine, and 15N3-melamine.

    PubMed

    Li, Xiu Qin; Zhang, Qing He; Yang, Zong; Li, Hong Mei; Huang, Dong Feng

    2017-05-01

    In this paper, the effect of isotope-labeled analogs on the liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) measurement was evaluated based on the comparison research of electrospray ionization responses (ESI) and matrix effect of melamine, 13 C 3 -melamine, 13 C 3 + 15 N 3 -melamine, and 15 N 3 -melamine. The isotope-labeled melamines had similar ionization efficiency with melamine in the electrospray ionization source, but the intensity of corresponding quantitative fragment ions had distinctive differences. Based on the density functional theory at the B3LYP/6-311+G** level, this phenomenon was explained very well. The rare cleavage pathways of melamine, which just could be exactly identified by 15 N-labeled melamines, resulted in the difference of quantitative fragment ions between 15 N-labeled melamines and melamine. The interaction of ESI response between melamine and isotope-labeled melamines was investigated using MRM monitor mode. 15 N-labeled melamine had significant ion inter-suppression effect on melamine, while 13 C-labeled melamine had little influence on melamine. Finally, the influence of different isotope-labeled melamines on the LC-IDMS result was evaluated using the IDMS correction factor (θ). Taking the determination of melamine in milk powder as an example, the matrix effects of different isotope-labeled melamines and melamine had notable difference and the impact of this difference on the measurement results depended on the concentrations of analyte and matrix solution. It was worth noting that 15 N 3 -melamine exhibited significant ion suppression to melamine in matrix solution. The deviation of the results from IDMS method might reach 59% using 15 N 3 -melamine as internal standard in special matrix solution. Graphical Abstract The comparison of ESI responses of melamine, 13 C 3 -melamine, 13 C 3 + 15 N 3 -melamine and 15 N 3 -melamine.

  12. Protein N- and C-Termini Identification Using Mass Spectrometry and Isotopic Labeling

    USDA-ARS?s Scientific Manuscript database

    A new method for protein N- and C-terminal analysis using mass spectrometry is introduced. A novel stable isotopic labeling scheme has been developed to identify terminal peptides generated from an enzyme digestion for the determination of both N- and C-termini of the protein. This method works dire...

  13. IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells

    PubMed Central

    Orlando, Ron; Lim, Jae-Min; Atwood, James A.; Angel, Peggi M.; Fang, Meng; Aoki, Kazuhiro; Alvarez-Manilla, Gerardo; Moremen, Kelley W.; York, William S.; Tiemeyer, Michael; Pierce, Michael; Dalton, Stephen; Wells, Lance

    2012-01-01

    Robust quantification is an essential component of comparative –omic strategies. In this regard, glycomics lags behind proteomics. Although various isotope-tagging and direct quantification methods have recently enhanced comparative glycan analysis, a cell culture labeling strategy, that could provide for glycomics the advantages that SILAC provides for proteomics, has not been described. Here we report the development of IDAWG, Isotopic Detection of Aminosugars With Glutamine, for the incorporation of differential mass tags into the glycans of cultured cells. In this method, culture media containing amide-15N-Gln is used to metabolically label cellular aminosugars with heavy nitrogen. Because the amide side chain of Gln is the sole source of nitrogen for the biosynthesis of GlcNAc, GalNAc, and sialic acid, we demonstrate that culturing mouse embryonic stems cells for 72 hours in the presence of amide-15N-Gln media results in nearly complete incorporation of 15N into N-linked and O-linked glycans. The isotopically heavy monosaccharide residues provide additional information for interpreting glycan fragmentation and also allow quantification in both full MS and MS/MS modes. Thus, IDAWG is a simple to implement, yet powerful quantitative tool for the glycomics toolbox. PMID:19449840

  14. Efficient Enzymatic Preparation of (13) N-Labelled Amino Acids: Towards Multipurpose Synthetic Systems.

    PubMed

    da Silva, Eunice S; Gómez-Vallejo, Vanessa; Baz, Zuriñe; Llop, Jordi; López-Gallego, Fernando

    2016-09-12

    Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Testing isotopic labeling with [¹³C₆]glucose as a method of advanced glycation sites identification.

    PubMed

    Kielmas, Martyna; Kijewska, Monika; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2012-12-01

    The Maillard reaction occurring between reducing sugars and reactive amino groups of biomolecules leads to the formation of a heterogeneous mixture of compounds: early, intermediate, and advanced glycation end products (AGEs). These compounds could be markers of certain diseases and of the premature aging process. Detection of Amadori products can be performed by various methods, including MS/MS techniques and affinity chromatography on immobilized boronic acid. However, the diversity of the structures of AGEs makes detection of these compounds more difficult. The aim of this study was to test a new method of AGE identification based on isotope (13)C labeling. The model protein (hen egg lysozyme) was modified with an equimolar mixture of [(12)C(6)]glucose and [(13)C(6)]glucose and then subjected to reduction of the disulfide bridges followed by tryptic hydrolysis. The digest obtained was analyzed by LC-MS. The glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. This method allowed identification of 38 early Maillard reaction products and five different structures of the end glycation products. This isotopic labeling technique combined with LC-MS is a sensitive method for identification of advanced glycation end products even if their chemical structure is unknown. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Independent valine and leucine isotope labeling in Escherichia coli protein overexpression systems.

    PubMed

    Lichtenecker, Roman J; Weinhäupl, Katharina; Reuther, Lukas; Schörghuber, Julia; Schmid, Walther; Konrat, Robert

    2013-11-01

    The addition of labeled α-ketoisovalerate to the growth medium of a protein-expressing host organism has evolved into a versatile tool to achieve concomitant incorporation of specific isotopes into valine- and leucine- residues. The resulting target proteins represent excellent probes for protein NMR analysis. However, as the sidechain resonances of these residues emerge in a narrow spectral range, signal overlap represents a severe limitation in the case of high-molecular-weight NMR probes. We present a protocol to eliminate leucine labeling by supplying the medium with unlabeled α-ketoisocaproate. The resulting spectra of a model protein exclusively feature valine signals of increased intensity, confirming the method to be a first example of independent valine and leucine labeling employing α-ketoacid precursor compounds.

  17. Stable isotope labeling-solid phase extraction-mass spectrometry analysis for profiling of thiols and aldehydes in beer.

    PubMed

    Zheng, Shu-Jian; Wang, Ya-Lan; Liu, Ping; Zhang, Zheng; Yu, Lei; Yuan, Bi-Feng; Feng, Yu-Qi

    2017-12-15

    In this study, we developed a strategy for profiling of thiols and aldehydes in beer samples by stable isotope labeling-solid phase extraction-liquid chromatography-double precursor ion scan/double neutral loss scan-mass spectrometry analysis (SIL-SPE-LC-DPIS/DNLS-MS). A pair of isotope reagents (ω-bromoacetonylquinolinium bromide, BQB; ω-bromoacetonylquinolinium-d 7 bromide, BQB-d 7 ) were used to label thiols; while for the aldehydes, a pair of isotope reagents (4-(2-(trimethylammonio) ethoxy) benzenaminium halide, 4-APC; 4-(2-(trimethylammonio) ethoxy) benzenaminium halide-d 4 , 4-APC-d 4 ) were used. The labeled thiols and aldehydes were extracted and purified with solid-phase extraction, respectively, followed by LC-MS analysis. Using the proposed SIL-SPE-LC-DPIS/DNLS-MS methods, 76 thiol and 25 aldehyde candidates were found in beer. Furthermore, we established SIL-SPE-LC-MRM-MS methods for the relative quantitation of thiols and aldehydes in different beer samples. The results showed that the contents of thiols and aldehydes are closely related to the brands and origins of beers. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. IsoMS: automated processing of LC-MS data generated by a chemical isotope labeling metabolomics platform.

    PubMed

    Zhou, Ruokun; Tseng, Chiao-Li; Huan, Tao; Li, Liang

    2014-05-20

    A chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform uses a chemical derivatization method to introduce a mass tag to all of the metabolites having a common functional group (e.g., amine), followed by LC-MS analysis of the labeled metabolites. To apply this platform to metabolomics studies involving quantitative analysis of different groups of samples, automated data processing is required. Herein, we report a data processing method based on the use of a mass spectral feature unique to the chemical labeling approach, i.e., any differential-isotope-labeled metabolites are detected as peak pairs with a fixed mass difference in a mass spectrum. A software tool, IsoMS, has been developed to process the raw data generated from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair filtering, and peak-pair intensity ratio calculation. The same peak pairs detected from multiple samples are then aligned to produce a CSV file that contains the metabolite information and peak ratios relative to a control (e.g., a pooled sample). This file can be readily exported for further data and statistical analysis, which is illustrated in an example of comparing the metabolomes of human urine samples collected before and after drinking coffee. To demonstrate that this method is reliable for data processing, five (13)C2-/(12)C2-dansyl labeled metabolite standards were analyzed by LC-MS. IsoMS was able to detect these metabolites correctly. In addition, in the analysis of a (13)C2-/(12)C2-dansyl labeled human urine, IsoMS detected 2044 peak pairs, and manual inspection of these peak pairs found 90 false peak pairs, representing a false positive rate of 4.4%. IsoMS for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS.

  19. Site-specific orientation of an α-helical peptide ovispirin-1 from isotope-labeled SFG spectroscopy.

    PubMed

    Ding, Bei; Laaser, Jennifer E; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T; Chen, Zhan

    2013-11-27

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single-isotope-labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138° from the surface normal, and the transition dipole of the isotope-labeled C═O group is tilted at 23° from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrate that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope-labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution.

  20. Site-specific Orientation of an α-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy

    PubMed Central

    Ding, Bei; Laaser, Jennifer E.; Liu, Yuwei; Wang, Pengrui; Zanni, Martin T.; Chen, Zhan

    2013-01-01

    Sum-frequency generation (SFG) vibrational spectroscopy is often used to probe the backbone structures and orientations of polypeptides at surfaces. Using the ovispirin-1 polypeptide at the solid/liquid interface of polystyrene, we demonstrate for the first time that SFG can probe the polarization response of a single isotope labeled residue. To interpret the spectral intensities, we simulated the spectra using an excitonic Hamiltonian approach. We show that the polarization dependence of either the label or the unlabeled amide I band alone does not provide sufficient structural constraints to obtain both the tilt and the twist of the ovispirin helix at a solid/liquid interface, but that both can be determined from the polarization dependence of the complete spectrum. For ovispirin, the detailed analysis of the polarized SFG experimental data shows that the helix axis is tilted at roughly 138 degrees from the surface normal, and the transition dipole of the isotope labeled C=O group is tilted at 23 degrees from the surface normal, with the hydrophobic region facing the polystyrene surface. We further demonstrated that the Hamiltonian approach is able to address the coupling effect and the structural disorder. For comparison, we also collected the FTIR spectrum of ovispirin under similar conditions, which reveals the enhanced sensitivity of SFG for structural studies of single monolayer peptide surfaces. Our study provides insight into how structural and environmental effects appear in SFG spectra of the amide I band and establishes that SFG of isotope labeled peptides will be a powerful technique for elucidating secondary structures with residue-by-residue resolution. PMID:24228619

  1. TRITIUM-LABELED COMPOUNDS VII. ISOTOPE EFFECTS IN THE OXIDATION OF d- MANNITOLS-C$sup 14$ AND d-MANNITOLS-t TO d-FRUCTOSES

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sniegoski, L.T.; Frush, H.L.; Isbell, H.S.

    1961-09-01

    D-Mannitols, labeled either with carbon-14 at C1, C2, or C3, or with tritium attached to C1, C2, or C3, were prepared. After oxidation by Acetobacter suboxydans, the distribution of radioactivity in each of the resulting labeled D- fructoses was determined. Labeled D-mannitol is unique among the hexitols in that it may be oxidized by A. suboxydans in either the labeled or the unlabeled part of the molecule. Except in the oxidation of D-mannitol-2-t, the competing reactions result in the formation of a mixture of D-fructoses, each having radioactivity in one of two different positions. Hence, the isotope effect, k*/ k,more » (where k* and k are, respectively, the rate constants for oxidation in the labeled and in the unlabeled part of the labeled emannitol molecule) is the ratio of the activities at the two positions of the product, D-fructose. The following isotope effects were found for the bacterial oxidation of labeled D-mannitols: for D-mannitol-2-C/sup 14/, k*/k = 0.93; for Dmannitol-2-t, k*/k = 0.23; and for D-mannitol-3-t, k*/k = 0.70. For D-mannitols labeled at other positions, no isotope effect was detected, since k*/k was unity. The large isotope effect for D-mannitol-2-t is indicative of rupture of the C2-H bond in the rate determining process. It is suggested that the secondary isotope effect for tritium at C3 indicates hyperconjugation of the C3 hydrogen atom in the activated enzyme-- substrate complex; the lack of such effect for tritium at C1 may be due to unfavorable steric conditions for hyperconjugation of the C1 hydrogen atoms in the complex. The following substances were prepared and their isotopic distributions determined: D-fructose1,6-C/sup 14/ and D-fructose-1,6-t (from 1- labeled D-mannitols); D-fructose-2,5-C/sup 14/ and D-fructose-5-t (from 2-labeled e-mannitols); and D-fructose-3,4-C/sup 14/ and D-fructose-3,4-t (from 3-labeled D- mannitols). A procedure, employing D-fructose-1,6-C/sup 14/ as an internal standard, was devised for the

  2. Quantification of isotope-labelled and unlabelled folates in plasma, ileostomy and food samples.

    PubMed

    Büttner, Barbara E; Öhrvik, Veronica E; Witthöft, Cornelia M; Rychlik, Michael

    2011-01-01

    New stable isotope dilution assays were developed for the simultaneous quantitation of [(13)C(5)]-labelled and unlabelled 5-methyltetrahydrofolic acid, 5-formyltetrahydrofolic acid, folic acid along with unlabelled tetrahydrofolic acid and 10-formylfolic acid in clinical samples deriving from human bioavailability studies, i.e. plasma, ileostomy samples, and food. The methods were based on clean-up by strong anion exchange followed by LC-MS/MS detection. Deuterated analogues of the folates were applied as the internal standards in the stable isotope dilution assays. Assay sensitivity was sufficient to detect all relevant folates in the respective samples as their limits of detection were below 0.62 nmol/L in plasma and below 0.73 μg/100 g in food or ileostomy samples. Quantification of the [(13)C(5)]-label in clinical samples offers the possibility to differentiate between folate from endogenous body pools and the administered dose when executing bioavailability trials.

  3. Characterization of Volatile Nylon 6.6 Thermal-Oxidative Degradation Products by Selective Isotopic Labeling and Cryo-GC/MS

    NASA Astrophysics Data System (ADS)

    Smith, Jonell N.; V. White, Gregory; White, Michael I.; Bernstein, Robert; Hochrein, James M.

    2012-09-01

    Aged materials, such as polymers, can exhibit modifications to their chemical structure and physical properties, which may render the material ineffective for its intended purpose. Isotopic labeling was used to characterize low-molecular weight volatile thermal-oxidative degradation products of nylon 6.6 in an effort to better understand and predict changes in the aged polymer. Headspace gas from aged (up to 243 d at 138 °C) nylon 6.6 monomers (adipic acid and 1,6-hexanediamine) and polymer were preconcentrated, separated, and detected using cryofocusing gas chromatography mass spectrometry (cryo-GC/MS). Observations regarding the relative concentrations observed in each chromatographic peak with respect to aging time were used in conjunction with mass spectra for samples aged under ambient air to determine the presence and identity of 18 degradation products. A comparison of the National Institute of Standards and Technology (NIST) library, unlabeled, and isotopically labeled mass spectra (C-13 or N-15) and expected fragmentation pathways of each degradation product were used to identify the location of isotopically labeled atoms within the product's chemical structure, which can later be used to determine the exact origin of the species. In addition, observations for unlabeled nylon 6.6 aged in an O-18 enriched atmosphere were used to determine if the source of oxygen in the applicable degradation products was from the gaseous environment or the polymer. Approximations for relative isotopic ratios of unlabeled to labeled products are reported, where appropriate.

  4. Systematic NMR Analysis of Stable Isotope Labeled Metabolite Mixtures in Plant and Animal Systems: Coarse Grained Views of Metabolic Pathways

    PubMed Central

    Chikayama, Eisuke; Suto, Michitaka; Nishihara, Takashi; Shinozaki, Kazuo; Hirayama, Takashi; Kikuchi, Jun

    2008-01-01

    Background Metabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling and analysis of metabolite mixtures in plant and animal systems. Methodology/Principal Findings The analysis method includes a stable isotope labeling technique for use in living organisms; a systematic method for simultaneously identifying a large number of metabolites by using a newly developed HSQC-based metabolite chemical shift database combined with heteronuclear multidimensional NMR spectroscopy; Principal Components Analysis; and a visualization method using a coarse-grained overview of the metabolic system. The database contains more than 1000 1H and 13C chemical shifts corresponding to 142 metabolites measured under identical physicochemical conditions. Using the stable isotope labeling technique in Arabidopsis T87 cultured cells and Bombyx mori, we systematically detected >450 HSQC peaks in each 13C-HSQC spectrum derived from model plant, Arabidopsis T87 cultured cells and the invertebrate animal model Bombyx mori. Furthermore, for the first time, efficient 13C labeling has allowed reliable signal assignment using analytical separation techniques such as 3D HCCH-COSY spectra in higher organism extracts. Conclusions/Significance Overall physiological changes could be detected and categorized in relation to a critical developmental phase change in B. mori by coarse-grained representations in which the organization of metabolic pathways related to a specific developmental phase was visualized on the basis of constituent changes of 56 identified metabolites. Based on the observed intensities of 13C atoms of

  5. Isotopic studies of metabolic systems by mass spectrometry: using Pascal's triangle to produce biological standards with fully controlled labeling patterns.

    PubMed

    Millard, Pierre; Massou, Stéphane; Portais, Jean-Charles; Létisse, Fabien

    2014-10-21

    Mass spectrometry (MS) is widely used for isotopic studies of metabolism in which detailed information about biochemical processes is obtained from the analysis of isotope incorporation into metabolites. The biological value of such experiments is dependent on the accuracy of the isotopic measurements. Using MS, isotopologue distributions are measured from the quantitative analysis of isotopic clusters. These measurements are prone to various biases, which can occur during the experimental workflow and/or MS analysis. The lack of relevant standards limits investigations of the quality of the measured isotopologue distributions. To meet that need, we developed a complete theoretical and experimental framework for the biological production of metabolites with fully controlled and predictable labeling patterns. This strategy is valid for different isotopes and different types of metabolisms and organisms, and was applied to two model microorganisms, Pichia augusta and Escherichia coli, cultivated on (13)C-labeled methanol and acetate as sole carbon source, respectively. The isotopic composition of the substrates was designed to obtain samples in which the isotopologue distribution of all the metabolites should give the binomial coefficients found in Pascal's triangle. The strategy was validated on a liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform by quantifying the complete isotopologue distributions of different intracellular metabolites, which were in close agreement with predictions. This strategy can be used to evaluate entire experimental workflows (from sampling to data processing) or different analytical platforms in the context of isotope labeling experiments.

  6. Application of isotope labeling experiments and (13)C flux analysis to enable rational pathway engineering.

    PubMed

    McAtee, Allison G; Jazmin, Lara J; Young, Jamey D

    2015-12-01

    Isotope labeling experiments (ILEs) and (13)C flux analysis provide actionable information for metabolic engineers to identify knockout, overexpression, and/or media optimization targets. ILEs have been used in both academic and industrial labs to increase product formation, discover novel metabolic functions in previously uncharacterized organisms, and enhance the metabolic efficiency of host cell factories. This review highlights specific examples of how ILEs have been used in conjunction with enzyme or metabolic engineering to elucidate host cell metabolism and improve product titer, rate, or yield in a directed manner. We discuss recent progress and future opportunities involving the use of ILEs and (13)C flux analysis to characterize non-model host organisms and to identify and subsequently eliminate wasteful byproduct pathways or metabolic bottlenecks. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. Development of High-Performance Chemical Isotope Labeling LC-MS for Profiling the Carbonyl Submetabolome.

    PubMed

    Zhao, Shuang; Dawe, Margot; Guo, Kevin; Li, Liang

    2017-06-20

    Metabolites containing a carbonyl group represent several important classes of molecules including various forms of ketones and aldehydes such as steroids and sugars. We report a high-performance chemical isotope labeling (CIL) LC-MS method for profiling the carbonyl submetabolome with high coverage and high accuracy and precision of relative quantification. This method is based on the use of dansylhydrazine (DnsHz) labeling of carbonyl metabolites to change their chemical and physical properties to such an extent that the labeled metabolites can be efficiently separated by reversed phase LC and ionized by electrospray ionization MS. In the analysis of six standards representing different carbonyl classes, acetaldehyde could be ionized only after labeling and MS signals were significantly increased for other 5 standards with an enhancement factor ranging from ∼15-fold for androsterone to ∼940-fold for 2-butanone. Differential 12 C- and 13 C-DnsHz labeling was developed for quantifying metabolic differences in comparative samples where individual samples were separately labeled with 12 C-labeling and spiked with a 13 C-labeled pooled sample, followed by LC-MS analysis, peak pair picking, and peak intensity ratio measurement. In the replicate analysis of a 1:1 12 C-/ 13 C-labeled human urine mixture (n = 6), an average of 2030 ± 39 pairs per run were detected with 1737 pairs in common, indicating the possibility of detecting a large number of carbonyl metabolites as well as high reproducibility of peak pair detection. The average RSD of the peak pair ratios was 7.6%, and 95.6% of the pairs had a RSD value of less than 20%, demonstrating high precision for peak ratio measurement. In addition, the ratios of most peak pairs were close to the expected value of 1.0 (e.g., 95.5% of them had ratios of between 0.67 and 1.5), showing the high accuracy of the method. For metabolite identification, a library of DnsHz-labeled standards was constructed, including 78 carbonyl

  8. Monitoring Microbial Mineralization Using Reverse Stable Isotope Labeling Analysis by Mid-Infrared Laser Spectroscopy

    PubMed Central

    2017-01-01

    Assessing the biodegradation of organic compounds is a frequent question in environmental science. Here, we present a sensitive, inexpensive, and simple approach to monitor microbial mineralization using reverse stable isotope labeling analysis (RIL) of dissolved inorganic carbon (DIC). The medium for the biodegradation assay contains regular organic compounds and 13C-labeled DIC with 13C atom fractions (x(13C)DIC) higher than natural abundance (typically 2–50%). The produced CO2 (x(13C) ≈ 1.11%) gradually dilutes the initial x(13C)DIC allowing to quantify microbial mineralization using mass-balance calculations. For 13C-enriched CO2 samples, a newly developed isotope ratio mid-infrared spectrometer was introduced with a precision of x(13C) < 0.006%. As an example for extremely difficult and slowly degradable compounds, CO2 production was close to the theoretical stoichiometry for anaerobic naphthalene degradation by a sulfate-reducing enrichment culture. Furthermore, we could measure the aerobic degradation of dissolved organic carbon (DOC) adsorbed to granular activated carbon in a drinking water production plant, which cannot be labeled with 13C. Thus, the RIL approach can be applied to sensitively monitor biodegradation of various organic compounds under anoxic or oxic conditions. PMID:28903553

  9. A cost-effective approach to produce 15N-labelled amino acids employing Chlamydomonas reinhardtii CC503.

    PubMed

    Nicolás Carcelén, Jesús; Marchante-Gayón, Juan Manuel; González, Pablo Rodríguez; Valledor, Luis; Cañal, María Jesús; Alonso, José Ignacio García

    2017-08-18

    The use of enriched stable isotopes is of outstanding importance in chemical metrology as it allows the application of isotope dilution mass spectrometry (IDMS). Primary methods based on IDMS ensure the quality of the analytical measurements and traceability of the results to the international system of units. However, the synthesis of isotopically labelled molecules from enriched stable isotopes is an expensive and a difficult task. Either chemical and biochemical methods to produce labelled molecules have been proposed, but so far, few cost-effective methods have been described. The aim of this study was to use the microalgae Chlamydomonas reinhardtii to produce, at laboratory scale, 15 N-labelled amino acids with a high isotopic enrichment. To do that, a culture media containing 15 NH 4 Cl was used. No kinetic isotope effect (KIE) was observed. The labelled proteins biosynthesized by the microorganism were extracted from the biomass and the 15 N-labelled amino acids were obtained after a protein hydrolysis with HCl. The use of the wall deficient strain CC503 cw92 mt+ is fit for purpose, as it only assimilates ammonia as nitrogen source, avoiding isotope contamination with nitrogen from the atmosphere or the reagents used in the culture medium, and enhancing the protein extraction efficiency compared to cell-walled wild type Chlamydomonas. The isotopic enrichment of the labelled amino acids was calculated from their isotopic composition measured by gas chromatography mass spectrometry (GC-MS). The average isotopic enrichment for the 16 amino acids characterized was 99.56 ± 0.05% and the concentration of the amino acids in the hydrolysate ranged from 18 to 90 µg/mL. Previously reported biochemical methods to produce isotopically labelled proteins have been applied in the fields of proteomics and fluxomics. For these approaches, low amounts of products are required and the isotopic enrichment of the molecules has never been properly determined. So far, only 13

  10. Intact stable isotope labeled plasma proteins from the SILAC-labeled HepG2 secretome.

    PubMed

    Mangrum, John B; Martin, Erika J; Brophy, Donald F; Hawkridge, Adam M

    2015-09-01

    The plasma proteome remains an attractive biospecimen for MS-based biomarker discovery studies. The success of these efforts relies on the continued development of quantitative MS-based proteomics approaches. Herein we report the use of the SILAC-labeled HepG2 secretome as a source for stable isotope labeled plasma proteins for quantitative LC-MS/MS measurements. The HepG2 liver cancer cell line secretes the major plasma proteins including serum albumin, apolipoproteins, protease inhibitors, coagulation factors, and transporters that represent some of the most abundant proteins in plasma. The SILAC-labeled HepG2 secretome was collected, spiked into human plasma (1:1 total protein), and then processed for LC-MS/MS analysis. A total of 62 and 56 plasma proteins were quantified (heavy:light (H/L) peptide pairs) from undepleted and depleted (serum albumin and IgG), respectively, with log2 H/L = ± 6. Major plasma proteins quantified included albumin, apolipoproteins (e.g., APOA1, APOA2, APOA4, APOB, APOC3, APOE, APOH, and APOM), protease inhibitors (e.g., A2M and SERPINs), coagulation factors (e.g., Factor V, Factor X, fibrinogen), and transport proteins (e.g., TTR). The average log2 H/L values for shared plasma proteins in both undepleted and depleted plasma samples were 0.43 and 0.44, respectively. This work further expands the SILAC strategy into MS-based biomarker discovery of clinical biospecimens. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Reconciling Estimates of Cell Proliferation from Stable Isotope Labeling Experiments

    PubMed Central

    Drylewicz, Julia; Elemans, Marjet; Zhang, Yan; Kelly, Elizabeth; Reljic, Rajko; Tesselaar, Kiki; de Boer, Rob J.; Macallan, Derek C.; Borghans, José A. M.; Asquith, Becca

    2015-01-01

    Stable isotope labeling is the state of the art technique for in vivo quantification of lymphocyte kinetics in humans. It has been central to a number of seminal studies, particularly in the context of HIV-1 and leukemia. However, there is a significant discrepancy between lymphocyte proliferation rates estimated in different studies. Notably, deuterated 2H2-glucose (D2-glucose) labeling studies consistently yield higher estimates of proliferation than deuterated water (D2O) labeling studies. This hampers our understanding of immune function and undermines our confidence in this important technique. Whether these differences are caused by fundamental biochemical differences between the two compounds and/or by methodological differences in the studies is unknown. D2-glucose and D2O labeling experiments have never been performed by the same group under the same experimental conditions; consequently a direct comparison of these two techniques has not been possible. We sought to address this problem. We performed both in vitro and murine in vivo labeling experiments using identical protocols with both D2-glucose and D2O. This showed that intrinsic differences between the two compounds do not cause differences in the proliferation rate estimates, but that estimates made using D2-glucose in vivo were susceptible to difficulties in normalization due to highly variable blood glucose enrichment. Analysis of three published human studies made using D2-glucose and D2O confirmed this problem, particularly in the case of short term D2-glucose labeling. Correcting for these inaccuracies in normalization decreased proliferation rate estimates made using D2-glucose and slightly increased estimates made using D2O; thus bringing the estimates from the two methods significantly closer and highlighting the importance of reliable normalization when using this technique. PMID:26437372

  12. Multi-Isotope Secondary Ion Mass Spectrometry Combining Heavy Water 2H with 15N Labeling As Complementary Tracers for Metabolic Heterogeneity at the Single-Cell Level

    NASA Astrophysics Data System (ADS)

    Kopf, S.; McGlynn, S.; Cowley, E.; Green, A.; Newman, D. K.; Orphan, V. J.

    2014-12-01

    Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system

  13. Evaluation of the impact of matrix effect on quantification of pesticides in foods by gas chromatography-mass spectrometry using isotope-labeled internal standards.

    PubMed

    Yarita, Takashi; Aoyagi, Yoshie; Otake, Takamitsu

    2015-05-29

    The impact of the matrix effect in GC-MS quantification of pesticides in food using the corresponding isotope-labeled internal standards was evaluated. A spike-and-recovery study of nine target pesticides was first conducted using paste samples of corn, green soybean, carrot, and pumpkin. The observed analytical values using isotope-labeled internal standards were more accurate for most target pesticides than that obtained using the external calibration method, but were still biased from the spiked concentrations when a matrix-free calibration solution was used for calibration. The respective calibration curves for each target pesticide were also prepared using matrix-free calibration solutions and matrix-matched calibration solutions with blank soybean extract. The intensity ratio of the peaks of most target pesticides to that of the corresponding isotope-labeled internal standards was influenced by the presence of the matrix in the calibration solution; therefore, the observed slope varied. The ratio was also influenced by the type of injection method (splitless or on-column). These results indicated that matrix-matching of the calibration solution is required for very accurate quantification, even if isotope-labeled internal standards were used for calibration. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Isotope Label-Aided Mass Spectrometry Reveals the Influence of Environmental Factors on Metabolism in Single Eggs of Fruit Fly

    PubMed Central

    Tseng, Te-Wei; Wu, June-Tai; Chen, Yu-Chie; Urban, Pawel L.

    2012-01-01

    In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar (13C6-glucose) for 12 h – either during the circadian day or the circadian night, at light or at dark. Third, eggs were obtained from the incubated female flies, and analyzed individually by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS): this yielded information about the extent of labeling with carbon-13. Since the incorporation of carbon-13 to uridine diphosphate glucose (UDP-glucose) in fruit fly eggs is very fast, the labeling of this metabolite was used as an indicator of the biosynthesis of metabolites flies/eggs during 12-h periods, which correspond to circadian day or circadian night. The results reveal that once the flies adapted to the 12-h-light/12-h-dark cycle, the incorporation of carbon-13 to UDP-glucose present in fruit fly eggs was not markedly altered by an acute perturbation to this cycle. This effect may be due to a relationship between biosynthesis of primary metabolites in developing eggs and an alteration to the intake of the labeled substrate – possibly related to the change of the feeding habit. Overall, the study shows the possibility of using MALDI-MS in conjunction with isotopic labeling of small metazoans to unravel the influence of environmental cues on primary metabolism. PMID:23185587

  15. High-efficiency dual labeling of influenza virus for single-virus imaging.

    PubMed

    Liu, Shu-Lin; Tian, Zhi-Quan; Zhang, Zhi-Ling; Wu, Qiu-Mei; Zhao, Hai-Su; Ren, Bin; Pang, Dai-Wen

    2012-11-01

    Many viruses invade host cells by entering the cells and releasing their genome for replication, which are remarkable incidents for viral infection. Therefore, the viral internal and external components should be simultaneously labeled and dynamically tracked at single-virus level for further understanding viral infection mechanisms. However, most of the previously reported methods have very low labeling efficiency and require considerable time and effort, which is laborious and inconvenient for researchers. In this work, we report a general strategy to high-efficiently label viral envelope and genome for single-virus imaging with quantum dots (QDs) and Syto 82, respectively. It was found that nearly all viral envelopes could be labeled with QDs with superior stability, which makes it possible to realize global and long-term tracking of single virus in individual cells. Effectively labeling their genome with Syto 82, about 90% of QDs-labeled viruses could be used to monitor the viral genome signal, which may provide valuable information for deeply studying viral genome transport. This is very important and meaningful to investigate the viral infection mechanism. Our labeling strategy has advantage in commonality, convenience and efficiency, which is expected to be widely used in biological research. Copyright © 2012 Elsevier Ltd. All rights reserved.

  16. High Yield Production and Radiochemical Isolation of Isotopically Pure Arsenic-72 and Novel Radioarsenic Labeling Strategies for the Development of Theranostic Radiopharmaceuticals.

    PubMed

    Ellison, Paul A; Barnhart, Todd E; Chen, Feng; Hong, Hao; Zhang, Yin; Theuer, Charles P; Cai, Weibo; Nickles, Robert J; DeJesus, Onofre T

    2016-01-20

    Radioisotopes of arsenic are of considerable interest to the field of nuclear medicine with unique nuclear and chemical properties making them well-suited for use in novel theranostic radiopharmaceuticals. However, progress must still be made in the production of isotopically pure radioarsenic and in its stable conjugation to biological targeting vectors. This work presents the production and irradiation of isotopically enriched (72)Ge(m) discs in an irrigation-cooled target system allowing for the production of isotopically pure (72)As with capability on the order of 10 GBq. A radiochemical separation procedure isolated the reactive trivalent radioarsenic in a small volume buffered aqueous solution, while reclaiming (72)Ge target material. The direct thiol-labeling of a monoclonal antibody resulted in a conjugate exhibiting exceptionally poor in vivo stability in a mouse model. This prompted further investigations to alternative radioarsenic labeling strategies, including the labeling of the dithiol-containing chelator dihydrolipoic acid, and thiol-modified mesoporous silica nanoparticles (MSN-SH). Radioarsenic-labeled MSN-SH showed exceptional in vivo stability toward dearsenylation.

  17. High Yield Production and Radiochemical Isolation of Isotopically Pure Arsenic-72 and Novel Radioarsenic Labeling Strategies for the Development of Theranostic Radiopharmaceuticals

    PubMed Central

    Ellison, Paul A.; Barnhart, Todd E.; Chen, Feng; Hong, Hao; Zhang, Yin; Theuer, Charles P.; Cai, Weibo; Nickles, Robert J.; DeJesus, Onofre T.

    2016-01-01

    Radioisotopes of arsenic are of considerable interest to the field of nuclear medicine with unique nuclear and chemical properties making them well-suited for use in novel theranostic radiopharmaceuticals. However, progress must still be made in the production of isotopically pure radioarsenic and in its stable conjugation to biological targeting vectors. This work presents the production and irradiation of isotopically enriched 72Ge(m) discs in an irrigation-cooled target system allowing for the production of isotopically pure 72As with capability on the order of 10 GBq. A radiochemical separation procedure isolated the reactive trivalent radioarsenic in a small volume buffered aqueous solution, while reclaiming 72Ge target material. The direct thiol-labeling of a monoclonal antibody resulted in a conjugate exhibiting exceptionally poor in vivo stability in a mouse model. This prompted further investigations to alternative radioarsenic labeling strategies, including the labeling of the dithiol-containing chelator dihydrolipoic acid, and thiol-modified mesoporous silica nanoparticles (MSN-SH). Radioarsenic-labeled MSN-SH showed exceptional in vivo stability toward dearsenylation. PMID:26646989

  18. Isotope-labeled cross-linkers and Fourier transform ion cyclotron resonance mass spectrometry for structural analysis of a protein/peptide complex.

    PubMed

    Ihling, Christian; Schmidt, Andreas; Kalkhof, Stefan; Schulz, Daniela M; Stingl, Christoph; Mechtler, Karl; Haack, Michael; Beck-Sickinger, Annette G; Cooper, Dermot M F; Sinz, Andrea

    2006-08-01

    For structural studies of proteins and their complexes, chemical cross-linking combined with mass spectrometry presents a promising strategy to obtain structural data of protein interfaces from low quantities of proteins within a short time. We explore the use of isotope-labeled cross-linkers in combination with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for a more efficient identification of cross-linker containing species. For our studies, we chose the calcium-independent complex between calmodulin and a 25-amino acid peptide from the C-terminal region of adenylyl cyclase 8 containing an "IQ-like motif." Cross-linking reactions between calmodulin and the peptide were performed in the absence of calcium using the amine-reactive, isotope-labeled (d0 and d4) cross-linkers BS3 (bis[sulfosuccinimidyl]suberate) and BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-gel digestion of excised gel bands from covalently cross-linked complexes resulted in complicated peptide mixtures, which were analyzed by nano-HPLC/nano-ESI-FTICR mass spectrometry. In cases where more than one reactive functional group, e.g., amine groups of lysine residues, is present in a sequence stretch, MS/MS analysis is a prerequisite for unambiguously identifying the modified residues. MS/MS experiments revealed two lysine residues in the central alpha-helix of calmodulin as well as three lysine residues both in the C-terminal and N-terminal lobes of calmodulin to be cross-linked with one single lysine residue of the adenylyl cyclase 8 peptide. Further cross-linking studies will have to be conducted to propose a structural model for the calmodulin/peptide complex, which is formed in the absence of calcium. The combination of using isotope-labeled cross-linkers, determining the accurate mass of intact cross-linked products, and verifying the amino acid sequences of cross-linked species by MS/MS presents a convenient approach that offers the perspective to obtain structural

  19. Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid.

    PubMed

    Geary, Bethany; Magee, Kieran; Cash, Phillip; Young, Iain S; Whitfield, Phillip D; Doherty, Mary K

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Multiplexed MRM-Based Protein Quantitation Using Two Different Stable Isotope-Labeled Peptide Isotopologues for Calibration.

    PubMed

    LeBlanc, André; Michaud, Sarah A; Percy, Andrew J; Hardie, Darryl B; Yang, Juncong; Sinclair, Nicholas J; Proudfoot, Jillaine I; Pistawka, Adam; Smith, Derek S; Borchers, Christoph H

    2017-07-07

    When quantifying endogenous plasma proteins for fundamental and biomedical research - as well as for clinical applications - precise, reproducible, and robust assays are required. Targeted detection of peptides in a bottom-up strategy is the most common and precise mass spectrometry-based quantitation approach when combined with the use of stable isotope-labeled peptides. However, when measuring protein in plasma, the unknown endogenous levels prevent the implementation of the best calibration strategies, since no blank matrix is available. Consequently, several alternative calibration strategies are employed by different laboratories. In this study, these methods were compared to a new approach using two different stable isotope-labeled standard (SIS) peptide isotopologues for each endogenous peptide to be quantified, enabling an external calibration curve as well as the quality control samples to be prepared in pooled human plasma without interference from endogenous peptides. This strategy improves the analytical performance of the assay and enables the accuracy of the assay to be monitored, which can also facilitate method development and validation.

  1. Carbon allocation belowground in Pinus pinaster using stable carbon isotope pulse labeling technique

    NASA Astrophysics Data System (ADS)

    Dannoura, M.; Bosc, A.; Chipeaux, C.; Sartore, M.; Lambrot, C.; Trichet, P.; Bakker, M.; Loustau, D.; Epron, D.

    2010-12-01

    Carbon allocation belowground competes with aboveground growth and biomass production. In the other hand, it contributes to resource acquisition such as nutrient, water and carbon sequestration in soil. Thus, a better characterization of carbon flow from plant to soil and its residence time within each compartment is an important issue for understanding and modeling forest ecosystem carbon budget. 13C pulse labeling of whole crown was conducted at 4 seasons to study the fate of assimilated carbon by photosynthesis into the root on 12 year old Pinus pinaster planted in the INRA domain of Pierroton. Maritime pine is the most widely planted species in South-West Europe. Stem, root and soil CO2 effluxes and their isotope composition were measured continuously by tunable diode laser absorption spectroscopy with a trace gas analyzer (TGA 100A; Campbell Scientific) coupled to flow-through chambers. 13CO2 recovery and peak were observed in respiration of each compartment after labeling. It appeared sequentially from top of stem to bottom, and to coarse root. The maximum velocity of carbon transfer was calculated as the difference in time lag of recovery between two positions on the trunk or on the root. It ranged between 0.08-0.2 m h-1 in stem and between 0.04-0.12 m h-1 in coarse root. This velocity was higher in warmer season, and the difference between time lag of recovery and peak increased after first frost. Photosynthates arrived underground 1.5 to 5 days after labeling, at similar time in soil CO2 effluxes and coarse root respiration. 0.08-1.4 g of carbon was respired per tree during first 20 days following labeling. It presented 0.6 -10% of 13C used for labeling and it is strongly related to seasons. The isotope signal was detected in fine root organs and microbial biomass by periodical core sampling. The peak was observed 6 days after labeling in early summer while it was delayed more than 10 days in autumn and winter with less amount of carbon allocated

  2. Carbon Allocation of 13CO2-labeled Photoassimilate in Larix gmelinii Saplings - A Physiological Basis for Isotope Dendroclimatology in Eastern Siberia.

    NASA Astrophysics Data System (ADS)

    Kagawa, A.; Sugimoto, A.; Maximov, T. C.

    2006-12-01

    Tree-ring density and widths have been successfully used to reconstruct summer temperatures in high- northern latitudes, although a discrepancy between tree-growth and temperature has been found for recent decades. The so-called "reduced sensitivity" of tree rings to summer temperatures has been observed especially strongly in northern Siberia (Briffa et al., 1998) and drought stress (increased water use efficiency) arose from global warming and/or increasing CO2 are suggested as causes (Barber et al. 2000, Saurer et al. 2004). By using carbon isotope ratio as an indicator of drought stress and ring-width/density as indicators of growth, we can clarify how drought stress caused by recent global warming affects wood formation of Siberian trees. However, isotope dendroclimatology is still in its infancy and our understanding of basic physiological processes of isotope signal transfer from leaves to tree rings is insufficient. In order to understand translocation, storage, and allocation of photoassimilate to different organs of trees, we pulse- labeled ten L. gmelinii growing in a continuous permafrost zone with stable 13CO2. We studied seasonal course of carbon allocation patterns of photoassimilate among needles, branches, stem and roots and also how spring, summer, and autumn photoassimilate is later used for both earlywood and latewood formation. About half of the carbon in new needles was derived from stored material. The starch pool in non- needle parts, which can be used for xylem formation, drew about 43 percent of its carbon from previous year's photoassimilate, suggesting that carbon storage is the key mechanism behind autocorrelation in (isotope) dendroclimatology. Analysis of intra-annual 13C of the tree rings formed after the labeling revealed that earlywood contained photoassimilate from the previous summer and autumn as well as from the current spring. Latewood was mainly composed of photoassimilate from the current year's summer/autumn, although it

  3. Efficient mixing of the solar nebula from uniform Mo isotopic composition of meteorites.

    PubMed

    Becker, Harry; Walker, Richard J

    2003-09-11

    The abundances of elements and their isotopes in our Galaxy show wide variations, reflecting different nucleosynthetic processes in stars and the effects of Galactic evolution. These variations contrast with the uniformity of stable isotope abundances for many elements in the Solar System, which implies that processes efficiently homogenized dust and gas from different stellar sources within the young solar nebula. However, isotopic heterogeneity has been recognized on the subcentimetre scale in primitive meteorites, indicating that these preserve a compositional memory of their stellar sources. Small differences in the abundance of stable molybdenum isotopes in bulk rocks of some primitive and differentiated meteorites, relative to terrestrial Mo, suggest large-scale Mo isotopic heterogeneity between some inner Solar System bodies, which implies physical conditions that did not permit efficient mixing of gas and dust. Here we report Mo isotopic data for bulk samples of primitive and differentiated meteorites that show no resolvable deviations from terrestrial Mo. This suggests efficient mixing of gas and dust in the solar nebula at least to 3 au from the Sun, possibly induced by magnetohydrodynamic instabilities. These mixing processes must have occurred before isotopic fractionation of gas-phase elements and volatility-controlled chemical fractionations were established.

  4. High efficiency labeling of glycoproteins on living cells

    PubMed Central

    Zeng, Ying; Ramya, T. N. C.; Dirksen, Anouk; Dawson, Philip E.; Paulson, James C.

    2010-01-01

    We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. PMID:19234450

  5. Modification degrees at specific sites on heparan sulphate: an approach to measure chemical modifications on biological molecules with stable isotope labelling

    PubMed Central

    Wu, Zhengliang L.; Lech, Miroslaw

    2005-01-01

    Chemical modification of biological molecules is a general mechanism for cellular regulation. A quantitative approach has been developed to measure the extent of modification on HS (heparan sulphates). Sulphation on HS by sulphotransferases leads to variable sulphation levels, which allows cells to tune their affinities to various extracellular proteins, including growth factors. With stable isotope labelling and HPLC-coupled MS, modification degrees at various O-sulphation sites could be determined. A bovine kidney HS sample was first saturated in vitro with 34S by an OST (O-sulphotransferase), then digested with nitrous acid and analysed with HPLC-coupled MS. The 34S-labelled oligosaccharides were identified based on their unique isotope clusters. The modification degrees at the sulphotransferase recognition sites were obtained by calculating the intensities of isotopic peaks in the isotope clusters. The modification degrees at 3-OST-1 and 6-OST-1 sites were examined in detail. This approach can also be used to study other types of chemical modifications on biological molecules. PMID:15743272

  6. Heavy isotope labeling study of the turnover of forskolin-stimulated adenylate cyclase in BC/sup 3/H1 cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.

    1987-06-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of adenylate cyclase in a nonfusing muscle cell line, the BC/sup 3/H1 cells. These cells contains an adenylate cyclase coupled to beta-adrenergic receptors and highly stimulated by forskolin, a potent activator of the enzyme. After transfer of the cells from normal medium to heavy medium (a medium containing heavy labeled amino acids, /sup 3/H, /sup 13/C, /sup 15/N), heavy isotope-labeled adenylate cyclase molecules progressively replace pre-existing light molecules. In sucrose gradient differential sedimentation, after a 5-day switch in heavy medium, the enzyme exhibited a higher massmore » (s = 8.40 +/- 0.03 S, n = 13) compared to the control enzyme. Indeed, the increase in the sedimentation coefficient of the heavy molecules was due to the synthesis of new molecules of adenylate cyclase labeled with heavy isotope amino acids since in the presence of cycloheximide, an inhibitor of protein synthesis, no change in the sedimentation pattern of the forskolin-stimulated adenylate cyclase occurred. After incorporation of heavy isotope amino acids in the adenylate cyclase molecules, the kinetics parameters of the enzyme did not change. However, adenylate cyclase from cells incubated with heavy medium exhibits an activity about 2-fold lower than control. After switching the cells to the heavy medium, the decrease of the activity of the enzyme occurred during the first 24 h and thereafter remained at a steady state for at least 4 days. In contrast, 24 h after the switch, the sedimentation coefficient of forskolin-stimulated adenylate cyclase was progressively shifted to a higher value.« less

  7. Synthesis of two potent glucocorticoid receptor agonists labeled with carbon-14 and stable isotopes.

    PubMed

    Latli, Bachir; Reeves, Jonathan T; Tan, Zhulin; Hrapchak, Matt; Song, Jinhua J; Busacca, Carl B; Senanayake, Chris H

    2015-01-01

    Two potent glucocorticoid receptor agonists were prepared labeled with carbon-14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon-14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5-amino-4-iodo-[2-(14)C]pyrimidine [(14)C]-(6), followed by a base-mediated cyclization (1) in 72% overall radiochemical yield. Carbon-14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [(14)C]-(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine-magnesium exchange and then electrophile trapping reaction with [(14)C]-carbon dioxide. A chiral auxiliary (S)-1-(4-methoxyphenyl)ethylamine was then coupled to this acid to give [(14)C]-(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [(14)C]-(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [(14)C]-(18) and finally removal of the chiral auxiliary gave [(14)C]-(2) in 7% overall yield. For stable isotope syntheses, [(13)C6]-(1) was obtained in three steps using [(13)C4]-(6) and trimethylsilylacetylene-[(13)C2] in 26% yield, while [(2)H5]-(2) was obtained by first preparing the iodopyridine [(2)H5]-(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [(2)H5]-(2) in 42% overall yield. Copyright © 2015 John Wiley & Sons, Ltd.

  8. Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

    PubMed

    Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2015-06-01

    Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

  9. Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b₅₅₈ (SdhC).

    PubMed

    Baureder, Michael; Hederstedt, Lars

    2011-11-01

    Cytochrome b₅₅₈ of the gram-positive bacterium Bacillussubtilis is the membrane anchor subunit of the succinate:quinone oxidoreductase of the citric acid cycle. The cytochrome consists of the SdhC polypeptide (202 residues) and two protoheme IX groups that function in transmembrane electron transfer to menaquinone. The general structure of the cytochrome is known from extensive experimental studies and by comparison to Wolinellasuccinogenes fumarate reductase for which the X-ray crystal structure has been determined. Solution state NMR can potentially be used to identify the quinone binding site(s) and study, e.g. redox-linked, dynamics of cytochrome b₅₅₈. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b₅₅₈ produced in Escherichia coli. We have also evaluated several detergents suitable for NMR for their effectiveness in maintaining the cytochrome solubilized and intact for days at room temperature. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Weaving a two-dimensional fishing net from titanoniobate nanosheets embedded with Fe3O4 nanocrystals for highly efficient capture and isotope labeling of phosphopeptides

    NASA Astrophysics Data System (ADS)

    Chen, Xueqin; Li, Siyuan; Zhang, Xiaoxia; Min, Qianhao; Zhu, Jun-Jie

    2015-03-01

    Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3O4 nanocrystals (Fe3O4-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe3O4 nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe3O4-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests.Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3

  11. Combining Raman and FT-IR spectroscopy with quantitative isotopic labeling for differentiation of E. coli cells at community and single cell levels.

    PubMed

    Muhamadali, Howbeer; Chisanga, Malama; Subaihi, Abdu; Goodacre, Royston

    2015-04-21

    There is no doubt that the contribution of microbially mediated bioprocesses toward maintenance of life on earth is vital. However, understanding these microbes in situ is currently a bottleneck, as most methods require culturing these microorganisms to suitable biomass levels so that their phenotype can be measured. The development of new culture-independent strategies such as stable isotope probing (SIP) coupled with molecular biology has been a breakthrough toward linking gene to function, while circumventing in vitro culturing. In this study, for the first time we have combined Raman spectroscopy and Fourier transform infrared (FT-IR) spectroscopy, as metabolic fingerprinting approaches, with SIP to demonstrate the quantitative labeling and differentiation of Escherichia coli cells. E. coli cells were grown in minimal medium with fixed final concentrations of carbon and nitrogen supply, but with different ratios and combinations of (13)C/(12)C glucose and (15)N/(14)N ammonium chloride, as the sole carbon and nitrogen sources, respectively. The cells were collected at stationary phase and examined by Raman and FT-IR spectroscopies. The multivariate analysis investigation of FT-IR and Raman data illustrated unique clustering patterns resulting from specific spectral shifts upon the incorporation of different isotopes, which were directly correlated with the ratio of the isotopically labeled content of the medium. Multivariate analysis results of single-cell Raman spectra followed the same trend, exhibiting a separation between E. coli cells labeled with different isotopes and multiple isotope levels of C and N.

  12. Global Potential of Energy Efficiency Standards and Labeling Programs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McNeil, Michael A; McNeil, Michael A.; Letschert, Virginie

    2008-06-15

    This report estimates the global potential reductions in greenhouse gas emissions by 2030 for energy efficiency improvements associated with equipment (appliances, lighting, and HVAC) in buildings by means of energy efficiency standards and labels (EES&L). A consensus has emerged among the world's scientists and many corporate and political leaders regarding the need to address the threat of climate change through emissions mitigation and adaptation. A further consensus has emerged that a central component of these strategies must be focused around energy, which is the primary generator of greenhouse gas emissions. Two important questions result from this consensus: 'what kinds ofmore » policies encourage the appropriate transformation to energy efficiency' and 'how much impact can these policies have'? This report aims to contribute to the dialogue surrounding these issues by considering the potential impacts of a single policy type, applied on a global scale. The policy addressed in this report is Energy Efficient Standards and Labeling (EES&L) for energy-consuming equipment, which has now been implemented in over 60 countries. Mandatory energy performance standards are important because they contribute positively to a nation's economy and provide relative certainty about the outcome (both timing and magnitudes). Labels also contribute positively to a nation's economy and importantly increase the awareness of the energy-consuming public. Other policies not analyzed here (utility incentives, tax credits) are complimentary to standards and labels and also contribute in significant ways to reducing greenhouse gas emissions. We believe the analysis reported here to be the first systematic attempt to evaluate the potential of savings from EES&L for all countries and for such a large set of products. The goal of the analysis is to provide an assessment that is sufficiently well-quantified and accurate to allow comparison and integration with other strategies under

  13. Rapid protein concentration, efficient fluorescence labeling and purification on a micro/nanofluidics chip.

    PubMed

    Wang, Chen; Ouyang, Jun; Ye, De-Kai; Xu, Jing-Juan; Chen, Hong-Yuan; Xia, Xing-Hua

    2012-08-07

    Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency.

  14. The synthesis of tritium, carbon-14 and stable isotope labelled selective estrogen receptor degraders.

    PubMed

    Bragg, Ryan A; Bushby, Nick; Ericsson, Cecilia; Kingston, Lee P; Ji, Hailong; Elmore, Charles S

    2016-09-01

    As part of a Medicinal Chemistry program aimed at developing an orally bioavailable selective estrogen receptor degrader, a number of tritium, carbon-14, and stable isotope labelled (E)-3-[4-(2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indol-1-yl)phenyl]prop-2-enoic acids were required. This paper discusses 5 synthetic approaches to this compound class. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Ionization enhancement in atmospheric pressure chemical ionization and suppression in electrospray ionization between target drugs and stable-isotope-labeled internal standards in quantitative liquid chromatography/tandem mass spectrometry.

    PubMed

    Liang, H R; Foltz, R L; Meng, M; Bennett, P

    2003-01-01

    The phenomena of ionization suppression in electrospray ionization (ESI) and enhancement in atmospheric pressure chemical ionization (APCI) were investigated in selected-ion monitoring and selected-reaction monitoring modes for nine drugs and their corresponding stable-isotope-labeled internal standards (IS). The results showed that all investigated target drugs and their co-eluting isotope-labeled IS suppress each other's ionization responses in ESI. The factors affecting the extent of suppression in ESI were investigated, including structures and concentrations of drugs, matrix effects, and flow rate. In contrast to the ESI results, APCI caused seven of the nine investigated target drugs and their co-eluting isotope-labeled IS to enhance each other's ionization responses. The mutual ionization suppression or enhancement between drugs and their isotope-labeled IS could possibly influence assay sensitivity, reproducibility, accuracy and linearity in quantitative liquid chromatography/mass spectrometry (LC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). However, calibration curves were linear if an appropriate IS concentration was selected for a desired calibration range to keep the response factors constant. Copyright 2003 John Wiley & Sons, Ltd.

  16. Stable isotope labeling tandem mass spectrometry (SILT) to quantify protein production and clearance rates

    PubMed Central

    Bateman, Randall J.; Munsell, Ling Y.; Chen, Xianghong; Holtzman, David M.; Yarasheski, Kevin E.

    2007-01-01

    In all biological systems, protein amount is a function of the rate of production and clearance. The speed of a response to a disturbance in protein homeostasis is determined by turnover rate. Quantifying alterations in protein synthesis and clearance rates is vital to understanding disease pathogenesis (e.g., aging, inflammation). No methods exist for quantifying production and clearance rates of low abundance (femtomole) proteins in vivo. We describe a novel, mass spectrometry-based method for quantitating low abundance protein synthesis and clearance rates in vitro and in vivo in animals and humans. The utility of this method is demonstrated with amyloid-beta (Aß), an important low abundance protein involved in Alzheimer's disease pathogenesis. We used in vivo stable isotope labeling, immunoprecipitation of Aß from cerebrospinal fluid, and quantitative liquid chromatography electrospray-ionization tandem mass spectrometry (LC-ESI-tandem MS) to quantify human Aß protein production and clearance rates. The method is sensitive and specific for stable isotope labeled amino acid incorporation into CNS (± 1% accuracy). This in vivo method can be used to identify pathophysiologic changes in protein metabolism; and may serve as a biomarker for monitoring disease risk, progression, or response to novel therapeutic agents. The technique is adaptable to other macromolecules, such as carbohydrates or lipids. PMID:17383190

  17. Cell-specific Labeling Enzymes for Analysis of Cell–Cell Communication in Continuous Co-culture*

    PubMed Central

    Tape, Christopher J.; Norrie, Ida C.; Worboys, Jonathan D.; Lim, Lindsay; Lauffenburger, Douglas A.; Jørgensen, Claus

    2014-01-01

    We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDCM.tub-KDEL) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (LyrM37-KDEL) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDCM.tub-KDEL and LyrM37-KDEL facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell–cell phospho-signaling experiments, we propose DDCM.tub-KDEL and LyrM37-KDEL as excellent enzymes for cell-specific labeling with amino acid precursors. PMID:24820872

  18. Measurement of skeletal muscle perfusion dynamics with pseudo-continuous arterial spin labeling (pCASL): Assessment of relative labeling efficiency at rest and during hyperemia, and comparison to pulsed arterial spin labeling (PASL).

    PubMed

    Englund, Erin K; Rodgers, Zachary B; Langham, Michael C; Mohler, Emile R; Floyd, Thomas F; Wehrli, Felix W

    2016-10-01

    To compare calf skeletal muscle perfusion measured with pulsed arterial spin labeling (PASL) and pseudo-continuous arterial spin labeling (pCASL) methods, and to assess the variability of pCASL labeling efficiency in the popliteal artery throughout an ischemia-reperfusion paradigm. At 3T, relative pCASL labeling efficiency was experimentally assessed in five subjects by measuring the signal intensity of blood in the popliteal artery just distal to the labeling plane immediately following pCASL labeling or control preparation pulses, or without any preparation pulses throughout separate ischemia-reperfusion paradigms. The relative label and control efficiencies were determined during baseline, hyperemia, and recovery. In a separate cohort of 10 subjects, pCASL and PASL sequences were used to measure reactive hyperemia perfusion dynamics. Calculated pCASL labeling and control efficiencies did not differ significantly between baseline and hyperemia or between hyperemia and recovery periods. Relative to the average baseline, pCASL label efficiency was 2 ± 9% lower during hyperemia. Perfusion dynamics measured with pCASL and PASL did not differ significantly (P > 0.05). Average leg muscle peak perfusion was 47 ± 20 mL/min/100g or 50 ± 12 mL/min/100g, and time to peak perfusion was 25 ± 3 seconds and 25 ± 7 seconds from pCASL and PASL data, respectively. Differences of further metrics parameterizing the perfusion time course were not significant between pCASL and PASL measurements (P > 0.05). No change in pCASL labeling efficiency was detected despite the almost 10-fold increase in average blood flow velocity in the popliteal artery. pCASL and PASL provide precise and consistent measurement of skeletal muscle reactive hyperemia perfusion dynamics. J. MAGN. RESON. IMAGING 2016;44:929-939. © 2016 International Society for Magnetic Resonance in Medicine.

  19. Efficient Site-Specific Labeling of Proteins via Cysteines

    PubMed Central

    Kim, Younggyu; Ho, Sam O.; Gassman, Natalie R.; Korlann, You; Landorf, Elizabeth V.; Collart, Frank R.; Weiss, Shimon

    2011-01-01

    Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70–90%, and specificities are better than ~95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis. PMID:18275130

  20. Efficient site-specific labeling of proteins via cysteines.

    PubMed

    Kim, Younggyu; Ho, Sam O; Gassman, Natalie R; Korlann, You; Landorf, Elizabeth V; Collart, Frank R; Weiss, Shimon

    2008-03-01

    Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

  1. In-depth Qualitative and Quantitative Profiling of Tyrosine Phosphorylation Using a Combination of Phosphopeptide Immunoaffinity Purification and Stable Isotope Dimethyl Labeling*

    PubMed Central

    Boersema, Paul J.; Foong, Leong Yan; Ding, Vanessa M. Y.; Lemeer, Simone; van Breukelen, Bas; Philp, Robin; Boekhorst, Jos; Snel, Berend; den Hertog, Jeroen; Choo, Andre B. H.; Heck, Albert J. R.

    2010-01-01

    Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative

  2. Iron physiological requirements in Chinese adults assessed by the stable isotope labeling technique.

    PubMed

    Cai, Jie; Ren, Tongxiang; Zhang, Yuhui; Wang, Zhilin; Gou, Lingyan; Huang, Zhengwu; Wang, Jun; Piao, Jianhua; Yang, Xiaoguang; Yang, Lichen

    2018-01-01

    Iron is a kind of essential trace mineral in the human body, while the studies on its physiological requirement are very limited recently, especially in China. And most studies were performed with the radioisotope tracer technique, which was harmful to health. This study aimed to first get the value of iron physiological requirements in Chinese adults assessed by the stable isotope labeling technique. Forty-four eligible young Chinese healthy adults were randomly recruited from the Bethune Military Medical College (Shijiazhuang, Hebei, China) between January 2010 and March 2011, and 19 subjects were included in the final data analysis. After adaptive diets and observation, subjects received 58 Fe intravenously. The baseline venous blood sample and general information were collected on day 0. Venous blood samples were also collected on day 14, 30, 60, 100, 120, 150, 240, 330, 425, 515, 605, 767, 1155, respectively. The blood samples were acid digested by a Microwave Digestion System and then analyzed by the MC-ICP-MS and Atomic Absorption Spectroscopy to get the abundance of Fe isotopes and the total iron concentration respectively. The circulation rate (the proportion of blood iron to whole body iron) could be calculated by the intake amount, background content and the peak isotope content. When the abundance changed stably, the iron physiological requirement could be calculated by the iron loss in a period of time. The abundance of 58 Fe reached its peak on day 14, and changed stably from day 425. The average circulation rate was 84%, with no significance difference between the 2 genders. The mean iron requirement in females was 1101.68 μg/d, and the mean requirement adjusted by body weight was 20.69 μg/kg.d. For males, the mean iron requirement was 959.9 μg/d, and the requirement adjusted by body weight was 14.04 μg/kg.d. Our study has obtained the data about the iron physiological requirements of Chinese adults using stable isotope labeling technique

  3. Application of isotopic labeling, and gas chromatography mass spectrometry, to understanding degradation products and pathways in the thermal-oxidative aging of Nylon 6.6

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    White, Gregory Von; Clough, Roger L.; Hochrein, James M.

    2013-12-01

    Nylon 6.6 containing 13C isotopic labels at specific positions along the macromolecular backbone has been subjected to extensive thermal-oxidative aging at 138 °C for time periods up to 243 days. In complementary experiments, unlabeled Nylon 6.6 was subjected to the same aging conditions under an atmosphere of 18O 2. Volatile organic degradation products were analyzed by cryofocusing gas chromatography mass spectrometry (cryo-GC/MS) to identify the isotopic labeling. The labeling results, combined with basic considerations of free radical reaction chemistry, provided insights to the origin of degradation species, with respect to the macromolecular structure. A number of inferences on chemical mechanismsmore » were drawn, based on 1) the presence (or absence) of the isotopic labels in the various products, 2) the location of the isotope within the product molecule, and 3) the relative abundance of products as indicated by large differences in peak intensities in the gas chromatogram. The overall degradation results can be understood in terms of free radical pathways originating from initial attacks on three different positions along the nylon chain which include hydrogen abstraction from: the (CH 2) group adjacent to the nitrogen atom, at the (CH 2) adjacent the carbonyl group, and direct radical attack on the carbonyl. Understanding the pathways which lead to Nylon 6.6 degradation ultimately provides new insight into changes that can be leveraged to detect and reduce early aging and minimize problems associated with material degradation.« less

  4. Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

    PubMed Central

    Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.

    2012-01-01

    Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account. PMID:22157354

  5. Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

    PubMed

    Aigbirhio, F I; Allwein, S; Anwar, A; Atzrodt, J; Audisio, D; Badman, G; Bakale, R; Berthon, F; Bragg, R; Brindle, K M; Bushby, N; Campos, S; Cant, A A; Chan, M Y T; Colbon, P; Cornelissen, B; Czarny, B; Derdau, V; Dive, V; Dunscombe, M; Eggleston, I; Ellis-Sawyer, K; Elmore, C S; Engstrom, P; Ericsson, C; Fairlamb, I J S; Georgin, D; Godfrey, S P; He, L; Hickey, M J; Huscroft, I T; Kerr, W J; Lashford, A; Lenz, E; Lewinton, S; L'Hermite, M M; Lindelöf, Å; Little, G; Lockley, W J S; Loreau, O; Maddocks, S; Marguerit, M; Mirabello, V; Mudd, R J; Nilsson, G N; Owens, P K; Pascu, S I; Patriarche, G; Pimlott, S L; Pinault, M; Plastow, G; Racys, D T; Reif, J; Rossi, J; Ruan, J; Sarpaki, S; Sephton, S M; Simonsson, R; Speed, D J; Sumal, K; Sutherland, A; Taran, F; Thuleau, A; Wang, Y; Waring, M; Watters, W H; Wu, J; Xiao, J

    2016-04-01

    The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK). Copyright © 2016 John Wiley & Sons, Ltd.

  6. Improving labeling efficiency in automatic quality control of MRSI data.

    PubMed

    Pedrosa de Barros, Nuno; McKinley, Richard; Wiest, Roland; Slotboom, Johannes

    2017-12-01

    To improve the efficiency of the labeling task in automatic quality control of MR spectroscopy imaging data. 28'432 short and long echo time (TE) spectra (1.5 tesla; point resolved spectroscopy (PRESS); repetition time (TR)= 1,500 ms) from 18 different brain tumor patients were labeled by two experts as either accept or reject, depending on their quality. For each spectrum, 47 signal features were extracted. The data was then used to run several simulations and test an active learning approach using uncertainty sampling. The performance of the classifiers was evaluated as a function of the number of patients in the training set, number of spectra in the training set, and a parameter α used to control the level of classification uncertainty required for a new spectrum to be selected for labeling. The results showed that the proposed strategy allows reductions of up to 72.97% for short TE and 62.09% for long TE in the amount of data that needs to be labeled, without significant impact in classification accuracy. Further reductions are possible with significant but minimal impact in performance. Active learning using uncertainty sampling is an effective way to increase the labeling efficiency for training automatic quality control classifiers. Magn Reson Med 78:2399-2405, 2017. © 2017 International Society for Magnetic Resonance in Medicine. © 2017 International Society for Magnetic Resonance in Medicine.

  7. Separation efficiency of the MASHA facility for short-lived mercury isotopes

    NASA Astrophysics Data System (ADS)

    Rodin, A. M.; Belozerov, A. V.; Chernysheva, E. V.; Dmitriev, S. N.; Gulyaev, A. V.; Gulyaeva, A. V.; Itkis, M. G.; Kliman, J.; Kondratiev, N. A.; Krupa, L.; Novoselov, A. S.; Oganessian, Yu. Ts.; Podshibyakin, A. V.; Salamatin, V. S.; Siváček, I.; Stepantsov, S. V.; Vanin, D. V.; Vedeneev, V. Yu.; Yukhimchuk, S. A.; Granja, C.; Pospisil, S.

    2014-06-01

    The mass-separator MASHA built to identify Super Heavy Elements by their mass-to-charge ratios is described. The results of the off- and on-line measurements of its separation efficiency are presented. In the former case four calibrated leaks of noble gases were used. In the latter the efficiency was measured via 284 MeV Ar beam and with using the hot catcher. The ECR ion source was used in both cases. The -radioactive isotopes of mercury produced in the complete fusion reaction Ar+SmHg+xn were detected at the mass-separator focal plane. The half-lives and the separation efficiency for the short-lived mercury isotopes were measured. Potentialities of the MEDIPIX detector system have been demonstrated for future use at the mass-separator MASHA.

  8. Energy-efficiency labels and standards: A guidebook for appliances, equipment and lighting

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McMahon, James E.; Wiel, Stephen

    2001-02-16

    Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and the United Nations Foundation (UNF) recognize the need to support policy makers in their efforts to implement energy-efficiency standards and labelingmore » programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This guidebook was prepared over the course of the past year with significant contribution from the authors and reviewers mentioned previously. Their diligent participation has made this the international guidance tool it was intended to be. The lead authors would also like to thank the following individuals for their support in the development, production, and distribution of the guidebook: Marcy Beck, Elisa Derby, Diana Dhunke, Ted Gartner, and Julie Osborn of Lawrence Berkeley National Laboratory as well as Anthony Ma of Bevilacqua-Knight, Inc. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards-setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design, development, implementation, maintenance, and evaluation of the

  9. Triple-Label β Liquid Scintillation Counting

    PubMed Central

    Bukowski, Thomas R.; Moffett, Tyler C.; Revkin, James H.; Ploger, James D.; Bassingthwaighte, James B.

    2010-01-01

    The detection of radioactive compounds by liquid scintillation has revolutionized modern biology, yet few investigators make full use of the power of this technique. Even though multiple isotope counting is considerably more difficult than single isotope counting, many experimental designs would benefit from using more than one isotope. The development of accurate isotope counting techniques enabling the simultaneous use of three β-emitting tracers has facilitated studies in our laboratory using the multiple tracer indicator dilution technique for assessing rates of transmembrane transport and cellular metabolism. The details of sample preparation, and of stabilizing the liquid scintillation spectra of the tracers, are critical to obtaining good accuracy. Reproducibility is enhanced by obtaining detailed efficiency/quench curves for each particular set of tracers and solvent media. The numerical methods for multiple-isotope quantitation depend on avoiding error propagation (inherent to successive subtraction techniques) by using matrix inversion. Experimental data obtained from triple-label β counting illustrate reproducibility and good accuracy even when the relative amounts of different tracers in samples of protein/electrolyte solutions, plasma, and blood are changed. PMID:1514684

  10. Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

    PubMed

    Lacour, William; Adjili, Salim; Blaising, Julie; Favier, Arnaud; Monier, Karine; Mezhoud, Sarra; Ladavière, Catherine; Place, Christophe; Pécheur, Eve-Isabelle; Charreyre, Marie-Thérèse

    2016-08-01

    Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Optimized small molecule antibody labeling efficiency through continuous flow centrifugal diafiltration.

    PubMed

    Cappione, Amedeo; Mabuchi, Masaharu; Briggs, David; Nadler, Timothy

    2015-04-01

    Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria.

    PubMed

    Chang, Shih Chieh; Galea, Charles A; Leung, Eleanor W W; Tajhya, Rajeev B; Beeton, Christine; Pennington, Michael W; Norton, Raymond S

    2012-10-01

    The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (T(EM)). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T(EM) cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni²⁺ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of ¹⁵N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a K(d) of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T(EM) cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of ¹⁵N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Application of SAIL phenylalanine and tyrosine with alternative isotope-labeling patterns for protein structure determination.

    PubMed

    Takeda, Mitsuhiro; Ono, Akira M; Terauchi, Tsutomu; Kainosho, Masatsune

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations.

  14. The impact of carbon-13 and deuterium on relative quantification of proteins using stable isotope diethyl labeling.

    PubMed

    Koehler, Christian J; Arntzen, Magnus Ø; Thiede, Bernd

    2015-05-15

    Stable isotopic labeling techniques are useful for quantitative proteomics. A cost-effective and convenient method for diethylation by reductive amination was established. The impact using either carbon-13 or deuterium on quantification accuracy and precision was investigated using diethylation. We established an effective approach for stable isotope labeling by diethylation of amino groups of peptides. The approach was validated using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nanospray liquid chromatography/electrospray ionization (nanoLC/ESI)-ion trap/orbitrap for mass spectrometric analysis as well as MaxQuant for quantitative data analysis. Reaction conditions with low reagent costs, high yields and minor side reactions were established for diethylation. Furthermore, we showed that diethylation can be applied to up to sixplex labeling. For duplex experiments, we compared diethylation in the analysis of the proteome of HeLa cells using acetaldehyde-(13) C(2)/(12) C(2) and acetaldehyde-(2) H(4)/(1) H(4). Equal numbers of proteins could be identified and quantified; however, (13) C(4)/(12) C(4) -diethylation revealed a lower variance of quantitative peptide ratios within proteins resulting in a higher precision of quantified proteins and less falsely regulated proteins. The results were compared with dimethylation showing minor effects because of the lower number of deuteriums. The described approach for diethylation of primary amines is a cost-effective and accurate method for up to sixplex relative quantification of proteomes. (13) C(4)/(12) C(4) -diethylation enables duplex quantification based on chemical labeling without using deuterium which reduces identification of false-negatives and increases the quality of the quantification results. Copyright © 2015 John Wiley & Sons, Ltd.

  15. Simple SPION Incubation as an Efficient Intracellular Labeling Method for Tracking Neural Progenitor Cells Using MRI

    PubMed Central

    D. M., Jayaseema; Lai, Jiann-Shiun; Hueng, Dueng-Yuan; Chang, Chen

    2013-01-01

    Cellular magnetic resonance imaging (MRI) has been well-established for tracking neural progenitor cells (NPC). Superparamagnetic iron oxide nanoparticles (SPIONs) approved for clinical application are the most common agents used for labeling. Conventionally, transfection agents (TAs) were added with SPIONs to facilitate cell labeling because SPIONs in the native unmodified form were deemed inefficient for intracellular labeling. However, compelling evidence also shows that simple SPION incubation is not invariably ineffective. The labeling efficiency can be improved by prolonged incubation and elevated iron doses. The goal of the present study was to establish simple SPION incubation as an efficient intracellular labeling method. To this end, NPCs derived from the neonatal subventricular zone were incubated with SPIONs (Feridex®) and then evaluated in vitro with regard to the labeling efficiency and biological functions. The results showed that, following 48 hours of incubation at 75 µg/ml, nearly all NPCs exhibited visible SPION intake. Evidence from light microscopy, electron microscopy, chemical analysis, and magnetic resonance imaging confirmed the effectiveness of the labeling. Additionally, biological assays showed that the labeled NPCs exhibited unaffected viability, oxidative stress, apoptosis and differentiation. In the demonstrated in vivo cellular MRI experiment, the hypointensities representing the SPION labeled NPCs remained observable throughout the entire tracking period. The findings indicate that simple SPION incubation without the addition of TAs is an efficient intracellular magnetic labeling method. This simple approach may be considered as an alternative approach to the mainstream labeling method that involves the use of TAs. PMID:23468856

  16. In Vivo Isotopic Labeling of Symbiotic Bacteria Involved in Cellulose Degradation and Nitrogen Recycling within the Gut of the Forest Cockchafer (Melolontha hippocastani).

    PubMed

    Alonso-Pernas, Pol; Bartram, Stefan; Arias-Cordero, Erika M; Novoselov, Alexey L; Halty-deLeon, Lorena; Shao, Yongqi; Boland, Wilhelm

    2017-01-01

    The guts of insects harbor symbiotic bacterial communities. However, due to their complexity, it is challenging to relate a specific symbiotic phylotype to its corresponding function. In the present study, we focused on the forest cockchafer ( Melolontha hippocastani ), a phytophagous insect with a dual life cycle, consisting of a root-feeding larval stage and a leaf-feeding adult stage. By combining in vivo stable isotope probing (SIP) with 13 C cellulose and 15 N urea as trophic links, with Illumina MiSeq (Illumina-SIP), we unraveled bacterial networks processing recalcitrant dietary components and recycling nitrogenous waste. The bacterial communities behind these processes change between larval and adult stages. In 13 C cellulose-fed insects, the bacterial families Lachnospiraceae and Enterobacteriaceae were isotopically labeled in larvae and adults, respectively. In 15 N urea-fed insects, the genera Burkholderia and Parabacteroides were isotopically labeled in larvae and adults, respectively. Additionally, the PICRUSt-predicted metagenome suggested a possible ability to degrade hemicellulose and to produce amino acids of, respectively, 13 C cellulose- and 15 N urea labeled bacteria. The incorporation of 15 N from ingested urea back into the insect body was confirmed, in larvae and adults, by isotope ratio mass spectrometry (IRMS). Besides highlighting key bacterial symbionts of the gut of M. hippocastani , this study provides example on how Illumina-SIP with multiple trophic links can be used to target microorganisms embracing different roles within an environment.

  17. Protected Amine Labels: A Versatile Molecular Scaffold for Multiplexed Nominal Mass and Sub-Da Isotopologue Quantitative Proteomic Reagents

    PubMed Central

    Ficarro, Scott B.; Biagi, Jessica M.; Wang, Jinhua; Scotcher, Jenna; Koleva, Rositsa I.; Card, Joseph D.; Adelmant, Guillaume; He, Huan; Askenazi, Manor; Marshall, Alan G.; Young, Nicolas L.; Gray, Nathanael S.; Marto, Jarrod A.

    2014-01-01

    We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (i) robust targeting of peptide N-termini and lysyl side chains, (ii) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (iii) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (iv) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally we provide exemplar data that extends the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers. PMID:24496597

  18. Amino Acid Selective 13C Labeling and 13C Scrambling Profile Analysis of Protein α and Side-Chain Carbons in Escherichia coli Utilized for Protein Nuclear Magnetic Resonance.

    PubMed

    Sugiki, Toshihiko; Furuita, Kyoko; Fujiwara, Toshimichi; Kojima, Chojiro

    2018-06-20

    Amino acid selective isotope labeling is an important nuclear magnetic resonance technique, especially for larger proteins, providing strong bases for the unambiguous resonance assignments and information concerning the structure, dynamics, and intermolecular interactions. Amino acid selective 15 N labeling suffers from isotope dilution caused by metabolic interconversion of the amino acids, resulting in isotope scrambling within the target protein. Carbonyl 13 C atoms experience less isotope scrambling than the main-chain 15 N atoms do. However, little is known about the side-chain 13 C atoms. Here, the 13 C scrambling profiles of the Cα and side-chain carbons were investigated for 15 N scrambling-prone amino acids, such as Leu, Ile, Tyr, Phe, Thr, Val, and Ala. The level of isotope scrambling was substantially lower in 13 Cα and 13 C side-chain labeling than in 15 N labeling. We utilized this reduced scrambling-prone character of 13 C as a simple and efficient method for amino acid selective 13 C labeling using an Escherichia coli cold-shock expression system and high-cell density fermentation. Using this method, the 13 C labeling efficiency was >80% for Leu and Ile, ∼60% for Tyr and Phe, ∼50% for Thr, ∼40% for Val, and 30-40% for Ala. 1 H- 15 N heteronuclear single-quantum coherence signals of the 15 N scrambling-prone amino acid were also easily filtered using 15 N-{ 13 Cα} spin-echo difference experiments. Our method could be applied to the assignment of the 55 kDa protein.

  19. Fast methodology for the reliable determination of nonylphenol in water samples by minimal labeling isotope dilution mass spectrometry.

    PubMed

    Fabregat-Cabello, Neus; Castillo, Ángel; Sancho, Juan V; González, Florenci V; Roig-Navarro, Antoni Francesc

    2013-08-02

    In this work we have developed and validated an accurate and fast methodology for the determination of 4-nonylphenol (technical mixture) in complex matrix water samples by UHPLC-ESI-MS/MS. The procedure is based on isotope dilution mass spectrometry (IDMS) in combination with isotope pattern deconvolution (IPD), which provides the concentration of the analyte directly from the spiked sample without requiring any methodological calibration graph. To avoid any possible isotopic effect during the analytical procedure the in-house synthesized (13)C1-4-(3,6-dimethyl-3-heptyl)phenol was used as labeled compound. This proposed surrogate was able to compensate the matrix effect even from wastewater samples. A SPE pre-concentration step together with exhaustive efforts to avoid contamination were included to reach the signal-to-noise ratio necessary to detect the endogenous concentrations present in environmental samples. Calculations were performed acquiring only three transitions, achieving limits of detection lower than 100ng/g for all water matrix assayed. Recoveries within 83-108% and coefficients of variation ranging from 1.5% to 9% were obtained. On the contrary a considerable overestimation was obtained with the most usual classical calibration procedure using 4-n-nonylphenol as internal standard, demonstrating the suitability of the minimal labeling approach. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Time-resolved Analysis of Proteome Dynamics by Tandem Mass Tags and Stable Isotope Labeling in Cell Culture (TMT-SILAC) Hyperplexing*

    PubMed Central

    Welle, Kevin A.; Zhang, Tian; Hryhorenko, Jennifer R.; Shen, Shichen; Qu, Jun; Ghaemmaghami, Sina

    2016-01-01

    Recent advances in mass spectrometry have enabled system-wide analyses of protein turnover. By globally quantifying the kinetics of protein clearance and synthesis, these methodologies can provide important insights into the regulation of the proteome under varying cellular and environmental conditions. To facilitate such analyses, we have employed a methodology that combines metabolic isotopic labeling (Stable Isotope Labeling in Cell Culture - SILAC) with isobaric tagging (Tandem Mass Tags - TMT) for analysis of multiplexed samples. The fractional labeling of multiple time-points can be measured in a single mass spectrometry run, providing temporally resolved measurements of protein turnover kinetics. To demonstrate the feasibility of the approach, we simultaneously measured the kinetics of protein clearance and accumulation for more than 3000 proteins in dividing and quiescent human fibroblasts and verified the accuracy of the measurements by comparison to established non-multiplexed approaches. The results indicate that upon reaching quiescence, fibroblasts compensate for lack of cellular growth by globally downregulating protein synthesis and upregulating protein degradation. The described methodology significantly reduces the cost and complexity of temporally-resolved dynamic proteomic experiments and improves the precision of proteome-wide turnover data. PMID:27765818

  1. Determination of chemical purity and isotopic composition of natural and carbon-13-labeled arsenobetaine bromide standards by quantitative(1)H-NMR.

    PubMed

    Le, Phuong-Mai; Ding, Jianfu; Leek, Donald M; Mester, Zoltan; Robertson, Gilles; Windust, Anthony; Meija, Juris

    2016-10-01

    In this study, we report the characterization of three arsenobetaine-certified reference materials by quantitative NMR. We have synthesized an arsenobetaine bromide high-purity standard of natural isotopic composition (ABET-1) and two carbon-13-labeled isotopic standards (BBET-1 and CBET-1). Assignments of the chemical purity and isotopic composition are not trivial in the case of arsenobetaine, and in this study we utilized quantitative(1)H-NMR techniques for the determination of the mass fractions (chemical purity). The isotopic purity of all three standards was also assessed by NMR from the carbon-13 satellite signals. The standards are non-hygroscopic, high-purity (ca. 0.99 g/g), and the carbon-13 enrichment for both isotopic standards is x((13)C)≈0.99. These standards are designed for use as primary calibrators for mass spectrometric determination of arsenobetaine in environmental samples.

  2. From position-specific isotope labeling towards soil fluxomics: a novel toolbox to assess the microbial impact on biogeochemical cycles

    NASA Astrophysics Data System (ADS)

    Apostel, C.; Dippold, M. A.; Kuzyakov, Y.

    2015-12-01

    Understanding the microbial impact on C and nutrient cycles is one of the most important challenges in terrestrial biogeochemistry. Transformation of low molecular weight organic substances (LMWOS) is a key step in all biogeochemical cycles because 1) all high molecular substances pass the LMWOS pool during their degradation and 2) only LMWOS can be taken up by microorganisms intact. Thus, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the microbial metabolic network and its control mechanism. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools but studies were nearly exclusively based on uniformly labeled substances. However, such tracers do not allow the differentiation of the intact use of the initial substances from its transformation to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of basic metabolites and quantification of isotope incorporation in CO2 and bulk soil enabled following the basic metabolic pathways of microorganisms. However, the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites like phospholipid fatty acids (PLFA) or amino sugars revealed new insights into the soil fluxome: First, it enables tracing specific anabolic pathways in diverse microbial communities in soils e.g. carbon starvation pathways versus pathways reflecting microbial growth. Second, it allows identification of specific pathways of individual functional microbial groups in soils in situ. Tracing metabolic pathways and understanding their regulating factors are crucial for soil C fluxomics i.e. the unravaling of the complex network of C transformations

  3. Envelope: interactive software for modeling and fitting complex isotope distributions.

    PubMed

    Sykes, Michael T; Williamson, James R

    2008-10-20

    An important aspect of proteomic mass spectrometry involves quantifying and interpreting the isotope distributions arising from mixtures of macromolecules with different isotope labeling patterns. These patterns can be quite complex, in particular with in vivo metabolic labeling experiments producing fractional atomic labeling or fractional residue labeling of peptides or other macromolecules. In general, it can be difficult to distinguish the contributions of species with different labeling patterns to an experimental spectrum and difficult to calculate a theoretical isotope distribution to fit such data. There is a need for interactive and user-friendly software that can calculate and fit the entire isotope distribution of a complex mixture while comparing these calculations with experimental data and extracting the contributions from the differently labeled species. Envelope has been developed to be user-friendly while still being as flexible and powerful as possible. Envelope can simultaneously calculate the isotope distributions for any number of different labeling patterns for a given peptide or oligonucleotide, while automatically summing these into a single overall isotope distribution. Envelope can handle fractional or complete atom or residue-based labeling, and the contribution from each different user-defined labeling pattern is clearly illustrated in the interactive display and is individually adjustable. At present, Envelope supports labeling with 2H, 13C, and 15N, and supports adjustments for baseline correction, an instrument accuracy offset in the m/z domain, and peak width. Furthermore, Envelope can display experimental data superimposed on calculated isotope distributions, and calculate a least-squares goodness of fit between the two. All of this information is displayed on the screen in a single graphical user interface. Envelope supports high-quality output of experimental and calculated distributions in PNG or PDF format. Beyond simply

  4. Automated LC-HRMS(/MS) Approach for the Annotation of Fragment Ions Derived from Stable Isotope Labeling-Assisted Untargeted Metabolomics

    PubMed Central

    2014-01-01

    Structure elucidation of biological compounds is still a major bottleneck of untargeted LC-HRMS approaches in metabolomics research. The aim of the present study was to combine stable isotope labeling and tandem mass spectrometry for the automated interpretation of the elemental composition of fragment ions and thereby facilitate the structural characterization of metabolites. The software tool FragExtract was developed and evaluated with LC-HRMS/MS spectra of both native 12C- and uniformly 13C (U-13C)-labeled analytical standards of 10 fungal substances in pure solvent and spiked into fungal culture filtrate of Fusarium graminearum respectively. Furthermore, the developed approach is exemplified with nine unknown biochemical compounds contained in F. graminearum samples derived from an untargeted metabolomics experiment. The mass difference between the corresponding fragment ions present in the MS/MS spectra of the native and U-13C-labeled compound enabled the assignment of the number of carbon atoms to each fragment signal and allowed the generation of meaningful putative molecular formulas for each fragment ion, which in turn also helped determine the elemental composition of the precursor ion. Compared to laborious manual analysis of the MS/MS spectra, the presented algorithm marks an important step toward efficient fragment signal elucidation and structure annotation of metabolites in future untargeted metabolomics studies. Moreover, as demonstrated for a fungal culture sample, FragExtract also assists the characterization of unknown metabolites, which are not contained in databases, and thus exhibits a significant contribution to untargeted metabolomics research. PMID:24965664

  5. Isotopic measurements (C,N,O) of detonation soot produced from labeled and unlabeled Composition B-3 indicate source of solid carbon residues

    NASA Astrophysics Data System (ADS)

    Podlesak, David; Manner, Virginia; Amato, Ronald; Dattelbaum, Dana; Gusavsen, Richard; Huber, Rachel

    2017-06-01

    Detonation of HE is an exothermic process whereby metastable complex molecules are converted to simple stable molecules such as H2 O, N2, CO, CO2, and solid carbon. The solid carbon contains various allotropes such as detonation nanodiamonds, graphite, and amorphous carbon. It is well known that certain HE formulations such as Composition B (60% RDX, 40% TNT) produce greater amounts of solid carbon than other more oxygen-balanced formulations. To develop a greater understanding of how formulation and environment influence solid carbon formation, we synthesized TNT and RDX with 13 C and 15 N at levels slightly above natural abundance levels. Synthesized RDX and TNT were mixed at a ratio of 60:40 to form Composition B and solid carbon residues were collected from detonations of isotopically-labeled as well as un-labelled Composition B. The raw HE and detonation residues were analyzed isotopically for C, N, O isotopic compositions. We will discuss differences between treatments groups as a function of formulation and environment. LA-UR - 17-21266.

  6. Analysis of the differentially expressed low molecular weight peptides in human serum via an N-terminal isotope labeling technique combining nano-liquid chromatography/matrix-assisted laser desorption/ionization mass spectrometry.

    PubMed

    Leng, Jiapeng; Zhu, Dong; Wu, Duojiao; Zhu, Tongyu; Zhao, Ningwei; Guo, Yinlong

    2012-11-15

    Peptidomics analysis of human serum is challenging due to the low abundance of serum peptides and interference from the complex matrix. This study analyzed the differentially expressed (DE) low molecular weight peptides in human serum integrating a DMPITC-based N-terminal isotope labeling technique with nano-liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (nano-LC/MALDI-MS). The workflow introduced a [d(6)]-4,6-dimethoxypyrimidine-2-isothiocyanate (DMPITC)-labeled mixture of aliquots from test samples as the internal standard. The spiked [d(0)]-DMPITC-labeled samples were separated by nano-LC then spotted on the MALDI target. Both quantitative and qualitative studies for serum peptides were achieved based on the isotope-labeled peaks. The DMPITC labeling technique combined with nano-LC/MALDI-MS not only minimized the errors in peptide quantitation, but also allowed convenient recognition of the labeled peptides due to the 6 Da mass difference. The data showed that the entire research procedure as well as the subsequent data analysis method were effective, reproducible, and sensitive for the analysis of DE serum peptides. This study successfully established a research model for DE serum peptides using DMPITC-based N-terminal isotope labeling and nano-LC/MALDI-MS. Application of the DMPITC-based N-terminal labeling technique is expected to provide a promising tool for the investigation of peptides in vivo, especially for the analysis of DE peptides under different biological conditions. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Cross-Course Collaboration in the Undergraduate Chemistry Curriculum: Isotopic Labeling with Sodium Borodeuteride in the Introductory Organic Chemistry Laboratory

    ERIC Educational Resources Information Center

    Kjonaas, Richard A.; Fitch, Richard W.; Noll, Robert J.

    2017-01-01

    A microscale isotopic labeling experiment is described for the introductory organic chemistry laboratory course wherein half of the students use sodium borohydride (NaBH[subscript 4]) and the other half use sodium borodeuteride (NaBD[subscript 4]) to reduce acetophenone to 1-phenylethanol and then compare spectral data. The cost is reasonable, and…

  8. Protein-based stable isotope probing.

    PubMed

    Jehmlich, Nico; Schmidt, Frank; Taubert, Martin; Seifert, Jana; Bastida, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Vogt, Carsten

    2010-12-01

    We describe a stable isotope probing (SIP) technique that was developed to link microbe-specific metabolic function to phylogenetic information. Carbon ((13)C)- or nitrogen ((15)N)-labeled substrates (typically with >98% heavy label) were used in cultivation experiments and the heavy isotope incorporation into proteins (protein-SIP) on growth was determined. The amount of incorporation provides a measure for assimilation of a substrate, and the sequence information from peptide analysis obtained by mass spectrometry delivers phylogenetic information about the microorganisms responsible for the metabolism of the particular substrate. In this article, we provide guidelines for incubating microbial cultures with labeled substrates and a protocol for protein-SIP. The protocol guides readers through the proteomics pipeline, including protein extraction, gel-free and gel-based protein separation, the subsequent mass spectrometric analysis of peptides and the calculation of the incorporation of stable isotopes into peptides. Extraction of proteins and the mass fingerprint measurements of unlabeled and labeled fractions can be performed in 2-3 d.

  9. Stable Isotope Labeling, in Vivo, of d- and l-Tryptophan Pools in Lemna gibba and the Low Incorporation of Label into Indole-3-Acetic Acid 1

    PubMed Central

    Baldi, Bruce G.; Maher, Barbara R.; Slovin, Janet Pernise; Cohen, Jerry D.

    1991-01-01

    We present evidence that the role of tryptophan and other potential intermediates in the pathways that could lead to indole derivatives needs to be reexamined. Two lines of Lemna gibba were tested for uptake of [15N-indole]-labeled tryptophan isomers and incorporation of that label into free indole-3-acetic acid (IAA). Both lines required levels of l-[15N]tryptophan 2 to 3 orders of magnitude over endogenous levels in order to obtain measurable incorporation of label into IAA. Labeled l-tryptophan was extractable from plant tissue after feeding and showed no measurable isomerization into d-tryptophan. d-[15N]tryptophan supplied to Lemna at rates of approximately 400 times excess of endogenous d-tryptophan levels (to yield an isotopic enrichment equal to that which allowed detection of the incorporation of l-tryptophan into IAA), did not result in measurable incorporation of label into free IAA. These results demonstrate that l-tryptophan is a more direct precursor to IAA than the d isomer and suggest (a) that the availability of tryptophan in vivo is not a limiting factor in the biosynthesis of IAA, thus implying that other regulatory mechanisms are in operation and (b) that l-tryptophan also may not be a primary precursor to IAA in plants. PMID:16668112

  10. Stable isotope, site-specific mass tagging for protein identification

    DOEpatents

    Chen, Xian

    2006-10-24

    Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

  11. Extrinsic labeling method may not accurately measure Fe absorption from cooked pinto beans (Phaseolus vulgaris): comparison of extrinsic and intrinsic labeling of beans.

    PubMed

    Jin, Fuxia; Cheng, Zhiqiang; Rutzke, Michael A; Welch, Ross M; Glahn, Raymond P

    2008-08-27

    Isotopic labeling of food has been widely used for the measurement of Fe absorption in determining requirements and evaluating the factors involved in Fe bioavailability. An extrinsic labeling technique will not accurately predict the total Fe absorption from foods unless complete isotopic exchange takes place between an extrinsically added isotope label and the intrinsic Fe of the food. We examined isotopic exchange in the case of both white beans and colored beans (Phaseolus vulgaris) with an in vitro digestion model. There are significant differences in (58)Fe/(56)Fe ratios between the sample digest supernatant and the pellet of extrinsically labeled pinto bean. The white bean digest shows significantly better equilibration of the extrinsic (58)Fe with the intrinsic (56)Fe. In contrast to the extrinsically labeled samples, both white and red beans labeled intrinsically with (58)Fe demonstrated consistent ratios of (58)Fe/(56)Fe in the bean meal, digest, supernatant, and pellet. It is possible that the polyphenolics in the bean seed coat may bind Fe and thus interfere with extrinsic labeling of the bean meals. These observations raise questions on the accuracy of studies that used extrinsic tags to measure Fe absorption from beans. Intrinsic labeling appears necessary to accurately measure Fe bioavailability from beans.

  12. Heterologous expression of Cenchritis muricatus protease inhibitor II (CmPI-II) in Pichia pastoris system: Purification, isotopic labeling and preliminary characterization.

    PubMed

    Cabrera-Muñoz, Aymara; Rojas, Laritza; Gil, Dayrom F; González-González, Yamile; Mansur, Manuel; Camejo, Ayamey; Pires, José R; Alonso-Del-Rivero Antigua, Maday

    2016-10-01

    Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Chemical Ligation of Folded Recombinant Proteins: Segmental Isotopic Labeling of Domains for NMR Studies

    NASA Astrophysics Data System (ADS)

    Xu, Rong; Ayers, Brenda; Cowburn, David; Muir, Tom W.

    1999-01-01

    A convenient in vitro chemical ligation strategy has been developed that allows folded recombinant proteins to be joined together. This strategy permits segmental, selective isotopic labeling of the product. The src homology type 3 and 2 domains (SH3 and SH2) of Abelson protein tyrosine kinase, which constitute the regulatory apparatus of the protein, were individually prepared in reactive forms that can be ligated together under normal protein-folding conditions to form a normal peptide bond at the ligation junction. This strategy was used to prepare NMR sample quantities of the Abelson protein tyrosine kinase-SH(32) domain pair, in which only one of the domains was labeled with 15N Mass spectrometry and NMR analyses were used to confirm the structure of the ligated protein, which was also shown to have appropriate ligand-binding properties. The ability to prepare recombinant proteins with selectively labeled segments having a single-site mutation, by using a combination of expression of fusion proteins and chemical ligation in vitro, will increase the size limits for protein structural determination in solution with NMR methods. In vitro chemical ligation of expressed protein domains will also provide a combinatorial approach to the synthesis of linked protein domains.

  14. Methyl-coenzyme M reductase from methanogenic archaea: isotope effects on label exchange and ethane formation with the homologous substrate ethyl-coenzyme M.

    PubMed

    Scheller, Silvan; Goenrich, Meike; Thauer, Rudolf K; Jaun, Bernhard

    2013-10-09

    Ethyl-coenzyme M (CH3CH2-S-CH2CH2-SO3(-), Et-S-CoM) serves as a homologous substrate for the enzyme methyl-coenzyme M reductase (MCR) resulting in the product ethane instead of methane. The catalytic reaction proceeds via an intermediate that already contains all six C-H bonds of the product. Because product release occurs after a second, rate-limiting step, many cycles of intermediate formation and reconversion to substrate occur before a substantial amount of ethane is released. In deuterated buffer, the intermediate becomes labeled, and C-H activation in the back reaction rapidly leads to labeled Et-S-CoM, which enables intermediate formation to be detected. Here, we present a comprehensive analysis of this pre-equilibrium. (2)H- and (13)C-labeled isotopologues of Et-S-CoM were used as the substrates, and the time course of each isotopologue was followed by NMR spectroscopy. A kinetic simulation including kinetic isotope effects allowed determination of the primary and α- and β-secondary isotope effects for intermediate formation and for the C-H/C-D bond activation in the ethane-containing intermediate. The values obtained are in accordance with those found for the native substrate Me-S-CoM (see preceding publication, Scheller, S.; Goenrich, M.; Thauer, R. K.; Jaun, B. J. Am. Chem. Soc. 2013, 135, DOI: 10.1021/ja406485z) and thus imply the same catalytic mechanism for both substrates. The experiment by Floss and co-workers, demonstrating a net inversion of configuration to chiral ethane with CH3CDT-S-CoM as the substrate, is compatible with the observed rapid isotope exchange if the isotope effects measured here are taken into account.

  15. Variability of 13C-labeling in plant leaves.

    PubMed

    Nguyen Tu, Thanh Thuy; Biron, Philippe; Maseyk, Kadmiel; Richard, Patricia; Zeller, Bernd; Quénéa, Katell; Alexis, Marie; Bardoux, Gérard; Vaury, Véronique; Girardin, Cyril; Pouteau, Valérie; Billiou, Daniel; Bariac, Thierry

    2013-09-15

    Plant tissues artificially labeled with (13)C are increasingly used in environmental studies to unravel biogeochemical and ecophysiological processes. However, the variability of (13)C-content in labeled tissues has never been carefully investigated. Hence, this study aimed at documenting the variability of (13)C-content in artificially labeled leaves. European beech and Italian ryegrass were subjected to long-term (13)C-labeling in a controlled-environment growth chamber. The (13)C-content of the leaves obtained after several months labeling was determined by isotope ratio mass spectrometry. The (13)C-content of the labeled leaves exhibited inter- and intra-leaf variability much higher than those naturally occurring in unlabeled plants, which do not exceed a few per mil. This variability was correlated with labeling intensity: the isotope composition of leaves varied in ranges of ca 60‰ and 90‰ for experiments that led to average leaf (13)C-content of ca +15‰ and +450‰, respectively. The reported variability of isotope composition in (13)C-enriched leaves is critical, and should be taken into account in subsequent experimental investigations of environmental processes using (13)C-labeled plant tissues. Copyright © 2013 John Wiley & Sons, Ltd.

  16. Quantitative detection of codeine in human plasma using surface-enhanced Raman scattering via adaptation of the isotopic labelling principle.

    PubMed

    Subaihi, Abdu; Muhamadali, Howbeer; Mutter, Shaun T; Blanch, Ewan; Ellis, David I; Goodacre, Royston

    2017-03-27

    In this study surface enhanced Raman scattering (SERS) combined with the isotopic labelling (IL) principle has been used for the quantification of codeine spiked into both water and human plasma. Multivariate statistical approaches were employed for the analysis of these SERS spectral data, particularly partial least squares regression (PLSR) which was used to generate models using the full SERS spectral data for quantification of codeine with, and without, an internal isotopic labelled standard. The PLSR models provided accurate codeine quantification in water and human plasma with high prediction accuracy (Q 2 ). In addition, the employment of codeine-d 6 as the internal standard further improved the accuracy of the model, by increasing the Q 2 from 0.89 to 0.94 and decreasing the low root-mean-square error of predictions (RMSEP) from 11.36 to 8.44. Using the peak area at 1281 cm -1 assigned to C-N stretching, C-H wagging and ring breathing, the limit of detection was calculated in both water and human plasma to be 0.7 μM (209.55 ng mL -1 ) and 1.39 μM (416.12 ng mL -1 ), respectively. Due to a lack of definitive codeine vibrational assignments, density functional theory (DFT) calculations have also been used to assign the spectral bands with their corresponding vibrational modes, which were in excellent agreement with our experimental Raman and SERS findings. Thus, we have successfully demonstrated the application of SERS with isotope labelling for the absolute quantification of codeine in human plasma for the first time with a high degree of accuracy and reproducibility. The use of the IL principle which employs an isotopolog (that is to say, a molecule which is only different by the substitution of atoms by isotopes) improves quantification and reproducibility because the competition of the codeine and codeine-d 6 for the metal surface used for SERS is equal and this will offset any difference in the number of particles under analysis or any fluctuations in

  17. Energy-Efficiency Labels and Standards: A Guidebook forAppliances, Equipment, and Lighting - 2nd Edition

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wiel, Stephen; McMahon, James E.

    2005-04-28

    Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and several other organizations identified on the cover of this guidebook recognize the need to support policy makers in their efforts tomore » implement energy-efficiency standards and labeling programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This second edition of the guidebook was prepared over the course of the past year, four years after the preparation of the first edition, with a significant contribution from the authors and reviewers mentioned previously. Their diligent participation helps maintain this book as the international guidance tool it has become. The lead authors would like to thank the members of the Communications Office of the Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory for their support in the development, production, and distribution of the guidebook. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design

  18. Status of China's Energy Efficiency Standards and Labels for Appliances and International Collaboration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, Nan

    2008-03-01

    China first adopted minimum energy performance standards (MEPS) in 1989. Today, there are standards for a wide range of domestic, commercial and selected industrial equipment. In 1999, China launched a voluntary endorsement label, which has grown to cover over 40 products including water-saving products (See Figure 1). Further, in 2005, China started a mandatory energy information label (also referred to as the 'Energy Label'). Today, the Energy Label is applied to four products including: air conditioners; household refrigerators; clothes washers; and unitary air conditioners (See Figure 2). MEPS and the voluntary endorsement labeling specifications have been updated and revised inmore » order to reflect technology improvements to those products in the market. These programs have had an important impact in reducing energy consumption of appliances in China. Indeed, China has built up a strong infrastructure to develop and implement product standards. Historically, however, the government's primary focus has been on the technical requirements for efficiency performance. Less attention has been paid to monitoring and enforcement with a minimal commitment of resources and little expansion of administrative capacity in this area. Thus, market compliance with both mandatory standards and labeling programs has been questionable and actual energy savings may have been undermined as a result. The establishment of a regularized monitoring system for tracking compliance with the mandatory standard and energy information label in China is a major area for program improvement. Over the years, the Collaborative Labeling and Appliance Standards Program (CLASP) has partnered with several Chinese institutions to promote energy-efficient products in China. CLASP, together with its implementing partner Lawrence Berkeley National Laboratory (LBNL), has assisted China in developing and updating the above-mentioned standards and labeling programs. Because of the increasing need for

  19. Scalable and efficient separation of hydrogen isotopes using graphene-based electrochemical pumping

    NASA Astrophysics Data System (ADS)

    Lozada-Hidalgo, M.; Zhang, S.; Hu, S.; Esfandiar, A.; Grigorieva, I. V.; Geim, A. K.

    2017-05-01

    Thousands of tons of isotopic mixtures are processed annually for heavy-water production and tritium decontamination. The existing technologies remain extremely energy intensive and require large capital investments. New approaches are needed to reduce the industry's footprint. Recently, micrometre-size crystals of graphene are shown to act as efficient sieves for hydrogen isotopes pumped through graphene electrochemically. Here we report a fully-scalable approach, using graphene obtained by chemical vapour deposition, which allows a proton-deuteron separation factor of around 8, despite cracks and imperfections. The energy consumption is projected to be orders of magnitude smaller with respect to existing technologies. A membrane based on 30 m2 of graphene, a readily accessible amount, could provide a heavy-water output comparable to that of modern plants. Even higher efficiency is expected for tritium separation. With no fundamental obstacles for scaling up, the technology's simplicity, efficiency and green credentials call for consideration by the nuclear and related industries.

  20. Improved efficiency of selective photoionization of palladium isotopes via autoionizing Rydberg states

    NASA Astrophysics Data System (ADS)

    Locke, Clayton R.; Kobayashi, Tohru; Midorikawa, Katsumi

    2017-01-01

    Odd-mass-selective ionization of palladium for purposes of resource recycling and management of long-lived fission products can be achieved by exploiting transition selection rules in a well-established three-step excitation process. In this conventional scheme, circularly polarized lasers of the same handedness excite isotopes via two intermediate 2D5/2 core states, and a third laser is then used for ionization via autoionizing Rydberg states. We propose an alternative excitation scheme via intermediate 2D3/2 core states before the autoionizing Rydberg state, improving ionization efficiency by over 130 times. We confirm high selectivity and measure odd-mass isotopes of >99.7(3)% of the total ionized product. We have identified and measured the relative ionization efficiency of the series of Rydberg states that converge to upper ionization limit of the 4 d 9(2D3/2) level, and identify the most efficient excitation is via the Rydberg state at 67668.18(10) cm-1.

  1. On structural identifiability analysis of the cascaded linear dynamic systems in isotopically non-stationary 13C labelling experiments.

    PubMed

    Lin, Weilu; Wang, Zejian; Huang, Mingzhi; Zhuang, Yingping; Zhang, Siliang

    2018-06-01

    The isotopically non-stationary 13C labelling experiments, as an emerging experimental technique, can estimate the intracellular fluxes of the cell culture under an isotopic transient period. However, to the best of our knowledge, the issue of the structural identifiability analysis of non-stationary isotope experiments is not well addressed in the literature. In this work, the local structural identifiability analysis for non-stationary cumomer balance equations is conducted based on the Taylor series approach. The numerical rank of the Jacobian matrices of the finite extended time derivatives of the measured fractions with respect to the free parameters is taken as the criterion. It turns out that only one single time point is necessary to achieve the structural identifiability analysis of the cascaded linear dynamic system of non-stationary isotope experiments. The equivalence between the local structural identifiability of the cascaded linear dynamic systems and the local optimum condition of the nonlinear least squares problem is elucidated in the work. Optimal measurements sets can then be determined for the metabolic network. Two simulated metabolic networks are adopted to demonstrate the utility of the proposed method. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Parallel labeling experiments and metabolic flux analysis: Past, present and future methodologies.

    PubMed

    Crown, Scott B; Antoniewicz, Maciek R

    2013-03-01

    Radioactive and stable isotopes have been applied for decades to elucidate metabolic pathways and quantify carbon flow in cellular systems using mass and isotope balancing approaches. Isotope-labeling experiments can be conducted as a single tracer experiment, or as parallel labeling experiments. In the latter case, several experiments are performed under identical conditions except for the choice of substrate labeling. In this review, we highlight robust approaches for probing metabolism and addressing metabolically related questions though parallel labeling experiments. In the first part, we provide a brief historical perspective on parallel labeling experiments, from the early metabolic studies when radioisotopes were predominant to present-day applications based on stable-isotopes. We also elaborate on important technical and theoretical advances that have facilitated the transition from radioisotopes to stable-isotopes. In the second part of the review, we focus on parallel labeling experiments for (13)C-metabolic flux analysis ((13)C-MFA). Parallel experiments offer several advantages that include: tailoring experiments to resolve specific fluxes with high precision; reducing the length of labeling experiments by introducing multiple entry-points of isotopes; validating biochemical network models; and improving the performance of (13)C-MFA in systems where the number of measurements is limited. We conclude by discussing some challenges facing the use of parallel labeling experiments for (13)C-MFA and highlight the need to address issues related to biological variability, data integration, and rational tracer selection. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.

    PubMed Central

    Zimmer, D P; Crothers, D M

    1995-01-01

    A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented. Images Fig. 2 Fig. 3 Fig. 4 PMID:7724521

  4. Combining position-specific 13C labeling with compound-specific isotope analysis: first steps towards soil fluxomics

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino

  5. Direct fluorescent labeling for efficient biological assessment of inhalable particles.

    PubMed

    Poudel, Bijay Kumar; Park, Jae Hong; Lim, Jiseok; Byeon, Jeong Hoon

    2017-10-01

    Labeling of aerosol particles with a radioactive, magnetic, or optical tracer has been employed to confirm particle localization in cell compartments, which has provided useful evidence for correlating toxic effects of inhaled particles. However, labeling requires several physicochemical steps to identify functionalities of the inner or outer surfaces of particles, and moreover, these steps can cause changes in size, surface charge, and bioactivity of the particles, resulting in misinterpretations regarding their toxic effects. This study addresses this challenging issue with a goal of introducing an efficient strategy for constantly supplying labeled aerosol particles in a single-pass configuration without any pre- or post-physicochemical batch treatments of aerosol particles. Carbon black (CB, simulating combustion-generated soot) or calcium carbonate (CC, simulating brake-wear fragments) particles were constantly produced via spark ablation or bubble bursting, respectively. These minute particles were incorporated with fluorescein isothiocyanate-poly(ethylene glycol) 2-aminoethyl ether acetic acid solution at the orifice of a collison atomizer to fabricate hybrid droplets. The droplets successively entered a diffusion dryer containing 254-nm UV irradiation; therefore, the droplets were dynamically stiffened by UV to form fluorescent probes on particles during solvent extraction in the dryer. Particle size distributions, morphologies, and surface charges before and after labeling were measured to confirm fluorescence labeling without significant changes in the properties. In vitro assays, including confocal imaging, were conducted to confirm the feasibility of the labeling approach without inducing significant differences in bioactivity compared with untreated CB or CC particles.

  6. Measurement of Enzyme Isotope Effects.

    PubMed

    Kholodar, Svetlana A; Ghosh, Ananda K; Kohen, Amnon

    2017-01-01

    Enzyme isotope effects, or the kinetic effects of "heavy" enzymes, refer to the effect of isotopically labeled protein residues on the enzyme's activity or physical properties. These effects are increasingly employed in the examination of the possible contributions of protein dynamics to enzyme catalysis. One hypothesis assumed that isotopic substitution of all 12 C, 14 N, and nonexchangeable 1 H by 13 C, 15 N, and 2 H, would slow down protein picosecond to femtosecond dynamics without any effect on the system's electrostatics following the Born-Oppenheimer approximation. It was suggested that reduced reaction rates reported for several "heavy" enzymes accords with that hypothesis. However, numerous deviations from the predictions of that hypothesis were also reported. Current studies also attempt to test the role of individual residues by site-specific labeling or by labeling a pattern of residues on activity. It appears that in several systems the protein's fast dynamics are indeed reduced in "heavy" enzymes in a way that reduces the probability of barrier crossing of its chemical step. Other observations, however, indicated that slower protein dynamics are electrostatically altered in isotopically labeled enzymes. Interestingly, these effects appear to be system dependent, thus it might be premature to suggest a general role of "heavy" enzymes' effect on catalysis. © 2017 Elsevier Inc. All rights reserved.

  7. Selected reaction monitoring (SRM) mass spectrometry without isotope labeling can be used for rapid protein quantification

    PubMed Central

    Zhi, Wenbo; Wang, Meiyao

    2014-01-01

    The validation of putative biomarker candidates has become the major bottle-neck in protein biomarker development. Conventional immunoaffinity methods are limited by the availability of antibodies and kits. Here we demonstrated the feasibility of using the selected reaction monitoring (SRM) without isotope labeling to achieve fast and reproducible quantification of serum proteins. The SRM/MRM assays for three standard serum proteins, including ceruloplasmin (CP), serum aymloid A (SAA) and sex hormone binding globulin (SHBG) have good linear ranges, generally 103 – 104. There are almost perfect correlations between SRM intensities and the loaded peptide amounts (R2 is usually ~0.99). Our data suggest that SRM/MRM is able to quantify proteins at 0.2 – 2 fmol level, which are comparable to the commercial ELISA/LUMINEX kits for these proteins. Excellent correlations between SRM/MRM and ELISA/LUMINEX assays were observed for SAA and SHBG (R2 = 0.928 and 0.851 respectively). The correlation between SRM/MRM and ELISA for CP is less desirable (R2 = 0.565). The reproducibility for SRM/MRM assays is generally very good but may depend on the proteins/peptides (R2 = 0.931 and 0.882 for SAA and SHBG, and 0.723 for CP). SRM/MRM assay without isotope labeling is a rapid and useful method for protein biomarker validation in a modest number of samples and is especially useful when other assays such as ELISA or Luminex beads are not available. PMID:21594933

  8. Classification-based quantitative analysis of stable isotope labeling by amino acids in cell culture (SILAC) data.

    PubMed

    Kim, Seongho; Carruthers, Nicholas; Lee, Joohyoung; Chinni, Sreenivasa; Stemmer, Paul

    2016-12-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit. We introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum. Our simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data. No matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Formation of Kokumi-Enhancing γ-Glutamyl Dipeptides in Parmesan Cheese by Means of γ-Glutamyltransferase Activity and Stable Isotope Double-Labeling Studies.

    PubMed

    Hillmann, Hedda; Behr, Jürgen; Ehrmann, Matthias A; Vogel, Rudi F; Hofmann, Thomas

    2016-03-02

    Recently, γ-glutamyl dipeptides (γ-GPs) were found to be responsible for the attractive kokumi flavor of Parmesan cheese (PC). Quantitation of γ-GPs and their parent amino acids in 13-, 24-, and 30-month ripened PC samples by LC-MS/MS and stable isotope dilution analysis (SIDA), in-cheese (13)C-labeling studies, followed by analysis of the γ-glutamyl transferase (GGT) activity revealed γ-GPs to be generated most efficiently after 24 months of ripening by a GGT-catalyzed transfer of the γ-glutamyl moiety of L-glutamine onto various acceptor amino acids released upon casein proteolysis. Following the identification of milk as a potential GGT source in PC, the functionality of the milk's GGT to generate the target γ-GPs was validated by stable isotope double-labeling (SIDL) experiments. Therefore, raw and heat-treated milk samples were incubated with L-glutamine-[U-(13)C] and acceptor amino acids (X) and the hetero- (γ-Glu-[(13)C5]-X) and homotranspeptidation products (γ-Glu-Gln-[(13)C10]) were quantitated by LC-MS/MS-SIDA using γ-Glu-Ala-[(13)C3] as the internal standard. High GGT activity to generate the γ-GPs and preference for L-phenylalanine and L-methionine as acceptor amino acids were found in raw milk and milk samples heat-treated for 10 min up to a maximum of 65 °C. In comparison, GGT activity and SIDL studies performed with inoculated Lactobacillus strains, including Lactobacillus harbinensis and Lactobacillus casei identified in PC by means of 16S rRNA gene sequencing, did not show any significant GGT activity and unequivocally demonstrated unpasteurized cow's milk, rather than microorganisms, as a key factor in γ-glutamyl dipeptide generation in Parmesan cheese.

  10. Phosphorus use efficiency by cotton measured through 32P isotope technique

    NASA Astrophysics Data System (ADS)

    Marcante, N. C.; Muraoka, T.; Camacho, M. A.; César, F. R. C. F.; Bruno, I. P.

    2012-04-01

    Deficiency of phosphorus (P) is the major limitation to agricultural production in the Brazilian Savannah (Cerrado), which is naturally poor in this nutrient. Most of the P applied by fertilizer in Cerrado soils are converted into low solubility forms and can not be easily absorbed by plants. This occurs for characteristics of adsorption, conditioned by the predominance of low pH and aluminum and iron oxides in the clay fraction. The development of genotypes and cultivars with greater capacity to grow up in soils with low P availability ('phosphorus efficiency') is interesting to improve the agriculture in these areas in a sustainable way. Cotton (Gossypium spp.) is the main product for the fibers used nationally and globally in the textile chain. This study aim was to evaluate the efficiency of absorption and utilization of P by cotton cultivars/genotypes grown in Cerrado soil by the isotopic dilution technique. The soil classified as Ultisols, was labeled with the radioisotope 32P.The experiment was conducted in a greenhouse in a completely randomized design factorial 2 x 17. Factors were considered two levels of P (insufficient = 20 mg kg-1 and sufficient = 120 mg kg-1) and 17 genetic materials of cotton recommended for Cerrado region. Phosphorus levels influenced significantly the shoots dry matter production, the P content and accumulation, the 32P specific activity, the L value and L value less seed cotton P by cultivars and genotypes. The hierarchical clustering analysis used to verify the similarities between the cultivars and genotypes of cotton, classified them into internally homogeneous groups and heterogeneous between different groups. Cultivars FMT 523, FM 910 and CNPA GO 2043 were the most responsive to phosphate fertilizer in sufficient level of P, while the genotype Barbadense 01 and cultivars FM 966LL, IPR Jataí, BRS Aroeira and BRS Buriti were most efficient absorbing P in soils with insufficient level.

  11. Quantitative Metabolome Analysis Based on Chromatographic Peak Reconstruction in Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry.

    PubMed

    Huan, Tao; Li, Liang

    2015-07-21

    Generating precise and accurate quantitative information on metabolomic changes in comparative samples is important for metabolomics research where technical variations in the metabolomic data should be minimized in order to reveal biological changes. We report a method and software program, IsoMS-Quant, for extracting quantitative information from a metabolomic data set generated by chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). Unlike previous work of relying on mass spectral peak ratio of the highest intensity peak pair to measure relative quantity difference of a differentially labeled metabolite, this new program reconstructs the chromatographic peaks of the light- and heavy-labeled metabolite pair and then calculates the ratio of their peak areas to represent the relative concentration difference in two comparative samples. Using chromatographic peaks to perform relative quantification is shown to be more precise and accurate. IsoMS-Quant is integrated with IsoMS for picking peak pairs and Zero-fill for retrieving missing peak pairs in the initial peak pairs table generated by IsoMS to form a complete tool for processing CIL LC-MS data. This program can be freely downloaded from the www.MyCompoundID.org web site for noncommercial use.

  12. Assessing of distribution, mobility and bioavailability of exogenous Pb in agricultural soils using isotopic labeling method coupled with BCR approach.

    PubMed

    Huang, Zhi-Yong; Xie, Hong; Cao, Ying-Lan; Cai, Chao; Zhang, Zhi

    2014-02-15

    The contamination of Pb in agricultural soils is one of the most important ecological problems, which potentially results in serious health risk on human health through food chain. Hence, the fate of exogenous Pb contaminated in agricultural soils is needed to be deeply explored. By spiking soils with the stable enriched isotopes of (206)Pb, the contamination of exogenous Pb(2+) ions in three agricultural soils sampled from the estuary areas of Jiulong River, China was simulated in the present study, and the distribution, mobility and bioavailability of exogenous Pb in the soils were investigated using the isotopic labeling method coupled with a four-stage BCR (European Community Bureau of Reference) sequential extraction procedure. Results showed that about 60-85% of exogenous Pb was found to distribute in reducible fractions, while the exogenous Pb in acid-extractable fractions was less than 1.0%. After planting, the amounts of exogenous Pb presenting in acid-extractable, reducible and oxidizable fractions in rhizospheric soils decreased by 60-66%, in which partial exogenous Pb was assimilated by plants while most of the metal might transfer downward due to daily watering and applying fertilizer. The results show that the isotopic labeling technique coupled with sequential extraction procedures enables us to explore the distribution, mobility and bioavailability of exogenous Pb contaminated in soils, which may be useful for the further soil remediation. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells.

    PubMed

    Liu, Baodong; Liu, Xiaoling; Lai, Weiyi; Wang, Hailin

    2017-06-06

    DNA N 6 -methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [ 15 N 5 ]-2'-deoxyadenosine ([ 15 N 5 ]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [ 15 N 5 ]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine 15 N, and further utilizes the purine salvage pathway to generate mainly both [ 15 N 4 ]-dA and [ 15 N 4 ]-2'-deoxyguanosine ([ 15 N 4 ]-dG) in mammalian genomes. However, [ 15 N 5 ]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [ 15 N 4 ]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.

  14. Shifts in rotifer life history in response to stable isotope enrichment: testing theories of isotope effects on organismal growth

    PubMed Central

    2017-01-01

    In ecology, stable isotope labelling is commonly used for tracing material transfer in trophic interactions, nutrient budgets and biogeochemical processes. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism growth and metabolism. This assumption is, however, challenged by theoretical considerations and experimental studies on kinetic isotope effects in vivo. Here, I demonstrate profound changes in life histories of the rotifer Brachionus plicatilis fed 15N-enriched algae (0.4–5.0 at%); i.e. at the enrichment levels commonly used in ecological studies. These findings support theoretically predicted effects of heavy isotope enrichment on growth, metabolism and ageing in biological systems and underline the importance of accounting for such effects when using stable isotope labelling in experimental studies. PMID:28405367

  15. An in-advance stable isotope labeling strategy for relative analysis of multiple acidic plant hormones in sub-milligram Arabidopsis thaliana seedling and a single seed.

    PubMed

    Sun, Xiaohong; Ouyang, Yue; Chu, Jinfang; Yan, Jing; Yu, Yan; Li, Xiaoqiang; Yang, Jun; Yan, Cunyu

    2014-04-18

    A sensitive and reliable in-advance stable isotope labeling strategy was developed for simultaneous relative quantification of 8 acidic plant hormones in sub-milligram amount of plant materials. Bromocholine bromide (BETA) and its deuterated counterpart D9-BETA were used to in-advance derivatize control and sample extracts individually, which were then combined and subjected to solid-phase extraction (SPE) purification followed by UPLC-MS/MS analysis. Relative quantification of target compounds was obtained by calculation of the peak area ratios of BETA/D9-BETA labeled plant hormones. The in-advance stable isotope labeling strategy realized internal standard-based relative quantification of multiple kinds of plant hormones independent of availability of internal standard of every analyte with enhanced sensitivity of 1-3 orders of magnitude. Meanwhile, the in-advance labeling contributes to higher sample throughput and more reliability. The method was successfully applied to determine 8 plant hormones in 0.8mg DW (dry weight) of seedlings and 4 plant hormones from single seed of Arabidopsis thaliana. The results show the potential of the method in relative quantification of multiple plant hormones in tiny plant tissues or organs, which will advance the knowledge of the crosstalk mechanism of plant hormones. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. The Label Matters: μPET Imaging of the Biodistribution of Low Molar Mass 89Zr and 18F-Labeled Poly(2-ethyl-2-oxazoline).

    PubMed

    Glassner, Mathias; Palmieri, Luca; Monnery, Bryn D; Verbrugghen, Thomas; Deleye, Steven; Stroobants, Sigrid; Staelens, Steven; Wyffels, Leonie; Hoogenboom, Richard

    2017-01-09

    Poly(2-alkyl-2-oxazoline)s (PAOx) have received increasing interest for biomedical applications. Therefore, it is of fundamental importance to gain an in-depth understanding of the biodistribution profile of PAOx. We report the biodistribution of poly(2-ethyl-2-oxazoline) (PEtOx) with a molar mass of 5 kDa radiolabeled with PET isotopes 89 Zr and 18 F. 18 F-labeled PEtOx is prepared by the strain-promoted azide-alkyne cycloaddition (SPAAC) of [ 18 F]fluoroethylazide to bicyclo[6.1.0]non-4-yne (BCN)-functionalized PEtOx as many common labeling strategies were found to be unsuccessful for PEtOx. 89 Zr-labeled PEtOx is prepared using desferrioxamine end-groups as a chelator. Five kDa PEtOx shows a significantly faster blood clearance compared to PEtOx of higher molar mass while uptake in the liver is lower, indicating a minor contribution of the liver in excretion of the 5 kDa PEtOx. While [ 18 F]-PEtOx displays a rapid and efficient clearance from the kidneys, 5 kDa [ 89 Zr]-Df-PEtOx is not efficiently cleared over the time course of the study, which is most likely caused by trapping of 89 Zr-labeled metabolites in the renal tubules and not the polymer itself, demonstrating the importance of selecting the appropriate label for biodistribution studies.

  17. Development And Evaluation Of Stable Isotope And Fluorescent Labeling And Detection Methodologies For Tracking Injected Bacteria During In Situ Bioremediation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mark E. Fuller; Tullis C. Onstott

    2003-12-17

    This report summarizes the results of a research project conducted to develop new methods to label bacterial cells so that they could be tracked and enumerated as they move in the subsurface after they are introduced into the groundwater (i.e., during bioaugmentation). Labeling methods based on stable isotopes of carbon (13C) and vital fluorescent stains were developed. Both approaches proved successful with regards to the ability to effectively label bacterial cells. Several methods for enumeration of fluorescently-labeled cells were developed and validated, including near-real time microplate spectrofluorometry that could be performed in the field. However, the development of a novelmore » enumeration method for the 13C-enriched cells, chemical reaction interface/mass spectrometry (CRIMS), was not successful due to difficulties with the proposed instrumentation. Both labeling methodologies were successfully evaluated and validated during laboratory- and field-scale bacterial transport experiments. The methods developed during this research should be useful for future bacterial transport work as well as other microbial ecology research in a variety of environments. A full bibliography of research articles and meeting presentations related to this project is included (including web links to abstracts and full text reprints).« less

  18. Status of the Local Enforcement of Energy Efficiency Standards and Labeling Program in China

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, Nan; Zheng, Nina; Fino-Chen, Cecilia

    2011-09-26

    As part of its commitment to promoting and improving the local enforcement of appliance energy efficiency standards and labeling, the China National Institute of Standardization (CNIS) launched the National and Local Enforcement of Energy Efficiency Standards and Labeling project on August 14, 2009. The project’s short-term goal is to expand the effort to improve enforcement of standards and labeling requirements to the entire country within three years, with a long-term goal of perfecting overall enforcement. For this project, Jiangsu, Shandong, Sichuan and Shanghai were selected as pilot locations. This report provides information on the local enforcement project’s recent background, activitiesmore » and results as well as comparison to previous rounds of check-testing in 2006 and 2007. In addition, the report also offers evaluation on the achievement and weaknesses in the local enforcement scheme and recommendations. The results demonstrate both improvement and some backsliding. Enforcement schemes are in place in all target cities and applicable national standards and regulations were followed as the basis for local check testing. Check testing results show in general high labeling compliance across regions with 100% compliance for five products, including full compliance for all three products tested in Jiangsu province and two out of three products tested in Shandong province. Program results also identified key weaknesses in labeling compliance in Sichuan as well as in the efficiency standards compliance levels for small and medium three-phase asynchronous motors and self-ballasted fluorescent lamps. For example, compliance for the same product ranged from as low as 40% to 100% with mixed results for products that had been tested in previous rounds. For refrigerators, in particular, the efficiency standards compliance rate exhibited a wider range of 50% to 100%, and the average rate across all tested models also dropped from 96% in 2007 to 63

  19. Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.

    PubMed

    Ono, Katsuhiko; Jung, Minkyung; Zhang, Tianli; Tsutsuki, Hiroyasu; Sezaki, Hiroshi; Ihara, Hideshi; Wei, Fan-Yan; Tomizawa, Kazuhito; Akaike, Takaaki; Sawa, Tomohiro

    2017-05-01

    Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to a reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases-CysE, CysK, and CysM-from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34 S-labeled L-cysteine from O-acetyl-L-serine and 34 S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur ( 34 S) and nitrogen ( 15 N) atoms was also achieved by performing enzyme reactions with 15 N-labeled L-serine, acetyl-CoA, and 34 S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, L-cysteine sulfonate, and L-selenocystine. We also prepared 34 S-labeled N-acetyl-L-cysteine (NAC) by incubating 34 S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low-molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34 S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology. Copyright © 2017 Elsevier Inc

  20. Reductive methods for isotopic labeling of antibiotics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Champney, W.S.

    1989-08-15

    Methods for the reductive methylation of the amino groups of eight different antibiotics using {sup 3}HCOH or H{sup 14}COH are presented. The reductive labeling of an additional seven antibiotics by NaB{sub 3}H{sub 4} is also described. The specific activity of the methyl-labeled drugs was determined by a phosphocellulose paper binding assay. Two quantitative assays for these compounds based on the reactivity of the antibiotic amino groups with fluorescamine and of the aldehyde and ketone groups with 2,4-dinitrophenylhydrazine are also presented. Data on the cellular uptake and ribosome binding of these labeled compounds are also presented.

  1. Quantitative isomer-specific N-glycan fingerprinting using isotope coded labeling and high performance liquid chromatography-electrospray ionization-mass spectrometry with graphitic carbon stationary phase.

    PubMed

    Michael, Claudia; Rizzi, Andreas M

    2015-02-27

    Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. NMR studies of two spliced leader RNAs using isotope labeling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lapham, J.; Crothers, D.M.

    1994-12-01

    Spliced leader RNAs are a class of RNA molecules (<200 nts) involved in the trans splicing of messenger RNA found in trypanosomes, nematodes, and other lower eukaryotes. The spliced leader RNA from the trypanosome Leptomonas Collosoma exists in two alternate structural forms with similar thermal stabilities. The 54 nucleotides on the 5{prime} end of the SL molecule is structurally independent from the 3{prime} half of the RNA, and displays the two structural forms. Furthermore, the favored of the two structures was shown to contain anomalous nuclease sensitivity and thermal stability features, which suggests that there may be tertiary interactions betweenmore » the splice site and other nucleotides in the 5{prime} end. Multidimensional NMR studies are underway to elucidate the structural elements present in the SL RNAs that give rise to their physical properties. Two spliced leader sequences have been studied. The first, the 54 nucleotides on the 5{prime} end of the L. Collosoma sequence, was selected because of earlier studies in our laboratory. The second sequence is the 5{prime} end of the trypanosome Crithidia Fasciculata, which was chosen because of its greater sequence homology to other SL sequences. Given the complexity of the NMR spectra for RNA molecules of this size, we have incorporated {sup 15}N/{sup 13}C-labeled nucleotides into the RNA. One of the techniques we have developed to simplify the spectra of these RNA molecules is isotope labeling of specific regions of the RNA. This has been especially helpful in assigning the secondary structure of molecules that may be able to adopt multiple conformations. Using this technique one can examine a part of the molecule without spectral interference from the unlabeled portion. We hope this approach will promote an avenue for studying the structure of larger RNAs in their native surroundings.« less

  3. The evaluation of new and isotopically labeled isoindoline nitroxides and an azaphenalene nitroxide for EPR oximetry

    PubMed Central

    Khan, Nadeem; Blinco, James P.; Bottle, Steven E.; Hosokawa, Kazuyuki; Swartz, Harold M.; Micallef, Aaron S.

    2011-01-01

    Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-(N,N,N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogues (2H12- and/or 2H12-15N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O2 concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O2 sensitivity. Labeling the nitroxides with 15N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation. PMID:21665499

  4. Chemical Isotope Labeling LC-MS for High Coverage and Quantitative Profiling of the Hydroxyl Submetabolome in Metabolomics.

    PubMed

    Zhao, Shuang; Luo, Xian; Li, Liang

    2016-11-01

    A key step in metabolomics is to perform accurate relative quantification of the metabolomes in comparative samples with high coverage. Hydroxyl-containing metabolites are an important class of the metabolome with diverse structures and physical/chemical properties; however, many of them are difficult to detect with high sensitivity. We present a high-performance chemical isotope labeling liquid chromatography mass spectrometry (LC-MS) technique for in-depth profiling of the hydroxyl submetabolome, which involves the use of acidic liquid-liquid extraction to enrich hydroxyl metabolites into ethyl acetate from an aqueous sample. After drying and then redissolving in acetonitrile, the metabolite extract is labeled using a base-activated 12 C- or 13 C-dansylation reaction. A fast step-gradient LC-UV method is used to determine the total concentration of labeled metabolites. On the basis of the concentration information, a 12 C-labeled individual sample is mixed with an equal mole amount of a 13 C-labeled pool or control for relative metabolite quantification. The 12 C-/ 13 C-labeled mixtures are individually analyzed by LC-MS, and the resultant peak pairs of labeled metabolites in MS are measured for relative quantification and metabolite identification. A standard library of 85 hydroxyl compounds containing MS, retention time, and MS/MS information was constructed for positive metabolite identification based on matches of two or all three of these parameters with those of an unknown. Using human urine as an example, we analyzed samples of 1:1 12 C-/ 13 C-labeled urine in triplicate with triplicate runs per sample and detected an average of 3759 ± 45 peak pairs or metabolites per run and 3538 ± 71 pairs per sample with 3093 pairs in common (n = 9). Out of the 3093 peak pairs, 2304 pairs (75%) could be positively or putatively identified based on metabolome database searches, including 20 pairs positively identified using the dansylated hydroxyl standards library

  5. Metabolic Flux Analysis in Isotope Labeling Experiments Using the Adjoint Approach.

    PubMed

    Mottelet, Stephane; Gaullier, Gil; Sadaka, Georges

    2017-01-01

    Comprehension of metabolic pathways is considerably enhanced by metabolic flux analysis (MFA-ILE) in isotope labeling experiments. The balance equations are given by hundreds of algebraic (stationary MFA) or ordinary differential equations (nonstationary MFA), and reducing the number of operations is therefore a crucial part of reducing the computation cost. The main bottleneck for deterministic algorithms is the computation of derivatives, particularly for nonstationary MFA. In this article, we explain how the overall identification process may be speeded up by using the adjoint approach to compute the gradient of the residual sum of squares. The proposed approach shows significant improvements in terms of complexity and computation time when it is compared with the usual (direct) approach. Numerical results are obtained for the central metabolic pathways of Escherichia coli and are validated against reference software in the stationary case. The methods and algorithms described in this paper are included in the sysmetab software package distributed under an Open Source license at http://forge.scilab.org/index.php/p/sysmetab/.

  6. Identification of degradation routes of metamitron in soil microcosms using 13C-isotope labeling.

    PubMed

    Wang, Shizong; Miltner, Anja; Nowak, Karolina M

    2017-01-01

    Metamitron is one of the most commonly used herbicide in sugar beet and flower bulb cultures. Numerous laboratory and field studies on sorption and degradation of metamitron were performed. Detailed biodegradation studies in soil using 13 C-isotope labeling are still missing. Therefore, we aimed at providing a detailed turnover mass balance of 13 C 6 -metamitron in soil microcosms over 80 days. In the biotic system, metamitron mineralized rapidly, and 13 CO 2 finally constituted 60% of the initial 13 C 6 -metamitron equivalents. In abiotic control experiments CO 2 rose to only 7.4% of the initial 13 C 6 -metamitron equivalents. The 13 C label from 13 C 6 -metamitron was incorporated into microbial amino acids that were ultimately stabilized in the soil organic matter forming presumably harmless biogenic residues. Finally, 13 C label from 13 C 6 -metamitron was distributed between the 13 CO 2 and the 13 C-biogenic residues indicating nearly complete biodegradation. The parallel increase of 13 C-alanine, 13 C-glutamate and 13 CO 2 indicates that metamitron was initially biodegraded via the desamino-metamitron route suggesting its relevance in the growth metabolism. In later phases of biodegradation, the "Rhodococcus route" was indicated by the low 13 CO 2 evolution and the high relevance of the pyruvate pathway, which aims at biomolecule synthesis and seems to be related to starvation. This is a first report on the detailed degradation route of metamitron in soil. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Combination of Mass Signal Amplification and Isotope-Labeled Alkanethiols for the Multiplexed Detection of miRNAs.

    PubMed

    Kang, Hyunook; Hong, Seol-Hye; Sung, Jiha; Yeo, Woon-Seok

    2017-08-04

    We report a fast and sensitive method for the multiplexed detection of miRNAs by combining mass signal amplification and isotope-labeled signal reporter molecules. In our strategy, target miRNAs are captured specifically by immobilized DNAs on gold nanoparticles (AuNPs), which carry a large number of small molecules, called amplification tags (Am-tags), as the reporter for the detection of target miRNAs. For multiplexed detection, we designed and synthesized four Am-tags containing 0, 4, 8, 12 isotopes so that they had same molecular properties but different molecular weights. By observing the mass signals of the Am-tags on AuNPs decorated along with different probe DNAs, four types of miRNAs in a sample could be easily discriminated, and the relative amounts of these miRNAs could be quantified. The practicability of our strategy was further verified by measuring the expression levels of two miRNAs in HUVECs in response to different CuSO 4 concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Synthesis of carbon-14 and stable isotope labeled Avagacestat: a novel gamma secretase inhibitor for the treatment of Alzheimer's disease.

    PubMed

    Burrell, Richard C; Easter, John A; Cassidy, Michael P; Gillman, Kevin W; Olson, Richard E; Bonacorsi, Samuel J

    2014-08-01

    Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)-2-(4-Chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%. Copyright © 2014 John Wiley & Sons, Ltd.

  9. Dehydrogenation and dehalogenation of amines in MALDI-TOF MS investigated by isotopic labeling.

    PubMed

    Kang, Chuanqing; Zhou, Yihan; Du, Zhijun; Bian, Zheng; Wang, Jianwei; Qiu, Xuepeng; Gao, Lianxun; Sun, Yuequan

    2013-12-01

    Secondary and tertiary amines have been reported to form [M-H](+) that correspond to dehydrogenation in matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this investigation, we studied the dehydrogenation of amines in MALDI-TOF MS by isotopic labeling. Aliphatic amines were labeled with deuterium on the methylene of an N-benzyl group, which resulted in the formation of [M-D](+) and [M-H](+) ions by dedeuteration and dehydrogenation, respectively. This method revealed the proton that was removed. The spectra of most tertiary amines with an N-benzyl group showed high-intensity [M-D](+) and [M-H](+) ion peaks, whereas those of secondary amines showed low-intensity ion peaks. Ratios between the peak intensities of [M-D](+) and [M-H](+) greater than 1 suggested chemoselective dehydrogenation at the N-benzyl groups. The presence of an electron donor group on the N-benzyl groups enhanced the selectivity. The dehalogenation of amines with an N-(4-halobenzyl) group was also observed alongside dehydrogenation. The amino ions from dehalogenation can undergo second dehydrogenation. These results provide the first direct evidence about the position at which dehydrogenation of an amine occurs and the first example of dehalogenation of haloaromatic compounds in MALDI-TOF MS. These results should be helpful in the structural identification and elucidation of synthetic and natural molecules. Copyright © 2013 John Wiley & Sons, Ltd.

  10. INCORPORATING CONCENTRATION DEPENDENCE IN STABLE ISOTOPE MIXING MODELS

    EPA Science Inventory

    Stable isotopes are often used as natural labels to quantify the contributions of multiple sources to a mixture. For example, C and N isotopic signatures can be used to determine the fraction of three food sources in a consumer's diet. The standard dual isotope, three source li...

  11. Sulphur tracer experiments in laboratory animals using 34S-labelled yeast.

    PubMed

    Martínez-Sierra, J Giner; Moreno Sanz, F; Herrero Espílez, P; Marchante Gayón, J M; Rodríguez Fernández, J; García Alonso, J I

    2013-03-01

    We have evaluated the use of (34)S-labelled yeast to perform sulphur metabolic tracer experiments in laboratory animals. The proof of principle work included the selection of the culture conditions for the preparation of sulphur labelled yeast, the study of the suitability of this labelled yeast as sulphur source for tracer studies using in vitro gastrointestinal digestion and the administration of the (34)S-labelled yeast to laboratory animals to follow the fate and distribution of (34)S in the organism. For in vitro gastrointestinal digestion, the combination of sodium dodecyl sulphate-polyacrylamide gel electrophoresis and high-performance liquid chromatography and inductively coupled plasma mass spectrometry (HPLC-ICP-MS) showed that labelled methionine, cysteine and other low molecular weight sulphur-containing biomolecules were the major components in the digested extracts of the labelled yeast. Next, in vivo kinetic experiments were performed in healthy Wistar rats after the oral administration of (34)S-labelled yeast. The isotopic composition of total sulphur in tissues, urine and faeces was measured by double-focusing inductively coupled plasma mass spectrometry after microwave digestion. It was observed that measurable isotopic enrichments were detected in all samples. Finally, initial investigations on sulphur isotopic composition of serum and urine samples by HPLC-ICP-MS have been carried out. For serum samples, no conclusive data were obtained. Interestingly, chromatographic analysis of urine samples showed differential isotope enrichment for several sulphur-containing biomolecules.

  12. Updating the procedure for metaiodobenzylguanidine labelling with iodine radioisotopes employed in industrial production.

    PubMed

    Franceschini, R; Mosca, R; Bonino, C

    1991-01-01

    The classical procedure used for the preparation of [125I]- and [131I]metaiodobenzylguanidine (MIBG) is the solid-phase isotopic exchange between MIBG and radioiodide. This reaction requires 1.5 hours at 160 degrees C to obtain maximum total labelling yields of 75-80%. Recently, the importance of rapid procedures for the preparation of 123I-MIBG has been highlighted. A highly efficient procedure for the industrial production of 123I-MIBG using ascorbic acid, tin sulfate and copper sulfate pentahydrate in 0.01 M sulfuric acid is reported. Sequential radio-TLC analysis of the labelling mixture shows that the labelling yield reaches 98% within 45 min at 100 degrees C. The specific activity of the 123I-MIBG produced in this manner is on the order of 100 Ci/mmol.

  13. Understanding N timing in corn yield and fertilizer N recovery: An insight from an isotopic labeled-N determination

    PubMed Central

    de Almeida, Rodrigo Estevam Munhoz; Pierozan Junior, Clovis; Lago, Bruno Cocco; Trivelin, Paulo Cesar Ocheuze

    2018-01-01

    Early fertilizer nitrogen (N) application on cover crops or their residues during the off-season is a practice adopted in Brazil subtropical conditions under no-tillage corn (Zea mays L.) systems. However, the effect of early N application on yield, plant N content, and N recovery efficiency (NRE) for corn is not yet well documented. Five fertilizer N timings in an oat-corn system were evaluated in two studies utilizing an isotopic-labeled N determination, 15N isotope. The N fertilization timings were: (i) oat tillering, (ii) 15 days before corn planting time, over the oat residues, (iii) at corn planting time, (iv) in-season at the three-leaf growth stage (V3), and (v) in-season split application at V3 and six-leaf (V6) growth stages. Based on the statistical analysis, the N fertilization timings were separated into three groups: 1) N-OATS, designated to N applied at oat; 2) N-PLANT, referred to pre-plant and planting N applications; and 3) N-CORN, designated to in-season corn N applications. Corn yield was not affected by the N fertilization timing. However, the N-CORN N fertilization timings enhanced NRE by 17% and 35% and final N recovery system (plant plus soil) by 16% and 24% all relative to N-OATS and N-PLANT groups, respectively. Overall, N-OATS resulted in the largest N derived from fertilizer (NDFF) amount in the deeper soil layer, in overall a delta of 10 kg N ha-1 relative to the rest of the groups. Notwithstanding corn yield was not affected, early N fertilization under subtropical conditions is not a viable option since NRE was diminished and the non-recovery N increased relative to the in-season N applications. PMID:29462178

  14. An Efficient Site-Specific Method for Irreversible Covalent Labeling of Proteins with a Fluorophore.

    PubMed

    Liu, Jiaquan; Hanne, Jeungphill; Britton, Brooke M; Shoffner, Matthew; Albers, Aaron E; Bennett, Jared; Zatezalo, Rachel; Barfield, Robyn; Rabuka, David; Lee, Jong-Bong; Fishel, Richard

    2015-11-19

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Förster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins.

  15. Practical strategies when using a stable isotope labeled microtracer for absolute bioavailability assessment: A case study of a high oral dose clinical candidate GDC-0810.

    PubMed

    Chen, Buyun; Lu, Pingping; Freeman, Dugan; Gao, Yang; Choo, Edna; DeMent, Kevin; Savage, Scott; Zhang, Kelly; Milanwoski, Dennis; Liu, Lichuan; Dean, Brian; Deng, Yuzhong

    2018-05-30

    The pH labile metabolite, hydrophobicity, high oral dose and systematic exposure of GDC-0810 posed tremendous challenges to develop a LC-MS method for a stable isotope labeled aBA study. In this study, we explored practical solutions to balance stability and sensitivity and to cope with the impact of high C p.o. to C i.v. ratio on the labeling selection and assay dynamic range. A [ 13 C 9 ] GDC-0810 was synthesized to minimize the isotopic interference between PO dose, internal standard and I.V. microtracer. A highly sensitive LC-MS assay was validated for quantitation of [ 13 C 9 ] GDC-0810 from 5 to 1250 pg/mL. The optimized method was applied to a proof of concept cynomolgus monkey aBA study and the bioavailability calculated using microtracer dosing and regular dosing were similar to each other. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Mass spectrometric measurements of norepinephrine synthesis in man from infusion of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Suzuki, T.; Sakoda, S.; Ueji, M.

    The kinetics of stable isotope-labelled L-threo-3,4-dihydroxyphenylserine (L-threo-DOPS), an immediate precursor of (-)-norepinephrine, was studied to investigate the pharmacologic mechanism of its therapeutic effect on orthostatic hypotension in familial amyloid polyneuropathy (FAP) and on akinesia and freezing in parkinsonism. (/sup 13/C,D)-L-threo-DOPS was synthesized, and 100 mg of the compound was infused for 2 h into two normal subjects, two FAP patients and two patients with the degenerative diseases of the central nervous system. Labelled and endogenous norepinephrine in urine and plasma was assayed simultaneously by gas chromatography/mass spectrometry. The results indicate that the increase in norepinephrine in biological fluids after administrationmore » of L-threo-DOPS is attributable mostly to norepinephrine derived from L-threo-DOPS, not to pre-formed endogenous norepinephrine released by L-threo-DOPS.« less

  17. Palladium-catalyzed double carbonylation using near stoichiometric carbon monoxide: expedient access to substituted 13C2-labeled phenethylamines.

    PubMed

    Nielsen, Dennis U; Neumann, Karoline; Taaning, Rolf H; Lindhardt, Anders T; Modvig, Amalie; Skrydstrup, Troels

    2012-07-20

    A novel and general approach for (13)C(2)- and (2)H-labeled phenethylamine derivatives has been developed, based on a highly convergent single-step assembly of the carbon skeleton. The efficient incorporation of two carbon-13 isotopes into phenethylamines was accomplished using a palladium-catalyzed double carbonylation of aryl iodides with near stoichiometric carbon monoxide.

  18. An aggregated perylene-based broad-spectrum, efficient and label-free quencher for multiplexed fluorescent bioassays.

    PubMed

    Liu, Tao; Hu, Rong; Lv, Yi-Fan; Wu, Yuan; Liang, Hao; Huan, Shuang-Yan; Zhang, Xiao-Bing; Tan, Weihong; Yu, Ru-Qin

    2014-08-15

    Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. The metabolic fate of doubly stable isotope labelled heat-killed Lactobacillus johnsonii in humans.

    PubMed

    Wutzke, K D; Berg, D; Haffner, D

    2008-02-01

    In this study, heat-killed Lactobacillus johnsonii (La1), doubly labelled with (13)C and (15)N (hk-dlLa1), was used to follow the metabolic fate after oral administration in humans. Experimental study. University of Rostock, Children's Hospital, Research Laboratory. Ten healthy adults aged 23-26 years. The subjects received 74.6 mg/kg body weight hk-dlLa1 and 10 g alpha-D-raffinose together with breakfast. A sample of venous blood was taken after 2 h. Expired air samples were taken over 14 h, whereas urine and faeces were collected over a period of 48 h. (13)C- and (15)N-enrichments were measured by isotope ratio mass spectrometry. Hydrogen concentrations were measured by electrochemical detection. The orocaecal transit time (OCTT) was reached after 3.4 h. After 2 h, (13)C- and (15)N-enrichment of fibrinogen amounted to 2 and 25 p.p.m. excess, respectively. The (13)CO(2)-exhalation amounted to 9.2% of the ingested dose. The urinary excretion of (13)C and (15)N was 2.1 and 10.4% of the ingested dose, respectively, whereas the faecal excretion was 47.9 and 43.7% of the ingested dose, respectively. In comparison to OCTT of 3.4 h, both stable isotopes appear after 30 min in breath and urine, indicating that hk-dlLa1 is rapidly digested in the small bowel before reaching the caecum. This is confirmed by (13)C-and (15)N-enrichments of blood plasma fractions. The ingestion of hk-dlLa1 led to a (13)C- and (15)N-excretion of 59.2 and 54.1% of the ingested dose, respectively, of both stable isotopes.

  20. Application of stable isotope tools for evaluating natural and stimulated biodegradation of organic pollutants in field studies.

    PubMed

    Fischer, Anko; Manefield, Mike; Bombach, Petra

    2016-10-01

    Stable isotope tools are increasingly applied for in-depth evaluation of biodegradation of organic pollutants at contaminated field sites. They can be divided into three methods i) determination of changes in natural abundance of stable isotopes using compound-specific stable isotope analysis (CSIA), ii) detection of incorporation of stable-isotope label from a stable-isotope labelled target compound into degradation and/or mineralisation products and iii) determination of stable-isotope label incorporation into biomarkers using stable isotope probing (SIP). Stable isotope tools have been applied as key monitoring tools for multiple-line-of-evidence-approaches (MLEA) for sensitive evaluation of pollutant biodegradation. This review highlights the application of CSIA, SIP and MLEA including stable isotope tools for assessing natural and stimulated biodegradation of organic pollutants in field studies dealing with soil and groundwater contaminations. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Development of isotope labeling liquid chromatography mass spectrometry for mouse urine metabolomics: quantitative metabolomic study of transgenic mice related to Alzheimer's disease.

    PubMed

    Peng, Jun; Guo, Kevin; Xia, Jianguo; Zhou, Jianjun; Yang, Jing; Westaway, David; Wishart, David S; Li, Liang

    2014-10-03

    Because of a limited volume of urine that can be collected from a mouse, it is very difficult to apply the common strategy of using multiple analytical techniques to analyze the metabolites to increase the metabolome coverage for mouse urine metabolomics. We report an enabling method based on differential isotope labeling liquid chromatography mass spectrometry (LC-MS) for relative quantification of over 950 putative metabolites using 20 μL of urine as the starting material. The workflow involves aliquoting 10 μL of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 μL of aliquot was taken from each sample to generate a pooled sample that was subjected to ¹³C-dansylation labeling. The ¹²C-labeled individual sample was mixed with an equal volume of the ¹³C-labeled pooled sample. The mixture was then analyzed by LC-MS to generate information on metabolite concentration differences among different individual samples. The interday repeatability for the LC-MS runs was assessed, and the median relative standard deviation over 4 days was 5.0%. This workflow was then applied to a metabolomic biomarker discovery study using urine samples obtained from the TgCRND8 mouse model of early onset familial Alzheimer's disease (FAD) throughout the course of their pathological deposition of beta amyloid (Aβ). It was showed that there was a distinct metabolomic separation between the AD prone mice and the wild type (control) group. As early as 15-17 weeks of age (presymptomatic), metabolomic differences were observed between the two groups, and after the age of 25 weeks the metabolomic alterations became more pronounced. The metabolomic changes at different ages corroborated well with the phenotype changes in this transgenic mice model. Several useful candidate biomarkers including methionine, desaminotyrosine, taurine, N1-acetylspermidine, and 5-hydroxyindoleacetic acid were identified. Some of them were found in previous

  2. Formation of nonextractable soil residues: A stable isotope approach

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Richnow, H.H.; Eschenback, A.; Mahro, B.

    1999-11-01

    Stable carbon isotopic measurements were employed to characterize the transformation of a {sup 13}C-labeled polycyclic aromatic hydrocarbon (PAH), anthracene, in a closed soil bioreactor system. The {sup 13}C-label was used to calculate a carbon mass balance including mineralization and the formation of nonextractable soil-bound residues. Similar results were obtained from {sup 13}C-labeled carbon and {sup 14}C-labeled carbon mass balance calculations for separate batch experiments with labeled anthracene. In concentration ranges typical for real PAH-contaminated sites, the sensitivity of the {sup 13}C tracer method meets the requirements of classical radiotracer experiments. Therefore, the authors balancing method based on stable isotope-labeled chemicalsmore » may supplement or substitute radiotracer experiments under many circumstances. One major advantage of using stable isotope-labeled tracers is the possible application in transformation studies where the use of radioactive substances is of environmental concern. The transformation of {sup 13}C-labeled PAH into nonextractable residues clearly depends on the metabolic activity of the soil microflora and occurs during an early phase of biodegradation. Successive contamination of the soil by anthracene leads to a progressive adaptation of the microflora to a complete mineralization of anthracene in the soil. The extent of residue formation is controlled by the capability of the microflora to degrade the contaminant. Results of long-term experiments indicate that nonextractable residues are relatively stable over time.« less

  3. Negative ion ESI-MS analysis of natural yellow dye flavonoids--An isotopic labelling study

    NASA Astrophysics Data System (ADS)

    McNab, Hamish; Ferreira, Ester S. B.; Hulme, Alison N.; Quye, Anita

    2009-07-01

    Flavonoids are amongst the most commonly used natural yellow colourants in paintings, as lakes, and in historical textiles as mordant dyes. In this paper, evidence from isotopically labelled substrates is used to propose negative ion electrospray collision induced decomposition mechanisms of flavones, flavonols and an isoflavone. These mechanisms include a retro-Diels-Alder fragmentation (observed for flavones and flavonols) and an M-122 fragmentation (characteristic of 3',4'-dihydroxyflavonols). In addition, the presence of a m/z 125 fragment ion is shown to be characteristic of 2'-hydroxyflavonols and an ion at m/z 149 is shown to be characteristic of 4'-hydroxyflavones. Applications of these methods are exemplified by the identification of a minor component of Dyer's camomile (Anthemis tinctoria L.) and the identification of the dye source in green threads sampled from an 18th Century Scottish tartan fragment.

  4. Isotopic labeling for the understanding of the alteration of limestone used in built cultural heritage

    NASA Astrophysics Data System (ADS)

    Saheb, Mandana; Chabas, Anne; Mertz, Jean-Didier; Rozenbaum, Olivier; Verney-Carron, Aurélie

    2015-04-01

    This project belongs to a specific work aiming at developing isotopic tools to better understand the alteration of materials used in the built cultural heritage. It is focused on the study of the alteration of limestone used in the facades of historic buildings subject to atmospheric polluted environment. Actually in the elevated parts of the buildings, water as rainfall (runoff or wet deposition) or in vapor form (condensation or dry deposition) is the main agent of alteration. Thus, the rock/water interactions need to be well understood to propose adapted solution to better preserve the buildings. To identify the water transfer within the porous limestone and locate the reaction preferential sites, two isotopic tracers (D and 18O) are used to monitor the alteration solution (D) and locate the zones containing the secondary phases (18O). The Saint-Maximin limestone used in many monuments in the suburbs of Paris (France) as a building and restoration stone has been specifically studied. Pristine materials, stones from monuments (monuments in the Paris area) and samples altered in laboratory constitute the analytical corpus to compare different stages of alteration. In a first step the stones are characterized at different scales to identify the alteration pattern (SEM-EDS, Raman microspectrometry, XRD, rugosimetry) and study the water transfers (X-ray tomography, mercury porosimetry, imbibition kinetics). The samples are then altered in the laboratory by realistic and controlled wet or dry deposition using isotopically labeled solutions to locate the reaction zones by SIMS. The multiscale characterization of the alteration pattern has allowed proposing alteration mechanisms linked to the properties of the stones and their location inside the building. Moreover, the location of the reactive zones inside the materials determined by the isotopic experiments helps examining the role of the evolution of porosity and formation of alteration products within the material

  5. Syntheses of halogen derivatives of L-tryptophan, L-tyrosine and L-phenylalanine labeled with hydrogen isotopes.

    PubMed

    Pająk, Małgorzata; Pałka, Katarzyna; Winnicka, Elżbieta; Kańska, Marianna

    2016-01-01

    Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L-tryptophan, i.e. 5'-bromo-[2-(3)H]-, 5'-bromo-[2-(2)H/(3)H]-, 5'-fluoro-[2-(3)H]-5'-fluoro-[2-(2)H/(3)H]-, 6'-fluoro-[2-(3)H]-, 6'-fluoro-[2-(2)H/(3)H]-L-tryptophan, as well as, L-tyrosine, i.e. 3'-fluoro-[2-(3)H]-, 3'-fluoro-[2-(2)H/(3)H]-, 3'-chloro-[2-(3)H]-, and 3'-chloro-[2-(2)H/(3)H]-L-tyrosine, and also L-phenylalanine, i.e. 2'-fluoro-[(3S)-(3)H]-, 2'-fluoro-[(3S)-(2)H/(3) H]-, 2'-chloro-[(3S)-(3)H]-, 2'-chloro-[(3S)-(2)H/(3)H]-, 4'-chloro-[(3S)-(3)H]-, and 4'-chloro-[(3S)-(2)H/(3)H]-L-phenylalanine were synthesized using enzymatic methods. Isotopomers of L-tryptophan were synthesized by coupling of halogenated indoles with S-methyl-L-cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L-tyrosine were obtained by the enzymatically supported exchange between halogenated L-tyrosine and isotopic water. Labeled halogenated isotopologues of L-Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D2O) with addition of HTO were used. Copyright © 2015 John Wiley & Sons, Ltd.

  6. Stable isotope methodology in the pharmacokinetic studies of androgenic steroids in humans.

    PubMed

    Shinohara, Y; Baba, S

    1990-04-01

    The use of stable isotopically labeled steroids combined with gas chromatography/mass spectrometry (GC/MS) has found a broad application in pharmacologic studies. Initially, stable isotopically labeled steroids served as the ideal analytic internal standard for GC/MS analysis; however, their in vivo use has expanded and has proven to be a powerful pharmacokinetic tool. We have successfully used stable isotope methodology to study the pharmacokinetic/bioavailability of androgens. The primary advantage of the technique is that endogenous and exogenous steroids with the same basic structure can be differentiated by using stable isotopically labeled analogs. The method was used to examine the pharmacokinetics of testosterone and testosterone propionate, and to clarify the influence of endogenous testosterone. Another advantage of the isotope methods is that steroidal drugs can be administered concomitantly in two formulations (e.g., solution and solid dosage). A single set of blood samples serves to describe the time course of the formulations being compared. This stable isotope coadministration technique was used to estimate the relative bioavailability of 17 alpha-methyltestosterone.

  7. Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

    PubMed

    Creek, Darren J; Mazet, Muriel; Achcar, Fiona; Anderson, Jana; Kim, Dong-Hyun; Kamour, Ruwida; Morand, Pauline; Millerioux, Yoann; Biran, Marc; Kerkhoven, Eduard J; Chokkathukalam, Achuthanunni; Weidt, Stefan K; Burgess, Karl E V; Breitling, Rainer; Watson, David G; Bringaud, Frédéric; Barrett, Michael P

    2015-03-01

    Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

  8. Simultaneous determination of the intravenous and oral pharmacokinetic parameters of D,L-verapamil using stable isotope-labelled verapamil.

    PubMed

    Eichelbaum, M; Somogyi, A; von Unruh, G E; Dengler, H J

    1981-01-01

    Following i.v. administration, the plasma concentration-time curve of verapamil could best be described by either a mono- or biexponential equation. Total plasma clearance (1.26 1/min) approached liver blood flow (1.51/min), so it can be concluded that its clearance is liver blood flow-dependent. Although absorption was almost complete after oral administration, absolute bioavailability (20%) was low, due to extensive hepatic first-pass metabolism. The approach using stable isotope-labelled and unlabelled drug permits simultaneous administration by the intravascular and extravascular routes, thus allowing determination of absolute bioavailability in a single experiment.

  9. Combining Solvent Isotope Effects with Substrate Isotope Effects in Mechanistic Studies of Alcohol and Amine Oxidation by Enzymes*

    PubMed Central

    Fitzpatrick, Paul F.

    2014-01-01

    Oxidation of alcohols and amines is catalyzed by multiple families of flavin-and pyridine nucleotide-dependent enzymes. Measurement of solvent isotope effects provides a unique mechanistic probe of the timing of the cleavage of the OH and NH bonds, necessary information for a complete description of the catalytic mechanism. The inherent ambiguities in interpretation of solvent isotope effects can be significantly decreased if isotope effects arising from isotopically labeled substrates are measured in combination with solvent isotope effects. The application of combined solvent and substrate (mainly deuterium) isotope effects to multiple enzymes is described here to illustrate the range of mechanistic insights that such an approach can provide. PMID:25448013

  10. Carbon isotope labeling in boreal forests to assess roles of fungal species in decomposition

    NASA Astrophysics Data System (ADS)

    Treseder, K. K.; Czimczik, C. I.; Trumbore, S. E.; Allison, S. D.

    2006-12-01

    We used 14C and 13C labeling to assess the in situ respiration of alanine-, starch-, and lignocellulose-derived carbon from the sporocarps of particular fungal species fruiting in a boreal forest in Alaska. By measuring isotopically-labeled respiration of sporocarps, which can be identified to species, we were able to attribute turnover of carbon compounds to specific fungal groups. Moreover, collection of sporocarp respiration is non-destructive, so we could return to the same sporocarps to collect a time series of measurements that spanned hours to days. We tested the hypotheses that alanine and starch turn over more quickly than lignocellulose, and that saprotrophic fungi would use starch-C and lignocellulose-C but ectomycorrhizal fungi would not. Small amounts of 14C-labeled alanine (about 100,000 permil) were dispensed into the soil within three meters of sporocarps of the ectomycorrhizal fungus Lactarius alnicola. Δ14CO2 values of sporocarp respiration climbed from 75.8 +/- 6.3 permil to 7855 +/- 3940 permil within one hour of additions, indicating that the fungus quickly acquired, transported, and transformed the alanine-C. In a separate approach, a mixture of 13C-labeled starch (about 15,000 permil) and 14C-labeled lignocellulose (about 36,000 permil) was applied in 9 m2 plots containing sporocarps of the ectomycorrhizal genera Phellodon and Sarcodon and the saprotrophic genera Lycoperdon and Polyporus. An unlabeled control plot was also established. We observed no detectable increase in 14CO2 or 13CO2 over a 144 hour period, suggesting that neither ectomycorrhizal nor saprotrophic fungi significantly broke down starch or lignocellulose during this time. The alanine experiment is one of the first to indicate that ectomycorrhizal fungi can influence the spatial distribution and storage of soil carbon over short time scales. This influence may be restricted to carbon of organic compounds like amino acids. In contrast, starch was not transformed quickly even

  11. Follow the Carbon: Isotopic Labeling Studies of Early Earth Aerosol.

    PubMed

    Hicks, Raea K; Day, Douglas A; Jimenez, Jose L; Tolbert, Margaret A

    2016-11-01

    Despite the faint young Sun, early Earth might have been kept warm by an atmosphere containing the greenhouse gases CH 4 and CO 2 in mixing ratios higher than those found on Earth today. Laboratory and modeling studies suggest that an atmosphere containing these trace gases could lead to the formation of organic aerosol haze due to UV photochemistry. Chemical mechanisms proposed to explain haze formation rely on CH 4 as the source of carbon and treat CO 2 as a source of oxygen only, but this has not previously been verified experimentally. In the present work, we use isotopically labeled precursor gases and unit-mass resolution (UMR) and high-resolution (HR) aerosol mass spectrometry to examine the sources of carbon and oxygen to photochemical aerosol formed in a CH 4 /CO 2 /N 2 atmosphere. UMR results suggest that CH 4 contributes 70-100% of carbon in the aerosol, while HR results constrain the value from 94% to 100%. We also confirm that CO 2 contributes approximately 10% of the total mass to the aerosol as oxygen. These results have implications for the geochemical interpretations of inclusions found in Archean rocks on Earth and for the astrobiological potential of other planetary atmospheres. Key Words: Atmosphere-Early Earth-Planetary atmospheres-Carbon dioxide-Methane. Astrobiology 16, 822-830.

  12. Using carbon isotope fractionation for an improved quantification of CH4 oxidation efficiency in Arctic peatlands

    NASA Astrophysics Data System (ADS)

    Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.

    2012-04-01

    Much research effort is focused on identifying global CH4 sources and sinks to estimate their current and potential strength in response to land-use change and global warming. Aerobic CH4 oxidation is regarded as the key process reducing the strength of CH4 emissions in wetlands, but is hitherto difficult to quantify. Recent studies quantify the efficiency of CH4 oxidation based on CH4 stable isotope signatures. The approach utilizes the fact that a significant isotope fractionation occurs when CH4 is oxidized. Moreover, it also considers isotope fractionation by diffusion. For field applications the 'open-system equation' is applied to determine the CH4 oxidation efficiency: fox = (δE - δP)/ (αox - αtrans) where fox is the fraction of CH4 oxidized; δE is δ13C of emitted CH4; δP is δ13C of produced CH4; αox is the isotopic fractionation factor of oxidation; αtrans is the isotopic fractionation factor of transport. We quantified CH4 oxidation in polygonal tundra soils of Russia's Lena River Delta analyzing depth profiles of CH4 concentrations and stable isotope signatures. Therefore, both fractionation factors αox and αtrans were determined for three polygon centers with differing water table positions and a polygon rim. While most previous studies on landfill cover soils have assumed a gas transport dominated by advection (αtrans = 1), other CH4 transport mechanisms as diffusion have to be considered in peatlands and αtrans exceeds a value of 1. At our study we determined αtrans = 1.013 ± 0.003 for CH4 when diffusion is the predominant transport mechanism. Furthermore, results showed that αox differs widely between sites and horizons (αox = 1.013 ± 0.012) and has to be determined for each case. The impact of both fractionation factors on the quantification of CH4 oxidation was estimated by considering both the potential diffusion rate at different water contents and potential oxidation rates. Calculations for a water saturated tundra soil

  13. Sulfonium Ion Derivatization, Isobaric Stable Isotope Labeling and Data Dependent CID- and ETD-MS/MS for Enhanced Phosphopeptide Quantitation, Identification and Phosphorylation Site Characterization

    PubMed Central

    Lu, Yali; Zhou, Xiao; Stemmer, Paul M.; Reid, Gavin E.

    2014-01-01

    An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded ‘fixed charge’ sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS3, and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S′-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of ‘light’ (S(CH3)2) and ‘heavy’ (S(CD3)2) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS3 or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency. PMID:21952753

  14. Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes

    PubMed Central

    Beckers, Veronique; Kiep, Katina; Becker, Horst; Bläsing, Oliver Ernst; Fuchs, Regine

    2016-01-01

    Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of 13CO2 and 15NH4NO3. The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, 13CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous 13CO2 and 15NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong 13C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping. PMID:26966172

  15. Development of chemical isotope labeling liquid chromatography mass spectrometry for silkworm hemolymph metabolomics.

    PubMed

    Shen, Weifeng; Han, Wei; Li, Yunong; Meng, Zhiqi; Cai, Leiming; Li, Liang

    2016-10-26

    Silkworm (Bombyx mori) is a very useful target insect for evaluation of endocrine disruptor chemicals (EDCs) due to mature breeding techniques, complete endocrine system and broad basic knowledge on developmental biology. Comparative metabolomics of silkworms with and without EDC exposure offers another dimension of studying EDCs. In this work, we report a workflow on metabolomic profiling of silkworm hemolymph based on high-performance chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) and demonstrate its application in studying the metabolic changes associated with the pesticide dichlorodiphenyltrichloroethane (DDT) exposure in silkworm. Hemolymph samples were taken from mature silkworms after growing on diet that contained DDT at four different concentrations (1, 0.1, 0.01, 0.001 ppm) as well as on diet without DDT as controls. They were subjected to differential 12 C-/ 13 C-dansyl labeling of the amine/phenol submetabolome, LC-UV quantification of the total amount of labeled metabolites for sample normalization, and LC-MS detection and relative quantification of individual metabolites in comparative samples. The total concentration of labeled metabolites did not show any significant change between four DDT-treatment groups and one control group. Multivariate statistical analysis of the metabolome data set showed that there was a distinct metabolomic separation between the five groups. Out of the 2044 detected peak pairs, 338 and 1471 metabolites have been putatively identified against the HMDB database and the EML library, respectively. 65 metabolites were identified by the dansyl library searching based on the accurate mass and retention time. Among the 65 identified metabolites, 33 positive metabolites had changes of greater than 1.20-fold or less than 0.83-fold in one or more groups with p-value of smaller than 0.05. Several useful biomarkers including serine, methionine, tryptophan, asymmetric dimethylarginine, N

  16. The Doubly Labeled Water Method for Measuring Human Energy Expenditure: Adaptations for Spaceflight

    NASA Technical Reports Server (NTRS)

    Schulz, Leslie O.

    1991-01-01

    It is essential to determine human energy requirements in space, and the doubly labeled water method has been identified as the most appropriate means of indirect calorimetry to meet this need. The method employs naturally occurring, stable isotopes of hydrogen (H-2, deuterium) and oxygen (O-18) which, after dosing, mix with body water. The deuterium is lost from the body as water while the O-18 is eliminated as both water and CO2. The difference between the two isotope elimination rates is therefore a measure of CO2 production and hence energy expenditure. Spaceflight will present a unique challenge to the application of the doubly labeled water method. Specifically, interpretation of doubly labeled water results assumes that the natural abundance or 'background' levels of the isotopes remain constant during the measurement interval. To address this issue, an equilibration model will be developed in an ongoing ground-based study. As energy requirements of women matched to counterparts in the Astronauts Corps are being determined by doubly labeled water, the baseline isotope concentration will be changed by consumption of 'simulated Shuttle water' which is artificially enriched. One group of subjects will be equilibrated on simulated Shuttle water prior to energy determinations by doubly labeled water while the others will consume simulated Shuttle water after dosing. This process will allow us to derive a prediction equation to mathematically model the effect of changing background isotope concentrations.

  17. A Low-Storage-Consumption XML Labeling Method for Efficient Structural Information Extraction

    NASA Astrophysics Data System (ADS)

    Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo

    Recently, labeling methods to extract and reconstruct the structural information of XML data, which are important for many applications such as XPath query and keyword search, are becoming more attractive. To achieve efficient structural information extraction, in this paper we propose C-DO-VLEI code, a novel update-friendly bit-vector encoding scheme, based on register-length bit operations combining with the properties of Dewey Order numbers, which cannot be implemented in other relevant existing schemes such as ORDPATH. Meanwhile, the proposed method also achieves lower storage consumption because it does not require either prefix schema or any reserved codes for node insertion. We performed experiments to evaluate and compare the performance and storage consumption of the proposed method with those of the ORDPATH method. Experimental results show that the execution times for extracting depth information and parent node labels using the C-DO-VLEI code are about 25% and 15% less, respectively, and the average label size using the C-DO-VLEI code is about 24% smaller, comparing with ORDPATH.

  18. Synthesis of stereoarray isotope labeled (SAIL) lysine via the "head-to-tail" conversion of SAIL glutamic acid.

    PubMed

    Terauchi, Tsutomu; Kamikawai, Tomoe; Vinogradov, Maxim G; Starodubtseva, Eugenia V; Takeda, Mitsuhiro; Kainosho, Masatsune

    2011-01-07

    A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-(2)H(4);1,2,3,4,5,6-(13)C(6);2,6-(15)N(2)]lysine, was synthesized by the "head-to-tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-(2)H(2);1,2,3,4,5-(13)C(5);2-(15)N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins.

  19. Kinetic 15N-isotope effects on algal growth

    NASA Astrophysics Data System (ADS)

    Andriukonis, Eivydas; Gorokhova, Elena

    2017-03-01

    Stable isotope labeling is a standard technique for tracing material transfer in molecular, ecological and biogeochemical studies. The main assumption in this approach is that the enrichment with a heavy isotope has no effect on the organism metabolism and growth, which is not consistent with current theoretical and empirical knowledge on kinetic isotope effects. Here, we demonstrate profound changes in growth dynamics of the green alga Raphidocelis subcapitata grown in 15N-enriched media. With increasing 15N concentration (0.37 to 50 at%), the lag phase increased, whereas maximal growth rate and total yield decreased; moreover, there was a negative relationship between the growth and the lag phase across the treatments. The latter suggests that a trade-off between growth rate and the ability to adapt to the high 15N environment may exist. Remarkably, the lag-phase response at 3.5 at% 15N was the shortest and deviated from the overall trend, thus providing partial support to the recently proposed Isotopic Resonance hypothesis, which predicts that certain isotopic composition is particularly favorable for living organisms. These findings confirm the occurrence of KIE in isotopically enriched algae and underline the importance of considering these effects when using stable isotope labeling in field and experimental studies.

  20. ICT: isotope correction toolbox.

    PubMed

    Jungreuthmayer, Christian; Neubauer, Stefan; Mairinger, Teresa; Zanghellini, Jürgen; Hann, Stephan

    2016-01-01

    Isotope tracer experiments are an invaluable technique to analyze and study the metabolism of biological systems. However, isotope labeling experiments are often affected by naturally abundant isotopes especially in cases where mass spectrometric methods make use of derivatization. The correction of these additive interferences--in particular for complex isotopic systems--is numerically challenging and still an emerging field of research. When positional information is generated via collision-induced dissociation, even more complex calculations for isotopic interference correction are necessary. So far, no freely available tools can handle tandem mass spectrometry data. We present isotope correction toolbox, a program that corrects tandem mass isotopomer data from tandem mass spectrometry experiments. Isotope correction toolbox is written in the multi-platform programming language Perl and, therefore, can be used on all commonly available computer platforms. Source code and documentation can be freely obtained under the Artistic License or the GNU General Public License from: https://github.com/jungreuc/isotope_correction_toolbox/ {christian.jungreuthmayer@boku.ac.at,juergen.zanghellini@boku.ac.at} Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  1. Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology.

    PubMed

    Armenta, Jenny M; Hoeschele, Ina; Lazar, Iulia M

    2009-07-01

    An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated.

  2. Pulsed 86Sr-labeling and NanoSIMS imaging to study coral biomineralization at ultra-structural length scales

    NASA Astrophysics Data System (ADS)

    Brahmi, C.; Domart-Coulon, I.; Rougée, L.; Pyle, D. G.; Stolarski, J.; Mahoney, J. J.; Richmond, R. H.; Ostrander, G. K.; Meibom, A.

    2012-09-01

    A method to label marine biocarbonates is developed based on a concentration enrichment of a minor stable isotope of a trace element that is a natural component of seawater, resulting in the formation of biocarbonate with corresponding isotopic enrichments. This biocarbonate is subsequently imaged with a NanoSIMS ion microprobe to visualize the locations of the isotopic marker on sub-micrometric length scales, permitting resolution of all ultra-structural details. In this study, a scleractinian coral, Pocillopora damicornis, was labeled 3 times with 86Sr-enhanced seawater for a period of 48 h with 5 days under normal seawater conditions separating each labeling event. Two non-specific cellular stress biomarkers, glutathione-S-transferase activity and porphyrin concentration plus carbonic anhydrase, an enzymatic marker involved in the physiology of carbonate biomineralization, as well as unchanged levels of zooxanthellae photosynthesis efficiency indicate that coral physiological processes are not affected by the 86Sr-enhancement. NanoSIMS images of the 86Sr/44Ca ratio in skeleton formed during the experiment allow for a determination of the average extension rate of the two major ultra-structural components of the coral skeleton: Rapid Accretion Deposits are found to form on average about 4.5 times faster than Thickening Deposits. The method opens up new horizons in the study of biocarbonate formation because it holds the potential to observe growth of calcareous structures such as skeletons, shells, tests, spines formed by a wide range of organisms under essentially unperturbed physiological conditions.

  3. Heavy atom labeled nucleotides for measurement of kinetic isotope effects.

    PubMed

    Weissman, Benjamin P; Li, Nan-Sheng; York, Darrin; Harris, Michael; Piccirilli, Joseph A

    2015-11-01

    Experimental analysis of kinetic isotope effects represents an extremely powerful approach for gaining information about the transition state structure of complex reactions not available through other methodologies. The implementation of this approach to the study of nucleic acid chemistry requires the synthesis of nucleobases and nucleotides enriched for heavy isotopes at specific positions. In this review, we highlight current approaches to the synthesis of nucleic acids enriched site specifically for heavy oxygen and nitrogen and their application in heavy atom isotope effect studies. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. DNA stable-isotope probing (DNA-SIP).

    PubMed

    Dunford, Eric A; Neufeld, Josh D

    2010-08-02

    DNA stable-isotope probing (DNA-SIP) is a powerful technique for identifying active microorganisms that assimilate particular carbon substrates and nutrients into cellular biomass. As such, this cultivation-independent technique has been an important methodology for assigning metabolic function to the diverse communities inhabiting a wide range of terrestrial and aquatic environments. Following the incubation of an environmental sample with stable-isotope labelled compounds, extracted nucleic acid is subjected to density gradient ultracentrifugation and subsequent gradient fractionation to separate nucleic acids of differing densities. Purification of DNA from cesium chloride retrieves labelled and unlabelled DNA for subsequent molecular characterization (e.g. fingerprinting, microarrays, clone libraries, metagenomics). This JoVE video protocol provides visual step-by-step explanations of the protocol for density gradient ultracentrifugation, gradient fractionation and recovery of labelled DNA. The protocol also includes sample SIP data and highlights important tips and cautions that must be considered to ensure a successful DNA-SIP analysis.

  5. Improved quantification of microbial CH4 oxidation efficiency in arctic wetland soils using carbon isotope fractionation

    NASA Astrophysics Data System (ADS)

    Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.

    2013-04-01

    Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the arctic will cause deeper permafrost thawing, followed by increased carbon mineralization and CH4 formation in water-saturated tundra soils, thus creating a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and δ13CH4 signatures were measured and the fractionation factors for the processes of oxidation (αox) and diffusion (αdiff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (such as landfill cover soils) have assumed a gas transport dominated by advection (αtrans = 1). In tundra soils, however, diffusion is the main gas transport mechanism and diffusive stable isotope fractionation should be considered alongside oxidative fractionation. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an αdiff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was αdiff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that αox differs widely between sites and horizons (mean αox = 1.017 ± 0.009) and needs to be determined on a case by case basis. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the

  6. Improved quantification of microbial CH4 oxidation efficiency in Arctic wetland soils using carbon isotope fractionation

    NASA Astrophysics Data System (ADS)

    Preuss, I.; Knoblauch, C.; Gebert, J.; Pfeiffer, E.-M.

    2012-12-01

    Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the Arctic will cause a deeper permafrost thawing followed by increased carbon mineralization and CH4 formation in water saturated tundra soils which might cause a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River Delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and δ13CH4-signatures were measured and the fractionation factors for the processes of oxidation (αox) and diffusion (αdiff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (e.g. landfill cover soils) have assumed a gas transport dominated by advection (αtrans = 1). In tundra soils, however, diffusion is the main gas transport mechanism, aside from ebullition. Hence, diffusive stable isotope fractionation has to be considered. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an αdiff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was αdiff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that αox differs widely between sites and horizons (mean αox, = 1.017 ± 0.009) and needs to be determined individually. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion

  7. Identification and characterization of isomeric N-glycans of human alfa-acid-glycoprotein by stable isotope labelling and ZIC-HILIC-MS in combination with exoglycosidase digestion.

    PubMed

    Mancera-Arteu, Montserrat; Giménez, Estela; Barbosa, José; Sanz-Nebot, Victòria

    2016-10-12

    In this study, a ZIC-HILIC-MS methodology for the analysis of N-glycan isomers was optimized to obtain greater detection sensitivity and thus identify more glycan structures in hAGP. In a second step, this method was combined with glycan reductive isotope labelling (GRIL) through [(12)C6]/[(13)C6]-aniline and exoglycosidase digestion to characterize the different glycan isomers. The GRIL method allows the peak areas resulting from two different labelled samples to be compared, since neither retention time shifts nor variations in the ionization of glycans between these samples are obtained. First, sialic acid linkage assignations were performed for most hAGP glycan isomers with α2-3 sialidase digestion. Bi-, tri- and tetraantennary glycan isomers with different terminal sialic acid linkages to galactose (α2-3 or α2-6) were assigned, and the potential of this technique for the structural characterization of isobaric isomers was therefore demonstrated. Furthermore, fucose linkage isomers of hAGP glycans were also characterized using this isotope-labelling approach in combination with α1-3,4 fucosidase and β1-4 galactosidase digestion. α1-3 antennary fucoses and α1-6 core fucosylation were detected in hAGP fucosylated glycans. These established methodologies can be extremely useful for patho-glycomic studies to characterize glycoproteins of biomedical interest and find novel glycan isomers that could be used as biomarkers in cancer research. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes.

    PubMed

    Dersch, Lisa Maria; Beckers, Veronique; Rasch, Detlev; Melzer, Guido; Bolten, Christoph; Kiep, Katina; Becker, Horst; Bläsing, Oliver Ernst; Fuchs, Regine; Ehrhardt, Thomas; Wittmann, Christoph

    2016-05-01

    Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of (13)CO2 and (15)NH4NO3 The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, (13)CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous (13)CO2 and (15)NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong (13)C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping. © 2016 American Society of Plant Biologists. All Rights Reserved.

  9. International Review of the Development and Implementation of Energy Efficiency Standards and Labeling Programs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, Nan; Zheng, Nina; Fridley, David

    2012-02-28

    Appliance energy efficiency standards and labeling (S&L) programs have been important policy tools for regulating the efficiency of energy-using products for over 40 years and continue to expand in terms of geographic and product coverage. The most common S&L programs include mandatory minimum energy performance standards (MEPS) that seek to push the market for efficient products, and energy information and endorsement labels that seek to pull the market. This study seeks to review and compare some of the earliest and most well-developed S&L programs in three countries and one region: the U.S. MEPS and ENERGY STAR, Australia MEPS and Energymore » Label, European Union MEPS and Ecodesign requirements and Energy Label and Japanese Top Runner programs. For each program, key elements of S&L programs are evaluated and comparative analyses across the programs undertaken to identify best practice examples of individual elements as well as cross-cutting factors for success and lessons learned in international S&L program development and implementation. The international review and comparative analysis identified several overarching themes and highlighted some common factors behind successful program elements. First, standard-setting and programmatic implementation can benefit significantly from a legal framework that stipulates a specific timeline or schedule for standard-setting and revision, product coverage and legal sanctions for non-compliance. Second, the different MEPS programs revealed similarities in targeting efficiency gains that are technically feasible and economically justified as the principle for choosing a standard level, in many cases at a level that no product on the current market could reach. Third, detailed survey data such as the U.S. Residential Energy Consumption Survey (RECS) and rigorous analyses provide a strong foundation for standard-setting while incorporating the participation of different groups of stakeholders further strengthen the

  10. An LC-MRM method for measuring intestinal triglyceride assembly using an oral stable isotope-labeled fat challenge.

    PubMed

    Li, Xiaofang; Parks, Elizabeth J; McLaren, David G; Lambert, Jennifer E; Cardasis, Helene L; Chappell, Derek L; McAvoy, Thomas; Salituro, Gino; Alon, Achilles; Dennie, Justin; Chakravarthy, Manu; Shankar, Sudha S; Laterza, Omar F; Lassman, Michael E

    2016-06-01

    A traditional oral fatty acid challenge assesses absorption of triacylglycerol (TG) into the periphery through the intestines, but cannot distinguish the composition or source of fatty acid in the TG. Stable isotope-labeled tracers combined with LC-MRM can be used to identify and distinguish TG synthesized with dietary and stored fatty acids. Concentrations of three abundant TGs (52:2, 54:3 and 54:4) were monitored for incorporation of one or two (2)H11-oleate molecules per TG. This method was subjected to routine assay validation and meets typical requirements for an assay to be used to support clinical studies. Calculations for the fractional appearance rate of TG in plasma are presented along with the intracellular enterocyte precursor pool for 12 study participants.

  11. Natural isotope correction of MS/MS measurements for metabolomics and (13)C fluxomics.

    PubMed

    Niedenführ, Sebastian; ten Pierick, Angela; van Dam, Patricia T N; Suarez-Mendez, Camilo A; Nöh, Katharina; Wahl, S Aljoscha

    2016-05-01

    Fluxomics and metabolomics are crucial tools for metabolic engineering and biomedical analysis to determine the in vivo cellular state. Especially, the application of (13)C isotopes allows comprehensive insights into the functional operation of cellular metabolism. Compared to single MS, tandem mass spectrometry (MS/MS) provides more detailed and accurate measurements of the metabolite enrichment patterns (tandem mass isotopomers), increasing the accuracy of metabolite concentration measurements and metabolic flux estimation. MS-type data from isotope labeling experiments is biased by naturally occurring stable isotopes (C, H, N, O, etc.). In particular, GC-MS(/MS) requires derivatization for the usually non-volatile intracellular metabolites introducing additional natural isotopes leading to measurements that do not directly represent the carbon labeling distribution. To make full use of LC- and GC-MS/MS mass isotopomer measurements, the influence of natural isotopes has to be eliminated (corrected). Our correction approach is analyzed for the two most common applications; (13)C fluxomics and isotope dilution mass spectrometry (IDMS) based metabolomics. Natural isotopes can have an impact on the calculated flux distribution which strongly depends on the substrate labeling and the actual flux distribution. Second, we show that in IDMS based metabolomics natural isotopes lead to underestimated concentrations that can and should be corrected with a nonlinear calibration. Our simulations indicate that the correction for natural abundance in isotope based fluxomics and quantitative metabolomics is essential for correct data interpretation. © 2015 Wiley Periodicals, Inc.

  12. Quantitative twoplex glycan analysis using 12C6 and 13C6 stable isotope 2-aminobenzoic acid labelling and capillary electrophoresis mass spectrometry.

    PubMed

    Váradi, Csaba; Mittermayr, Stefan; Millán-Martín, Silvia; Bones, Jonathan

    2016-12-01

    Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13 C 6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12 C 6 'light' and 13 C 6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.

  13. Iridium-Catalysed ortho-Directed Deuterium Labelling of Aromatic Esters--An Experimental and Theoretical Study on Directing Group Chemoselectivity.

    PubMed

    Devlin, Jennifer; Kerr, William J; Lindsay, David M; McCabe, Timothy J D; Reid, Marc; Tuttle, Tell

    2015-06-25

    Herein we report a combined experimental and theoretical study on the deuterium labelling of benzoate ester derivatives, utilizing our developed iridium N-heterocyclic carbene/phosphine catalysts. A range of benzoate esters were screened, including derivatives with electron-donating and -withdrawing groups in the para- position. The substrate scope, in terms of the alkoxy group, was studied and the nature of the catalyst counter-ion was shown to have a profound effect on the efficiency of isotope exchange. Finally, the observed chemoselectivity was rationalized by rate studies and theoretical calculations, and this insight was applied to the selective labelling of benzoate esters bearing a second directing group.

  14. Energy Efficiency standards and labels provide a solid foundationfor economic growth, climate change mitigation, and regional trade

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wiel, Stephen; Egan, Christine; della Cava, Mirka

    2006-09-01

    Governments around the world have increasingly beenimplementing energy efficiency standards and labeling programs for thepast 30 years. There has been an especially rapid growth in the numberand extent of these programs over the past 15 years. By the end of 2005,62 countries had adopted 1818 separate standards or labels covering 82products. The impact has been dramatic. This paper describes: (1) thebenefits that can be obtained through this policy, (2) which countriesare implementing standards and labels and for which products, (3) theimpacts such programs have been having in some countries, and (4) recentprogress through regional cooperation and alignment.

  15. Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system.

    PubMed

    Ikeya, Teppei; Takeda, Mitsuhiro; Yoshida, Hitoshi; Terauchi, Tsutomu; Jee, Jun-Goo; Kainosho, Masatsune; Güntert, Peter

    2009-08-01

    Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13C/15N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional "through-bond" spectrum (and 2D HSQC spectra) in addition to the 13C-edited and 15N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods.

  16. Monitoring CO[subscript 2] Fixation Using GC-MS Detection of a [superscript 13]C-Label

    ERIC Educational Resources Information Center

    Hammond, Daniel G.; Bridgham, April; Reichert, Kara; Magers, Martin

    2010-01-01

    Much of our understanding of metabolic pathways has resulted from the use of chemical and isotopic labels. In this experiment, a heavy isotope of carbon, [superscript 13]C, is used to label the product of the well-known RuBisCO enzymatic reaction. This is a key reaction in photosynthesis that converts inorganic carbon to organic carbon; a process…

  17. Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (INLIGHT): A Novel Glycan Relative Quantification Strategy

    PubMed Central

    Walker, S. Hunter; Taylor, Amber D.; Muddiman, David C.

    2013-01-01

    The INLIGHT strategy for the sample preparation, data analysis, and relative quantification of N-linked glycans is presented. Glycans are derivatized with either natural (L) or stable-isotope labeled (H) hydrazide reagents and analyzed using reversed phase liquid chromatography coupled online to a Q Exactive mass spectrometer. A simple glycan ladder, maltodextrin, is first used to demonstrate the relative quantification strategy in samples with negligible analytical and biological variability. It is shown that after a molecular weight correction due to isotopic overlap and a post-acquisition normalization of the data to account for both the systematic variability, a plot of the experimental H:L ratio vs. the calculated H:L ratio exhibits a correlation of unity for maltodextrin samples mixed in different ratios. We also demonstrate that the INLIGHT approach can quantify species over four orders of magnitude in ion abundance. The INLIGHT strategy is further demonstrated in pooled human plasma, where it is shown that the post-acquisition normalization is more effective than using a single spiked-in internal standard. Finally, changes in glycosylation are able to be detected in complex biological matrices, when spiked with a glycoprotein. The ability to spike in a glycoprotein and detect change at the glycan level validates both the sample preparation and data analysis strategy, making INLIGHT an invaluable relative quantification strategy for the field of glycomics. PMID:23860851

  18. Mixed-Isotope Labeling with LC-IMS-MS for Characterization of Protein–Protein Interactions by Chemical Cross-Linking

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Merkley, Eric D.; Baker, Erin S.; Crowell, Kevin L.

    2013-02-20

    Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides can provide insights into protein structure and protein-protein interactions. However, cross-linked peptides are by necessity of low stoichometry and have different physicochemical properties than linear peptides, routine unambiguous identification of the cross-linked peptides has remained difficult. To address this challenge, we demonstrated the use of liquid chromatography and ion mobility separations coupled with mass spectrometry in combination with a heavy-isotope labeling method. The combination of mixed-isotope cross-linking and ion mobility provided unique and easily interpretable spectral multiplet features for the intermolecular cross-linked peptides. Applicationmore » of the method to two different homodimeric proteins - SrfN, a virulence factor from Salmonella Typhimurium and SO_2176, a protein of unknown function from Shewanella oneidensis- revealed several cross-linked peptides from both proteins that were identified with a low false discovery rate (estimated using a decoy approach). A greater number of cross-linked peptides were identified using ion mobility drift time information in the analysis than when the data were summed across the drift time dimension before analysis. The identified cross-linked peptides migrated more quickly in the ion mobility drift tube than the unmodified peptides.« less

  19. Multiple tag labeling method for DNA sequencing

    DOEpatents

    Mathies, Richard A.; Huang, Xiaohua C.; Quesada, Mark A.

    1995-01-01

    A DNA sequencing method described which uses single lane or channel electrophoresis. Sequencing fragments are separated in said lane and detected using a laser-excited, confocal fluorescence scanner. Each set of DNA sequencing fragments is separated in the same lane and then distinguished using a binary coding scheme employing only two different fluorescent labels. Also described is a method of using radio-isotope labels.

  20. Compensation of matrix effects in gas chromatography-mass spectrometry analysis of pesticides using a combination of matrix matching and multiple isotopically labeled internal standards.

    PubMed

    Tsuchiyama, Tomoyuki; Katsuhara, Miki; Nakajima, Masahiro

    2017-11-17

    In the multi-residue analysis of pesticides using GC-MS, the quantitative results are adversely affected by a phenomenon known as the matrix effect. Although the use of matrix-matched standards is considered to be one of the most practical solutions to this problem, complete removal of the matrix effect is difficult in complex food matrices owing to their inconsistency. As a result, residual matrix effects can introduce analytical errors. To compensate for residual matrix effects, we have developed a novel method that employs multiple isotopically labeled internal standards (ILIS). The matrix effects of ILIS and pesticides were evaluated in spiked matrix extracts of various agricultural commodities, and the obtained data were subjected to simple statistical analysis. Based on the similarities between the patterns of variation in the analytical response, a total of 32 isotopically labeled compounds were assigned to 338 pesticides as internal standards. It was found that by utilizing multiple ILIS, residual matrix effects could be effectively compensated. The developed method exhibited superior quantitative performance compared with the common single-internal-standard method. The proposed method is more feasible for regulatory purposes than that using only predetermined correction factors and is considered to be promising for practical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Quantification of dynamic soil - vegetation feedbacks following an isotopically labelled precipitation pulse

    NASA Astrophysics Data System (ADS)

    Piayda, Arndt; Dubbert, Maren; Siegwolf, Rolf; Cuntz, Matthias; Werner, Christiane

    2017-04-01

    The presence of vegetation alters hydrological cycles of ecosystems. Complex plant-soil interactions govern the fate of precipitation input and water transitions through ecosystem compartments. Disentangling these interactions is a major challenge in the field of ecohydrology and pivotal foundation for understanding the carbon cycle of semi-arid ecosystems. Stable water isotopes can be used in this context as tracer to quantify water movement through soil-vegetation-atmosphere interfaces. The aim of this study is to disentangle vegetation effects on soil water infiltration and distribution as well as dynamics of soil evaporation and grassland water-use in a Mediterranean cork-oak woodland during dry conditions. An irrigation experiment using δ18O-labeled water was carried out in order to quantify distinct effects of tree and herbaceous vegetation on infiltration and distribution of event water in the soil profile. Dynamic responses of soil and herbaceous vegetation fluxes to precipitation regarding event water-use, water uptake depth plasticity and contribution to ecosystem evapotranspiration were quantified. Total water loss to the atmosphere from bare soil was as high as from vegetated soil, utilizing large amounts of unproductive water loss for biomass production, carbon sequestration and nitrogen fixation. During the experiment no adjustments of main root water uptake depth to changes of water availability could be observed, rendering light to medium precipitation events under dry conditions useless. This forces understory plants to compete with adjacent trees for soil water in deeper soil layers. Thus understory plants are faster subject to chronic drought, leading to premature senescence at the onset of drought. Despite this water competition, the presence of Cork oak trees fosters infiltration to large degrees. That reduces drought stress, caused by evapotranspiration, due to favourable micro climatic conditions under tree crown shading. This study

  2. Quantification of dynamic soil-vegetation feedbacks following an isotopically labelled precipitation pulse

    NASA Astrophysics Data System (ADS)

    Piayda, Arndt; Dubbert, Maren; Siegwolf, Rolf; Cuntz, Matthias; Werner, Christiane

    2017-05-01

    The presence of vegetation alters hydrological cycles of ecosystems. Complex plant-soil interactions govern the fate of precipitation input and water transitions through ecosystem compartments. Disentangling these interactions is a major challenge in the field of ecohydrology and a pivotal foundation for understanding the carbon cycle of semi-arid ecosystems. Stable water isotopes can be used in this context as tracer to quantify water movement through soil-vegetation-atmosphere interfaces. The aim of this study is to disentangle vegetation effects on soil water infiltration and distribution as well as dynamics of soil evaporation and grassland water use in a Mediterranean cork oak woodland during dry conditions. An irrigation experiment using δ18O labelled water was carried out in order to quantify distinct effects of tree and herbaceous vegetation on the infiltration and distribution of event water in the soil profile. Dynamic responses of soil and herbaceous vegetation fluxes to precipitation regarding event water use, water uptake depth plasticity, and contribution to ecosystem soil evaporation and transpiration were quantified. Total water loss to the atmosphere from bare soil was as high as from vegetated soil, utilizing large amounts of unproductive evaporation for transpiration, but infiltration rates decreased. No adjustments of main root water uptake depth to changes in water availability could be observed during the experiment. This forces understorey plants to compete with adjacent trees for water in deeper soil layers at the onset of summer. Thus, understorey plants are subjected to chronic water deficits faster, leading to premature senescence at the onset of drought. Despite this water competition, the presence of cork oak trees fosters infiltration and reduces evapotranspirative water losses from the understorey and the soil, both due to altered microclimatic conditions under crown shading. This study highlights complex soil-plant-atmosphere and

  3. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

    NASA Astrophysics Data System (ADS)

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

    2016-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded "heavy" and "light" GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the "heavy" and "light" peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.

  4. 15N-metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

    PubMed

    Lanquar, Viviane; Kuhn, Lauriane; Lelièvre, Françoise; Khafif, Mehdi; Espagne, Christelle; Bruley, Christophe; Barbier-Brygoo, Hélène; Garin, Jérôme; Thomine, Sébastien

    2007-03-01

    An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

  5. influx_s: increasing numerical stability and precision for metabolic flux analysis in isotope labelling experiments.

    PubMed

    Sokol, Serguei; Millard, Pierre; Portais, Jean-Charles

    2012-03-01

    The problem of stationary metabolic flux analysis based on isotope labelling experiments first appeared in the early 1950s and was basically solved in early 2000s. Several algorithms and software packages are available for this problem. However, the generic stochastic algorithms (simulated annealing or evolution algorithms) currently used in these software require a lot of time to achieve acceptable precision. For deterministic algorithms, a common drawback is the lack of convergence stability for ill-conditioned systems or when started from a random point. In this article, we present a new deterministic algorithm with significantly increased numerical stability and accuracy of flux estimation compared with commonly used algorithms. It requires relatively short CPU time (from several seconds to several minutes with a standard PC architecture) to estimate fluxes in the central carbon metabolism network of Escherichia coli. The software package influx_s implementing this algorithm is distributed under an OpenSource licence at http://metasys.insa-toulouse.fr/software/influx/. Supplementary data are available at Bioinformatics online.

  6. [Microbial synthesis of deuterium labelled L-phenylalanine with different levels of isotopic enrichment by facultative methylotrophic bacterium Brevibacterium methylicum with RMP assimilation of carbon].

    PubMed

    Mosin, O V; Shvets, V I; Skladnev, D A; Ignatov, I

    2014-01-01

    The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions.

  7. Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes.

    PubMed

    Lo, Andy; Weiner, Joel H; Li, Liang

    2013-09-17

    Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Deciphering Systemic Wound Responses of the Pumpkin Extrafascicular Phloem by Metabolomics and Stable Isotope-Coded Protein Labeling1[C][W

    PubMed Central

    Gaupels, Frank; Sarioglu, Hakan; Beckmann, Manfred; Hause, Bettina; Spannagl, Manuel; Draper, John; Lindermayr, Christian; Durner, Jörg

    2012-01-01

    In cucurbits, phloem latex exudes from cut sieve tubes of the extrafascicular phloem (EFP), serving in defense against herbivores. We analyzed inducible defense mechanisms in the EFP of pumpkin (Cucurbita maxima) after leaf damage. As an early systemic response, wounding elicited transient accumulation of jasmonates and a decrease in exudation probably due to partial sieve tube occlusion by callose. The energy status of the EFP was enhanced as indicated by increased levels of ATP, phosphate, and intermediates of the citric acid cycle. Gas chromatography coupled to mass spectrometry also revealed that sucrose transport, gluconeogenesis/glycolysis, and amino acid metabolism were up-regulated after wounding. Combining ProteoMiner technology for the enrichment of low-abundance proteins with stable isotope-coded protein labeling, we identified 51 wound-regulated phloem proteins. Two Sucrose-Nonfermenting1-related protein kinases and a 32-kD 14-3-3 protein are candidate central regulators of stress metabolism in the EFP. Other proteins, such as the Silverleaf Whitefly-Induced Protein1, Mitogen Activated Protein Kinase6, and Heat Shock Protein81, have known defensive functions. Isotope-coded protein labeling and western-blot analyses indicated that Cyclophilin18 is a reliable marker for stress responses of the EFP. As a hint toward the induction of redox signaling, we have observed delayed oxidation-triggered polymerization of the major Phloem Protein1 (PP1) and PP2, which correlated with a decline in carbonylation of PP2. In sum, wounding triggered transient sieve tube occlusion, enhanced energy metabolism, and accumulation of defense-related proteins in the pumpkin EFP. The systemic wound response was mediated by jasmonate and redox signaling. PMID:23085839

  9. Isotope-encoded Carboxyl Group Footprinting for Mass Spectrometry-based Protein Conformational Studies

    PubMed Central

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; Gross, Michael L.

    2015-01-01

    We report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the Orange Carotenoid Protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy” and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting. PMID:26384685

  10. Correlated optical and isotopic nanoscopy

    NASA Astrophysics Data System (ADS)

    Saka, Sinem K.; Vogts, Angela; Kröhnert, Katharina; Hillion, François; Rizzoli, Silvio O.; Wessels, Johannes T.

    2014-04-01

    The isotopic composition of different materials can be imaged by secondary ion mass spectrometry. In biology, this method is mainly used to study cellular metabolism and turnover, by pulsing the cells with marker molecules such as amino acids labelled with stable isotopes (15N, 13C). The incorporation of the markers is then imaged with a lateral resolution that can surpass 100 nm. However, secondary ion mass spectrometry cannot identify specific subcellular structures like organelles, and needs to be correlated with a second technique, such as fluorescence imaging. Here, we present a method based on stimulated emission depletion microscopy that provides correlated optical and isotopic nanoscopy (COIN) images. We use this approach to study the protein turnover in different organelles from cultured hippocampal neurons. Correlated optical and isotopic nanoscopy can be applied to a variety of biological samples, and should therefore enable the investigation of the isotopic composition of many organelles and subcellular structures.

  11. Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling

    PubMed Central

    Nishiyama, Yusuke; Endo, Yuki; Nemoto, Takahiro; Yamauchi, Kazuo; Asakura, Tetsuo; Takeda, Mitsuhiro; Terauchi, Tsutomu; Kainosho, Masatsune; Ishii, Yoshitaka

    2015-01-01

    We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems. PMID:25856081

  12. Fate of 3H-thymidine labelled myogenic cells in regeneration of muscle isografts.

    PubMed

    Gutmann, E; Mares, V; Stichová, J

    1976-03-05

    Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later. In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The presen experiments provide a direct proof of utilization of donor satelite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process.

  13. Bacterial Production of Site Specific 13C Labeled Phenylalanine and Methodology for High Level Incorporation into Bacterially Expressed Recombinant Proteins

    PubMed Central

    Ramaraju, Bhargavi; McFeeters, Hana; Vogler, Bernhard; McFeeters, Robert L.

    2016-01-01

    Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific 13C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct 13C chemical shifts and multiple magnetically equivalent 1H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with 13C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated 1H-13C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study. PMID:28028744

  14. IRMS detection of testosterone manipulated with 13C labeled standards in human urine by removing the labeled 13C.

    PubMed

    Wang, Jingzhu; Yang, Rui; Yang, Wenning; Liu, Xin; Xing, Yanyi; Xu, Youxuan

    2014-12-10

    Isotope ratio mass spectrometry (IRMS) is applied to confirm testosterone (T) abuse by determining the carbon isotope ratios (δ(13)C value). However, (13)C labeled standards can be used to control the δ(13)C value and produce manipulated T which cannot be detected by the current method. A method was explored to remove the (13)C labeled atom at C-3 from the molecule of androsterone (Andro), the metabolite of T in urine, to produce the resultant (A-nor-5α-androstane-2,17-dione, ANAD). The difference in δ(13)C values between Andro and ANAD (Δδ(13)CAndro-ANAD, ‰) would change significantly in case manipulated T is abused. Twenty-one volunteers administered T manipulated with different (13)C labeled standards. The collected urine samples were analyzed with the established method, and the maximum value of Δδ(13)CAndro-ANAD post ingestion ranged from 3.0‰ to 8.8‰. Based on the population reference, the cut-off value of Δδ(13)CAndro-ANAD for positive result was suggested as 1.2‰. The developed method could be used to detect T manipulated with 3-(13)C labeled standards. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Standard addition with internal standardisation as an alternative to using stable isotope labelled internal standards to correct for matrix effects-Comparison and validation using liquid chromatography-​tandem mass spectrometric assay of vitamin D.

    PubMed

    Hewavitharana, Amitha K; Abu Kassim, Nur Sofiah; Shaw, Paul Nicholas

    2018-06-08

    With mass spectrometric detection in liquid chromatography, co-eluting impurities affect the analyte response due to ion suppression/enhancement. Internal standard calibration method, using co-eluting stable isotope labelled analogue of each analyte as the internal standard, is the most appropriate technique available to correct for these matrix effects. However, this technique is not without drawbacks, proved to be expensive because separate internal standard for each analyte is required, and the labelled compounds are expensive or require synthesising. Traditionally, standard addition method has been used to overcome the matrix effects in atomic spectroscopy and was a well-established method. This paper proposes the same for mass spectrometric detection, and demonstrates that the results are comparable to those with the internal standard method using labelled analogues, for vitamin D assay. As conventional standard addition procedure does not address procedural errors, we propose the inclusion of an additional internal standard (not co-eluting). Recoveries determined on human serum samples show that the proposed method of standard addition yields more accurate results than the internal standardisation using stable isotope labelled analogues. The precision of the proposed method of standard addition is superior to the conventional standard addition method. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Simultaneous determination of chlorantraniliprole and cyantraniliprole in fruits, vegetables and cereals using ultra-high-performance liquid chromatography-tandem mass spectrometry with the isotope-labelled internal standard method.

    PubMed

    Pan, Xinglu; Dong, Fengshou; Xu, Jun; Liu, Xingang; Chen, Zenglong; Liu, Na; Chen, Xixi; Tao, Yan; Zhang, Hongjun; Zheng, Yongquan

    2015-05-01

    A reliable and sensitive isotope-labelled internal standard method for simultaneous determination of chlorantraniliprole and cyantraniliprole in fruits (apple and grape), vegetables (cucumber and tomato) and cereals (rice and wheat) using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed. Isotope-labelled internal standards were effective in compensating for the loss in the pretreatment and overcoming the matrix effect. The analytes were extracted with acetonitrile and cleaned up with different kinds of sorbents. The determination of the target compounds was achieved in less than 4 min using a T3 column combined with an electrospray ionization source in positive mode. The overall average relative recoveries in all matrices at three spiking levels (10, 20 and 50 μg kg(-1)) ranged from 95.5 to 106.2 %, with all relative standard deviations being less than 14.4 % for all analytes. The limits of detection did not exceed 0.085 μg kg(-1) and the limits of quantification were below 0.28 μg kg(-1) in all matrices. The method was demonstrated to be convenient and accurate for the routine monitoring of chlorantraniliprole and cyantraniliprole in fruits, vegetables and cereals.

  17. Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

    PubMed

    Hattori, Yoshikazu; Heidenreich, David; Ono, Yuki; Sugiki, Toshihiko; Yokoyama, Kei-Ichi; Suzuki, Ei-Ichiro; Fujiwara, Toshimichi; Kojima, Chojiro

    2017-08-01

    The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15 N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19 F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19 F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19 F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19 F-labeling method was 3.5-fold more sensitive than 15 N-labeling, and could be combined with other chemical modification techniques such as lysine 13 C-methylation. 13 C-dimethylated- 19 F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

  18. Calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry assays and its application in supporting microdose absolute bioavailability studies.

    PubMed

    Gu, Huidong; Wang, Jian; Aubry, Anne-Françoise; Jiang, Hao; Zeng, Jianing; Easter, John; Wang, Jun-sheng; Dockens, Randy; Bifano, Marc; Burrell, Richard; Arnold, Mark E

    2012-06-05

    A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

  19. Isotope-Encoded Carboxyl Group Footprinting for Mass Spectrometry-Based Protein Conformational Studies

    DOE PAGES

    Zhang, Hao; Liu, Haijun; Blankenship, Robert E.; ...

    2015-09-18

    Here, we report an isotope-encoding method coupled with carboxyl-group footprinting to monitor protein conformational changes. The carboxyl groups of aspartic/glutamic acids and of the C-terminus of proteins can serve as reporters for protein conformational changes when labeled with glycine ethyl ester (GEE) mediated by carbodiimide. In the new development, isotope-encoded “heavy” and “light” GEE are used to label separately the two states of the orange carotenoid protein (OCP) from cyanobacteria. Two samples are mixed (1:1 ratio) and analyzed by a single LC-MS/MS experiment. The differences in labeling extent between the two states are represented by the ratio of the “heavy”more » and “light” peptides, providing information about protein conformational changes. Combining isotope-encoded MS quantitative analysis and carboxyl-group footprinting reduces the time of MS analysis and improves the sensitivity of GEE and other footprinting.« less

  20. Use of stable isotope-labelled cells to identify active grazers of picocyanobacteria in ocean surface waters.

    PubMed

    Frias-Lopez, Jorge; Thompson, Anne; Waldbauer, Jacob; Chisholm, Sallie W

    2009-02-01

    Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web.

  1. OpenMebius: an open source software for isotopically nonstationary 13C-based metabolic flux analysis.

    PubMed

    Kajihata, Shuichi; Furusawa, Chikara; Matsuda, Fumio; Shimizu, Hiroshi

    2014-01-01

    The in vivo measurement of metabolic flux by (13)C-based metabolic flux analysis ((13)C-MFA) provides valuable information regarding cell physiology. Bioinformatics tools have been developed to estimate metabolic flux distributions from the results of tracer isotopic labeling experiments using a (13)C-labeled carbon source. Metabolic flux is determined by nonlinear fitting of a metabolic model to the isotopic labeling enrichment of intracellular metabolites measured by mass spectrometry. Whereas (13)C-MFA is conventionally performed under isotopically constant conditions, isotopically nonstationary (13)C metabolic flux analysis (INST-(13)C-MFA) has recently been developed for flux analysis of cells with photosynthetic activity and cells at a quasi-steady metabolic state (e.g., primary cells or microorganisms under stationary phase). Here, the development of a novel open source software for INST-(13)C-MFA on the Windows platform is reported. OpenMebius (Open source software for Metabolic flux analysis) provides the function of autogenerating metabolic models for simulating isotopic labeling enrichment from a user-defined configuration worksheet. Analysis using simulated data demonstrated the applicability of OpenMebius for INST-(13)C-MFA. Confidence intervals determined by INST-(13)C-MFA were less than those determined by conventional methods, indicating the potential of INST-(13)C-MFA for precise metabolic flux analysis. OpenMebius is the open source software for the general application of INST-(13)C-MFA.

  2. Stable-isotope-based labeling of styrene-degrading microorganisms in biofilters.

    PubMed

    Alexandrino, M; Knief, C; Lipski, A

    2001-10-01

    Deuterated styrene ([(2)H(8)]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [(2)H(8)]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [(2)H(8)]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.

  3. Overcoming interference with the detection of a stable isotopically labeled microtracer in the evaluation of beclabuvir absolute bioavailability using a concomitant microtracer approach.

    PubMed

    Jiang, Hao; Titsch, Craig; Zeng, Jianing; Jones, Barry; Joyce, Philip; Gandhi, Yash; Turley, Wesley; Burrell, Richard; Aubry, Anne F; Arnold, Mark E

    2017-09-05

    The oral absolute bioavailability of beclabuvir in healthy subjects was determined using a microdose (100μg) of the stable isotopically labeled tracer via intravenous (IV) infusion started after oral dosing of beclabuvir (150mg). To simultaneously analyze the concentrations of the IV microtracer ([ 13 C 6 ]beclabuvir) and beclabuvir in plasma samples, a liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) method was initially developed. Surprisingly beclabuvir significantly interfered with the IV microtracer detection when using the selected reaction monitoring (SRM) in the assay. An interfering component from the drug substance was observed using a high resolution mass spectrometer (HRMS). The mass-to-charge (m/z) of the interfering component was -32ppm different from the nominal value for the IV microtracer and thus could not be differentiated in the SRM assay by the unit mass resolution. To overcome this interference, we evaluated two approaches by either monitoring an alternative product ion using the SRM assay or isolating the interfering component using the parallel reaction monitoring (PRM) assay on the HRMS. This case study has demonstrated two practical approaches for overcoming interferences with the detection of stable isotopically labeled IV microtracers in the evaluation of absolute bioavailability, which provides users the flexibility in using either LC-MS/MS or HRMS to mitigate unpredicted interferences in the assay to support microtracer absolute bioavailability studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. New trends and applications in carboxylation for isotope chemistry.

    PubMed

    Bragg, Ryan A; Sardana, Malvika; Artelsmair, Markus; Elmore, Charles S

    2018-05-08

    Carboxylations are an important method for the incorporation of isotopically labeled 14 CO 2 into molecules. This manuscript will review labeled carboxylations since 2010 and will present a perspective on the potential of recent unlabeled methodology for labeled carboxylations. The perspective portion of the manuscript is broken into 3 major sections based on product type, arylcarboxylic acids, benzylcarboxylic acids, and alkyl carboxylic acids, and each of those sections is further subdivided by substrate. © 2018 AstraZeneca. Journal of Labelled Compounds and Radiopharmaceuticals Published by John Wiley & Sons, Ltd.

  5. SIMSISH Technique Does Not Alter the Apparent Isotopic Composition of Bacterial Cells

    PubMed Central

    Chapleur, Olivier; Wu, Ting-Di; Guerquin-Kern, Jean-Luc; Mazéas, Laurent; Bouchez, Théodore

    2013-01-01

    In order to identify the function of uncultured microorganisms in their environment, the SIMSISH method, combining in situ hybridization (ISH) and nanoscale secondary ion mass spectrometry (nanoSIMS) imaging, has been proposed to determine the quantitative uptake of specific labelled substrates by uncultured microbes at the single cell level. This technique requires the hybridization of rRNA targeted halogenated DNA probes on fixed and permeabilized microorganisms. Exogenous atoms are introduced into cells and endogenous atoms removed during the experimental procedures. Consequently differences between the original and the apparent isotopic composition of cells may occur. In the present study, the influence of the experimental procedures of SIMSISH on the isotopic composition of carbon in E. coli cells was evaluated with nanoSIMS and compared to elemental analyser-isotopic ratio mass spectrometer (EA-IRMS) measurements. Our results show that fixation and hybridization have a very limited, reproducible and homogeneous influence on the isotopic composition of cells. Thereby, the SIMSISH procedure minimizes the contamination of the sample by exogenous atoms, thus providing a means to detect the phylogenetic identity and to measure precisely the carbon isotopic composition at the single cell level. This technique was successfully applied to a complex sample with double bromine – iodine labelling targeting a large group of bacteria and a specific archaea to evaluate their specific 13C uptake during labelled methanol anaerobic degradation. PMID:24204855

  6. Benzylic rearrangement stable isotope labeling for quantitation of guanidino and ureido compounds in thyroid tissues by liquid chromatography-electrospray ionization mass spectrometry.

    PubMed

    Fan, Ruo-Jing; Guan, Qing; Zhang, Fang; Leng, Jia-Peng; Sun, Tuan-Qi; Guo, Yin-Long

    2016-02-18

    Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, l-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (l-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p < 0.05) between the para-carcinoma and carcinoma thyroid tissue samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards and Labeling Programs for Clothes Washers, Water Dispensers, Vending Machines and CFLs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fridley, David; Zheng, Nina; Zhou, Nan

    Since the late 1970s, energy labeling programs and mandatory energy performance standards have been used in many different countries to improve the efficiency levels of major residential and commercial equipment. As more countries and regions launch programs covering a greater range of products that are traded worldwide, greater attention has been given to harmonizing the specific efficiency criteria in these programs and the test methods for measurements. For example, an international compact fluorescent light (CFL) harmonization initiative was launched in 2006 to focus on collaboration between Australia, China, Europe and North America. Given the long history of standards and labelingmore » programs, most major energy-consuming residential appliances and commercial equipment are already covered under minimum energy performance standards (MEPS) and/or energy labels. For these products, such as clothes washers and CFLs, harmonization may still be possible when national MEPS or labeling thresholds are revised. Greater opportunity for harmonization exists in newer energy-consuming products that are not commonly regulated but are under consideration for new standards and labeling programs. This may include commercial products such as water dispensers and vending machines, which are only covered by MEPS or energy labels in a few countries or regions. As China continues to expand its appliance standards and labeling programs and revise existing standards and labels, it is important to learn from recent international experiences with efficiency criteria and test procedures for the same products. Specifically, various types of standards and labeling programs already exist in North America, Europe and throughout Asia for products in China's 2010 standards and labeling programs, namely clothes washers, water dispensers, vending machines and CFLs. This report thus examines similarities and critical differences in energy efficiency values, test procedure specifications and

  8. Household adoption of energy and water-efficient appliances: An analysis of attitudes, labelling and complementary green behaviours in selected OECD countries.

    PubMed

    Dieu-Hang, To; Grafton, R Quentin; Martínez-Espiñeira, Roberto; Garcia-Valiñas, Maria

    2017-07-15

    Using a household-based data set of more than 12,000 households from 11 OECD countries, we analyse the factors underlying the decision by households to adopt energy-efficient and water-efficient equipment. We evaluate the roles of both attitudes and labelling schemes on the adoption of energy and water-efficient equipment, and also the interaction and complementarity between energy and water conservation behaviours. Our findings show: one, 'green' social norms and favourable attitudes towards the environment are associated with an increased likelihood of households' adoption of energy and water-efficient appliances; two, households' purchase decisions are positively affected by their awareness, understanding, and trust of labelling schemes; and three, there is evidence of complementarity between energy conservation and water conservation behaviours. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Efficient synthesis of a fluorine-18 labeled biotin derivative.

    PubMed

    Claesener, Michael; Breyholz, Hans-Jörg; Hermann, Sven; Faust, Andreas; Wagner, Stefan; Schober, Otmar; Schäfers, Michael; Kopka, Klaus

    2012-11-01

    The natural occurring vitamin biotin, also known as vitamin H or vitamin B(7), plays a major role in various metabolic reactions. Caused by its high binding affinity to the protein avidin with a dissociation constant of about 10(-15)M the biotin-avidin system was extensively examined for multiple applications. We have synthesized a fluorine-18 labeled biotin derivative [(18)F]4 for a potential application in positron emission tomography (PET). Mesylate precursor 3 was obtained by an efficient two-step reaction via a copper catalyzed azide-alkyne cycloaddition (CuAAC) from easily accessible starting materials. [(18)F]4 was successfully synthesized by a nucleophilic radiofluorination of precursor 3. A biodistribution study by means of small-animal PET imaging in wt-mice was performed and serum stability was examined. Compound [(18)F]4 was obtained from precursor compound 3 with an average specific activity of 16GBq/μmol within 45min and a radiochemical yield of 45±5% (decay corrected). [(18)F]4 demonstrated only negligible decomposition in human serum. A qualitative binding study revealed the high affinity of the synthesized biotin derivative to avidin. Blocking experiments with native biotin showed that binding was site-specific. Biodistribution studies showed that [(18)F]4 was cleared quickly and efficiently from the body by hepatobiliary and renal elimination. An efficient synthesis for [(18)F]4 was established. In vivo characteristics were determined and demonstrated the pharmacokinetic behaviour of [(18)F]4. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Expression and purification of isotopically labeled peptide inhibitors and substrates of cAMP-dependant protein kinase A for NMR analysis.

    PubMed

    Masterson, Larry R; Bortone, Nadia; Yu, Tao; Ha, Kim N; Gaffarogullari, Ece C; Nguyen, Oanh; Veglia, Gianluigi

    2009-04-01

    Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis.

  11. A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation.

    PubMed

    Cheng, Dongwan; Zheng, Li; Hou, Junjie; Wang, Jifeng; Xue, Peng; Yang, Fuquan; Xu, Tao

    2015-01-01

    The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.

  12. High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.

    PubMed

    Lechene, Claude; Hillion, Francois; McMahon, Greg; Benson, Douglas; Kleinfeld, Alan M; Kampf, J Patrick; Distel, Daniel; Luyten, Yvette; Bonventre, Joseph; Hentschel, Dirk; Park, Kwon Moo; Ito, Susumu; Schwartz, Martin; Benichou, Gilles; Slodzian, Georges

    2006-01-01

    Secondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials. The new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse. MIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

  13. Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

    PubMed Central

    2016-01-01

    Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

  14. Quantifying the effect of plant growth on litter decomposition using a novel, triple-isotope label approach

    NASA Astrophysics Data System (ADS)

    Ernakovich, J. G.; Baldock, J.; Carter, T.; Davis, R. A.; Kalbitz, K.; Sanderman, J.; Farrell, M.

    2017-12-01

    Microbial degradation of plant detritus is now accepted as a major stabilizing process of organic matter in soils. Most of our understanding of the dynamics of decomposition come from laboratory litter decay studies in the absence of plants, despite the fact that litter decays in the presence of plants in many native and managed systems. There is growing evidence that living plants significantly impact the degradation and stabilization of litter carbon (C) due to changes in the chemical and physical nature of soils in the rhizosphere. For example, mechanistic studies have observed stimulatory effects of root exudates on litter decomposition, and greenhouse studies have shown that living plants accelerate detrital decay. Despite this, we lack a quantitative understanding of the contribution of living plants to litter decomposition and how interactions of these two sources of C build soil organic matter (SOM). We used a novel triple-isotope approach to determine the effect of living plants on litter decomposition and C cycling. In the first stage of the experiment, we grew a temperate grass commonly used for forage, Poa labillardieri, in a continuously-labelled atmosphere of 14CO2 fertilized with K15NO3, such that the grass biomass was uniformly labelled with 14C and 15N. In the second stage, we constructed litter decomposition mescososms with and without a living plant to test for the effect of a growing plant on litter decomposition. The 14C/15N litter was decomposed in a sandy clay loam while a temperate forage grass, Lolium perenne, grew in an atmosphere of enriched 13CO2. The fate of the litter-14C/15N and plant-13C was traced into soil mineral fractions and dissolved organic matter (DOM) over the course of nine weeks using four destructive harvests of the mesocosms. Our preliminary results suggest that living plants play a major role in the degradation of plant litter, as litter decomposition was greater, both in rate and absolute amount, for soil mesocosms

  15. Validation of the doubly labeled water method using off-axis integrated cavity output spectroscopy and isotope ratio mass spectrometry.

    PubMed

    Melanson, Edward L; Swibas, Tracy; Kohrt, Wendy M; Catenacci, Vicki A; Creasy, Seth A; Plasqui, Guy; Wouters, Loek; Speakman, John R; Berman, Elena S F

    2018-02-01

    When the doubly labeled water (DLW) method is used to measure total daily energy expenditure (TDEE), isotope measurements are typically performed using isotope ratio mass spectrometry (IRMS). New technologies, such as off-axis integrated cavity output spectroscopy (OA-ICOS) provide comparable isotopic measurements of standard waters and human urine samples, but the accuracy of carbon dioxide production (V̇co 2 ) determined with OA-ICOS has not been demonstrated. We compared simultaneous measurement V̇co 2 obtained using whole-room indirect calorimetry (IC) with DLW-based measurements from IRMS and OA-ICOS. Seventeen subjects (10 female; 22 to 63 yr) were studied for 7 consecutive days in the IC. Subjects consumed a dose of 0.25 g H 2 18 O (98% APE) and 0.14 g 2 H 2 O (99.8% APE) per kilogram of total body water, and urine samples were obtained on days 1 and 8 to measure average daily V̇co 2 using OA-ICOS and IRMS. V̇co 2 was calculated using both the plateau and intercept methods. There were no differences in V̇co 2 measured by OA-ICOS or IRMS compared with IC when the plateau method was used. When the intercept method was used, V̇co 2 using OA-ICOS did not differ from IC, but V̇co 2 measured using IRMS was significantly lower than IC. Accuracy (~1-5%), precision (~8%), intraclass correlation coefficients ( R = 0.87-90), and root mean squared error (30-40 liters/day) of V̇co 2 measured by OA-ICOS and IRMS were similar. Both OA-ICOS and IRMS produced measurements of V̇co 2 with comparable accuracy and precision compared with IC.

  16. Visualizing Microbial Biogeochemistry: NanoSIMS and Stable Isotope Probing (Invited)

    NASA Astrophysics Data System (ADS)

    Pett-Ridge, J.; Weber, P. K.

    2009-12-01

    Linking phylogenetic information to function in microbial communities is a key challenge for microbial ecology. Isotope-labeling experiments provide a useful means to investigate the ecophysiology of microbial populations and cells in the environment and allow measurement of nutrient transfers between cell types, symbionts and consortia. The combination of Nano-Secondary Ion Mass Spectrometry (NanoSIMS) analysis, in situ labeling and high resolution microscopy allows isotopic analysis to be linked to phylogeny and morphology and holds great promise for fine-scale studies of microbial systems. In NanoSIMS analysis, samples are sputtered with an energetic primary beam (Cs+, O-) liberating secondary ions that are separated by the mass spectrometer and detected in a suite of electron multipliers. Five isotopic species may be analyzed concurrently with spatial resolution as fine as 50nm. A high sensitivity isotope ratio ‘map’ can then be generated for the analyzed area. NanoSIMS images of 13C, 15N and Mo (a nitrogenase co-factor) localization in diazotrophic cyanobacteria show how cells differentially allocate resources within filaments and allow calculation of nutrient uptake rates on a cell by cell basis. Images of AM fungal hyphae-root and cyanobacteria-rhizobia associations indicate the mobilization and sharing (stealing?) of newly fixed C and N. In a related technique, “El-FISH”, stable isotope labeled biomass is probed with oligonucleotide-elemental labels and then imaged by NanoSIMS. In microbial consortia and cyanobacterial mats, this technique helps link microbial structure and function simultaneously even in systems with unknown and uncultivated microbes. Finally, the combination of re-engineered universal 16S oligonucleotide microarrays with NanoSIMS analyses may allow microbial identity to be linked to functional roles in complex systems such as mats and cellulose degrading hindgut communities. These newly developed methods provide correlated

  17. Determination of thiol metabolites in human urine by stable isotope labeling in combination with pseudo-targeted mass spectrometry analysis

    NASA Astrophysics Data System (ADS)

    Liu, Ping; Qi, Chu-Bo; Zhu, Quan-Fei; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-02-01

    Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1-11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines.

  18. USE OF OXYGEN-18 ISOTOPE LABELING FOR MEASUREMENT OF OXIDATIVE STRESS

    EPA Science Inventory

    Oxygen-18 (18-O) labeling provides a sensitive means for quantifying oxygen
    binding that occurs during in vivo oxidations. Oxidants (ozone, nitrogen
    oxides, hydrogen peroxide, etc.) are first synthesized using 18-O, then cells
    or tissues are exposed to the labeled ...

  19. Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions

    PubMed Central

    Evans, Erin M.; Freund, Dana M.; Sondervan, Veronica M.; Cohen, Jerry D.; Hegeman, Adrian D.

    2018-01-01

    In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies. PMID:29904627

  20. Metabolic Patterns in Spirodela polyrhiza Revealed by 15N Stable Isotope Labeling of Amino Acids in Photoautotrophic, Heterotrophic, and Mixotrophic Growth Conditions.

    PubMed

    Evans, Erin M; Freund, Dana M; Sondervan, Veronica M; Cohen, Jerry D; Hegeman, Adrian D

    2018-01-01

    In this study we describe a [ 15 N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for 17 of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of <100% [ 15 N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

  1. Metabolic patterns in Spirodela polyrhiza revealed by 15N stable isotope labeling of amino acids in photoautotrophic, heterotrophic, and mixotrophic growth conditions

    NASA Astrophysics Data System (ADS)

    Evans, Erin M.; Freund, Dana M.; Sondervan, Veronica M.; Cohen, Jerry D.; Hegeman, Adrian D.

    2018-05-01

    In this study we describe a [15N] stable isotopic labeling study of amino acids in Spirodela polyrhiza (common duckweed) grown under three different light and carbon input conditions which represent unique potential metabolic modes. Plants were grown with a light cycle, either with supplemental sucrose (mixotrophic) or without supplemental sucrose (photoautotrophic) and in the dark with supplemental sucrose (heterotrophic). Labeling patterns, pool sizes (both metabolically active and inactive), and kinetics/turnover rates were estimated for fifteen of the proteinogenic amino acids. Estimation of these parameters followed several overall trends. First, most amino acids showed plateaus in labeling patterns of less than 100% [15N]-labeling, indicating the possibility of a large proportion of amino acids residing in metabolically inactive metabolite pools. Second, total pool sizes appear largest in the dark (heterotrophic) condition, whereas active pool sizes appeared to be largest in the light with sucrose (mixotrophic) growth condition. In contrast turnover measurements based on pool size were highest overall in the light with sucrose experiment, with the exception of leucine/isoleucine, lysine, and arginine, which all showed higher turnover in the dark. K-means clustering analysis also revealed more rapid turnover in the light treatments with many amino acids clustering in lower-turnover groups. Emerging insights from other research were also supported, such as the prevalence of alternate pathways for serine metabolism in non-photosynthetic cells. These data provide extensive novel information on amino acid pool size and kinetics in S. polyrhiza and can serve as groundwork for future metabolic studies.

  2. Relative quantitation of glycosylation variants by stable isotope labeling of enzymatically released N-glycans using [12C]/[13C] aniline and ZIC-HILIC-ESI-TOF-MS.

    PubMed

    Giménez, Estela; Sanz-Nebot, Victòria; Rizzi, Andreas

    2013-09-01

    Glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ∼1-5 % for major and ∼5-10 % for minor N-glycans). In a second step, zwitterionic-hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (μZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine α1-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples.

  3. Isotope-labeled aspartate sidechain as a non-perturbing infrared probe: Application to investigate the dynamics of a carboxylate buried inside a protein

    NASA Astrophysics Data System (ADS)

    Abaskharon, Rachel M.; Brown, Stephen P.; Zhang, Wenkai; Chen, Jianxin; Smith, Amos B.; Gai, Feng

    2017-09-01

    Because of their negatively charged carboxylates, aspartate and glutamate are frequently found at the active or binding site of proteins. However, studying a specific carboxylate in proteins that contain multiple aspartates and/or glutamates via infrared spectroscopy is difficult due to spectral overlap. We show, herein, that isotopic-labeling of the aspartate sidechain can overcome this limitation as the resultant 13COO- asymmetric stretching vibration resides in a transparent region of the protein IR spectrum. Applicability of this site-specific vibrational probe is demonstrated by using it to assess the dynamics of an aspartate ion buried inside a small protein via two-dimensional infrared spectroscopy.

  4. The Sensitivity of Atmospheric Water Isotopes to Entrainment and Precipitation Efficiency in a Bulk Plume Model of Convection

    NASA Astrophysics Data System (ADS)

    Duan, S.; Wright, J. S.; Romps, D. M.

    2016-12-01

    Atmospheric water isotopes have been proposed as potentially powerful constraints on the physics of convective clouds and parameterizations of convective processes in models. We have previously derived an analytical model of water vapor (H2O) and one of its heavy isotopes (HDO) in convective environments based on a bulk-plume convective water budget in radiative convective equilibrium. This analytical model provides a useful starting point for examining the joint responses of water vapor and its isotopic composition to changes in convective parameters; however, certain idealistic assumptions are required to make the model analytically solvable. Here, we develop a more flexible numerical framework that enables a wider range of model configurations and includes additional isotopic tracers. This model provides a bridge between Rayleigh distillation, which is simple but inflexible, and more complicated convection schemes and cloud resolving models, which are more realistic but also more difficult to perturb and interpret. Application of realistic in-cloud water profiles in our model produces vertical distributions of δD that qualitatively match satellite observations from the Tropospheric Emission Spectrometer (TES). We test the sensitivity of water vapor and its isotopic composition to a wide range of perturbations in the model parameters and their vertical profiles. In this presentation, we focus especially on establishing constraints for convective entrainment and precipitation efficiency. We conclude by discussing the potential application of this model as part of a larger water isotope toolkit for use with offline diagnostics provided by reanalyses and GCMs.

  5. Considerations in the development of the utility of stable isotopes in science, medicine, and agriculture, and environmental studies

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Matwiyoff, N.A.

    1976-01-01

    The prospects for the broad scale development of the utility of stable isotopes in science, medicine, agriculture, and environmental studies are considered with emphasis on the current status of isotope production, synthesis of isotopically labelled compounds, and analytical techniques.

  6. LC/MS Method for the Determination of Stable Isotope Labeled Promethazine in Human Plasma

    NASA Technical Reports Server (NTRS)

    Zuwei, Wang; Boyd, Jason; Berens, Kurt L.; Putcha, Lakshmi

    2004-01-01

    Promethazine (PMZ) is taken by astronauts orally (PO), intramuscularly (IM) or rectally (PR) for space motion sickness. LC/MS method was developed with off-line solid phase extraction to measure plasma concentrations of PMZ given as stable isotope-labeled (SIL) formulations by the three different routes of administration simultaneously. Samples (0.5ml) were loaded on to Waters Oasis HLB co-polymer cartridges and eluted with 1.0 mL methanol. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 6 min. Acetonitrile/ ammonium acetate (30 mM) in water (3:2, v/v), pH 5.6 plus or minus 0.1, was used as the mobile phase for separation. Concentrations of PMZ, PMZ-d4 and PMZ-d7 and chlorpromazine (internal standard) were determined using a Micromass ZMD single quadrupole mass spectrometer with Electrospray Ionization (ESI). ESI mass spectra were acquired in positive ion mode with selected ion monitoring of [M+ H]dot plus. The method is rapid, reproducible and the assay specific parameters are listed in a table. A novel, sensitive and specific method for the measurement of PMZ and SIL PMZ in human plasma is reported.

  7. Biodegradation and mineralization of isotopically labeled TNT and RDX in anaerobic marine sediments.

    PubMed

    Ariyarathna, Thivanka; Vlahos, Penny; Smith, Richard W; Fallis, Stephen; Groshens, Thomas; Tobias, Craig

    2017-05-01

    The lack of knowledge on the fate of explosive compounds 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), particularly in marine ecosystems, constrains the application of bioremediation techniques in explosive-contaminated coastal sites. The authors present a comparative study on anaerobic biodegradation and mineralization of 15 N-nitro group isotopically labeled TNT and RDX in organic carbon-rich, fine-grained marine sediment with native microbial assemblages. Separate sediment slurry experiments were carried out for TNT and RDX at 23°C for 16 d. Dissolved and sediment-sorbed fractions of parent and transformation products, isotopic compositions of sediment, and mineralization products of the dissolved inorganic N pool ( 15 NH 4 + , 15 NO 3 - , 15 NO 2 - , and 15 N 2 ) were measured. The rate of TNT removal from the aqueous phase was faster (0.75 h -1 ) than that of RDX (0.37 h -1 ), and 15 N accumulation in sediment was higher in the TNT (13%) than the RDX (2%) microcosms. Mono-amino-dinitrotoluenes were identified as intermediate biodegradation products of TNT. Two percent of the total spiked TNT-N is mineralized to dissolved inorganic N through 2 different pathways: denitration as well as deamination and formation of NH 4 + , facilitated by iron and sulfate reducing bacteria in the sediments. The majority of the spiked TNT-N (85%) is in unidentified pools by day 16. Hexahydro-1,3,5-trinitro-1,3,5-triazine (10%) biodegrades to nitroso derivatives, whereas 13% of RDX-N in nitro groups is mineralized to dissolved inorganic N anaerobically by the end of the experiment. The primary identified mineralization end product of RDX (40%) is NH 4 + , generated through either deamination or mono-denitration, followed by ring breakdown. A reasonable production of N 2 gas (13%) was seen in the RDX system but not in the TNT system. Sixty-eight percent of the total spiked RDX-N is in an unidentified pool by day 16 and may include unquantified

  8. Isotope labeling to determine the dynamics of metabolic response in CHO cell perfusion bioreactors using MALDI-TOF-MS.

    PubMed

    Karst, Daniel J; Steinhoff, Robert F; Kopp, Marie R G; Soos, Miroslav; Zenobi, Renato; Morbidelli, Massimo

    2017-11-01

    The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C 6 glucose containing feed media during constant operation at 20 × 10 6 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C 6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C 6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017. © 2017 American Institute of Chemical Engineers.

  9. Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.

    PubMed

    von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana

    2013-10-01

    The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

  10. Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring

    PubMed Central

    Tang, Hsin-Yao; Beer, Lynn A.; Barnhart, Kurt T.; Speicher, David W.

    2011-01-01

    Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves, quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1-D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μl serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers. PMID:21726088

  11. Rapid verification of candidate serological biomarkers using gel-based, label-free multiple reaction monitoring.

    PubMed

    Tang, Hsin-Yao; Beer, Lynn A; Barnhart, Kurt T; Speicher, David W

    2011-09-02

    Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) has emerged as a promising platform for verification of serological candidate biomarkers. However, cost and time needed to synthesize and evaluate stable isotope peptides, optimize spike-in assays, and generate standard curves quickly becomes unattractive when testing many candidate biomarkers. In this study, we demonstrate that label-free multiplexed MRM-MS coupled with major protein depletion and 1D gel separation is a time-efficient, cost-effective initial biomarker verification strategy requiring less than 100 μL of serum. Furthermore, SDS gel fractionation can resolve different molecular weight forms of targeted proteins with potential diagnostic value. Because fractionation is at the protein level, consistency of peptide quantitation profiles across fractions permits rapid detection of quantitation problems for specific peptides from a given protein. Despite the lack of internal standards, the entire workflow can be highly reproducible, and long-term reproducibility of relative protein abundance can be obtained using different mass spectrometers and LC methods with external reference standards. Quantitation down to ~200 pg/mL could be achieved using this workflow. Hence, the label-free GeLC-MRM workflow enables rapid, sensitive, and economical initial screening of large numbers of candidate biomarkers prior to setting up SID-MRM assays or immunoassays for the most promising candidate biomarkers.

  12. International experiences in assessing vitamin A status and applying the vitamin A-labeled isotope dilution method.

    PubMed

    Lopez-Teros, Veronica; Chileshe, Justin; Idohou-Dossou, Nicole; Fajarwati, Tetra; Medoua Nama, Gabriel; Newton, Sam; Vinod Kumar, Malavika; Wang, Zhixu; Wasantwisut, Emorn; Hunt, Janet R

    2014-01-01

    Inadequate vitamin A (VA) nutrition continues to be a major problem worldwide, and many interventions being implemented to improve VA status in various populations need to be evaluated. The interpretation of results after an intervention depends greatly on the method selected to assess VA status. To evaluate the effect of an intervention on VA status, researchers in Cameroon, India, Indonesia, Mexico, Senegal and Zambia have used serum retinol as an indicator, and have not always found improvement in response to supplementation. One problem is that homeostatic control of serum retinol may mask positive effects of treatment in that changes in concentration are observed only when status is either moderately to severely depleted or excessive. Because VA is stored mainly in the liver, measurements of hepatic VA stores are the “gold standard” for assessing VA status. Dose response tests such as the relative dose response (RDR) and the modified relative dose response (MRDR), allow a qualitative assessment of VA liver stores. On the other hand, the use of the vitamin A-labeled isotope dilution (VALID) technique, (using 13C or 2H-labeled retinyl acetate) serves as an indirect method to quantitatively estimate total body and liver VA stores. Countries including Cameroon, China, Ghana, Mexico, Thailand and Zambia are now applying the VALID method to sensitively assess changes in VA status during interventions, or to estimate a population’s dietary requirement for VA. Transition to the use of more sensitive biochemical indicators of VA status such as the VALID technique is needed to effectively assess interventions in populations where mild to moderate VA deficiency is more prevalent than severe deficiency.

  13. Plasma Protein Turnover Rates in Rats Using Stable Isotope Labeling, Global Proteomics, and Activity-Based Protein Profiling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Smith, Jordan Ned; Tyrrell, Kimberly J.; Hansen, Joshua R.

    Protein turnover is important for general health on cellular and organism scales providing a strategy to replace old, damaged, or dysfunctional proteins. Protein turnover also informs of biomarker kinetics, as a better understanding of synthesis and degradation of proteins increases the clinical utility of biomarkers. Here, turnover rates of plasma proteins in rats were measured in vivo using a pulse-chase stable isotope labeling experiment. During the pulse, rats (n=5) were fed 13C6-labeled lysine (“heavy”) feed for 23 days to label proteins. During the chase, feed was changed to an unlabeled equivalent feed (“light”), and blood was repeatedly sampled from ratsmore » over 10 time points for 28 days. Plasma samples were digested with trypsin, and analyzed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). MaxQuant was used to identify peptides and proteins, and quantify heavy:light lysine ratios. A system of ordinary differential equations was used to calculate protein turnover rates. Using this approach, 273 proteins were identified, and turnover rates were quantified for 157 plasma proteins with half-lives ranging 0.3-103 days. For the ~70 most abundant proteins, variability in turnover rates among rats was low (median coefficient of variation: 0.09). Activity-based protein profiling was applied to pooled plasma samples to enrich serine hydrolases using a fluorophosphonate (FP2) activity-based probe. This enrichment resulted in turnover rates for an additional 17 proteins. This study is the first to measure global plasma protein turnover rates in rats in vivo, measure variability of protein turnover rates in any animal model, and utilize activity-based protein profiling for enhancing measurements of targeted, low-abundant proteins, such as those commonly used as biomarkers. Measured protein turnover rates will be important for understanding of the role of protein turnover in cellular and organism health as well as increasing the utility of

  14. Accuracy of delta 18O isotope ratio measurements on the same sample by continuous-flow isotope-ratio mass spectrometry

    USDA-ARS?s Scientific Manuscript database

    The doubly labeled water method is considered the reference method to measure energy expenditure. Conventional mass spectrometry requires a separate aliquot of the same sample to be prepared and analyzed separately. With continuous-flow isotope-ratio mass spectrometry, the same sample could be analy...

  15. Development of isotope labeling LC-MS for human salivary metabolomics and application to profiling metabolome changes associated with mild cognitive impairment.

    PubMed

    Zheng, Jiamin; Dixon, Roger A; Li, Liang

    2012-12-18

    Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential (13)C/(12)C isotope dansylation labeling method, combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), is described for quantitative profiling of the human salivary metabolome. New strategies are presented to optimize the sample preparation and LC-MS detection processes. The strategies allow the use of as little of 5 μL of saliva sample as a starting material to determine the concentration changes of an average of 1058 ion pairs or putative metabolites in comparative saliva samples. The overall workflow consists of several steps including acetone-induced protein precipitation, (12)C-dansylation labeling of the metabolites, and LC-UV measurement of the total concentration of the labeled metabolites in individual saliva samples. A pooled sample was prepared from all the individual samples and labeled with (13)C-dansylation to serve as a reference. Using this metabolome profiling method, it was found that compatible metabolome results could be obtained after saliva samples were stored in tubes normally used for genetic material collection at room temperature, -20 °C freezer, and -80 °C freezer over a period of 1 month, suggesting that many saliva samples already collected in genomic studies could become a valuable resource for metabolomics studies, although the effect of much longer term of storage remains to be determined. Finally, the developed method was applied for analyzing the metabolome changes of two different groups: normal healthy older adults and comparable older adults with mild cognitive impairment (MCI). Top-ranked 18 metabolites successfully distinguished the two groups, among which seven metabolites were putatively identified while one metabolite, taurine, was definitively identified.

  16. Using label-free screening technology to improve efficiency in drug discovery.

    PubMed

    Halai, Reena; Cooper, Matthew A

    2012-02-01

    Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

  17. Seasonal patterns of carbon allocation to respiratory pools in 60-yr-old deciduous (Fagus sylvatica) and evergreen (Picea abies) trees assessed via whole-tree stable carbon isotope labeling.

    PubMed

    Kuptz, Daniel; Fleischmann, Frank; Matyssek, Rainer; Grams, Thorsten E E

    2011-07-01

    • The CO(2) efflux of adult trees is supplied by recent photosynthates and carbon (C) stores. The extent to which these C pools contribute to growth and maintenance respiration (R(G) and R(M), respectively) remains obscure. • Recent photosynthates of adult beech (Fagus sylvatica) and spruce (Picea abies) trees were labeled by exposing whole-tree canopies to (13) C-depleted CO(2). Label was applied three times during the year (in spring, early summer and late summer) and changes in the stable C isotope composition (δ(13) C) of trunk and coarse-root CO(2) efflux were quantified. • Seasonal patterns in C translocation rate (CTR) and fractional contribution of label to CO(2) efflux (F(Label-Max)) were found. CTR was fastest during early summer. In beech, F(Label-Max) was lowest in spring and peaked in trunks during late summer (0.6 ± 0.1, mean ± SE), whereas no trend was observed in coarse roots. No seasonal dynamics in F(Label-Max) were found in spruce. • During spring, the R(G) of beech trunks was largely supplied by C stores. Recent photosynthates supplied growth in early summer and refilled C stores in late summer. In spruce, CO(2) efflux was constantly supplied by a mixture of stored (c. 75%) and recent (c. 25%) C. The hypothesis that R(G) is exclusively supplied by recent photosynthates was rejected for both species. © 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.

  18. Determination of thiol metabolites in human urine by stable isotope labeling in combination with pseudo-targeted mass spectrometry analysis

    PubMed Central

    Liu, Ping; Qi, Chu-Bo; Zhu, Quan-Fei; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-01-01

    Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1–11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines. PMID:26888486

  19. Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry

    PubMed Central

    Taylor, Gordon T.; Suter, Elizabeth A.; Li, Zhuo Q.; Chow, Stephanie; Stinton, Dallyce; Zaliznyak, Tatiana; Beaupré, Steven R.

    2017-01-01

    A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm−1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/x¯), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements. PMID:28824580

  20. Efficient 3He/4He separation in a nanoporous graphenylene membrane.

    PubMed

    Qu, Yuanyuan; Li, Feng; Zhao, Mingwen

    2017-08-16

    Helium-3 is a precious noble gas, which is essential in many advanced technologies such as cryogenics, isotope labeling and nuclear weapons. The current imbalance of 3 He demand and supply shortage leads to the search for an efficient membrane with high performance for 3 He separation. In this study, based on first-principles calculations, we demonstrated that highly efficient 3 He harvesting can be achieved in a nanoporous graphenylene membrane with industrially-acceptable selectivity and permeance. The quantum tunneling effect leads to 3 He harvesting with high efficiency via kinetic sieving. Both the quantum tunneling effect and zero-point energy (ZPE) determine the 3 He/ 4 He separation via thermally-driven equilibrium sieving, where the ZPE effect dominates efficient 3 He/ 4 He separation between two reservoirs. The quantum effects revealed in this work suggest that the nanoporous graphenylene membrane is promising for efficient 3 He harvesting that can be exploited for industrial applications.

  1. Informational Aspects of Isotopic Diversity in Biology and Medicine

    NASA Astrophysics Data System (ADS)

    Berezin, Alexander A.

    2004-10-01

    Use of stable and radioactive isotopes in biology and medicine is intensive, yet informational aspects of isotopes as such are largely neglected (A.A.Berezin, J.Theor.Biol.,1992). Classical distinguishability (``labelability'') of isotopes allows for pattern generation dynamics. Quantum mechanically advantages of isotopicity (diversity of stable isotopes) arise from (almost perfect) degeneracy of various isotopic configurations; this in turn allows for isotopic sweeps (hoppings) by resonance neutron tunneling (Eccles mechanism). Isotopic variations of de Broglie wavelength affect quantum tunneling, diffusivity, magnetic interactions (e.g. by Lorentz force), etc. Ergodicity principle (all isoenergetic states are eventually accessed) implies possibility of fast scanning of library of morphogenetic patterns (cf metaphors of universal ``Platonic'' Library of Patterns: e.g. J.L.Borges, R.Sheldrake) with subsequent Darwinian reinforcement (e.g. by targeted mutations) of evolutionary advantageous patterns and structures. Isotopic shifts in organisms, from viruses and protozoa to mammalians, (e.g. DNA with enriched or depleted C-13) are tools to elucidate possible informational (e.g. Shannon entropy) role of isotopicity in genetic (e.g. evolutionary and morphological), dynamical (e.g. physiological and neurological) as well as medical (e.g. carcinogenesis, aging) aspects of biology and medicine.

  2. An analysis by metabolic labelling of the encephalomyocarditis virus ribosomal frameshifting efficiency and stimulators.

    PubMed

    Ling, Roger; Firth, Andrew E

    2017-08-01

    Programmed -1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift -1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3'-adjacent to a 'slippery' heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a trans-acting virus protein. Thus, in contrast to other examples of -1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3'-adjacent stem-loop structure, but is not appreciably affected by the 'StopGo' sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46-76 % efficient.

  3. Rare isotope studies involving catalytic oxidation of CO over platinum-tin oxide

    NASA Technical Reports Server (NTRS)

    Upchurch, Billy T.; Wood, George M., Jr.; Hess, Robert V.; Hoyt, Ronald F.

    1987-01-01

    Results of studies utilizing normal and rare oxygen isotopes in the catalytic oxidation of carbon monoxide over a platinum-tin oxide catalyst substrate are presented. Chemisorption of labeled carbon monoxide on the catalyst followed by thermal desorption yielded a carbon dioxide product with an oxygen-18 composition consistent with the formation of a carbonate-like intermediate in the chemisorption process. The efficacy of a method developed for the oxygen-18 labeling of the platinum-tin oxide catalyst surface for use in closed cycle pulsed care isotope carbon dioxide lasers is demonstrated for the equivalent of 10 to the 6th power pulses at 10 pulses per second.

  4. Synthesis and preliminary biological evaluations of fluorescent or 149Promethium labeled Trastuzumab-polyethylenimine

    DOE PAGES

    Fitzsimmons, Jonathan; Nayak, Tapan; Cutler, Cathy; ...

    2015-12-30

    Radioimmunotherapy utilize a targeting antibody coupled to a therapeutic isotope to target and treat a tumor or disease. In this study we examine the synthesis and cell binding of a polymer scaffold containing a radiotherapeutic isotope and a targeting antibody. Methods: The multistep synthesis of a fluorescent or 149Promethium-labeled Trastuzumab-polyethyleneimine (PEI), Trastuzumab, or PEI is described. In vitro uptake, internalization and/or the binding affinity to the Her2/neu expressing human breast adenocarcinoma SKBr3 cells was investigated with the labeled compounds. Fluorescent-labeled Trastuzumab-PEI was internalized more into cells at 2 and 18 h than fluorescent-labeled Trastuzumab or PEI. The fluorescent-labeled Trastuzumab wasmore » concentrated on the cell surface at 2 and 18 h and the labeled PEI had minimal uptake. DOTA-PEI was prepared and contained an average of 16 chelates per PEI; the compound was radio-labeled with 149Promethium and conjugated to Trastuzumab. The purified 149Pm-DOTA-PEI-Trastuzumab had a radiochemical purity of 96.7% and a specific activity of 0.118 TBq/g. The compound demonstrated a dissociation constant for the Her2/neu receptor of 20.30 ± 6.91 nM. In conclusion, the results indicate the DOTA-PEI-Trastuzumab compound has potential as a targeted therapeutic carrier, and future in vivo studies should be performed.« less

  5. Assessing the dietary bioavailability of metals associated with natural particles: Extending the use of the reverse labeling approach to zinc

    USGS Publications Warehouse

    Croteau, Marie-Noele; Cain, Daniel J.; Fuller, Christopher C.

    2017-01-01

    We extend the use of a novel tracing technique to quantify the bioavailability of zinc (Zn) associated with natural particles using snails enriched with a less common Zn stable isotope. Lymnaea stagnalis is a model species that has relatively fast Zn uptake rates from the dissolved phase, enabling their rapid enrichment in 67Zn during the initial phase of labeling. Isotopically enriched snails were subsequently exposed to algae mixed with increasing amounts of metal-rich particles collected from two acid mine drainage impacted rivers. Zinc bioavailability from the natural particles was inferred from calculations of 66Zn assimilation into the snail’s soft tissues. Zinc assimilation efficiency (AE) varied from 28% for the Animas River particles to 45% for the Snake River particles, indicating that particle-bound, or sorbed Zn, was bioavailable from acid mine drainage wastes. The relative binding strength of Zn sorption to the natural particles was inversely related to Zn bioavailability; a finding that would not have been possible without using the reverse labeling approach. Differences in the chemical composition of the particles suggest that their geochemical properties may influence the extent of Zn bioavailability.

  6. Assessing the Dietary Bioavailability of Metals Associated with Natural Particles: Extending the Use of the Reverse Labeling Approach to Zinc.

    PubMed

    Croteau, Marie-Noële; Cain, Daniel J; Fuller, Christopher C

    2017-03-07

    We extend the use of a novel tracing technique to quantify the bioavailability of zinc (Zn) associated with natural particles using snails enriched with a less common Zn stable isotope. Lymnaea stagnalis is a model species that has relatively fast Zn uptake rates from the dissolved phase, enabling their rapid enrichment in 67 Zn during the initial phase of labeling. Isotopically enriched snails were subsequently exposed to algae mixed with increasing amounts of metal-rich particles collected from two acid mine drainage impacted rivers. Zinc bioavailability from the natural particles was inferred from calculations of 66 Zn assimilation into the snail's soft tissues. Zinc assimilation efficiency (AE) varied from 28% for the Animas River particles to 45% for the Snake River particles, indicating that particle-bound, or sorbed Zn, was bioavailable from acid mine drainage wastes. The relative binding strength of Zn sorption to the natural particles was inversely related to Zn bioavailability; a finding that would not have been possible without using the reverse labeling approach. Differences in the chemical composition of the particles suggest that their geochemical properties may influence the extent of Zn bioavailability.

  7. WaterSense Labeled New Homes

    EPA Pesticide Factsheets

    Homes built to meet EPA's specification can earn the WaterSense label. EPA criteria include WaterSense labeled plumbing fixtures, efficient hot water delivery systems, water-smart landscape design, and other features.

  8. A Method to Determine 18O Kinetic Isotope Effects in the Hydrolysis of Nucleotide Triphosphates

    PubMed Central

    Du, Xinlin; Ferguson, Kurt; Sprang, Stephen R.

    2007-01-01

    A method to determine 18O kinetic isotope effects (KIE) in the hydrolysis of GTP is described that is generally applicable to reactions involving other nucleotide triphosphates. Internal competition, wherein the substrate of the reaction is a mixture of 18O-labeled and unlabeled nucleotides, is employed and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18O at sites of mechanistic interest also contains 13C at all carbon positions, while the 16O-nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink™ interface (ThermoFinnigan). Carbon isotope ratios can be determined with accuracy and precision greater than 0.04%, and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1333-catalyzed hydrolysis of [β18O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (< 0.1%). A single KIE measurement can be conducted in 25 minutes with less than 5 μg nucleotide reaction product. PMID:17963711

  9. Assessing compartmentalized flux in lipid metabolism with isotopes

    DOE PAGES

    Allen, Doug K.

    2016-03-18

    Metabolism in plants takes place across multiple cell types and within distinct organelles. The distributions equate to spatial heterogeneity; though the limited means to experimentally assess metabolism frequently involve homogenizing tissues and mixing metabolites from different locations.Most current isotope investigations of metabolism therefore lack the ability to resolve spatially distinct events. Recognition of this limitation has resulted in inspired efforts to advance metabolic flux analysis and isotopic labeling techniques. Though a number of these efforts have been applied to studies in central metabolism; recent advances in instrumentation and techniques present an untapped opportunity to make similar progress in lipid metabolismmore » where the use of stable isotopes has been more limited. These efforts will benefit from sophisticated radiolabeling reports that continue to enrich our knowledge on lipid biosynthetic pathways and provide some direction for stable isotope experimental design and extension of MFA. Evidence for this assertion is presented through the review of several elegant stable isotope studies and by taking stock of what has been learned from radioisotope investigations when spatial aspects of metabolism were considered. The studies emphasize that glycerolipid production occurs across several locations with assembly of lipids in the ER or plastid, fatty acid biosynthesis occurring in the plastid, and the generation of acetyl-CoA and glycerol-3-phosphate taking place at multiple sites. Considering metabolism in this context underscores the cellular and subcellular organization that is important to enhanced production of glycerolipids in plants. An attempt is made to unify salient features from a number of reports into a diagrammatic model of lipid metabolism and propose where stable isotope labeling experiments and further flux analysis may help address questions in the field.« less

  10. Assessing compartmentalized flux in lipid metabolism with isotopes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Allen, Doug K.

    Metabolism in plants takes place across multiple cell types and within distinct organelles. The distributions equate to spatial heterogeneity; though the limited means to experimentally assess metabolism frequently involve homogenizing tissues and mixing metabolites from different locations.Most current isotope investigations of metabolism therefore lack the ability to resolve spatially distinct events. Recognition of this limitation has resulted in inspired efforts to advance metabolic flux analysis and isotopic labeling techniques. Though a number of these efforts have been applied to studies in central metabolism; recent advances in instrumentation and techniques present an untapped opportunity to make similar progress in lipid metabolismmore » where the use of stable isotopes has been more limited. These efforts will benefit from sophisticated radiolabeling reports that continue to enrich our knowledge on lipid biosynthetic pathways and provide some direction for stable isotope experimental design and extension of MFA. Evidence for this assertion is presented through the review of several elegant stable isotope studies and by taking stock of what has been learned from radioisotope investigations when spatial aspects of metabolism were considered. The studies emphasize that glycerolipid production occurs across several locations with assembly of lipids in the ER or plastid, fatty acid biosynthesis occurring in the plastid, and the generation of acetyl-CoA and glycerol-3-phosphate taking place at multiple sites. Considering metabolism in this context underscores the cellular and subcellular organization that is important to enhanced production of glycerolipids in plants. An attempt is made to unify salient features from a number of reports into a diagrammatic model of lipid metabolism and propose where stable isotope labeling experiments and further flux analysis may help address questions in the field.« less

  11. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    PubMed Central

    Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  12. Assimilation efficiency for sediment-sorbed benzo(a)pyrene by Diporeia spp.

    USGS Publications Warehouse

    Lydy, M.J.; Landrum, P.F.

    1993-01-01

    Two methods are currently available for determining contaminant assimilation efficiencies (AE) from ingested material in benthic invertebrates. These methods were compared using the Great Lakes amphipod Diporeia spp. and [14C]benzo(a)pyrene (BaP) sorbed to Florissant sediment (< 63 µm). The first approach, the direct measurement method, uses total organic carbon as a tracer and yielded AE values ranging from 45.9~50.4%. The second approach, the dual-labeled method, uses 51Cr as a non-assimilated tracer and did not yield AE values for our data. The inability of the dual-labeled approach to estimate AEs was due, in part, to the selective feeding by Diporeia resulting in a failure of the non-assimilated tracer (51Cr) to track with the assimilated tracer ([14C]BaP). The failure of the dual-labeled approach was not a result of an uneven distribution of the labels among particle size classes, but more likely resulted from differential sorption of the two isotopically labeled materials to particles of differing composition. The [14C]BaP apparently sorbs to organic particles that are selectively ingested, while the 51Cr apparently sorbs to particles which are selectively excluded by Diporeia. The dual-labeled approach would be a viable and easier experimental approach for determining AE values if the characteristics that govern selective feeding can be determined.

  13. Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

    NASA Astrophysics Data System (ADS)

    Zhu, Zhikai; Go, Eden P.; Desaire, Heather

    2014-06-01

    N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

  14. Catalytic-site design for inverse heavy-enzyme isotope effects in human purine nucleoside phosphorylase

    PubMed Central

    Harijan, Rajesh K.; Zoi, Ioanna; Antoniou, Dimitri; Schwartz, Steven D.; Schramm, Vern L.

    2017-01-01

    Heavy-enzyme isotope effects (15N-, 13C-, and 2H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect (kchem light/kchem heavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect (kchem light/kchem heavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre–steady-state chemistry was slowed 32-fold in F159Y PNP. Pre–steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage. PMID:28584087

  15. DNA Hybridization: Nonradioactive Labeling Now Available for the Laboratory.

    ERIC Educational Resources Information Center

    Freeman, Lenore Gardner

    1984-01-01

    The advantages of DNA hybridization procedures for classroom and clinical use can now be realized by the recent development of nonradioactive DNA labeling/detection procedures. These methods (which are described) can replace the use of isotopes in standard DNA hybridization procedures. (JN)

  16. Isotope-coded ESI-enhancing derivatization reagents for differential analysis, quantification and profiling of metabolites in biological samples by LC/MS: A review.

    PubMed

    Higashi, Tatsuya; Ogawa, Shoujiro

    2016-10-25

    The analysis of the qualitative and quantitative changes of metabolites in body fluids and tissues yields valuable information for the diagnosis, pathological analysis and treatment of many diseases. Recently, liquid chromatography/electrospray ionization-(tandem) mass spectrometry [LC/ESI-MS(/MS)] has been widely used for these purposes due to the high separation capability of LC, broad coverage of ESI for various compounds and high specificity of MS(/MS). However, there are still two major problems to be solved regarding the biological sample analysis; lack of sensitivity and limited availability of stable isotope-labeled analogues (internal standards, ISs) for most metabolites. Stable isotope-coded derivatization (ICD) can be the answer for these problems. By the ICD, different isotope-coded moieties are introduced to the metabolites and one of the resulting derivatives can serve as the IS, which minimize the matrix effects. Furthermore, the derivatization can improve the ESI efficiency, fragmentation property in the MS/MS and chromatographic behavior of the metabolites, which lead to a high sensitivity and specificity in the various detection modes. Based on this background, this article reviews the recently-reported isotope-coded ESI-enhancing derivatization (ICEED) reagents, which are key components for the ICD-based LC/MS(/MS) studies, and their applications to the detection, identification, quantification and profiling of metabolites in human and animal samples. The LC/MS(/MS) using the ICEED reagents is the powerful method especially for the differential analysis (relative quantification) of metabolites in two comparative samples, simultaneous quantification of multiple metabolites whose stable isotope-labeled ISs are not available, and submetabolome profiling. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Exploiting Diffusion Barrier and Chemical Affinity of Metal-Organic Frameworks for Efficient Hydrogen Isotope Separation.

    PubMed

    Kim, Jin Yeong; Balderas-Xicohténcatl, Rafael; Zhang, Linda; Kang, Sung Gu; Hirscher, Michael; Oh, Hyunchul; Moon, Hoi Ri

    2017-10-25

    Deuterium plays a pivotal role in industrial and scientific research, and is irreplaceable for various applications such as isotope tracing, neutron moderation, and neutron scattering. In addition, deuterium is a key energy source for fusion reactions. Thus, the isolation of deuterium from a physico-chemically almost identical isotopic mixture is a seminal challenge in modern separation technology. However, current commercial approaches suffer from extremely low separation efficiency (i.e., cryogenic distillation, selectivity of 1.5 at 24 K), requiring a cost-effective and large-scale separation technique. Herein, we report a highly effective hydrogen isotope separation system based on metal-organic frameworks (MOFs) having the highest reported separation factor as high as ∼26 at 77 K by maximizing synergistic effects of the chemical affinity quantum sieving (CAQS) and kinetic quantum sieving (KQS). For this purpose, the MOF-74 system having high hydrogen adsorption enthalpies due to strong open metal sites is chosen for CAQS functionality, and imidazole molecules (IM) are employed to the system for enhancing the KQS effect. To the best of our knowledge, this work is not only the first attempt to implement two quantum sieving effects, KQS and CAQS, in one system, but also provides experimental validation of the utility of this system for practical industrial usage by isolating high-purity D 2 through direct selective separation studies using 1:1 D 2 /H 2 mixtures.

  18. Metabolic flux analysis using 13C peptide label measurements

    USDA-ARS?s Scientific Manuscript database

    13C metabolic flux analysis (MFA) has become the experimental method of choice to investigate cellular metabolism. MFA has established flux maps of central metabolism for dozens of microbes, cell cultures, and plant seeds. Steady-state MFA utilizes isotopic labeling measurements of amino acids obtai...

  19. Shape-Controlled Synthesis of Isotopic Yttrium-90-Labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging.

    PubMed

    Paik, Taejong; Chacko, Ann-Marie; Mikitsh, John L; Friedberg, Joseph S; Pryma, Daniel A; Murray, Christopher B

    2015-09-22

    Isotopically labeled nanomaterials have recently attracted much attention in biomedical research, environmental health studies, and clinical medicine because radioactive probes allow the elucidation of in vitro and in vivo cellular transport mechanisms, as well as the unambiguous distribution and localization of nanomaterials in vivo. In addition, nanocrystal-based inorganic materials have a unique capability of customizing size, shape, and composition; with the potential to be designed as multimodal imaging probes. Size and shape of nanocrystals can directly influence interactions with biological systems, hence it is important to develop synthetic methods to design radiolabeled nanocrystals with precise control of size and shape. Here, we report size- and shape-controlled synthesis of rare earth fluoride nanocrystals doped with the β-emitting radioisotope yttrium-90 ((90)Y). Size and shape of nanocrystals are tailored via tight control of reaction parameters and the type of rare earth hosts (e.g., Gd or Y) employed. Radiolabeled nanocrystals are synthesized in high radiochemical yield and purity as well as excellent radiolabel stability in the face of surface modification with different polymeric ligands. We demonstrate the Cerenkov radioluminescence imaging and magnetic resonance imaging capabilities of (90)Y-doped GdF3 nanoplates, which offer unique opportunities as a promising platform for multimodal imaging and targeted therapy.

  20. Chemical Isotope Labeling LC-MS for Monitoring Disease Progression and Treatment in Animal Models: Plasma Metabolomics Study of Osteoarthritis Rat Model

    NASA Astrophysics Data System (ADS)

    Chen, Deying; Su, Xiaoling; Wang, Nan; Li, Yunong; Yin, Hua; Li, Liang; Li, Lanjuan

    2017-01-01

    We report a chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) method generally applicable for tracking metabolomic changes from samples collected in an animal model for studying disease development and treatment. A rat model of surgically induced osteoarthritis (OA) was used as an example to illustrate the workflow and technical performance. Experimental duplicate analyses of 234 plasma samples were carried out using dansylation labeling LC-MS targeting the amine/phenol submetabolome. These samples composed of 39 groups (6 rats per group) were collected at multiple time points with sham operation, OA control group, and OA rats with treatment, separately, using glucosamine/Celecoxib and three traditional Chinese medicines (Epimedii folium, Chuanxiong Rhizoma and Bushen-Huoxue). In total, 3893 metabolites could be detected and 2923 of them were consistently detected in more than 50% of the runs. This high-coverage submetabolome dataset could be used to track OA progression and treatment. Many differentiating metabolites were found and 11 metabolites including 2-aminoadipic acid, saccharopine and GABA were selected as potential biomarkers of OA progression and OA treatment. This study illustrates that CIL LC-MS is a very useful technique for monitoring incremental metabolomic changes with high coverage and accuracy for studying disease progression and treatment in animal models.

  1. Chemical Isotope Labeling LC-MS for Monitoring Disease Progression and Treatment in Animal Models: Plasma Metabolomics Study of Osteoarthritis Rat Model

    PubMed Central

    Chen, Deying; Su, Xiaoling; Wang, Nan; Li, Yunong; Yin, Hua; Li, Liang; Li, Lanjuan

    2017-01-01

    We report a chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) method generally applicable for tracking metabolomic changes from samples collected in an animal model for studying disease development and treatment. A rat model of surgically induced osteoarthritis (OA) was used as an example to illustrate the workflow and technical performance. Experimental duplicate analyses of 234 plasma samples were carried out using dansylation labeling LC-MS targeting the amine/phenol submetabolome. These samples composed of 39 groups (6 rats per group) were collected at multiple time points with sham operation, OA control group, and OA rats with treatment, separately, using glucosamine/Celecoxib and three traditional Chinese medicines (Epimedii folium, Chuanxiong Rhizoma and Bushen-Huoxue). In total, 3893 metabolites could be detected and 2923 of them were consistently detected in more than 50% of the runs. This high-coverage submetabolome dataset could be used to track OA progression and treatment. Many differentiating metabolites were found and 11 metabolites including 2-aminoadipic acid, saccharopine and GABA were selected as potential biomarkers of OA progression and OA treatment. This study illustrates that CIL LC-MS is a very useful technique for monitoring incremental metabolomic changes with high coverage and accuracy for studying disease progression and treatment in animal models. PMID:28091618

  2. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant tissue isotope labeling

    USDA-ARS?s Scientific Manuscript database

    Tracing heavy stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O o...

  3. Quantifying plant phenotypes with isotopic labeling and metabolic flux analysis

    USDA-ARS?s Scientific Manuscript database

    Analyses of metabolic flux using stable isotopes in plants have traditionally been restricted to tissues with presumed homogeneous cell populations such as developing seeds, cell suspensions, or cultured roots and root tips. It is now possible to describe these and other more complex tissues such a...

  4. Multiple isotope effects with alternative dinucleotide substrates as a probe of the malic enzyme reaction

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Weiss, P.M.; Urbauer, J.L.; Cleland, W.W.

    1991-06-11

    Deuterium isotope effects and {sup 13}C isotope effects with deuterium- and protium-labeled malate have been obtained for both NAD- and NADP-malic enzymes by using a variety of alternative dinucleotide substrates. With nicotinamide-containing dinucleotides as the oxidizing substrate, the {sup 13}C effect decreases when deuterated malate is the substrate compared to the value obtained with protium-labeled malate. These data are consistent with a stepwise chemical mechanism in which hydride transfer precedes decarboxylation of the oxalacetate intermediate as previously proposed. When dinucleotide substrates such as thio-NAD, 3-nicotinamide rings are used, the {sup 13}C effect increases when deuterated malate is the substrate comparedmore » to the value obtained with protium-labeled malate. These data, at face value, are consistent with a change in mechanism from stepwise to concerted for the oxidative decarboxylation portion of the mechanism. However, the increase in the deuterium isotope effect from 1.5 to 3 with a concomitant decrease in the {sup 13}C isotope effect from 1.034 to 1.003 as the dinucleotide substrate is changed suggests that the reaction may still be stepwise with the non-nicotinamide dinucleotides. A more likely explanation is that a {beta}-secondary {sup 13}C isotope effect accompanies hydride transfer as a result of hyperconjugation of the {beta}-carboxyl of malate as the transition state for the hydride transfer step is approached.« less

  5. Use of a multi-isotope and multi-tracer approach including organic matter isotopes for quantifying nutrient contributions from agricultural vs wastewater sources

    NASA Astrophysics Data System (ADS)

    Kendall, C.; Silva, S. R.; Young, M. B.

    2013-12-01

    While nutrient isotopes are a well-established tool for quantifying nutrients inputs from agricultural vs wastewater treatment plant (WWTP) sources, we have found that combining nutrient isotopes with the C, N, and S isotopic compositions of dissolved and particulate organic matter, as part of a comprehensive multi-isotope and multi-tracer approach, is a much more diagnostic approach. The main reasons why organic matter C-N-S isotopes are a useful adjunct to studies of nutrient sources and biogeochemical processes are that the dissolved and particulate organic matter associated with (1) different kinds of animals (e.g., humans vs cows) often have distinctive isotopic compositions reflecting the different diets of the animals, and (2) the different processes associated with the different land uses (e.g., in the WWTP or associated with different crop types) often result in significant differences in the isotopic compositions of the organics. The analysis of the δ34S of particulate organic matter (POM) and dissolved organic matter (DOM) has been found to be especially useful for distinguishing and quantifying water, nutrient, and organic contributions from different land uses in aquatic systems where much of the organic matter is aquatic in origin. In such environments, the bacteria and algae incorporate S from sulfate and sulfide that is isotopically labeled by the different processes associated with different land uses. We have found that there is ~35 permil range in δ34S of POM along the river-estuary continuum in the San Joaquin/Sacramento River basin, with low values associated with sulfate reduction in the upstream wetlands and high values associated with tidal inputs of marine water into the estuary. Furthermore, rice agriculture results in relatively low δ34S values whereas WWTP effluent in the Sacramento River produces distinctly higher values than upstream of the WWTP, presumably because SO2 is used to treat chlorinated effluent. The fish living

  6. An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

    PubMed

    Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

    2013-06-07

    In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies.

  7. Consensus structures of the Mo(v) sites of sulfite-oxidizing enzymes derived from variable frequency pulsed EPR spectroscopy, isotopic labelling and DFT calculations.

    PubMed

    Enemark, John H

    2017-10-10

    Sulfite-oxidizing enzymes from eukaryotes and prokaryotes have five-coordinate distorted square-pyramidal coordination about the molybdenum atom. The paramagnetic Mo(v) state is easily generated, and over the years four distinct CW EPR spectra have been identified, depending upon enzyme source and the reaction conditions, namely high and low pH (hpH and lpH), phosphate inhibited (P i ) and sulfite (or blocked). Extensive studies of these paramagnetic forms of sulfite-oxidizing enzymes using variable frequency pulsed electron spin echo (ESE) spectroscopy, isotopic labeling and density functional theory (DFT) calculations have led to the consensus structures that are described here. Errors in some of the previously proposed structures are corrected.

  8. A new combined method of stable isotope-labeling derivatization-ultrasound-assisted dispersive liquid-liquid microextraction for the determination of neurotransmitters in rat brain microdialysates by ultra high performance liquid chromatography tandem mass spectrometry.

    PubMed

    Zheng, Longfang; Zhao, Xian-En; Zhu, Shuyun; Tao, Yanduo; Ji, Wenhua; Geng, Yanling; Wang, Xiao; Chen, Guang; You, Jinmao

    2017-06-01

    In this work, for the first time, a new hyphenated technique of stable isotope-labeling derivatization-ultrasound-assisted dispersive liquid-liquid microextraction has been developed for the simultaneous determination of monoamine neurotransmitters (MANTs) and their biosynthesis precursors and metabolites. The developed method was based on ultra high performance liquid chromatography tandem mass spectrometry detection using multiple-reaction monitoring mode. A pair of mass spectrometry sensitizing reagents, d 0 -10-methyl-acridone-2-sulfonyl chloride and d 3 -10-methyl-acridone-2-sulfonyl chloride, as stable isotope probes was utilized to facilely label neurotransmitters, respectively. The heavy labeled MANTs standards were prepared and used as internal standards for quantification to minimize the matrix effects in mass spectrometry analysis. Low toxic bromobenzene (extractant) and acetonitrile (dispersant) were utilized in microextraction procedure. Under the optimized conditions, good linearity was observed with the limits of detection (S/N>3) and limits of quantification (S/N>10) in the range of 0.002-0.010 and 0.015-0.040nmol/L, respectively. Meanwhile, it also brought acceptable precision (4.2-8.8%, peak area RSDs %) and accuracy (recovery, 96.9-104.1%) results. This method was successfully applied to the simultaneous determination of monoamine neurotransmitters and their biosynthesis precursors and metabolites in rat brain microdialysates of Parkinson's disease and normal rats. This provided a new method for the neurotransmitters related studies in the future. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Quantitative Proteomics by Metabolic Labeling of Model Organisms*

    PubMed Central

    Gouw, Joost W.; Krijgsveld, Jeroen; Heck, Albert J. R.

    2010-01-01

    In the biological sciences, model organisms have been used for many decades and have enabled the gathering of a large proportion of our present day knowledge of basic biological processes and their derailments in disease. Although in many of these studies using model organisms, the focus has primarily been on genetics and genomics approaches, it is important that methods become available to extend this to the relevant protein level. Mass spectrometry-based proteomics is increasingly becoming the standard to comprehensively analyze proteomes. An important transition has been made recently by moving from charting static proteomes to monitoring their dynamics by simultaneously quantifying multiple proteins obtained from differently treated samples. Especially the labeling with stable isotopes has proved an effective means to accurately determine differential expression levels of proteins. Among these, metabolic incorporation of stable isotopes in vivo in whole organisms is one of the favored strategies. In this perspective, we will focus on methodologies to stable isotope label a variety of model organisms in vivo, ranging from relatively simple organisms such as bacteria and yeast to Caenorhabditis elegans, Drosophila, and Arabidopsis up to mammals such as rats and mice. We also summarize how this has opened up ways to investigate biological processes at the protein level in health and disease, revealing conservation and variation across the evolutionary tree of life. PMID:19955089

  10. Isotope Tales: Remaining Problems, Unsolvable Questions, and Gentle Successes

    NASA Astrophysics Data System (ADS)

    fogel, marilyn; bradley, christina; newsome, seth; filipp, fabian

    2014-05-01

    Earth's biomes function and adapt today as climate changes and ecosystems and the organisms within them adapt. Stable isotope biogeochemistry has had a major influence in understanding climate perturbations and continues to be an active area of research on many fronts. Banking on the success of compound specific stable isotope analyses of amino acids, nitrogen, carbon, and hydrogen isotopes continue to reveal subtle shifts in oceanic food webs and metabolic changes in microbes, plants, and animals. A biochemical understanding of exactly how organisms process and partition stable isotopes during metabolism remains unsolved, but is required if this field is to move beyond description to quantitation. Although the patterns of carbon and nitrogen isotopes are fairly well established in the common amino acids, we need to consider specifics: How do shifting metabolic pathways (metabolomics) influence the outcome of stable isotope partitioning? What influence does the gut microflora in animals have on isotopic labeling? What are the intramolecular isotope patterns of common amino acids and what do they tell us? What can be learned with other isotope systems, such as hydrogen? Results and ideas of how to move forward in this field will be presented starting at the molecular level and ending with ecosystems.

  11. Capillary absorption spectrometer and process for isotopic analysis of small samples

    DOEpatents

    Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.; Moran, James J.; Newburn, Matthew K.; Blake, Thomas A.

    2016-03-29

    A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The method also permits sampling in vivo permitting real-time ambient studies of microbial communities.

  12. Capillary absorption spectrometer and process for isotopic analysis of small samples

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Alexander, M. Lizabeth; Kelly, James F.; Sams, Robert L.

    A capillary absorption spectrometer and process are described that provide highly sensitive and accurate stable absorption measurements of analytes in a sample gas that may include isotopologues of carbon and oxygen obtained from gas and biological samples. It further provides isotopic images of microbial communities that allow tracking of nutrients at the single cell level. It further targets naturally occurring variations in carbon and oxygen isotopes that avoids need for expensive isotopically labeled mixtures which allows study of samples taken from the field without modification. The process also permits sampling in vivo permitting real-time ambient studies of microbial communities.

  13. Energy expenditure in space flight (doubly labelled water method) (8-IML-1)

    NASA Technical Reports Server (NTRS)

    Parsons, Howard G.

    1992-01-01

    The objective of the Energy Expenditure in Space Flight (ESS) experiment is to demonstrate and evaluate the doubly labeled water method of measuring the energy expended by crew members during approximately 7 days in microgravity. The doubly labeled water technique determines carbon dioxide production which is then used to calculate energy expenditure. The method relies on the equilibrium between oxygen in respiratory carbon dioxide and oxygen in body water. Because of this equilibrium, the kinetic of water turnover and respiration are interdependent. Under normal conditions, man contains small but significant amounts of deuterium and oxygen 18. Deuterium is eliminated from the body as water while oxygen 18 is eliminated as water and carbon dioxide. The difference in the turnover rates in the two isotopes is proportional to the carbon dioxide production. Deliberately enriching the total body water with both of these isotopes allows the isotope turnovers to be accurately measured in urine, plasma, or saliva samples. The samples are taken to the laboratory for analysis using an ion-ratio spectrometer.

  14. Realistic Fasting Does Not Affect Stable Isotope Levels of a Metabolically Efficient Salamander

    EPA Science Inventory

    Stable isotopes are commonly used to examine various aspects of animal ecology. The use of stable isotopes generally proceeds under the implicit assumption that resource use is the only factor driving variation in stable isotope levels; however, a wealth of studies demonstrate a...

  15. Ideal regularization for learning kernels from labels.

    PubMed

    Pan, Binbin; Lai, Jianhuang; Shen, Lixin

    2014-08-01

    In this paper, we propose a new form of regularization that is able to utilize the label information of a data set for learning kernels. The proposed regularization, referred to as ideal regularization, is a linear function of the kernel matrix to be learned. The ideal regularization allows us to develop efficient algorithms to exploit labels. Three applications of the ideal regularization are considered. Firstly, we use the ideal regularization to incorporate the labels into a standard kernel, making the resulting kernel more appropriate for learning tasks. Next, we employ the ideal regularization to learn a data-dependent kernel matrix from an initial kernel matrix (which contains prior similarity information, geometric structures, and labels of the data). Finally, we incorporate the ideal regularization to some state-of-the-art kernel learning problems. With this regularization, these learning problems can be formulated as simpler ones which permit more efficient solvers. Empirical results show that the ideal regularization exploits the labels effectively and efficiently. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Flow-through SIP - A novel stable isotope probing approach limiting cross-feeding

    NASA Astrophysics Data System (ADS)

    Mooshammer, Maria; Kitzinger, Katharina; Schintlmeister, Arno; Kjedal, Henrik; Nielsen, Jeppe Lund; Nielsen, Per; Wagner, Michael

    2017-04-01

    Stable isotope probing (SIP) is a widely applied tool to link specific microbial populations to metabolic processes in the environment without the prerequisite of cultivation, which has greatly advanced our understanding of the role of microorganisms in biogeochemical cycling. SIP relies on tracing specific isotopically labeled substrates (e.g., 13C, 15N, 18O) into cellular biomarkers, such as DNA, RNA or phospholipid fatty acids, and is considered to be a robust technique to identify microbial populations that assimilate the labeled substrate. However, cross-feeding can occur when labeled metabolites are released from a primary consumer and then used by other microorganisms. This leads to erroneous identification of organisms that are not directly responsible for the process of interest, but are rather connected to primary consumers via a microbial food web. Here, we introduce a new approach that has the potential to eliminate the effect of cross-feeding in SIP studies and can thus also be used to distinguish primary consumers from other members of microbial food webs. In this approach, a monolayer of microbial cells are placed on a filter membrane, and labeled substrates are supplied by a continuous flow. By means of flow-through, labeled metabolites and degradation products are constantly removed, preventing secondary consumption of the substrate. We present results from a proof-of-concept experiment using nitrifiers from activated sludge as model system, in which we used fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes for identification of nitrifiers in combination with nanoscale secondary ion mass spectrometry (NanoSIMS) for visualization of isotope incorporation at the single-cell level. Our results show that flow-through SIP is a promising approach to significantly reduce cross-feeding and secondary substrate consumption in SIP experiments.

  17. Preparation of ⁶⁸Ga-labelled DOTA-peptides using a manual labelling approach for small-animal PET imaging.

    PubMed

    Romero, Eduardo; Martínez, Alfonso; Oteo, Marta; García, Angel; Morcillo, Miguel Angel

    2016-01-01

    (68)Ga-DOTA-peptides are a promising PET radiotracers used in the detection of different tumours types due to their ability for binding specifically receptors overexpressed in these. Furthermore, (68)Ga can be produced by a (68)Ge/(68)Ga generator on site which is a very good alternative to cyclotron-based PET isotopes. Here, we describe a manual labelling approach for the synthesis of (68)Ga-labelled DOTA-peptides based on concentration and purification of the commercial (68)Ga/(68)Ga generator eluate using an anion exchange-cartridge. (68)Ga-DOTA-TATE was used to image a pheochromocytoma xenograft mouse model by a microPET/CT scanner. The method described provides satisfactory results, allowing the subsequent (68)Ga use to label DOTA-peptides. The simplicity of the method along with its implementation reduced cost, makes it useful in preclinical PET studies. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Use of doubly labeled water technique in soldiers training for jungle warfare

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forbes-Ewan, C.H.; Morrissey, B.L.; Gregg, G.C.

    1989-07-01

    The doubly labeled water method was used to estimate the energy expended by four members of an Australian Army platoon (34 soldiers) engaged in training for jungle warfare. Each subject received an oral isotope dose sufficient to raise isotope levels by 200-250 ({sup 18}O) and 100-120 ppm ({sup 2}H). The experimental period was 7 days. Concurrently, a factorial estimate of the energy expenditure of the platoon was conducted. Also, a food intake-energy balance study was conducted for the platoon. Mean daily energy expenditure by the doubly labeled water method was 4,750 kcal (range 4,152-5,394 kcal). The factorial estimate of meanmore » daily energy expenditure was 4,535 kcal. Because of inherent inaccuracies in the food intake-energy balance technique, we were able to conclude only that energy expenditure, as measured by this method, was greater than the estimated mean daily intake of 4,040 kcal. The doubly labeled water technique was well tolerated, is noninvasive, and appears to be suitable in a wide range of field applications.« less

  19. The fate of (13)C-labelled and non-labelled inulin predisposed to large bowel fermentation in rats.

    PubMed

    Butts, Christine A; Paturi, Gunaranjan; Tavendale, Michael H; Hedderley, Duncan; Stoklosinski, Halina M; Herath, Thanuja D; Rosendale, Douglas; Roy, Nicole C; Monro, John A; Ansell, Juliet

    2016-04-01

    The fate of stable-isotope (13)C labelled and non-labelled inulin catabolism by the gut microbiota was assessed in a healthy rat model. Sprague-Dawley male rats were randomly assigned to diets containing either cellulose or inulin, and were fed these diets for 3 days. On day (d) 4, rats allocated to the inulin diet received (13)C-labelled inulin. The rats were then fed the respective non-labelled diets (cellulose or inulin) until sampling (d4, d5, d6, d7, d10 and d11). Post feeding of (13)C-labelled substrate, breath analysis showed that (13)C-inulin cleared from the host within a period of 36 hours. Faecal (13)C demonstrated the clearance of inulin from gut with a (13)C excess reaching maximum at 24 hours (d5) and then declining gradually. There were greater variations in caecal organic acid concentrations from d4 to d6, with higher concentrations of acetic, butyric and propionic acids observed in the rats fed inulin compared to those fed cellulose. Inulin influenced caecal microbial glycosidase activity, increased colon crypt depth, and decreased the faecal output and polysaccharide content compared to the cellulose diet. In summary, the presence of inulin in the diet positively influenced large bowel microbial fermentation.

  20. Novel methodology for labelling mesoporous silica nanoparticles using the 18F isotope and their in vivo biodistribution by positron emission tomography

    NASA Astrophysics Data System (ADS)

    Rojas, Santiago; Gispert, Juan Domingo; Menchón, Cristina; Baldoví, Herme G.; Buaki-Sogo, Mireia; Rocha, Milagros; Abad, Sergio; Victor, Victor Manuel; García, Hermenegildo; Herance, José Raúl

    2015-03-01

    Nanoparticles have been proposed for several biomedical applications due to their potential as drug carriers, diagnostic and therapeutic agents. However, only a few of them have been approved for their use in humans. In order to gauge the potential applicability of a specific type of nanoparticle, in vivo biodistribution studies to characterize their pharmacokinetic properties are essential. In this regard, mesoporous silica nanoparticles (30-130 nm) have been functionalized with amino groups in order to react with N-succinimidyl 4-[18F]fluorobenzoate and thus anchor the 18F positron emission isotope by using a novel and easy labelling strategy. In vivo biodistribution was characterized in mice after intravenous administration of radiolabelled nanoparticles by positron emission tomography. Our results indicated that radiolabelled mesoporous silica nanoparticles were excreted into bile and urine and accumulated mainly in the organs of the reticuloendothelial system and lungs.

  1. Determination of Multiple φ-Torsion Angles in Proteins by Selective and Extensive 13C Labeling and Two-Dimensional Solid-State NMR

    NASA Astrophysics Data System (ADS)

    Hong, Mei

    1999-08-01

    We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive 13C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective 13C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-13C]glucose preferentially labels the ends of the side chains, while [2-13C]glycerol labels the Cα of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles φ simultaneously, using an isotropic-anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively 13C labeled protein were performed using 15N-13C 2D correlation spectroscopy. From the time dependence of the 15N-13C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective 13C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.

  2. Evaluation of Drosophila metabolic labeling strategies for in vivo quantitative proteomic analyses with applications to early pupa formation and amino acid starvation.

    PubMed

    Chang, Ying-Che; Tang, Hong-Wen; Liang, Suh-Yuen; Pu, Tsung-Hsien; Meng, Tzu-Ching; Khoo, Kay-Hooi; Chen, Guang-Chao

    2013-05-03

    Although stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics was first developed as a cell culture-based technique, stable isotope-labeled amino acids have since been successfully introduced in vivo into select multicellular model organisms by manipulating the feeding diets. An earlier study by others has demonstrated that heavy lysine labeled Drosophila melanogaster can be derived by feeding with an exclusive heavy lysine labeled yeast diet. In this work, we have further evaluated the use of heavy lysine and/or arginine for metabolic labeling of fruit flies, with an aim to determine its respective quantification accuracy and versatility. In vivo conversion of heavy lysine and/or heavy arginine to several nonessential amino acids was observed in labeled flies, leading to distorted isotope pattern and underestimated heavy to light ratio. These quantification defects can nonetheless be rectified at protein level using the normalization function. The only caveat is that such a normalization strategy may not be suitable for every biological application, particularly when modified peptides need to be individually quantified at peptide level. In such cases, we showed that peptide ratios calculated from the summed intensities of all isotope peaks are less affected by the heavy amino acid conversion and therefore less sequence-dependent and more reliable. Applying either the single Lys8 or double Lys6/Arg10 metabolic labeling strategy to flies, we quantitatively mapped the proteomic changes during the onset of metamorphosis and upon amino acid deprivation. The expression of a number of steroid hormone 20-hydroxyecdysone regulated proteins was found to be changed significantly during larval-pupa transition, while several subunits of the V-ATPase complex and components regulating actomyosin were up-regulated under starvation-induced autophagy conditions.

  3. Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope-assisted labeling.

    PubMed

    Haviland, Julia A; Tonelli, Marco; Haughey, Dermot T; Porter, Warren P; Assadi-Porter, Fariba M

    2012-08-01

    Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, type 2 diabetes mellitus, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy and cavity ring-down spectroscopy to analyze serial plasma samples and real-time breath measurements following selective (13)C-isotope-assisted labeling. These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. This novel diagnostics approach is fast, noninvasive, and sensitive for determining specific pathway utilization, and provides a direct translational application in the health care field. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Measurement of endogenous versus exogenous formaldehyde-induced DNA-protein crosslinks in animal tissues by stable isotope labeling and ultrasensitive mass spectrometry

    PubMed Central

    Lai, Yongquan; Yu, Rui; Hartwell, Hadley J.; Moeller, Benjamin C.; Bodnar, Wanda M.; Swenberg, James A.

    2016-01-01

    DNA-protein crosslinks (DPCs) arise from a wide range of endogenous and exogenous chemicals, such as chemotherapeutic drugs and formaldehyde. Importantly, recent identification of aldehydes as endogenous genotoxins in Fanconi anemia has provided new insight into disease causation. Due to their bulky nature, DPCs pose severe threats to genome stability, but previous methods to measure formaldehyde-induced DPCs were incapable of discriminating between endogenous and exogenous sources of chemical. In this study, we developed methods that provide accurate and distinct measurements of both exogenous and endogenous DPCs in a structurally-specific manner. We exposed experimental animals to stable isotope-labeled formaldehyde ([13CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous (13CD2-labeled) DPCs. We found that exogenous DPCs readily accumulated in nasal respiratory tissues, but were absent in tissues distant to the site of contact. This observation together with the finding that endogenous formaldehyde-induced DPCs were present in all tissues examined suggests that endogenous DPCs may be responsible for increased risks of bone marrow toxicity and leukemia. Furthermore, the slow rate of DPC repair provided evidence for persistence of DPCs. In conclusion, our method for measuring endogenous and exogenous DPCs presents a new perspective for the potential health risks inflicted by endogenous formaldehyde, and may inform improved disease prevention and treatment strategies. PMID:26984759

  5. Influence of 13C isotopic labeling location of 13C DNP of acetate using TEMPO free radical

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Lumata, Lloyd

    2015-03-01

    Dynamic nuclear polarization (DNP) via the dissolution method enhances the liquid-state magnetic resonance (NMR or MRI) signals of insensitive nuclear spins by at least 10,000-fold. The basis for all these signal enhancements at room temperature is the polarization transfer from the electrons to nuclear spins at cryogenic temperature and high magnetic field. In this work, we have studied the influence of the location of 13C isotopic labeling on the DNP of sodium acetate at 3.35 T and 1.4 K using a wide ESR linewidth free radical 4-oxo-TEMPO. The carbonyl [1-13C]acetate spins produced a polarization level that is almost twice that of the methyl [2-13C]acetate spins. On the other hand, the polarization of the methyl 13C spins doubled to reach the level of [1-13C]acetate when the methyl group was deuterated. Meanwhile, the solid-state nuclear relaxation of these samples are the same and do not correlate with the polarization levels. These behavior implies that the nuclear relaxation for these samples is dominated by the contribution from the free radicals and the polarization levels can be explained by a thermodynamic picture of DNP.

  6. Global combined precursor isotopic labeling and isobaric tagging (cPILOT) approach with selective MS(3) acquisition.

    PubMed

    Evans, Adam R; Robinson, Renã A S

    2013-11-01

    Recently, we reported a novel proteomics quantitation scheme termed "combined precursor isotopic labeling and isobaric tagging (cPILOT)" that allows for the identification and quantitation of nitrated peptides in as many as 12-16 samples in a single experiment. cPILOT offers enhanced multiplexing and posttranslational modification specificity, however excludes global quantitation for all peptides present in a mixture and underestimates reporter ion ratios similar to other isobaric tagging methods due to precursor co-isolation. Here, we present a novel chemical workflow for cPILOT that can be used for global tagging of all peptides in a mixture. Specifically, through low pH precursor dimethylation of tryptic or LysC peptides followed by high pH tandem mass tags, the same reporter ion can be used twice in a single experiment. Also, to improve triple-stage mass spectrometry (MS(3) ) data acquisition, a selective MS(3) method that focuses on product selection of the y1 fragment of lysine-terminated peptides is incorporated into the workflow. This novel cPILOT workflow has potential for global peptide quantitation that could lead to enhanced sample multiplexing and increase the number of quantifiable spectra obtained from MS(3) acquisition methods. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. An efficient climate model with water isotope physics: NEEMY

    NASA Astrophysics Data System (ADS)

    Hu, J.; Emile-Geay, J.

    2015-12-01

    This work describes the development of an isotope-enabled atmosphere-ocean global climate model, NEEMY. This is a model of intermediate complexity, which can run 100 model years in 30 hours using 33 CPUs. The atmospheric component is the SPEEDY-IER (Molteni et al. 2003; Dee et al. 2015a), which is a water isotope-enabled (with equilibrium and kinetic fractionation schemes in precipitation, evaporation and soil moisture) simplified atmospheric general circulation model, with T30 horizontal resolution and 8 vertical layers. The oceanic component is NEMO 3.4 (Madec 2008), a state-of-the-art oceanic model (~2° horizontal resolution and 31 vertical layers) with an oceanic isotope module (a passive tracer scheme). A 1000-year control run shows that NEEMY is stable and its energy is conserved. The mean state is comparable to that of CMIP3-era CGCMs, though much cheaper to run. Atmospheric teleconnections such as the NAO and PNA are simulated very well. NEEMY also simulates the oceanic meridional overturning circulation well. The tropical climate variability is weaker than observations, and the climatology exhibits a double ITCZ problem despite bias corrections. The standard deviation of the monthly mean Nino3.4 index is 0.61K, compared to 0.91K in observations (Reynolds et al. 2002). We document similarities and differences with a close cousin, SPEEDY-NEMO (Kucharski et al. 2015). With its fast speed and relatively complete physical processes, NEEMY is suitable for paleoclimate studies ; we will present some forced simulations of the past millennium and their use in forward-modeling climate proxies, via proxy system models (PSMs, Dee et al 2015b). References Dee, S., D. Noone, N. Buenning, J. Emile-Geay, and Y. Zhou, 2015a: SPEEDY-IER: A fast atmospheric GCM with water isotope physics. J. Geophys. Res. Atmos., 120: 73-91. doi:10.1002/2014JD022194. Dee, S. G., J. Emile-Geay, M. N. Evans, Allam, A., D. M. Thompson, and E. J. Steig, 2015b: PRYSM: an open-source framework

  8. LABELLING OF NUCLEIC ACID WITH RADIOACTIVE ISOTOPES AND THEIR APPLICATION (in Yugoslavian)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Becarevic, A.; Hudnik-Plevnik, T.; Glisin, V.

    The DNA of the liver and spleen of rats was labeled with P/sup 32/. The specific activity obtained was high enough to be able to follow the fate of these acids after injection in irradiated and non-irradiated rats. The results show that, after injection in the rats, the labeled kids were degraded into small fragmnts, which were utilized as building blocks for the synthesis of the big molecules of the cells. (auth)

  9. Enhanced sensitivity of DNA- and rRNA-based stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.

    PubMed

    Lueders, Tillmann; Manefield, Mike; Friedrich, Michael W

    2004-01-01

    Stable isotope probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.

  10. Metabolic De-Isotoping for Improved LC-MS Characterization of Modified RNAs

    NASA Astrophysics Data System (ADS)

    Wetzel, Collin; Li, Siwei; Limbach, Patrick A.

    2014-07-01

    Mapping, sequencing, and quantifying individual noncoding ribonucleic acids (ncRNAs), including post-transcriptionally modified nucleosides, by mass spectrometry is a challenge that often requires rigorous sample preparation prior to analysis. Previously, we have described a simplified method for the comparative analysis of RNA digests (CARD) that is applicable to relatively complex mixtures of ncRNAs. In the CARD approach for transfer RNA (tRNA) analysis, two complete sets of digestion products from total tRNA are compared using the enzymatic incorporation of 16O/18O isotopic labels. This approach allows one to rapidly screen total tRNAs from gene deletion mutants or comparatively sequence total tRNA from two related bacterial organisms. However, data analysis can be challenging because of convoluted mass spectra arising from the natural 13C and 15 N isotopes present in the ribonuclease-digested tRNA samples. Here, we demonstrate that culturing in 12C-enriched/13C-depleted media significantly reduces the isotope patterns that must be interpreted during the CARD experiment. Improvements in data quality yield a 35 % improvement in detection of tRNA digestion products that can be uniquely assigned to particular tRNAs. These mass spectral improvements lead to a significant reduction in data processing attributable to the ease of spectral identification of labeled digestion products and will enable improvements in the relative quantification of modified RNAs by the 16O/18O differential labeling approach.

  11. Isotopically Nonstationary Metabolic Flux Analysis (INST-MFA) of Photosynthesis and Photorespiration in Plants.

    PubMed

    Ma, Fangfang; Jazmin, Lara J; Young, Jamey D; Allen, Doug K

    2017-01-01

    Photorespiration is a central component of photosynthesis; however to better understand its role it should be viewed in the context of an integrated metabolic network rather than a series of individual reactions that operate independently. Isotopically nonstationary 13 C metabolic flux analysis (INST-MFA), which is based on transient labeling studies at metabolic steady state, offers a comprehensive platform to quantify plant central metabolism. In this chapter, we describe the application of INST-MFA to investigate metabolism in leaves. Leaves are an autotrophic tissue, assimilating CO 2 over a diurnal period implying that the metabolic steady state is limited to less than 12 h and thus requiring an INST-MFA approach. This strategy results in a comprehensive unified description of photorespiration, Calvin cycle, sucrose and starch synthesis, tricarboxylic acid (TCA) cycle, and amino acid biosynthetic fluxes. We present protocols of the experimental aspects for labeling studies: transient 13 CO 2 labeling of leaf tissue, sample quenching and extraction, mass spectrometry (MS) analysis of isotopic labeling data, measurement of sucrose and amino acids in vascular exudates, and provide details on the computational flux estimation using INST-MFA.

  12. Learning classification models with soft-label information.

    PubMed

    Nguyen, Quang; Valizadegan, Hamed; Hauskrecht, Milos

    2014-01-01

    Learning of classification models in medicine often relies on data labeled by a human expert. Since labeling of clinical data may be time-consuming, finding ways of alleviating the labeling costs is critical for our ability to automatically learn such models. In this paper we propose a new machine learning approach that is able to learn improved binary classification models more efficiently by refining the binary class information in the training phase with soft labels that reflect how strongly the human expert feels about the original class labels. Two types of methods that can learn improved binary classification models from soft labels are proposed. The first relies on probabilistic/numeric labels, the other on ordinal categorical labels. We study and demonstrate the benefits of these methods for learning an alerting model for heparin induced thrombocytopenia. The experiments are conducted on the data of 377 patient instances labeled by three different human experts. The methods are compared using the area under the receiver operating characteristic curve (AUC) score. Our AUC results show that the new approach is capable of learning classification models more efficiently compared to traditional learning methods. The improvement in AUC is most remarkable when the number of examples we learn from is small. A new classification learning framework that lets us learn from auxiliary soft-label information provided by a human expert is a promising new direction for learning classification models from expert labels, reducing the time and cost needed to label data.

  13. Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli.

    PubMed

    Delli-Bovi, Teegan A; Spalding, Maroya D; Prigge, Sean T

    2010-10-11

    Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography.

  14. Effects of sample injection amount and time-of-flight mass spectrometric detection dynamic range on metabolome analysis by high-performance chemical isotope labeling LC-MS.

    PubMed

    Zhou, Ruokun; Li, Liang

    2015-04-06

    The effect of sample injection amount on metabolome analysis in a chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) platform was investigated. The performance of time-of-flight (TOF) mass spectrometers with and without a high-dynamic-range (HD) detection system was compared in the analysis of (12)C2/(13)C2-dansyl labeled human urine samples. An average of 1635 ± 21 (n = 3) peak pairs or putative metabolites was detected using the HD-TOF-MS, compared to 1429 ± 37 peak pairs from a conventional or non-HD TOF-MS. In both instruments, signal saturation was observed. However, in the HD-TOF-MS, signal saturation was mainly caused by the ionization process, while in the non-HD TOF-MS, it was caused by the detection process. To extend the MS detection range in the non-HD TOF-MS, an automated switching from using (12)C to (13)C-natural abundance peaks for peak ratio calculation when the (12)C peaks are saturated has been implemented in IsoMS, a software tool for processing CIL LC-MS data. This work illustrates that injecting an optimal sample amount is important to maximize the metabolome coverage while avoiding the sample carryover problem often associated with over-injection. A TOF mass spectrometer with an enhanced detection dynamic range can also significantly increase the number of peak pairs detected. In chemical isotope labeling (CIL) LC-MS, relative metabolite quantification is done by measuring the peak ratio of a (13)C2-/(12)C2-labeled peak pair for a given metabolite present in two comparative samples. The dynamic range of peak ratio measurement does not need to be very large, as only subtle changes of metabolite concentrations are encountered in most metabolomic studies where relative metabolome quantification of different groups of samples is performed. However, the absolute concentrations of different metabolites can be very different, requiring a technique to provide a wide detection dynamic range to allow the detection of as

  15. An Overview of Advanced SILAC-Labeling Strategies for Quantitative Proteomics.

    PubMed

    Terzi, F; Cambridge, S

    2017-01-01

    Comparative, quantitative mass spectrometry of proteins provides great insight to protein abundance and function, but some molecular characteristics related to protein dynamics are not so easily obtained. Because the metabolic incorporation of stable amino acid isotopes allows the extraction of distinct temporal and spatial aspects of protein dynamics, the SILAC methodology is uniquely suited to be adapted for advanced labeling strategies. New SILAC strategies have emerged that allow deeper foraging into the complexity of cellular proteomes. Here, we review a few advanced SILAC-labeling strategies that have been published during last the years. Among them, different subsaturating-labeling as well as dual-labeling schemes are most prominent for a range of analyses including those of neuronal proteomes, secretion, or cell-cell-induced stimulations. These recent developments suggest that much more information can be gained from proteomic analyses if the labeling strategies are specifically tailored toward the experimental design. © 2017 Elsevier Inc. All rights reserved.

  16. Primary deuterium and tritium isotope effects upon V/K in the liver alcohol dehydrogenase reaction with ethanol.

    PubMed

    Damgaard, S E

    1981-09-29

    The primary isotope effect upon V/K when ethanol stereospecifically labeled with deuterium or tritium is oxidized by liver alcohol dehydrogenase has been measured between pH 6 and 9. The deuterium isotope effect was obtained with high reproducibility by the use of two different radioactive tracers, viz. 14C and 3H, to follow the rate of acetaldehyde formation from deuterium-labeled ethanol and normal ethanol, respectively. Synthesis of the necessary labeled compounds is described in this and earlier work referred to. V/K isotope effects for both tritium and deuterium have been measured with three different coenzymes, NAD+, thio-NAD+, and acetyl-NAD+. With NAD+ at pH 7, D(V/K) was 3.0 and T(V/K) was 6.5. With increasing pH, these values decreased to 1.5 and 2.5 at pH 9. The intrinsic isotope effect evaluated by the method of Northrop [Northrop, D.B. (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M, H., & Northrop, D. B., Eds.) pp 112-152, University Park Press, Baltimore] varies little with pH. It amounts to about 10 with NAD+ and about 5 with the coenzyme analogues. Commitment functions and their dependence upon pH calculated in this connection appear to be in agreement with known kinetic parameters of liver alcohol dehydrogenase. This assay method was also applied in vivo in the rat. Being a noninvasive method because only trace amounts of isotopes are needed, it may yield information about alternative routes of ethanol oxidation in vivo. In naive rats at low concentrations of ethanol, it confirms the discrete role of the non alcohol dehydrogenase systems.

  17. Optimal tracers for parallel labeling experiments and 13C metabolic flux analysis: A new precision and synergy scoring system.

    PubMed

    Crown, Scott B; Long, Christopher P; Antoniewicz, Maciek R

    2016-11-01

    13 C-Metabolic flux analysis ( 13 C-MFA) is a widely used approach in metabolic engineering for quantifying intracellular metabolic fluxes. The precision of fluxes determined by 13 C-MFA depends largely on the choice of isotopic tracers and the specific set of labeling measurements. A recent advance in the field is the use of parallel labeling experiments for improved flux precision and accuracy. However, as of today, no systemic methods exist for identifying optimal tracers for parallel labeling experiments. In this contribution, we have addressed this problem by introducing a new scoring system and evaluating thousands of different isotopic tracer schemes. Based on this extensive analysis we have identified optimal tracers for 13 C-MFA. The best single tracers were doubly 13 C-labeled glucose tracers, including [1,6- 13 C]glucose, [5,6- 13 C]glucose and [1,2- 13 C]glucose, which consistently produced the highest flux precision independent of the metabolic flux map (here, 100 random flux maps were evaluated). Moreover, we demonstrate that pure glucose tracers perform better overall than mixtures of glucose tracers. For parallel labeling experiments the optimal isotopic tracers were [1,6- 13 C]glucose and [1,2- 13 C]glucose. Combined analysis of [1,6- 13 C]glucose and [1,2- 13 C]glucose labeling data improved the flux precision score by nearly 20-fold compared to widely use tracer mixture 80% [1- 13 C]glucose +20% [U- 13 C]glucose. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  18. Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.

    PubMed

    Rajesh, Mathur; Wang, Gang; Jones, Roger; Tretyakova, Natalia

    2005-02-15

    The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene

  19. 18O-labeled proteome reference as global internal standards for targeted quantification by selected reaction monitoring-mass spectrometry.

    PubMed

    Kim, Jong-Seo; Fillmore, Thomas L; Liu, Tao; Robinson, Errol; Hossain, Mahmud; Champion, Boyd L; Moore, Ronald J; Camp, David G; Smith, Richard D; Qian, Wei-Jun

    2011-12-01

    Selected reaction monitoring (SRM)-MS is an emerging technology for high throughput targeted protein quantification and verification in biomarker discovery studies; however, the cost associated with the application of stable isotope-labeled synthetic peptides as internal standards can be prohibitive for screening a large number of candidate proteins as often required in the preverification phase of discovery studies. Herein we present a proof of concept study using an (18)O-labeled proteome reference as global internal standards (GIS) for SRM-based relative quantification. The (18)O-labeled proteome reference (or GIS) can be readily prepared and contains a heavy isotope ((18)O)-labeled internal standard for every possible tryptic peptide. Our results showed that the percentage of heavy isotope ((18)O) incorporation applying an improved protocol was >99.5% for most peptides investigated. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into the labeled mouse plasma reference. Reliable quantification was observed with high reproducibility (i.e. coefficient of variance <10%) for analyte concentrations that were set at 100-fold higher or lower than those of the GIS based on the light ((16)O)/heavy ((18)O) peak area ratios. The utility of (18)O-labeled GIS was further illustrated by accurate relative quantification of 45 major human plasma proteins. Moreover, quantification of the concentrations of C-reactive protein and prostate-specific antigen was illustrated by coupling the GIS with standard additions of purified protein standards. Collectively, our results demonstrated that the use of (18)O-labeled proteome reference as GIS provides a convenient, low cost, and effective strategy for relative quantification of a large number of candidate proteins in biological or clinical samples using SRM.

  20. Fluorinase: a tool for the synthesis of ¹⁸F-labeled sugars and nucleosides for PET.

    PubMed

    Onega, Mayca; Winkler, Margit; O'Hagan, David

    2009-08-01

    There is an increasing interest in the preparation of (18)F-labeled radiopharmaceuticals with potential applications in PET for medicinal imaging. Appropriate synthetic methods require a quick and efficient route in which to incorporate the (18)F into a ligand, due to the relatively short half-life of the (18)F isotope. Enzymatic methods are rare in this area; however, the discovery of a fluorinating enzyme from Streptomyces cattleya (EC 2.5.1.63) has opened up the possibility of the enzymatic synthesis and formation of C-(18)F bonds from the [(18)F]fluoride ion. In this article, the development of enzymatic preparations of (18)F-labeled sugars and nucleosides as potential radiotracers using the fluorinase from S. cattleya for PET applications is reviewed. Enzymatic reactions are not traditional in PET synthesis, but this enzyme has some attractive features. The enzyme is available in an overexpressed form from Escherichia coli and it is relatively stable and can be easily purified and manipulated. Most notably, it utilizes [(18)F] fluoride, the form of the isotope normally generated by the cyclotron and usually in very high specific radioactivity. The disadvantage with the enzyme is that it is substrate specific; however, when the fluorinase is used in combination biotransformations with a second or third enzyme, then a range of radiolabeled nucleosides and ribose sugars can be prepared. The fluorinase enzyme has emerged as a curiosity from biosynthesis studies, but it now has some potential as a new catalyst for (18)F incorporation for PET syntheses. The focus is now on delivering a user-friendly catalyst to the PET synthesis community and establishing a clinical role for some of the (18)F-labeled molecules available using this technology.

  1. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    PubMed

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

  2. Carbon "Quantum" Dots for Fluorescence Labeling of Cells.

    PubMed

    Liu, Jia-Hui; Cao, Li; LeCroy, Gregory E; Wang, Ping; Meziani, Mohammed J; Dong, Yiyang; Liu, Yuanfang; Luo, Pengju G; Sun, Ya-Ping

    2015-09-02

    The specifically synthesized and selected carbon dots of relatively high fluorescence quantum yields were evaluated in their fluorescence labeling of cells. For the cancer cell lines, the cellular uptake of the carbon dots was generally efficient, resulting in the labeling of the cells with bright fluorescence emissions for both one- and two-photon excitations from predominantly the cell membrane and cytoplasm. In the exploration on labeling the live stem cells, the cellular uptake of the carbon dots was relatively less efficient, though fluorescence emissions could still be adequately detected in the labeled cells, with the emissions again predominantly from the cell membrane and cytoplasm. This combined with the observed more efficient internalization of the same carbon dots by the fixed stem cells might suggest some significant selectivity of the stem cells toward surface functionalities of the carbon dots. The needs and possible strategies for more systematic and comparative studies on the fluorescence labeling of different cells, including especially live stem cells, by carbon dots as a new class of brightly fluorescent probes are discussed.

  3. 78 FR 8362 - Energy Labeling Rule

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-06

    ... FEDERAL TRADE COMMISSION 16 CFR Part 305 RIN 3084-AB15] Energy Labeling Rule AGENCY: Federal Trade... furnace or central air conditioner meets applicable Department of Energy regional efficiency standards..., 20580. SUPPLEMENTARY INFORMATION: I. Background The Commission's Energy Labeling Rule (``Rule'') (16 CFR...

  4. 10 CFR 431.30 - Applicability of labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Applicability of labeling requirements. 431.30 Section 431.30 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability of labeling requirements. The...

  5. The anatomy of a laser label

    USDA-ARS?s Scientific Manuscript database

    Laser labeling of fruits and vegetables is an efficient alternative to adhesive tags. The advantages of this system are numerous. In general the label consists of alphanumerical characters formed by laser generated pinhole depressions that penetrate the produce’s surface creating visible markings. H...

  6. Incorporation of {sup 13}C-labeled intermediates into developing lignin revealed by analytical pyrolysis and CuO oxidation in combination with IRM-GC-MS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eglinton, T.I.; Goni, M.A.; Boon, J.J.

    1995-12-31

    Tissue samples from Ginkgo shoots (Ginkgo biloba L.) and Rice grass (Oryzasitiva sp.) incubated in the presence of {sup 13}C-labeled substrates such as coniferin (postulated to be biosynthetic intermediates in lignin biosynthesis) were studied using thermal and chemical dissociation methods in combination with molecular-level isotopic measurements. The aim of the study was (1) to investigate dissociation mechanisms, and (2) to examine and quantify the proportions of labeled material incorporated within each sample. Isotopic analysis of specific dissociation products revealed the presence of the label in its original positions, and only within lignin-derived (phenolic) products. Moreover, the distribution and isotopic compositionmore » of the dissociation products strongly suggest an origin from newly-formed lignin. These results clearly indicate that there is no {open_quotes}scrambling{close_quotes} of carbon atoms as a result of the dissociation process, thereby lending support to this analytical approach. In addition, the data provide confidence in the selective labeling approach for elucidation of the structure and biosynthesis of lignin.« less

  7. Copper-catalyzed oxidative desulfurization-oxygenation of thiocarbonyl compounds using molecular oxygen: an efficient method for the preparation of oxygen isotopically labeled carbonyl compounds.

    PubMed

    Shibahara, Fumitoshi; Suenami, Aiko; Yoshida, Atsunori; Murai, Toshiaki

    2007-06-21

    A novel copper-catalyzed oxidative desulfurization reaction of thiocarbonyl compounds, using molecular oxygen as an oxidant and leading to formation of carbonyl compounds, has been developed, and the utility of the process is demonstrated by its application to the preparation of a carbonyl-18O labeled sialic acid derivative.

  8. Labeled trees and the efficient computation of derivations

    NASA Technical Reports Server (NTRS)

    Grossman, Robert; Larson, Richard G.

    1989-01-01

    The effective parallel symbolic computation of operators under composition is discussed. Examples include differential operators under composition and vector fields under the Lie bracket. Data structures consisting of formal linear combinations of rooted labeled trees are discussed. A multiplication on rooted labeled trees is defined, thereby making the set of these data structures into an associative algebra. An algebra homomorphism is defined from the original algebra of operators into this algebra of trees. An algebra homomorphism from the algebra of trees into the algebra of differential operators is then described. The cancellation which occurs when noncommuting operators are expressed in terms of commuting ones occurs naturally when the operators are represented using this data structure. This leads to an algorithm which, for operators which are derivations, speeds up the computation exponentially in the degree of the operator. It is shown that the algebra of trees leads naturally to a parallel version of the algorithm.

  9. Quantifying dispersal rates and distances in North American martens: a test of enriched isotope labeling

    Treesearch

    Jonathan N. Pauli; Winston P. Smith; Merav Ben-David

    2012-01-01

    Advances in the application of stable isotopes have allowed the quantitative evaluation of previously cryptic ecological processes. In particular, researchers have utilized the predictable spatial patterning in natural abundance of isotopes to better understand animal dispersal and migration. However, quantifying dispersal via natural abundance alone has proven to be...

  10. Peptides Labeled with Pyridinium Salts for Sensitive Detection and Sequencing by Electrospray Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Waliczek, Mateusz; Kijewska, Monika; Rudowska, Magdalena; Setner, Bartosz; Stefanowicz, Piotr; Szewczuk, Zbigniew

    2016-11-01

    Mass spectrometric analysis of trace amounts of peptides may be problematic due to the insufficient ionization efficiency resulting in limited sensitivity. One of the possible ways to overcome this problem is the application of ionization enhancers. Herein we developed new ionization markers based on 2,4,6-triphenylpyridinium and 2,4,6-trimethylpyridinium salts. Using of inexpensive and commercially available pyrylium salt allows selective derivatization of primary amino groups, especially those sterically unhindered, such as ɛ-amino group of lysine. The 2,4,6-triphenylpyridinium modified peptides generate in MS/MS experiments an abundant protonated 2,4,6-triphenylpyridinium ion. This fragment is a promising reporter ion for the multiple reactions monitoring (MRM) analysis. In addition, the fixed positive charge of the pyridinium group enhances the ionization efficiency. Other advantages of the proposed ionization enhancers are the simplicity of derivatization of peptides and the possibility of convenient incorporation of isotopic labels into derivatized peptides.

  11. Efficient organ localization using multi-label convolutional neural networks in thorax-abdomen CT scans

    NASA Astrophysics Data System (ADS)

    Efrain Humpire-Mamani, Gabriel; Arindra Adiyoso Setio, Arnaud; van Ginneken, Bram; Jacobs, Colin

    2018-04-01

    Automatic localization of organs and other structures in medical images is an important preprocessing step that can improve and speed up other algorithms such as organ segmentation, lesion detection, and registration. This work presents an efficient method for simultaneous localization of multiple structures in 3D thorax-abdomen CT scans. Our approach predicts the location of multiple structures using a single multi-label convolutional neural network for each orthogonal view. Each network takes extra slices around the current slice as input to provide extra context. A sigmoid layer is used to perform multi-label classification. The output of the three networks is subsequently combined to compute a 3D bounding box for each structure. We used our approach to locate 11 structures of interest. The neural network was trained and evaluated on a large set of 1884 thorax-abdomen CT scans from patients undergoing oncological workup. Reference bounding boxes were annotated by human observers. The performance of our method was evaluated by computing the wall distance to the reference bounding boxes. The bounding boxes annotated by the first human observer were used as the reference standard for the test set. Using the best configuration, we obtained an average wall distance of 3.20~+/-~7.33 mm in the test set. The second human observer achieved 1.23~+/-~3.39 mm. For all structures, the results were better than those reported in previously published studies. In conclusion, we proposed an efficient method for the accurate localization of multiple organs. Our method uses multiple slices as input to provide more context around the slice under analysis, and we have shown that this improves performance. This method can easily be adapted to handle more organs.

  12. Measurement of Endogenous versus Exogenous Formaldehyde-Induced DNA-Protein Crosslinks in Animal Tissues by Stable Isotope Labeling and Ultrasensitive Mass Spectrometry.

    PubMed

    Lai, Yongquan; Yu, Rui; Hartwell, Hadley J; Moeller, Benjamin C; Bodnar, Wanda M; Swenberg, James A

    2016-05-01

    DNA-protein crosslinks (DPC) arise from a wide range of endogenous and exogenous chemicals, such as chemotherapeutic drugs and formaldehyde. Importantly, recent identification of aldehydes as endogenous genotoxins in Fanconi anemia has provided new insight into disease causation. Because of their bulky nature, DPCs pose severe threats to genome stability, but previous methods to measure formaldehyde-induced DPCs were incapable of discriminating between endogenous and exogenous sources of chemical. In this study, we developed methods that provide accurate and distinct measurements of both exogenous and endogenous DPCs in a structurally specific manner. We exposed experimental animals to stable isotope-labeled formaldehyde ([(13)CD2]-formaldehyde) by inhalation and performed ultrasensitive mass spectrometry to measure endogenous (unlabeled) and exogenous ((13)CD2-labeled) DPCs. We found that exogenous DPCs readily accumulated in nasal respiratory tissues but were absent in tissues distant to the site of contact. This observation, together with the finding that endogenous formaldehyde-induced DPCs were present in all tissues examined, suggests that endogenous DPCs may be responsible for increased risks of bone marrow toxicity and leukemia. Furthermore, the slow rate of DPC repair provided evidence for the persistence of DPCs. In conclusion, our method for measuring endogenous and exogenous DPCs presents a new perspective for the potential health risks inflicted by endogenous formaldehyde and may inform improved disease prevention and treatment strategies. Cancer Res; 76(9); 2652-61. ©2016 AACR. ©2016 American Association for Cancer Research.

  13. Development and implementation of energy efficiency standards and labeling programs in China: Progress and challenges

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhou, Nan; Khanna, Nina Zheng; Fridley, David

    Over the last twenty years, with growing policy emphasis on improving energy efficiency and reducing environmental pollution and carbon emissions, China has implemented a series of new minimum energy performance standards (MEPS) and mandatory and voluntary energy labels to improve appliance energy efficiency. As China begins planning for the next phase of standards and labeling (S&L) program development under the 12th Five Year Plan, an evaluation of recent program developments and future directions is needed to identify gaps that still exist when compared with international best practices. The review of China’s S&L program development and implementation in comparison with majormore » findings from international experiences reveal that there are still areas of improvement, particularly when compared to success factors observed across leading international S&L program. China currently lacks a formalized regulatory process for standard-setting and do not have any legal or regulatory guidance on elements of S&L development such as stakeholder participation or the issue of legal precedence between conflicting national, industrial and local standards. Consequently, China’s laws regarding standard-setting and management of the mandatory energy label program could be updated, as they have not been amended or revised recently and no longer reflects the current situation. While China uses similar principles for choosing target products as the U.S., Australia, EU and Japan, including high energy-consumption, mature industry and testing procedure and stakeholder support, recent MEPS revisions have generally aimed at only eliminating the bottom 20% efficiency of the market. Setting a firm principle based on maximizing energy savings that are technically feasible and economically justified may help improve the stringency of China’s MEPS program and reduce the need for frequent revisions. China also lacks robust survey data and relies primarily on market research data in

  14. Qualitative metabolome analysis of human cerebrospinal fluid by 13C-/12C-isotope dansylation labeling combined with liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry.

    PubMed

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by (13)C-dansyl and (12)C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner. © American Society for Mass Spectrometry, 2011

  15. Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

  16. On-target labeling of intracellular metabolites combined with chemical mapping of individual hyphae revealing cytoplasmic relocation of isotopologues.

    PubMed

    Hu, Jie-Bi; Chen, Yu-Chie; Urban, Pawel L

    2012-06-05

    A microscale analytical platform integrating microbial cell culture, isotopic labeling, along with visual and mass spectrometric imaging with single-cell resolution has been developed and applied in the monitoring of cellular metabolism in fungal mycelium. The method implements open chips with a two-dimensional surface pattern composed of hydrophobic and hydrophilic zones. Two hydrophilic islands are used as medium reservoirs, while the hydrophobic area constitutes the support for the growing aerial hyphae, which do not have direct contact with the medium. The first island, containing (12)C(6)-glucose medium, was initially inoculated with the mycelium (Neurospora crassa), and following the initial incubation period, the hyphae progressed toward the second medium island, containing an isotopically labeled substrate ((13)C(6)-glucose). The (13)C atoms were gradually incorporated into cellular metabolites, which was revealed by MALDI-MS. The fate of the chitin-biosynthesis precursor, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), was monitored by recording mass spectra with characteristic isotopic patterns, which indicated the presence of various (12)C/(13)C isotopologues. The method enabled mapping the (13)C-labeled UDP-GlcNAc in fungal mycelium and recording its redistribution in hyphae, directly on the chip.

  17. Evidencing 98 secondary metabolites of Penicillium verrucosum using substrate isotopic labeling and high-resolution mass spectrometry.

    PubMed

    Hautbergue, Thaïs; Puel, Olivier; Tadrist, Souria; Meneghetti, Lauriane; Péan, Michel; Delaforge, Marcel; Debrauwer, Laurent; Oswald, Isabelle P; Jamin, Emilien L

    2017-12-15

    Industrial applications of fungal compounds, coupled with the emergence of fungal threats to natural ecosystems and public health, have increased interest in filamentous fungi. Among all pathogenic fungi, Penicillium verrucosum is one of the most common mold-infecting stored cereals in temperate regions. However, it is estimated that 80% of fungal secondary metabolites remain unknown. To detect new P. verrucosum compounds, an untargeted metabolomic approach was applied to fungus grown on wheat grains labeled with stable isotopes: (i) natural grains (99% 12 C); (ii) grains enriched with 97% of 13 C; and (iii) grains enriched with 53% of 13 C and 97% of 15 N. Analyses performed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) enabled the specific detection of fungal metabolites, and the unambiguous characterization of their chemical formulas. In this way, 98 secondary metabolites were detected and their chemical formulas were determined. Of these, only 18 identifications could be made based on databases, the literature and mass spectrometry fragmentation experiments, with the result that 80 were totally unknown. Molecular networks were generated to analyze these results, leading to the characterization by MS n experiments of a new fungisporin produced by P. verrucosum. More generally, this article provides precise mass spectrometric data about all these compounds for further studies of the Penicillium metabolome. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Profiling and relative quantification of phosphatidylethanolamine based on acetone stable isotope derivatization.

    PubMed

    Wang, Xiang; Wei, Fang; Xu, Ji-Qu; Lv, Xin; Dong, Xu-Yan; Han, Xianlin; Quek, Siew-Young; Huang, Feng-Hong; Chen, Hong

    2016-01-01

    Phosphatidylethanolamine (PE) is considered to be one of the pivotal lipids for normal cellular function as well as disease initiation and progression. In this study, a simple, efficient, reliable, and inexpensive method for the qualitative analysis and relative quantification of PE, based on acetone stable isotope derivatization combined with double neutral loss scan-shotgun electrospray ionization tandem-quadrupole mass spectrometry analysis (ASID-DNLS-Shotgun ESI-MS/MS), was developed. The ASID method led to alkylation of the primary amino groups of PE with an isopropyl moiety. The use of acetone (d0-acetone) and deuterium-labeled acetone (d6-acetone) introduced a 6 Da mass shift that was ideally suited for relative quantitative analysis, and enhanced sensitivity for mass analysis. The DNLS model was introduced to simultaneously analyze the differential derivatized PEs by shotgun ESI-MS/MS with high selectivity and accuracy. The reaction specificity, labeling efficiency, and linearity of the ASID method were thoroughly evaluated in this study. Its excellent applicability was validated by qualitative and relative quantitative analysis of PE species presented in liver samples from rats fed different diets. Using the ASID-DNLS-Shotgun ESI-MS/MS method, 45 PE species from rat livers have been identified and quantified in an efficient manner. The level of total PEs tended to decrease in the livers of rats on high fat diets compared with controls. The levels of PE 32:1, 34:3, 34:2, 36:3, 36:2, 42:10, plasmalogen PE 36:1 and lyso PE 22:6 were significantly reduced, while levels of PE 36:1 and lyso PE 16:0 increased. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry.

    PubMed

    Wei, Guor-Jien; Sheen, Jenn-Feng; Lu, Wen-Chien; Hwang, Lucy Sun; Ho, Chi-Tang; Lin, Ching-I

    2013-05-29

    Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate.

  20. Utilizing Stable Isotopes and Isotopic Anomalies to Study Early Solar System Formation Processes

    NASA Technical Reports Server (NTRS)

    Simon, Justin

    2017-01-01

    Chondritic meteorites contain a diversity of particle components, i.e., chondrules and calcium-, aluminum-rich refractory inclusions (CAIs), that have survived since the formation of the Solar System. The chemical and isotopic compositions of these materials provide a record of the conditions present in the protoplanetary disk where they formed and can aid our understanding of the processes and reservoirs in which solids formed in the solar nebula, an important step leading to the accretion of planetesimals. Isotopic anomalies associated with nucleosynthetic processes are observed in these discrete materials, and can be compared to astronomical observations and astrophysical formation models of stars and more recently proplyds. The existence and size of these isotopic anomalies are typically thought to reflect a significant state of isotopic heterogeneity in the earliest Solar System, likely left over from molecular cloud heterogeneities on the grain scale, but some could also be due to late stellar injection. The homogenization of these isotopic anomalies towards planetary values can be used to track the efficiency and timescales of disk wide mixing,

  1. Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

    PubMed

    Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

    2010-01-01

    Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

  2. Comparative proteomics of two life cycle stages of stable isotope-labeled Trypanosoma brucei reveals novel components of the parasite's host adaptation machinery.

    PubMed

    Butter, Falk; Bucerius, Ferdinand; Michel, Margaux; Cicova, Zdenka; Mann, Matthias; Janzen, Christian J

    2013-01-01

    Trypanosoma brucei developed a sophisticated life cycle to adapt to different host environments. Although developmental differentiation of T. brucei has been the topic of intensive research for decades, the mechanisms responsible for adaptation to different host environments are not well understood. We developed stable isotope labeling by amino acids in cell culture in trypanosomes to compare the proteomes of two different life cycle stages. Quantitative comparison of 4364 protein groups identified many proteins previously not known to be stage-specifically expressed. The identification of stage-specific proteins helps to understand how parasites adapt to different hosts and provides new insights into differences in metabolism, gene regulation, and cell architecture. A DEAD-box RNA helicase, which is highly up-regulated in the bloodstream form of this parasite and which is essential for viability and proper cell cycle progression in this stage is described as an example.

  3. Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

    PubMed

    Giménez, Estela; Balmaña, Meritxell; Figueras, Joan; Fort, Esther; de Bolós, Carme; Sanz-Nebot, Victòria; Peracaula, Rosa; Rizzi, Andreas

    2015-03-25

    In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for

  4. Novel and non-traditional use of stable isotope tracers to study metal bioavailability from natural particles

    USGS Publications Warehouse

    Croteau, Marie-Noële; Cain, Daniel J.; Fuller, Christopher C.

    2013-01-01

    We devised a novel tracing approach that involves enriching test organisms with a stable metal isotope of low natural abundance prior to characterizing metal bioavailability from natural inorganic particles. In addition to circumventing uncertainties associated with labeling natural particles and distinguishing background metals, the proposed "reverse labeling" technique overcomes many drawbacks inherent to using radioisotope tracers. Specifically, we chronically exposed freshwater snails (Lymnaea stagnalis) to synthetic water spiked with Cu that was 99.4% 65Cu to increase the relative abundance of 65Cu in the snail’s tissues from 32% to >80%. The isotopically enriched snails were then exposed to benthic algae mixed with Cu-bearing Fe–Al particles collected from the Animas River (Colorado), an acid mine drainage impacted river. We used 63Cu to trace Cu uptake from the natural particles and inferred their bioavailability from calculation of Cu assimilation into tissues. Cu assimilation from these particles was 44%, indicating that 44% of the particulate Cu was absorbed by the invertebrate. This demonstrates that inorganic particulate Cu can be bioavailable. The reverse labeling approach shows great potential in various scientific areas such as environmental contamination and nutrition for addressing questions involving uptake of an element that naturally has multiple isotopes.

  5. Selective detection of carbon-13, nitrogen-15, and deuterium labeled metabolites by capillary gas chromatography-chemical reaction interface/mass spectrometry

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chace, D.H.; Abramson, F.P.

    1989-12-15

    We have applied a new chemical reaction interface/mass spectrometer technique (CRIMS) to the selective detection of 13C-, 15N-, and 2H-labeled phenytoin and its metabolites in urine following separation by capillary gas chromatography. The microwave-powered chemical reaction interface converts materials from their original forms into small molecules whose mass spectra serve to identify and quantify the nuclides that make up each analyte. The presence of each element is followed by monitoring the isotopic variants of CO2, NO, or H2 that are produced by the chemical reaction interface. Chromatograms showing only enriched 13C and 15N were produced by subtracting the abundance ofmore » naturally occurring isotopes from the observed M + 1 signal. A selective chromatogram of 2H (D) was obtained by measuring HD at m/z 3.0219 with a resolution of 2000. Metabolites representing less than 1.5% of the total labeled compounds could be identified in the chromatogram. Detection limits from urine of 380 pg/mL of a 15N-labeled metabolite, 7 ng/mL of a 13C-labeled metabolite, and 16 ng/mL of a deuterium labeled metabolite were determined at a signal to noise ratio of 2. Depending on the isotope examined, a linear dynamic range of 250-1000 was observed using CRIMS. To identify many of these labeled peaks (metabolites), the chromatographic analysis was repeated with the chemical reaction interface turned off and mass spectra obtained at the retention times found in the CRIMS experiment. CRIMS is a new analytical method that appears to be particularly useful for metabolism studies.« less

  6. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach

    PubMed Central

    Bennett, Bryson D; Yuan, Jie; Kimball, Elizabeth H; Rabinowitz, Joshua D

    2009-01-01

    This protocol provides a method for quantitating the intracellular concentrations of endogenous metabolites in cultured cells. The cells are grown in stable isotope-labeled media to near-complete isotopic enrichment and then extracted in organic solvent containing unlabeled internal standards in known concentrations. The ratio of endogenous metabolite to internal standard in the extract is determined using mass spectrometry (MS). The product of this ratio and the unlabeled standard amount equals the amount of endogenous metabolite present in the cells. The cellular concentration of the metabolite can then be calculated on the basis of intracellular volume of the extracted cells. The protocol is exemplified using Escherichia coli and primary human fibroblasts fed uniformly with 13C-labeled carbon sources, with detection of 13C-assimilation by liquid chromatography–tandem MS. It enables absolute quantitation of several dozen metabolites over ~1 week of work. PMID:18714298

  7. NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans*

    PubMed Central

    Rhoads, Timothy W.; Prasad, Aman; Kwiecien, Nicholas W.; Merrill, Anna E.; Zawack, Kelson; Westphall, Michael S.; Schroeder, Frank C.; Kimble, Judith; Coon, Joshua J.

    2015-01-01

    The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress. PMID:26392051

  8. A diagram retrieval method with multi-label learning

    NASA Astrophysics Data System (ADS)

    Fu, Songping; Lu, Xiaoqing; Liu, Lu; Qu, Jingwei; Tang, Zhi

    2015-01-01

    In recent years, the retrieval of plane geometry figures (PGFs) has attracted increasing attention in the fields of mathematics education and computer science. However, the high cost of matching complex PGF features leads to the low efficiency of most retrieval systems. This paper proposes an indirect classification method based on multi-label learning, which improves retrieval efficiency by reducing the scope of compare operation from the whole database to small candidate groups. Label correlations among PGFs are taken into account for the multi-label classification task. The primitive feature selection for multi-label learning and the feature description of visual geometric elements are conducted individually to match similar PGFs. The experiment results show the competitive performance of the proposed method compared with existing PGF retrieval methods in terms of both time consumption and retrieval quality.

  9. Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation

    PubMed Central

    Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.

    2012-01-01

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP®) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known “click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC “click chemistry” for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥ 98%. The subsequent CuI catalyzed “click” reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 5–20% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative

  10. Bidirectional reaction steps in metabolic networks: I. Modeling and simulation of carbon isotope labeling experiments.

    PubMed

    Wiechert, W; de Graaf, A A

    1997-07-05

    The extension of metabolite balancing with carbon labeling experiments, as described by Marx et al. (Biotechnol. Bioeng. 49: 11-29), results in a much more detailed stationary metabolic flux analysis. As opposed to basic metabolite flux balancing alone, this method enables both flux directions of bidirectional reaction steps to be quantitated. However, the mathematical treatment of carbon labeling systems is much more complicated, because it requires the solution of numerous balance equations that are bilinear with respect to fluxes and fractional labeling. In this study, a universal modeling framework is presented for describing the metabolite and carbon atom flux in a metabolic network. Bidirectional reaction steps are extensively treated and their impact on the system's labeling state is investigated. Various kinds of modeling assumptions, as usually made for metabolic fluxes, are expressed by linear constraint equations. A numerical algorithm for the solution of the resulting linear constrained set of nonlinear equations is developed. The numerical stability problems caused by large bidirectional fluxes are solved by a specially developed transformation method. Finally, the simulation of carbon labeling experiments is facilitated by a flexible software tool for network synthesis. An illustrative simulation study on flux identifiability from available flux and labeling measurements in the cyclic pentose phosphate pathway of a recombinant strain of Zymomonas mobilis concludes this contribution.

  11. Variability in mesophyll conductance between barley genotypes, and effects on transpiration efficiency and carbon isotope discrimination.

    PubMed

    Barbour, Margaret M; Warren, Charles R; Farquhar, Graham D; Forrester, Guy; Brown, Hamish

    2010-07-01

    Leaf internal, or mesophyll, conductance to CO(2) (g(m)) is a significant and variable limitation of photosynthesis that also affects leaf transpiration efficiency (TE). Genotypic variation in g(m) and the effect of g(m) on TE were assessed in six barley genotypes (four Hordeum vulgare and two H. bulbosum). Significant variation in g(m) was found between genotypes, and was correlated with photosynthetic rate. The genotype with the highest g(m) also had the highest TE and the lowest carbon isotope discrimination as recorded in leaf tissue (Delta(p)). These results suggest g(m) has unexplored potential to provide TE improvement within crop breeding programmes.

  12. Doubly labelled water assessment of energy expenditure: principle, practice, and promise.

    PubMed

    Westerterp, Klaas R

    2017-07-01

    The doubly labelled water method for the assessment of energy expenditure was first published in 1955, application in humans started in 1982, and it has become the gold standard for human energy requirement under daily living conditions. The method involves enriching the body water of a subject with heavy hydrogen ( 2 H) and heavy oxygen ( 18 O), and then determining the difference in washout kinetics between both isotopes, being a function of carbon dioxide production. In practice, subjects get a measured amount of doubly labelled water ( 2 H 2 18 O) to increase background enrichment of body water for 18 O of 2000 ppm with at least 180 ppm and background enrichment of body water for 2 H of 150 ppm with 120 ppm. Subsequently, the difference between the apparent turnover rates of the hydrogen and oxygen of body water is assessed from blood-, saliva-, or urine samples, collected at the start and end of the observation interval of 1-3 weeks. Samples are analyzed for 18 O and 2 H with isotope ratio mass spectrometry. The doubly labelled water method is the indicated method to measure energy expenditure in any environment, especially with regard to activity energy expenditure, without interference with the behavior of the subjects. Applications include the assessment of energy requirement from total energy expenditure, validation of dietary assessment methods and validation of physical activity assessment methods with doubly labelled water measured energy expenditure as reference, and studies on body mass regulation with energy expenditure as a determinant of energy balance.

  13. Manifold regularized matrix completion for multi-label learning with ADMM.

    PubMed

    Liu, Bin; Li, Yingming; Xu, Zenglin

    2018-05-01

    Multi-label learning is a common machine learning problem arising from numerous real-world applications in diverse fields, e.g, natural language processing, bioinformatics, information retrieval and so on. Among various multi-label learning methods, the matrix completion approach has been regarded as a promising approach to transductive multi-label learning. By constructing a joint matrix comprising the feature matrix and the label matrix, the missing labels of test samples are regarded as missing values of the joint matrix. With the low-rank assumption of the constructed joint matrix, the missing labels can be recovered by minimizing its rank. Despite its success, most matrix completion based approaches ignore the smoothness assumption of unlabeled data, i.e., neighboring instances should also share a similar set of labels. Thus they may under exploit the intrinsic structures of data. In addition, the matrix completion problem can be less efficient. To this end, we propose to efficiently solve the multi-label learning problem as an enhanced matrix completion model with manifold regularization, where the graph Laplacian is used to ensure the label smoothness over it. To speed up the convergence of our model, we develop an efficient iterative algorithm, which solves the resulted nuclear norm minimization problem with the alternating direction method of multipliers (ADMM). Experiments on both synthetic and real-world data have shown the promising results of the proposed approach. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Pharmacokinetics of stable isotopically labeled L-histidine in humans and the assessment of in vivo histidine ammonia lyase activities.

    PubMed

    Furuta, T; Okamiya, K; Shibasaki, H; Kasuya, Y

    1996-01-01

    The pharmacokinetics of L-histidine in humans has been investigated to evaluate the in vivo histidine ammonia lyase system for the conversion of L-histidine to urocanic acid. Two healthy volunteers (subjects A and B) received a single 100-mg oral dose of L-[3,3-2H2,1',3'-15N2]histidine. Blood and urine samples were obtained over 24 hr after the administration and analyzed by stable isotope dilution ms. Labeled L-histidine was rapidly absorbed, and a maximum plasma concentration of L-histidine was observed at 30 min (1057.6 ng/ml) in subject A and at 60 min (1635.6 ng/ml) in subject B after oral administration. Pharmacokinetic parameters were calculated based on a two-compartment model. Labeled L-histidine in subject A (t1/2 = 1.0 hr) was eliminated approximately twice faster than that in subject B (t1/2 = 1.9 hr). Total body clearances were 70.0 liters/hr in subject A and 30.0 liters/hr in subject B. The low ratios of the renal clearance to the total body clearance (1.04% for subject A and 0.43% for subject B) indicated that most of L-histidine was eliminated via the nonrenal processes. L-Histidine was rapidly metabolized to urocanic acid. Maximum plasma concentrations of urocanic acid were 59.61 ng/ml at 30 min for subject A and 46.10 ng/ml at 60 min for subject B. The slope of the plot of urinary excretion rate of urocanic acid vs. the plasma concentration of unchanged L-histidine was demonstrated to reflect the metabolic clearance of L-histidine to urocanic acid. The method of evaluating the in vivo human histidine ammonia lyase activities discussed in this study offers a significant value with regard to the biochemical and clinical elucidations of the heterogeneity of histidinemia.

  15. Perspective: next generation isotope-aided methods for protein NMR spectroscopy.

    PubMed

    Kainosho, Masatsune; Miyanoiri, Yohei; Terauchi, Tsutomu; Takeda, Mitsuhiro

    2018-06-22

    In this perspective, we describe our efforts to innovate the current isotope-aided NMR methodology to investigate biologically important large proteins and protein complexes, for which only limited structural information could be obtained by conventional NMR approaches. At the present time, it is widely believed that only backbone amide and methyl signals are amenable for investigating such difficult targets. Therefore, our primary mission is to disseminate our novel knowledge within the biological NMR community; specifically, that any type of NMR signals other than methyl and amide groups can be obtained, even for quite large proteins, by optimizing the transverse relaxation properties by isotope labeling methods. The idea of "TROSY by isotope labeling" has been cultivated through our endeavors aiming to improve the original stereo-array isotope labeling (SAIL) method (Kainosho et al., Nature 440:52-57, 2006). The SAIL TROSY methods subsequently culminated in the successful observations of individual NMR signals for the side-chain aliphatic and aromatic 13 CH groups in large proteins, as exemplified by the 82 kDa single domain protein, malate synthase G. Meanwhile, the expected role of NMR spectroscopy in the emerging integrative structural biology has been rapidly shifting, from structure determination to the acquisition of biologically relevant structural dynamics, which are poorly accessible by X-ray crystallography or cryo-electron microscopy. Therefore, the newly accessible NMR probes, in addition to the methyl and amide signals, will open up a new horizon for investigating difficult protein targets, such as membrane proteins and supramolecular complexes, by NMR spectroscopy. We briefly introduce our latest results, showing that the protons attached to 12 C-atoms give profoundly narrow 1 H-NMR signals even for large proteins, by isolating them from the other protons using the selective deuteration. The direct 1 H observation methods exhibit the highest

  16. A label-free and high-efficient GO-based aptasensor for cancer cells based on cyclic enzymatic signal amplification.

    PubMed

    Xiao, Kunyi; Liu, Juan; Chen, Hui; Zhang, Song; Kong, Jilie

    2017-05-15

    A label-free and high-efficient graphene oxide (GO)-based aptasensor was developed for the detection of low quantity cancer cells based on cell-triggered cyclic enzymatic signal amplification (CTCESA). In the absence of target cells, hairpin aptamer probes (HAPs) and dye-labeled linker DNAs stably coexisted in solution, and the fluorescence was quenched by the GO-based FÖrster resonance energy transfer (FRET) process. In the presence of target cells, the specific binding of HAPs with the target cells triggered a conformational alternation, which resulted in linker DNA complementary pairing and cleavage by nicking endonuclease-strand scission cycles. Consequently, more cleaved fragments of linker DNAs with more the terminal labeled dyes could show the enhanced fluorescence because these cleaved DNA fragments hardly combine with GOs and prevent the FRET process. Fluorescence analysis demonstrated that this GO-based aptasensor exhibited selective and sensitive response to the presence of target CCRF-CEM cells in the concentration range from 50 to 10 5 cells. The detection limit of this method was 25 cells, which was approximately 20 times lower than the detection limit of normal fluorescence aptasensors without amplification. With high sensitivity and specificity, it provided a simple and cost-effective approach for early cancer diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Digestive selection underlies differential utilization of phytoplankton and sedimentary organics by infaunal bivalves: Experiments with cockles (Cerastoderma edule) using cross-labelled mixed diets.

    PubMed

    Navarro, Enrique; Méndez, Soco; Urrutia, Miren Begoñe; Arambalza, Udane; Ibarrola, Irrintzi

    2016-09-01

    Differential utilization of phytoplankton and detrital particles present in natural sediments of mud-flats was studied in a series of experiments performed on the infaunal bivalve Cerastoderma edule. In order to assess digestive selection, parameters of food processing (organic ingestion rate: OIR, gross absorption efficiency: GAE and gut passage time: GPT) were recorded for each organic component in different combinations of food particles radio-labelled with (14)C. Experimental design included the use of both labelled diets of a sole organic component and cross-labelled diets; i.e., mixed suspensions presenting alternatively labelled one of the various components tested: phytoplankton cells, sedimentary organic particles and particulate detritus from vascular salt-marsh plants. Preferential absorption of phytoplankton was accounted for by absorption efficiency values that were two-fold those for sedimentary detritus when recorded with mixed diets of both organic components. Two factors contributed to this difference: a) higher digestibility of microalgae, measured as the ratio of GAE to GPT, and b) faster gut passage of detrital particles that results from digestive selection likely involving the preferential incorporation of phytoplankton into the digestive gland. However, when diets based on a sole organic component (either phytoplankton or detritus) were compared, larger GPT were recorded for detrital particles that enabled improving GAE of this rather refractory food. Overall results of these experiments are consistent with most studies in trophic ecology based on stable isotopes enrichment, concerning both the diversity of trophic sources used by marine bivalves and its preferential utilization of phytoplankton over phyto-detritus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. 15N-labeled glycine synthesis.

    PubMed

    Tavares, Claudinéia R O; Bendassolli, José A; Coelho, Fernando; Sant'ana Filho, Carlos R; Prestes, Clelber V

    2006-09-01

    This work describes a method for 15N-isotope-labeled glycine synthesis, as well as details about a recovery line for nitrogen residues. To that effect, amination of alpha-haloacids was performed, using carboxylic chloroacetic acid and labeled aqueous ammonia (15NH3). Special care was taken to avoid possible 15NH3 losses, since its production cost is high. In that respect, although the purchase cost of the 13N-labeled compound (radioactive) is lower, the stable tracer produced constitutes an important tool for N cycling studies in living organisms, also minimizing labor and environmental hazards, as well as time limitation problems in field studies. The tests were carried out with three replications, and variable 15NH3aq volumes in the reaction were used (50, 100, and 150 mL), in order to calibrate the best operational condition; glycine masses obtained were 1.7, 2, and 3.2 g, respectively. With the development of a system for 15NH3 recovery, it was possible to recover 71, 83, and 87% of the ammonia initially used in the synthesis. With the required adaptations, the same system was used to recover methanol, and 75% of the methanol initially used in the amino acid purification process were recovered.

  19. Quantitative proteome analysis using isobaric peptide termini labeling (IPTL).

    PubMed

    Arntzen, Magnus O; Koehler, Christian J; Treumann, Achim; Thiede, Bernd

    2011-01-01

    The quantitative comparison of proteome level changes across biological samples has become an essential feature in proteomics that remains challenging. We have recently introduced isobaric peptide termini labeling (IPTL), a novel strategy for isobaric quantification based on the derivatization of peptide termini with complementary isotopically labeled reagents. Unlike non-isobaric quantification methods, sample complexity at the MS level is not increased, providing improved sensitivity and protein coverage. The distinguishing feature of IPTL when comparing it to more established isobaric labeling methods (iTRAQ and TMT) is the presence of quantification signatures in all sequence-determining ions in MS/MS spectra, not only in the low mass reporter ion region. This makes IPTL a quantification method that is accessible to mass spectrometers with limited capabilities in the low mass range. Also, the presence of several quantification points in each MS/MS spectrum increases the robustness of the quantification procedure.

  20. Software LS-MIDA for efficient mass isotopomer distribution analysis in metabolic modelling.

    PubMed

    Ahmed, Zeeshan; Zeeshan, Saman; Huber, Claudia; Hensel, Michael; Schomburg, Dietmar; Münch, Richard; Eisenreich, Wolfgang; Dandekar, Thomas

    2013-07-09

    The knowledge of metabolic pathways and fluxes is important to understand the adaptation of organisms to their biotic and abiotic environment. The specific distribution of stable isotope labelled precursors into metabolic products can be taken as fingerprints of the metabolic events and dynamics through the metabolic networks. An open-source software is required that easily and rapidly calculates from mass spectra of labelled metabolites, derivatives and their fragments global isotope excess and isotopomer distribution. The open-source software "Least Square Mass Isotopomer Analyzer" (LS-MIDA) is presented that processes experimental mass spectrometry (MS) data on the basis of metabolite information such as the number of atoms in the compound, mass to charge ratio (m/e or m/z) values of the compounds and fragments under study, and the experimental relative MS intensities reflecting the enrichments of isotopomers in 13C- or 15 N-labelled compounds, in comparison to the natural abundances in the unlabelled molecules. The software uses Brauman's least square method of linear regression. As a result, global isotope enrichments of the metabolite or fragment under study and the molar abundances of each isotopomer are obtained and displayed. The new software provides an open-source platform that easily and rapidly converts experimental MS patterns of labelled metabolites into isotopomer enrichments that are the basis for subsequent observation-driven analysis of pathways and fluxes, as well as for model-driven metabolic flux calculations.

  1. Simultaneous determination of creatinine and creatine in human serum by double-spike isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS).

    PubMed

    Fernández-Fernández, Mario; Rodríguez-González, Pablo; Añón Álvarez, M Elena; Rodríguez, Felix; Menéndez, Francisco V Álvarez; García Alonso, J Ignacio

    2015-04-07

    This work describes the first multiple spiking isotope dilution procedure for organic compounds using (13)C labeling. A double-spiking isotope dilution method capable of correcting and quantifying the creatine-creatinine interconversion occurring during the analytical determination of both compounds in human serum is presented. The determination of serum creatinine may be affected by the interconversion between creatine and creatinine during sample preparation or by inefficient chemical separation of those compounds by solid phase extraction (SPE). The methodology is based on the use differently labeled (13)C analogues ((13)C1-creatinine and (13)C2-creatine), the measurement of the isotopic distribution of creatine and creatinine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the application of multiple linear regression. Five different lyophilized serum-based controls and two certified human serum reference materials (ERM-DA252a and ERM-DA253a) were analyzed to evaluate the accuracy and precision of the proposed double-spike LC-MS/MS method. The methodology was applied to study the creatine-creatinine interconversion during LC-MS/MS and gas chromatography-mass spectrometry (GC-MS) analyses and the separation efficiency of the SPE step required in the traditional gas chromatography-isotope dilution mass spectrometry (GC-IDMS) reference methods employed for the determination of serum creatinine. The analysis of real serum samples by GC-MS showed that creatine-creatinine separation by SPE can be a nonquantitative step that may induce creatinine overestimations up to 28% in samples containing high amounts of creatine. Also, a detectable conversion of creatine into creatinine was observed during sample preparation for LC-MS/MS. The developed double-spike LC-MS/MS improves the current state of the art for the determination of creatinine in human serum by isotope dilution mass spectrometry (IDMS), because corrections are made for all the possible errors

  2. Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard.

    PubMed

    Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

    2015-04-15

    Protein NP24 is a thaumatin-like protein contained in tomato (Lycopersicon esculentum Mill.). This protein is reported to be a putative tomato allergen and is listed as a food allergen in Structural Database of Allergenic Proteins (SDAP). In this research, we developed the quantitative analysis of NP24 by employing the protein absolute quantification (AQUA) technology composed of stable isotope-labelled internal standard (SIIS) peptide (GQTWVINAPR[(13)C6,(15)N4]) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). A linear relationship (r(2)>0.99) was found throughout the concentration range (2.0-500 fmol/μL). The coefficients of variation (CVs) measured on each of the five days when NP24 contained in the tomato skin was analysed did not exceed 13%. Our developed assay of NP24 will contribute to the allergological examination of tomato and its derived products. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Biomedical research applications of electromagnetically separated enriched stable isotopes

    NASA Astrophysics Data System (ADS)

    Lambrecht, R. M.

    The current and projected annual requirements through 1985 for stable isotopes enriched by electromagnetic separation methods were reviewed for applications in various types of biomedical research: (1) medical radiosiotope production, labeled compounds, and potential radio-pharmaceuticals; (2) nutrition, food science, and pharmacology: (3) metallobiochemistry and environmental toxicology; (4) nuclear magnetic resonance, electron paramagnetic resonance, and moessbauer spectroscopy in biochemical, biophysical, and biomedical research; and (5) miscellaneous advances in radioactive and nonradioactive tracer technology. Radioisotopes available from commercial sources or routinely used in clinical nuclear medicine were excluded. Current requirements for enriched stable isotopes in biomedical research are not being satisfied. Severe shortages exist for Mg 26, Ca 43, Zn 70, Se 76, Se 77, Se 78, Pd 102, Cd 111, Cd 113, and Os 190. Many interesting and potentially important investigations in biomedical research require small quantities of specific elements at high isotopic enrichments.

  4. The calibration of the intramolecular nitrogen isotope distribution in nitrous oxide measured by isotope ratio mass spectrometry.

    PubMed

    Westley, Marian B; Popp, Brian N; Rust, Terri M

    2007-01-01

    Two alternative approaches for the calibration of the intramolecular nitrogen isotope distribution in nitrous oxide using isotope ratio mass spectrometry have yielded a difference in the 15N site preference (defined as the difference between the delta15N of the central and end position nitrogen in NNO) of tropospheric N2O of almost 30 per thousand. One approach is based on adding small amounts of labeled 15N2O to the N2O reference gas and tracking the subsequent changes in m/z 30, 31, 44, 45 and 46, and this yields a 15N site preference of 46.3 +/- 1.4 per thousand for tropospheric N2O. The other involves the synthesis of N2O by thermal decomposition of isotopically characterized ammonium nitrate and yields a 15N site preference of 18.7 +/- 2.2 per thousand for tropospheric N2O. Both approaches neglect to fully account for isotope effects associated with the formation of NO+ fragment ions from the different isotopic species of N2O in the ion source of a mass spectrometer. These effects vary with conditions in the ion source and make it impossible to reproduce a calibration based on the addition of isotopically enriched N2O on mass spectrometers with different ion source configurations. These effects have a much smaller impact on the comparison of a laboratory reference gas with N2O synthesized from isotopically characterized ammonium nitrate. This second approach was successfully replicated and leads us to advocate the acceptance of the site preference value 18.7 +/- 2.2 per thousand for tropospheric N2O as the provisional community standard until further independent calibrations are developed and validated. We present a technique for evaluating the isotope effects associated with fragment ion formation and revised equations for converting ion signal ratios into isotopomer ratios. Copyright 2007 John Wiley & Sons, Ltd.

  5. Quantitative Peptidomics with Five-plex Reductive Methylation labels

    NASA Astrophysics Data System (ADS)

    Tashima, Alexandre K.; Fricker, Lloyd D.

    2017-12-01

    Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

  6. Quantitative Peptidomics with Five-plex Reductive Methylation labels

    NASA Astrophysics Data System (ADS)

    Tashima, Alexandre K.; Fricker, Lloyd D.

    2018-05-01

    Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

  7. Stable isotope tracer dilution for quantifying very low-density lipoprotein-triacylglycerol kinetics in man.

    PubMed

    Sidossis, Labros S; Magkos, Faidon; Mittendorfer, Bettina; Wolfe, Robert R

    2004-08-01

    A number of approaches have been employed in the past to measure very low-density lipoprotein (VLDL) triacylglycerol (TG) kinetics in humans in vivo, varying in the selection of tracer and mode of administration. All, however, make use of labeled TG precursors and more or less complicated mathematical models to derive the kinetic parameters of interest. The aim of the present study was to develop a conceptually straightforward method, based on the traditional tracer infusion technique, for quantifying VLDL-TG production rates in man using stable isotopes. Our approach involves ingestion of [U-13C3]glycerol to endogenously label the glycerol in VLDL-TG, plasmapheresis, isolation of the newly 13C-labeled VLDL from plasma, and administration within the next 2-3 days via a primed constant autologous reinfusion. This procedure produces enough tracer for a priming dose plus 2-3 h of infusion. In the physiological conditions examined (basal and hyperglycemic states, fat- and carbohydrate-rich diets), with almost 3-fold ranging VLDL-TG pool sizes, a steady state in plasma VLDL-TG glycerol tracer-to-tracee ratio was readily achieved within 2 h. Consequently, calculations are made according to the isotope dilution principle, thus avoiding assumptions implicit in more complicated models. The stable isotope VLDL-TG tracer dilution method offers an alternative and reliable tool for the determination of endogenous VLDL-TG kinetics in man under a variety of metabolic states. Copyright 2003 Elsevier Ltd.

  8. Preparation of Labeled Aflatoxins with High Specific Activities

    PubMed Central

    Hsieh, D. P. H.; Mateles, R. I.

    1971-01-01

    Resting cells of Aspergillus parasiticus ATCC 15517 were used to prepare highly labeled aflatoxins from labeled acetate. High synthetic activity in growing cells was evidenced only during 40 to 70 hr of incubation. Glucose was required for high incorporation efficiency, whereas the concentration of the labeled acetate determined the specific activity of the product. When labeled acetate was continuously added to maintain a concentration near but not exceeding 10 mm, in a culture containing 30 g of glucose per liter, 2% of its labels could be recovered in the purified aflatoxins which have a specific activity more than three times that of the labeled acetate. PMID:4329435

  9. A Coding Method for Efficient Subgraph Querying on Vertex- and Edge-Labeled Graphs

    PubMed Central

    Zhu, Lei; Song, Qinbao; Guo, Yuchen; Du, Lei; Zhu, Xiaoyan; Wang, Guangtao

    2014-01-01

    Labeled graphs are widely used to model complex data in many domains, so subgraph querying has been attracting more and more attention from researchers around the world. Unfortunately, subgraph querying is very time consuming since it involves subgraph isomorphism testing that is known to be an NP-complete problem. In this paper, we propose a novel coding method for subgraph querying that is based on Laplacian spectrum and the number of walks. Our method follows the filtering-and-verification framework and works well on graph databases with frequent updates. We also propose novel two-step filtering conditions that can filter out most false positives and prove that the two-step filtering conditions satisfy the no-false-negative requirement (no dismissal in answers). Extensive experiments on both real and synthetic graphs show that, compared with six existing counterpart methods, our method can effectively improve the efficiency of subgraph querying. PMID:24853266

  10. Variation in the carbon and oxygen isotope composition of plant biomass and its relationship to water-use efficiency at the leaf- and ecosystem-scales in a northern Great Plains grassland.

    PubMed

    Flanagan, Lawrence B; Farquhar, Graham D

    2014-02-01

    Measurements of the carbon (δ(13) Cm ) and oxygen (δ(18) Om ) isotope composition of C3 plant tissue provide important insights into controls on water-use efficiency. We investigated the causes of seasonal and inter-annual variability in water-use efficiency in a grassland near Lethbridge, Canada using stable isotope (leaf-scale) and eddy covariance measurements (ecosystem-scale). The positive relationship between δ(13) Cm and δ(18) Om values for samples collected during 1998-2001 indicated that variation in stomatal conductance and water stress-induced changes in the degree of stomatal limitation of net photosynthesis were the major controls on variation in δ(13) Cm and biomass production during this time. By comparison, the lack of a significant relationship between δ(13) Cm and δ(18) Om values during 2002, 2003 and 2006 demonstrated that water stress was not a significant limitation on photosynthesis and biomass production in these years. Water-use efficiency was higher in 2000 than 1999, consistent with expectations because of greater stomatal limitation of photosynthesis and lower leaf ci /ca during the drier conditions of 2000. Calculated values of leaf-scale water-use efficiency were 2-3 times higher than ecosystem-scale water-use efficiency, a difference that was likely due to carbon lost in root respiration and water lost during soil evaporation that was not accounted for by the stable isotope measurements. © 2013 John Wiley & Sons Ltd.

  11. Incorporation of isotopic, fluorescent, and heavy-atom-modified nucleotides into RNAs by position-selective labeling of RNA.

    PubMed

    Liu, Yu; Holmstrom, Erik; Yu, Ping; Tan, Kemin; Zuo, Xiaobing; Nesbitt, David J; Sousa, Rui; Stagno, Jason R; Wang, Yun-Xing

    2018-05-01

    Site-specific incorporation of labeled nucleotides is an extremely useful synthetic tool for many structural studies (e.g., NMR, electron paramagnetic resonance (EPR), fluorescence resonance energy transfer (FRET), and X-ray crystallography) of RNA. However, specific-position-labeled RNAs >60 nt are not commercially available on a milligram scale. Position-selective labeling of RNA (PLOR) has been applied to prepare large RNAs labeled at desired positions, and all the required reagents are commercially available. Here, we present a step-by-step protocol for the solid-liquid hybrid phase method PLOR to synthesize 71-nt RNA samples with three different modification applications, containing (i) a 13 C 15 N-labeled segment; (ii) discrete residues modified with Cy3, Cy5, or biotin; or (iii) two iodo-U residues. The flexible procedure enables a wide range of downstream biophysical analyses using precisely localized functionalized nucleotides. All three RNAs were obtained in <2 d, excluding time for preparing reagents and optimizing experimental conditions. With optimization, the protocol can be applied to other RNAs with various labeling schemes, such as ligation of segmentally labeled fragments.

  12. Photosynthetic carbon metabolism in seagrasses C-labeling evidence for the c(3) pathway.

    PubMed

    Andrews, T J; Abel, K M

    1979-04-01

    The delta(13)C values of several seagrasses were considerably less negative than those of terrestrial C(3) plants and tended toward those of terrestrial C(4) plants. However, for Thalassia hemprichii (Ehrenb.) Aschers and Halophila spinulosa (R. Br.) Aschers, phosphoglycerate and other C(3) cycle intermediates predominated among the early labeled products of photosynthesis in (14)C-labeled seawater (more than 90% at the earliest times) and the labeling pattern at longer times was brought about by the operation of the C(3) pathway. Malate and aspartate together accounted for only a minor fraction of the total fixed label at all times and the kinetic data of this labeling were not at all consistent with these compounds being early intermediates in seagrass photosynthesis. Pulse-chase (14)C-labeling studies further substantiated these conclusions. Significant labeling of photorespiratory intermediates was observed in all experiments. The kinetics of total fixation of label during some steady-state and pulse-chase experiments suggested that there may be an intermediate pool of inorganic carbon of variable size closely associated with the leaves, either externally or internally. Such a pool may be one cause for the C(4)-like carbon isotope ratios of seagrasses.

  13. Optimization and validation of FePro cell labeling method.

    PubMed

    Janic, Branislava; Rad, Ali M; Jordan, Elaine K; Iskander, A S M; Ali, Md M; Varma, N Ravi S; Frank, Joseph A; Arbab, Ali S

    2009-06-11

    Current method to magnetically label cells using ferumoxides (Fe)-protamine (Pro) sulfate (FePro) is based on generating FePro complexes in a serum free media that are then incubated overnight with cells for the efficient labeling. However, this labeling technique requires long (>12-16 hours) incubation time and uses relatively high dose of Pro (5-6 microg/ml) that makes large extracellular FePro complexes. These complexes can be difficult to clean with simple cell washes and may create low signal intensity on T2* weighted MRI that is not desirable. The purpose of this study was to revise the current labeling method by using low dose of Pro and adding Fe and Pro directly to the cells before generating any FePro complexes. Human tumor glioma (U251) and human monocytic leukemia cell (THP-1) lines were used as model systems for attached and suspension cell types, respectively and dose dependent (Fe 25 to 100 microg/ml and Pro 0.75 to 3 microg/ml) and time dependent (2 to 48 h) labeling experiments were performed. Labeling efficiency and cell viability of these cells were assessed. Prussian blue staining revealed that more than 95% of cells were labeled. Intracellular iron concentration in U251 cells reached approximately 30-35 pg-iron/cell at 24 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. However, comparable labeling was observed after 4 h across the described FePro concentrations. Similarly, THP-1 cells achieved approximately 10 pg-iron/cell at 48 h when labeled with 100 microg/ml of Fe and 3 microg/ml of Pro. Again, comparable labeling was observed after 4 h for the described FePro concentrations. FePro labeling did not significantly affect cell viability. There was almost no extracellular FePro complexes observed after simple cell washes. To validate and to determine the effectiveness of the revised technique, human T-cells, human hematopoietic stem cells (hHSC), human bone marrow stromal cells (hMSC) and mouse neuronal stem cells (mNSC C17

  14. Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides

    PubMed Central

    Thakur, Chandar S.; Brown, Margaret E.; Sama, Jacob N.; Jackson, Melantha E.

    2010-01-01

    Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2813-y) contains supplementary material, which is available to authorized users. PMID:20730533

  15. Isotope ratio mass spectrometry in nutrition research

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Luke, A.H.

    Many of the biochemical pathways and processes that form the foundation of modern nutrition research was elucidated using stable isotopes as physiological tracers. Since the discovery of stable isotopes, improvements and innovations in mass spectrometry and chromatography have led to greatly expanded applications. This research project was designed to evaluate gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) as a tool for isotopic tracer studies and to delineate the operational parameters for the analysis of {sup 13}C-labeled cholesterol, leucine and {alpha}-ketoisocaproate. The same isotope ratio mass spectrometer was then used as the base instrument for the ratio mass spectrometer was then usedmore » as the base instrument for the development of two additional inlet systems: a continuous-flow inlet for the analyses of {sup 13}C and {sup 18}O as CO{sub 2} and a filament inlet for on-line combustion and isotopic analysis of non-volatile organic compounds. Each of these three inlets was evaluated and their utility in nutrition research illustrated. GC/C/IRMS was used to analyze cholesterol, leucine and {alpha}-ketoisocaproate with good accuracy, precision and little isotopic memory. For all three compounds the detection limits achieved well surpassed currently used technologies. For compounds that can be well separated by GC, GC/C/IRMS is a valuable analytical tool. The continuous-flow inlet provided good accuracy and precision for measurements of {sup 13}CO{sub 2} from breath tests and {sup 18}O as CO{sub 2} from total energy expenditure tests. Most importantly, the continuous-flow inlet increased sample throughput by at least a factor of three over conventional analytical techniques. The filament inlet provided accurate and precise {sup 13}C ratio measurements of both natural abundance and enriched standards of non-volatile organic compounds of physiological interest.« less

  16. Target analyte quantification by isotope dilution LC-MS/MS directly referring to internal standard concentrations--validation for serum cortisol measurement.

    PubMed

    Maier, Barbara; Vogeser, Michael

    2013-04-01

    Isotope dilution LC-MS/MS methods used in the clinical laboratory typically involve multi-point external calibration in each analytical series. Our aim was to test the hypothesis that determination of target analyte concentrations directly derived from the relation of the target analyte peak area to the peak area of a corresponding stable isotope labelled internal standard compound [direct isotope dilution analysis (DIDA)] may be not inferior to conventional external calibration with respect to accuracy and reproducibility. Quality control samples and human serum pools were analysed in a comparative validation protocol for cortisol as an exemplary analyte by LC-MS/MS. Accuracy and reproducibility were compared between quantification either involving a six-point external calibration function, or a result calculation merely based on peak area ratios of unlabelled and labelled analyte. Both quantification approaches resulted in similar accuracy and reproducibility. For specified analytes, reliable analyte quantification directly derived from the ratio of peak areas of labelled and unlabelled analyte without the need for a time consuming multi-point calibration series is possible. This DIDA approach is of considerable practical importance for the application of LC-MS/MS in the clinical laboratory where short turnaround times often have high priority.

  17. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL... be marked clearly with the following information: (i) The motor's nominal full load efficiency (as of...

  18. Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols

    DOEpatents

    Martinez, Rodolfo A [Santa Fe, NM; Unkefer, Clifford J [Los Alamos, NM; Alvarez, Marc A [Santa Fe, NM

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  19. Influence of liquid structure on diffusive isotope separation in molten silicates and aqueous solutions

    NASA Astrophysics Data System (ADS)

    Watkins, James M.; DePaolo, Donald J.; Ryerson, Frederick J.; Peterson, Brook T.

    2011-06-01

    Molecular diffusion in natural volcanic liquids discriminates between isotopes of major ions (e.g., Fe, Mg, Ca, and Li). Although isotope separation by diffusion is expected on theoretical grounds, the dependence on mass is highly variable for different elements and in different media. Silicate liquid diffusion experiments using simple liquid compositions were carried out to further probe the compositional dependence of diffusive isotopic discrimination and its relationship to liquid structure. Two diffusion couples consisting of the mineral constituents anorthite (CaAl 2Si 2O 8; denoted AN), albite (NaAlSi 3O 8; denoted AB), and diopside (CaMgSi 2O 6; denoted DI) were held at 1450 °C for 2 h and then quenched to ambient pressure and temperature. Major-element as well as Ca and Mg isotope profiles were measured on the recovered quenched glasses. In both experiments, Ca diffuses rapidly with respect to Si. In the AB-AN experiment, D Ca/ D Si ≈ 20 and the efficiency of isotope separation for Ca is much greater than in natural liquid experiments where D Ca/ D Si ≈ 1. In the AB-DI experiment, D Ca/ D Si ≈ 6 and the efficiency of isotope separation is between that of the natural liquid experiments and the AB-AN experiment. In the AB-DI experiment, D Mg/ D Si ≈ 1 and the efficiency of isotope separation for Mg is smaller than it is for Ca yet similar to that observed for Mg in natural liquids. The results from the experiments reported here, in combination with results from natural volcanic liquids, show clearly that the efficiency of diffusive separation of Ca isotopes is systematically related to the solvent-normalized diffusivity - the ratio of the diffusivity of the cation ( D Ca) to the diffusivity of silicon ( D Si). The results on Ca isotopes are consistent with available data on Fe, Li, and Mg isotopes in silicate liquids, when considered in terms of the parameter D cation/ D Si. Cations diffusing in aqueous solutions display a similar relationship

  20. Solid-phase Microextraction (SPME) with Stable Isotope Calibration for Measuring Bioavailability of Hydrophobic Organic Contaminants

    PubMed Central

    Cui, Xinyi; Bao, Lianjun; Gan, Jay

    2014-01-01

    Solid-phase microextraction (SPME) is a biomimetic tool ideally suited for measuring bioavailability of hydrophobic organic compounds (HOCs) in sediment and soil matrices. However, conventional SPME sampling requires the attainment of equilibrium between the fiber and sample matrix, which may take weeks or months, greatly limiting its applicability. In this study, we explored the preloading of polydimethylsiloxane fiber with stable isotope labeled analogs (SI-SPME) to circumvent the need for long sampling time, and evaluated the performance of SI-SPME against the conventional equilibrium SPME (Eq-SPME) using a range of sediments and conditions. Desorption of stable isotope-labeled analogs and absorption of PCB-52, PCB-153, bifenthrin and cis-permethrin were isotropic, validating the assumption for SI-SPME. Highly reproducible preloading was achieved using acetone-water (1:4, v/v) as the carrier. Compared to Eq-SPME that required weeks or even months, the fiber concentrations (Cf) under equilibrium could be reliably estimated by SI-SPME in 1 d under agitated conditions or 20 d under static conditions in spiked sediments. The Cf values predicted by SI-SPME were statistically identical to those determined by Eq-SPME. The SI-SPME method was further applied successfully to field sediments contaminated with PCB 52, PCB 153, and bifenthrin. The increasing availability of stable isotope labeled standards and mass spectrometry nowadays makes SI-SPME highly feasible, allowing the use of SPME under non-equilibrium conditions with much shorter or flexible sampling time. PMID:23930601

  1. Unraveling the Nature of Active Sites in Ethanol Electro-oxidation by Site-Specific Marking of a Pt Catalyst with Isotope-Labeled 13CO.

    PubMed

    Farias, Manuel J S; Cheuquepán, William; Tanaka, Auro A; Feliu, Juan M

    2018-03-15

    This works deals with the identification of preferential site-specific activation at a model Pt surface during a multiproduct reaction. The (110)-type steps of a Pt(332) surface were selectively marked by attaching isotope-labeled 13 CO molecules to them, and ethanol oxidation was probed by in situ Foureir transfrom infrared spectroscopy in order to precisely determine the specific sites at which CO 2 , acetic acid, and acetaldehyde were preferentially formed. The (110) steps were active for splitting the C-C bond, but unexpectedly, we provide evidence that the pathway of CO 2 formation was preferentially activated at (111) terraces, rather than at (110) steps. Acetaldehyde was formed at (111) terraces at potentials comparable to those for CO 2 formation also at (111) terraces, while the acetic acid formation pathway became active only when the (110) steps were released by the oxidation of adsorbed 13 CO, at potentials higher than for the formation of CO 2 at (111) terraces of the stepped surface.

  2. Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.

    PubMed

    Gregorius, Barbara; Jakoby, Thomas; Schaumlöffel, Dirk; Tholey, Andreas

    2013-05-21

    Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

  3. Temporal and Spatial Variances in Arterial Spin-Labeling Are Inversely Related to Large-Artery Blood Velocity.

    PubMed

    Robertson, A D; Matta, G; Basile, V S; Black, S E; Macgowan, C K; Detre, J A; MacIntosh, B J

    2017-08-01

    The relationship between extracranial large-artery characteristics and arterial spin-labeling MR imaging may influence the quality of arterial spin-labeling-CBF images for older adults with and without vascular pathology. We hypothesized that extracranial arterial blood velocity can explain between-person differences in arterial spin-labeling data systematically across clinical populations. We performed consecutive pseudocontinuous arterial spin-labeling and phase-contrast MR imaging on 82 individuals (20-88 years of age, 50% women), including healthy young adults, healthy older adults, and older adults with cerebral small vessel disease or chronic stroke infarcts. We examined associations between extracranial phase-contrast hemodynamics and intracranial arterial spin-labeling characteristics, which were defined by labeling efficiency, temporal signal-to-noise ratio, and spatial coefficient of variation. Large-artery blood velocity was inversely associated with labeling efficiency ( P = .007), temporal SNR ( P < .001), and spatial coefficient of variation ( P = .05) of arterial spin-labeling, after accounting for age, sex, and group. Correction for labeling efficiency on an individual basis led to additional group differences in GM-CBF compared to correction using a constant labeling efficiency. Between-subject arterial spin-labeling variance was partially explained by extracranial velocity but not cross-sectional area. Choosing arterial spin-labeling timing parameters with on-line knowledge of blood velocity may improve CBF quantification. © 2017 by American Journal of Neuroradiology.

  4. Determination of urea kinetics by isotope dilution with [13C]urea and gas chromatography-isotope ratio mass spectrometry (GC-IRMS) analysis.

    PubMed

    Kloppenburg, W D; Wolthers, B G; Stellaard, F; Elzinga, H; Tepper, T; de Jong, P E; Huisman, R M

    1997-07-01

    1. Stable urea isotopes can be used to study urea kinetics in humans. The use of stable urea isotopes for studying urea kinetic parameters in humans on a large scale is hampered by the high costs of the labelled material. We devised a urea dilution for measurement of the distribution volume, production rate and clearance of urea in healthy subjects and renal failure patients using the inexpensive single labelled [13C]urea isotope with subsequent analysis by headspace chromatography-isotope ratio MS (GC-IRMS) of the [13C]urea enrichment. 2. The method involves measurement of the molar percentage excess of [13C]urea in plasma samples taken over a 4 h period after an intravenous bolus injection of [13C]urea. During the sample processing procedure, the plasma samples together with calibration samples containing a known molar percentage excess of [13C]urea are acidified with phosphoric acid to remove endogenous CO2, and are subsequently incubated with urease to convert the urea present in the plasma samples into CO2. The 13C enrichment of the generated CO2 is analysed by means of GC-IRMS. This method allows measurement of the molar percentage excess of [13C]urea to an accuracy of 0.02%. 3. Reproducibility studies showed that the sample processing procedure [within-run coefficient of variation (CV) < 2.8% and between-run CV < 8.8%] and the GC-IRMS analysis (within-day CV < 1.3% and between-day CV < 1.3%) could be repeated with good reproducibility. 4. In clinical urea kinetic studies in a healthy subject and in a renal failure patient without residual renal function, reproducible values of the distribution volume, production rate and clearance of urea were determined using minimal amounts of [13C]urea (25-50 mg). 5. Because only low [13C]urea enrichments are needed in this urea dilution method using GC-IRMS analysis, the costs of urea kinetic studies are reduced considerably, especially in patients with renal failure.

  5. Biosynthesis and characterization of ¹⁵N₆-labeled phomopsin A, a lupin associated mycotoxin produced by Diaporthe toxica.

    PubMed

    Schloß, Svenja; Wedell, Ines; Koch, Matthias; Rohn, Sascha; Maul, Ronald

    2015-06-15

    The hepatotoxin phomopsin A (PHO-A), a secondary metabolite mainly produced by the fungus Diaporthe toxica, occurs predominantly on sweet lupins. Along with the growing interest in sweet lupins for food and feed commodities, concerns have been raised about fungal infestations, and consequently, about the determination of PHO-A. High performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) represents the most suitable analytical technique for sensitive and selective detection of mycotoxins including PHO-A. However, isotopic labeled substances are needed as internal standards for a reliable and convenient quantification. As no isotope standard for PHO-A is currently available, a biosynthesis of fully (15)N6-labeled PHO-A was established by cultivation of D. toxica on defined media containing Na(15)NO3 and (15)N-labeled yeast extract as the only nitrogen sources. The identity of (15)N6-PHO-A was confirmed by high resolution mass spectrometry. The new (15)N6-labeled standard will facilitate the method development for PHO-A including a more accurate quantification by LC-MS/MS. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Novel isotopic N, N-Dimethyl Leucine (iDiLeu) Reagents Enable Absolute Quantification of Peptides and Proteins Using a Standard Curve Approach

    NASA Astrophysics Data System (ADS)

    Greer, Tyler; Lietz, Christopher B.; Xiang, Feng; Li, Lingjun

    2015-01-01

    Absolute quantification of protein targets using liquid chromatography-mass spectrometry (LC-MS) is a key component of candidate biomarker validation. One popular method combines multiple reaction monitoring (MRM) using a triple quadrupole instrument with stable isotope-labeled standards (SIS) for absolute quantification (AQUA). LC-MRM AQUA assays are sensitive and specific, but they are also expensive because of the cost of synthesizing stable isotope peptide standards. While the chemical modification approach using mass differential tags for relative and absolute quantification (mTRAQ) represents a more economical approach when quantifying large numbers of peptides, these reagents are costly and still suffer from lower throughput because only two concentration values per peptide can be obtained in a single LC-MS run. Here, we have developed and applied a set of five novel mass difference reagents, isotopic N, N-dimethyl leucine (iDiLeu). These labels contain an amine reactive group, triazine ester, are cost effective because of their synthetic simplicity, and have increased throughput compared with previous LC-MS quantification methods by allowing construction of a four-point standard curve in one run. iDiLeu-labeled peptides show remarkably similar retention time shifts, slightly lower energy thresholds for higher-energy collisional dissociation (HCD) fragmentation, and high quantification accuracy for trypsin-digested protein samples (median errors <15%). By spiking in an iDiLeu-labeled neuropeptide, allatostatin, into mouse urine matrix, two quantification methods are validated. The first uses one labeled peptide as an internal standard to normalize labeled peptide peak areas across runs (<19% error), whereas the second enables standard curve creation and analyte quantification in one run (<8% error).

  7. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    PubMed Central

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

  8. Osmium isotopes suggest fast and efficient mixing in the oceanic upper mantle.

    NASA Astrophysics Data System (ADS)

    Bizimis, Michael; Salters, Vincent

    2010-05-01

    The depleted upper mantle (DUM; the source of MORB) is thought to represent the complementary reservoir of continental crust extraction. Previous studies have calculated the "average" DUM composition based on the geochemistry of MORB. However the Nd isotope compositions of abyssal peridotites have been shown to extend to more depleted compositions than associated MORB. While this argues for the presence of both relatively depleted and enriched material within the upper mantle, the extent of compositional variability, length scales of heterogeneity and timescales of mixing in the upper mantle are not well constrained. Model calculations show that 2Ga is a reasonable mean age of depletion for DUM while Hf - Nd isotopes show the persistence of a depleted terrestrial reservoir by the early Archean (3.5-3.8Ga). U/Pb zircon ages of crustal rocks show three distinct peaks at 1.2, 1.9, and 2.7Ga and these are thought to represent the ages of three major crustal growth events. A fundamental question therefore is whether the present day upper mantle retains a memory of multiple ancient depletion events, or has been effectively homogenized. This has important implications for the nature of convection and time scales of survival of heterogeneities in the upper mantle. Here we compare published Os isotope data from abyssal peridotites and ophiolitic Os-Ir alloys with new data from Hawaiian spinel peridotite xenoliths. The Re-Os isotope system has been shown to yield useful depletion age information in peridotites, so we use it here to investigate the distribution of Re-depletion ages (TRD) in these mantle samples as a proxy for the variability of DUM. The probability density functions (PDF) of TRD from osmiridiums, abyssal and Hawaiian peridotites are all remarkably similar and show a distinct peak at 1.2-1.3 Ga (errors for TRD are set at 0.2Ga to suppress statistically spurious age peaks). The Hawaiian peridotites further show a distinct peak at 1.9-2Ga, but no oceanic mantle

  9. Tracing photosynthetic carbon in leaves with nanoSIMS after 13CO2 labelling

    NASA Astrophysics Data System (ADS)

    Dannoura, Masako; Takeuchi, Miyuki; Kominami, Yuji; Takanashi, Satoru; Kenichi, Yoshimura; Ataka, Mioko

    2015-04-01

    To understand the carbon allocation of the tree and forest ecosystem, it is important to consider the residence time of carbon in different pools at suitable time scales. For example the carbon used for respiration will stay a few minutes to a few days in the tree, the carbon used for storage or structure of leaves will stay months to years, and the carbon used for wood structure, it will stay over the whole lifespan of the tree. The leaves are the entrance of carbon in trees where it can be used for foliage growth and maintenance or exported to the other organs or the other forest ecosystem compartments. Tracing carbon isotope using NanoSIMS technique is one of useful methods to estimate where and how long the carbon stay in the tree organs. In this study, 13CO2 pulse labelling were conducted and 13C was measured by IRMS to see the amount of C remaining in the leaves with time.NanoSIMS was used to localize where the labelled C remained within the leaf tissue. Twice labelling were done on branches of Quercus serrata at FFPRI(Forest and Forest Products research Institute) in Kyoto, Japan. The first labelling was in 30 April 2012 when the leaves start flushing and the second one was in 29 May 2012 when the leaves were completely deployed. For both labelling experiment, one branch was selected and covered with transparent plastic bag. CO2 concentration was recorded with IRGA and air temperature inside the chamber was monitored. Then 13CO2 was injected into the bag, and after 1 hour, the bag was removed and the branch was again exposed to ambient air. Leaves were collected before and 10-12 times after labelling and their isotope compositions were measured by IRMS. The leaf collected just after labelling and 6 days after labelling were used for NanoSIMS observation. Samples for nanoSIMS were preserved in glutaraldehyde and then embed in epoxy resin. The sliced sample were placed on the silicon wafer and observed by NanoSIMS 50L(Cameca, France). The 13C was highest just

  10. Efficient Photochemical Dihydrogen Generation Initiated by a Bimetallic Self-Quenching Mechanism

    DOE PAGES

    Chambers, Matthew B.; Kurtz, Daniel A.; Pitman, Catherine L.; ...

    2016-09-27

    Artificial photosynthesis relies on coupling light absorption with chemical fuel generation. A mechanistic study of visible light-driven H 2 production from [Cp*Ir(bpy)H] + (1) has revealed a new, highly efficient pathway for integrating light absorption with bond formation. The net reaction of 1 with a proton source produces H 2, but the rate of excited state quenching is surprisingly acid-independent and displays no observable deuterium kinetic isotopic effect. Time-resolved photoluminescence and labeling studies are consistent with diffusion-limited bimetallic self-quenching by electron transfer. Accordingly, the quantum yield of H 2 release nearly reaches unity as the concentration of 1 increases. Furthermore,more » this unique pathway for photochemical H 2 generation provides insight into transformations catalyzed by 1.« less

  11. A novel method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies

    NASA Astrophysics Data System (ADS)

    Wu, Dianming; Kampf, Christopher; Pöschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Sörgel, Matthias

    2014-05-01

    We developed a new method (gas-phase stripping-derivatization coupled to LC-MS) to measure the 15N atom percent excess (APE) of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye by the well-known Griess reaction in the Long Path Absorption Photometer (LOPAP). The reaction solutions containing the dye are collected at the outflow of the LOPAP, purified by solid-phase extraction and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The unlabeled azo dye (C18H19O2N5S) with a monoisotopic molecular mass of 369.41 g mol-1 can be detected as its protonated molecular ion ([M+H+], M) by HPLC-MS at a retention time of 2.8 min. Due to the natural isotope distribution M + 0, M + 1, M + 2, and M + 3 ions were considered for the calculation of the 15N APE. The optimal working range was found to be between 20 and 50% for the 15N/14N ratio. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method has been applied for the measurement of HO15NO emissions from soil in a dynamic chamber with and without spiking 15N labeled urea. Our results confirm biogenic HONO emissions from soil as HO15NO was measured after addition of 15N urea.

  12. Using Power Spectrum Analysis to Evaluate 18O-Water Labeling Data Acquired from Low Resolution Mass Spectrometers

    PubMed Central

    Sadygov, Rovshan G.; Zhao, Yingxin; Haidacher, Sigmund J.; Starkey, Jonathan M.; Tilton, Ronald G.; Denner, Larry

    2010-01-01

    We describe a method for ratio estimations in 18O-water labeling experiments acquired from low resolution isotopically resolved data. The method is implemented in a software package specifically designed for use in experiments making use of zoom-scan mode data acquisition. Zoom-scan mode data allows commonly used ion trap mass spectrometers to attain isotopic resolution, which make them amenable to use in labeling schemes such as 18O-water labeling, but algorithms and software developed for high resolution instruments may not be appropriate for the lower resolution data acquired in zoom-scan mode. The use of power spectrum analysis is proposed as a general approach which may be uniquely suited to these data types. The software implementation uses power spectrum to remove high-frequency noise, and band-filter contributions from co-eluting species of differing charge states. From the elemental composition of a peptide sequence we generate theoretical isotope envelopes of heavy-light peptide pairs in five different ratios; these theoretical envelopes are correlated with the filtered experimental zoom scans. To automate peptide quantification in high-throughput experiments, we have implemented our approach in a computer program, MassXplorer. We demonstrate the application of MassXplorer to two model mixtures of known proteins, and to a complex mixture of mouse kidney cortical extract. Comparison with another algorithm for ratio estimations demonstrates the increased precision and automation of MassXplorer. PMID:20568695

  13. FITC labeled silica nanoparticles as efficient cell tags: uptake and photostability study in endothelial cells.

    PubMed

    Veeranarayanan, Srivani; Poulose, Aby Cheruvathoor; Mohamed, Sheikh; Aravind, Athulya; Nagaoka, Yutaka; Yoshida, Yasuhiko; Maekawa, Toru; Kumar, D Sakthi

    2012-03-01

    The use of fluorescent nanomaterials has gained great importance in the field of medical imaging. Many traditional imaging technologies have been reported utilizing dyes in the past. These methods face drawbacks due to non-specific accumulation and photobleaching of dyes. We studied the uptake and internalization of two different sized (30 nm and 100 nm) FITC labeled silica nanoparticles in Human umbilical vein endothelial cell line. These nanomaterials show high biocompatability and are highly photostable inside live cells for increased period of time in comparison to the dye alone. To our knowledge, we report for the first time the use of 30 nm fluorescent silica nanoparticles as efficient endothelial tags along with the well studied 100 nm particles. We also have emphasized the good photostability of these materials in live cells.

  14. Proteomic validation of protease drug targets: pharmacoproteomics of matrix metalloproteinase inhibitor drugs using isotope-coded affinity tag labelling and tandem mass spectrometry.

    PubMed

    Butler, G S; Overall, C M

    2007-01-01

    We illustrate the use of quantitative proteomics, namely isotope-coded affinity tag labelling and tandem mass spectrometry, to assess the targets and effects of the blockade of matrix metalloproteinases by an inhibitor drug in a breast cancer cell culture system. Treatment of MT1-MMP-transfected MDA-MB-231 cells with AG3340 (Prinomastat) directly affected the processing a multitude of matrix metalloproteinase substrates, and indirectly altered the expression of an array of other proteins with diverse functions. Therefore, broad spectrum blockade of MMPs has wide-ranging biological consequences. In this human breast cancer cell line, secreted substrates accumulated uncleaved in the conditioned medium and plasma membrane protein substrates were retained on the cell surface, due to reduced processing and shedding of these proteins (cell surface receptors, growth factors and bioactive molecules) to the medium in the presence of the matrix metalloproteinase inhibitor. Hence, proteomic investigation of drug-perturbed cellular proteomes can identify new protease substrates and at the same time provides valuable information for target validation, drug efficacy and potential side effects prior to commitment to clinical trials.

  15. Desired and Undesired Effects of Energy Labels--An Eye-Tracking Study.

    PubMed

    Waechter, Signe; Sütterlin, Bernadette; Siegrist, Michael

    2015-01-01

    Saving energy is an important pillar for the mitigation of climate change. Electric devices (e.g., freezer and television) are an important player in the residential sector in the final demand for energy. Consumers' purchase decisions are therefore crucial to successfully reach the energy-efficiency goals. Putting energy labels on products is often considered an adequate way of empowering consumers to make informed purchase decisions. Consequently, this approach should contribute to reducing overall energy consumption. The effectiveness of its measurement depends on consumers' use and interpretation of the information provided. Despite advances in energy efficiency and a mandatory labeling policy, final energy consumption per capita is in many countries still increasing. This paper provides a systematic analysis of consumers' reactions to one of the most widely used eco-labels, the European Union (EU) energy label, by using eye-tracking methodology as an objective measurement. The study's results partially support the EU's mandatory policy, showing that the energy label triggers attention toward energy information in general. However, the energy label's effect on consumers' actual product choices seems to be rather low. The study's results show that the currently used presentation format on the label is insufficient. The findings suggest that it does not facilitate the integration of energy-related information. Furthermore, the current format can attract consumers to focus more on energy-efficiency information, leading them to disregard information about actual energy consumption. As a result, the final energy consumption may increase because excellent ratings on energy efficiency (e.g., A++) do not automatically imply little consumption. Finally, implications for policymakers and suggestions for further research are discussed.

  16. Isotope separation apparatus and method

    DOEpatents

    Feldman, Barry J.

    1985-01-01

    The invention relates to an improved method and apparatus for laser isotope separation by photodeflection. A molecular beam comprising at least two isotopes to be separated intersects, preferably substantially perpendicular to one broad side of the molecular beam, with a laser beam traveling in a first direction. The laser beam is reflected back through the molecular beam, preferably in a second direction essentially opposite to the first direction. Because the molecules in the beam occupy various degenerate energy levels, if the laser beam comprises chirped pulses comprising selected wavelengths, the laser beam will very efficiently excite substantially all unexcited molecules and will cause stimulated emission of substantially all excited molecules of a selected one of the isotopes in the beam which such pulses encounter. Excitation caused by first direction chirped pulses moves molecules of the isotope excited thereby in the first direction. Stimulated emission of excited molecules of the isotope is brought about by returning chirped pulses traveling in the second direction. Stimulated emission moves emitting molecules in a direction opposite to the photon emitted. Because emitted photons travel in the second direction, emitting molecules move in the first direction. Substantial molecular movement of essentially all the molecules containing the one isotope is accomplished by a large number of chirped pulse-molecule interactions. A beam corer collects the molecules in the resulting enriched divergent portions of the beam.

  17. Nitrate dynamics in natural plants: insights based on the concentration and natural isotope abundances of tissue nitrate

    PubMed Central

    Liu, Xue-Yan; Koba, Keisuke; Makabe, Akiko; Liu, Cong-Qiang

    2014-01-01

    The dynamics of nitrate (NO−3), a major nitrogen (N) source for natural plants, has been studied mostly through experimental N addition, enzymatic assay, isotope labeling, and genetic expression. However, artificial N supply may not reasonably reflect the N strategies in natural plants because NO−3 uptake and reduction may vary with external N availability. Due to abrupt application and short operation time, field N addition, and isotopic labeling hinder the elucidation of in situ NO−3-use mechanisms. The concentration and natural isotopes of tissue NO−3 can offer insights into the plant NO−3 sources and dynamics in a natural context. Furthermore, they facilitate the exploration of plant NO−3 utilization and its interaction with N pollution and ecosystem N cycles without disturbing the N pools. The present study was conducted to review the application of the denitrifier method for concentration and isotope analyses of NO−3 in plants. Moreover, this study highlights the utility and advantages of these parameters in interpreting NO−3 sources and dynamics in natural plants. We summarize the major sources and reduction processes of NO−3 in plants, and discuss the implications of NO−3 concentration in plant tissues based on existing data. Particular emphasis was laid on the regulation of soil NO−3 and plant ecophysiological functions in interspecific and intra-plant NO−3 variations. We introduce N and O isotope systematics of NO−3 in plants and discuss the principles and feasibilities of using isotopic enrichment and fractionation factors; the correlation between concentration and isotopes (N and O isotopes: δ18O and Δ17O); and isotope mass-balance calculations to constrain sources and reduction of NO−3 in possible scenarios for natural plants are deliberated. Finally, we offer a preliminary framework of intraplant δ18O-NO−3 variation, and summarize the uncertainties in using tissue NO−3 parameters to interpret plant NO−3 utilization

  18. Nitrate dynamics in natural plants: insights based on the concentration and natural isotope abundances of tissue nitrate.

    PubMed

    Liu, Xue-Yan; Koba, Keisuke; Makabe, Akiko; Liu, Cong-Qiang

    2014-01-01

    The dynamics of nitrate (NO(-) 3), a major nitrogen (N) source for natural plants, has been studied mostly through experimental N addition, enzymatic assay, isotope labeling, and genetic expression. However, artificial N supply may not reasonably reflect the N strategies in natural plants because NO(-) 3 uptake and reduction may vary with external N availability. Due to abrupt application and short operation time, field N addition, and isotopic labeling hinder the elucidation of in situ NO(-) 3-use mechanisms. The concentration and natural isotopes of tissue NO(-) 3 can offer insights into the plant NO(-) 3 sources and dynamics in a natural context. Furthermore, they facilitate the exploration of plant NO(-) 3 utilization and its interaction with N pollution and ecosystem N cycles without disturbing the N pools. The present study was conducted to review the application of the denitrifier method for concentration and isotope analyses of NO(-) 3 in plants. Moreover, this study highlights the utility and advantages of these parameters in interpreting NO(-) 3 sources and dynamics in natural plants. We summarize the major sources and reduction processes of NO(-) 3 in plants, and discuss the implications of NO(-) 3 concentration in plant tissues based on existing data. Particular emphasis was laid on the regulation of soil NO(-) 3 and plant ecophysiological functions in interspecific and intra-plant NO(-) 3 variations. We introduce N and O isotope systematics of NO(-) 3 in plants and discuss the principles and feasibilities of using isotopic enrichment and fractionation factors; the correlation between concentration and isotopes (N and O isotopes: δ(18)O and Δ(17)O); and isotope mass-balance calculations to constrain sources and reduction of NO(-) 3 in possible scenarios for natural plants are deliberated. Finally, we offer a preliminary framework of intraplant δ(18)O-NO(-) 3 variation, and summarize the uncertainties in using tissue NO(-) 3 parameters to interpret

  19. Use of phenyl/tetrazolyl-functionalized magnetic microspheres and stable isotope labeled internal standards for significant reduction of matrix effect in determination of nine fluoroquinolones by liquid chromatography-quadrupole linear ion trap mass spectrometry.

    PubMed

    Xu, Fei; Liu, Feng; Wang, Chaozhan; Wei, Yinmao

    2018-02-01

    In this study, the strategy of unique adsorbent combined with isotope labeled internal standards was used to significantly reduce the matrix effect for the enrichment and analysis of nine fluoroquinolones in a complex sample by liquid chromatography coupled to quadrupole linear ion trap mass spectrometry (LC-QqQ LIT -MS/MS). The adsorbent was prepared conveniently by functionalizing Fe 3 O 4 @SiO 2 microspheres with phenyl and tetrazolyl groups, which could adsorb fluoroquinolones selectively via hydrophobic, electrostatic, and π-π interactions. The established magnetic solid-phase extraction (MSPE) method as well as using stable isotope labeled internal standards in the next MS/MS detection was able to reduce the matrix effect significantly. In the process of LC-QqQ LIT -MS/MS analysis, the precursor and product ions of the analytes were monitored quantitatively and qualitatively on a QTrap system equipped simultaneously with the multiple reaction monitoring (MRM) and enhanced product ion (EPI) scan. Subsequently, the enrichment method combined with LC-QqQ LIT -MS/MS demonstrated good analytical features in terms of linearity (7.5-100.0 ng mL -1 , r > 0.9960), satisfactory recoveries (88.6%-118.3%) with RSDs < 12.0%, LODs = 0.5 μg kg -1 and LOQs = 1.5 μg kg -1 for all tested analytes. Finally, the developed MSPE-LC-QqQ LIT -MS/MS method had been successfully applied to real pork samples for food-safety risk monitoring in Ningxia Province, China. Graphical abstract Mechanism of reducing matrix effect through the as-prepared adsorbent.

  20. Regioselective and stereospecific deuteration of bioactive aza compounds by the use of ruthenium nanoparticles.

    PubMed

    Pieters, Grégory; Taglang, Céline; Bonnefille, Eric; Gutmann, Torsten; Puente, Céline; Berthet, Jean-Claude; Dugave, Christophe; Chaudret, Bruno; Rousseau, Bernard

    2014-01-03

    An efficient H/D exchange method allowing the deuteration of pyridines, quinolines, indoles, and alkyl amines with D2 in the presence of Ru@PVP nanoparticles is described. By a general and simple procedure involving mild reaction conditions and simple filtration to recover the labeled product, the isotopic labeling of 22 compounds proceeded in good yield with high chemo- and regioselectivity. The viability of this procedure was demonstrated by the labeling of eight biologically active compounds. Remarkably, enantiomeric purity was conserved in the labeled compounds, even though labeling took place in the vicinity of the stereogenic center. The level of isotopic enrichment observed is suitable for metabolomic studies in most cases. This approach is also perfectly adapted to tritium labeling because it uses a gas as an isotopic source. Besides these applications to molecules of biological interest, this study reveals a rich and underestimated chemistry on the surface of ruthenium nanoparticles. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Rationale and evidence for menu-labeling legislation.

    PubMed

    Roberto, Christina A; Schwartz, Marlene B; Brownell, Kelly D

    2009-12-01

    Menu-labeling legislation is a proposed public health intervention for poor diet and obesity that requires chain restaurants to provide nutrition information on menus and menu boards. The restaurant industry has strongly opposed menu-labeling legislation. Using scientific evidence, this paper counters industry arguments against menu labeling by demonstrating that consumers want chain restaurant nutrition information to be disclosed; the current methods of providing nutrition information are inadequate; the expense of providing nutrition information is minimal; the government has the legal right to mandate disclosure of information; consumers have the right to know nutrition information; a lack of information reduces the efficiency of a market economy; and menu labeling has the potential to make a positive public health impact.

  2. In-situ Isotopic Analysis at Nanoscale using Parallel Ion Electron Spectrometry: A Powerful New Paradigm for Correlative Microscopy

    NASA Astrophysics Data System (ADS)

    Yedra, Lluís; Eswara, Santhana; Dowsett, David; Wirtz, Tom

    2016-06-01

    Isotopic analysis is of paramount importance across the entire gamut of scientific research. To advance the frontiers of knowledge, a technique for nanoscale isotopic analysis is indispensable. Secondary Ion Mass Spectrometry (SIMS) is a well-established technique for analyzing isotopes, but its spatial-resolution is fundamentally limited. Transmission Electron Microscopy (TEM) is a well-known method for high-resolution imaging down to the atomic scale. However, isotopic analysis in TEM is not possible. Here, we introduce a powerful new paradigm for in-situ correlative microscopy called the Parallel Ion Electron Spectrometry by synergizing SIMS with TEM. We demonstrate this technique by distinguishing lithium carbonate nanoparticles according to the isotopic label of lithium, viz. 6Li and 7Li and imaging them at high-resolution by TEM, adding a new dimension to correlative microscopy.

  3. Complementary Constraints from Carbon (13C) and Nitrogen (15N) Isotopes on the Efficiency of the Glacial Ocean's Soft-Tissue Biological Pump

    NASA Astrophysics Data System (ADS)

    Schmittner, A.; Somes, C. J.

    2016-12-01

    A three-dimensional, process-based model of the ocean's carbon and nitrogen cycles, including 13C and 15N isotopes, is used to explore effects of idealized changes in the soft-tissue biological pump. Results are presented from one preindustrial control run and six simulations of the Last Glacial Maximum (LGM) with increasing values of the spatially constant maximum phytoplankton growth rate μmax, which mimicks iron fertilization. The default LGM simulation, without increasing μmax and with a shallower and weaker Atlantic Meridional Overturning Circulation and increased sea ice cover, leads to 280 Pg more respired organic carbon (Corg) than the pre-industrial control. Dissolved oxygen in the thermocline increase, which reduces water column denitrification and nitrogen fixation, thus increasing the ocean's fixed nitrogen inventory and decreasing δ15NNO3. This simulation already fits observed carbon and nitrogen isotopes relatively well, but it overestimates deep ocean δ13CDIC and underestimates δ15NNO3 at high latitudes. Increasing μmax enhances Corg and lowers deep ocean δ13CDIC, improving the fit. Modest increases in μmax result in higher subpolar δ15NNO3 due to enhanced local nutrient utilization, and better agreement with reconstructions. Large increases in nutrient utilization are inconsistent with nitrogen isotopes although they still fit the carbon isotopes reasonably well. The best fitting models with modest increases in μmax reproduce major features of the glacial δ13CDIC, δ15N, and oxygen reconstructions while simulating increased Corg by 510-670 Pg. These results are consistent with the idea that the soft-tissue pump was more efficient during the LGM. Both circulation and biological nutrient utilization contribute. However, these conclusions are preliminary given our idealized experiments, which do not consider changes in benthic denitrification and spatially inhomogenous changes in aeolian iron fluxes. The analysis illustrates interactions

  4. Proteomic analysis of common bean stem under drought stress using in-gel stable isotope labeling.

    PubMed

    Zadražnik, Tanja; Egge-Jacobsen, Wolfgang; Meglič, Vladimir; Šuštar-Vozlič, Jelka

    2017-02-01

    Drought is an abiotic stress that strongly influences plant growth, development and productivity. Proteome changes in the stem of the drought-tolerant common bean (Phaseolus vulgaris L.) cultivar Tiber have were when the plants were exposed to drought. Five-week-old plants were subjected to water deficit by withholding irrigation for 7, 12 and 17days, whereas control plants were regularly irrigated. Relative water content (RWC) of leaves, as an indicator of the degree of cell and tissue hydration, showed the highest statistically significant differences between control and drought-stressed plants after 17days of treatment, where RWC remained at 90% for control and declined to 45% for stressed plants. Plants exposed to drought for 17days and control plants at the same developmental stage were included in quantitative proteomic analysis using in-gel stable isotope labeling of proteins in combination with mass spectrometry. The quantified proteins were grouped into several functional groups, mainly into energy metabolism, photosynthesis, proteolysis, protein synthesis and proteins related to defense and stress. 70kDa heat shock protein showed the greatest increase in abundance under drought of all the proteins, suggesting its role in protecting plants against stress by re-establishing normal protein conformations and thus cellular homeostasis. The abundance of proteins involved in protein synthesis also increased under drought stress, important for recovery of damaged proteins involved in the plant cell's metabolic activities. Other important proteins in this study were related to proteolysis and folding, which are necessary for maintaining proper cellular protein homeostasis. Taken together, these results reveal the complexity of pathways involved in the drought stress response in common bean stems and enable comparison with the results of proteomic analysis of leaves, thus providing important information to further understand the biochemical and molecular mechanisms

  5. Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

    PubMed Central

    Persson, Xuan-Mai T.; Błachnio-Zabielska, Agnieszka Urszula; Jensen, Michael D.

    2010-01-01

    Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of 13C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of 13C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment. PMID:20526002

  6. Neonatal Respiratory Diseases in the Newborn Infant: Novel Insights from Stable Isotope Tracer Studies.

    PubMed

    Carnielli, Virgilio P; Giorgetti, Chiara; Simonato, Manuela; Vedovelli, Luca; Cogo, Paola

    2016-01-01

    Respiratory distress syndrome is a common problem in preterm infants and the etiology is multifactorial. Lung underdevelopment, lung hypoplasia, abnormal lung water metabolism, inflammation, and pulmonary surfactant deficiency or disfunction play a variable role in the pathogenesis of respiratory distress syndrome. High-quality exogenous surfactant replacement studies and studies on surfactant metabolism are available; however, the contribution of surfactant deficiency, alteration or dysfunction in selected neonatal lung conditions is not fully understood. In this article, we describe a series of studies made by applying stable isotope tracers to the study of surfactant metabolism and lung water. In a first set of studies, which we call 'endogenous studies', using stable isotope-labelled intravenous surfactant precursors, we showed the feasibility of measuring surfactant synthesis and kinetics in infants using several metabolic precursors including plasma glucose, plasma fatty acids and body water. In a second set of studies, named 'exogenous studies', using stable isotope-labelled phosphatidylcholine tracer given endotracheally, we could estimate surfactant disaturated phosphatidylcholine pool size and half-life. Very recent studies are focusing on lung water and on the endogenous biosynthesis of the surfactant-specific proteins. Information obtained from these studies in infants will help to better tailor exogenous surfactant treatment in neonatal lung diseases. © 2016 S. Karger AG, Basel.

  7. Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169*

    PubMed Central

    Allen, James W.; DiRusso, Concetta C.; Black, Paul N.

    2017-01-01

    Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U-13C]glucose, 13CO2, or D2O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions. PMID:27903654

  8. Carbon and Acyl Chain Flux during Stress-induced Triglyceride Accumulation by Stable Isotopic Labeling of the Polar Microalga Coccomyxa subellipsoidea C169.

    PubMed

    Allen, James W; DiRusso, Concetta C; Black, Paul N

    2017-01-06

    Deriving biofuels and other lipoid products from algae is a promising future technology directly addressing global issues of atmospheric CO 2 balance. To better understand the metabolism of triglyceride synthesis in algae, we examined their metabolic origins in the model species, Coccomyxa subellipsoidea C169, using stable isotopic labeling. Labeling patterns arising from [U- 13 C]glucose, 13 CO 2 , or D 2 O supplementation were analyzed by GC-MS and/or LC-MS over time courses during nitrogen starvation to address the roles of catabolic carbon recycling, acyl chain redistribution, and de novo fatty acid (FA) synthesis during the expansion of the lipid bodies. The metabolic origin of stress-induced triglyceride was found to be a continuous 8:2 ratio between de novo synthesized FA and acyl chain transfer from pre-stressed membrane lipids with little input from lipid remodeling. Membrane lipids were continually synthesized with associated acyl chain editing during nitrogen stress, in contrast to an overall decrease in total membrane lipid. The incorporation rates of de novo synthesized FA into lipid classes were measured over a time course of nitrogen starvation. The synthesis of triglycerides, phospholipids, and galactolipids followed a two-stage pattern where nitrogen starvation resulted in a 2.5-fold increase followed by a gradual decline. Acyl chain flux into membrane lipids was dominant in the first stage followed by triglycerides. These data indicate that the level of metabolic control that determines acyl chain flux between membrane lipids and triglycerides during nitrogen stress relies primarily on the Kennedy pathway and de novo FA synthesis with limited, defined input from acyl editing reactions. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Quantifying nitrous oxide production pathways in wastewater treatment systems using isotope technology - A critical review.

    PubMed

    Duan, Haoran; Ye, Liu; Erler, Dirk; Ni, Bing-Jie; Yuan, Zhiguo

    2017-10-01

    Nitrous oxide (N 2 O) is an important greenhouse gas and an ozone-depleting substance which can be emitted from wastewater treatment systems (WWTS) causing significant environmental impacts. Understanding the N 2 O production pathways and their contribution to total emissions is the key to effective mitigation. Isotope technology is a promising method that has been applied to WWTS for quantifying the N 2 O production pathways. Within the scope of WWTS, this article reviews the current status of different isotope approaches, including both natural abundance and labelled isotope approaches, to N 2 O production pathways quantification. It identifies the limitations and potential problems with these approaches, as well as improvement opportunities. We conclude that, while the capabilities of isotope technology have been largely recognized, the quantification of N 2 O production pathways with isotope technology in WWTS require further improvement, particularly in relation to its accuracy and reliability. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Protein quantification using a cleavable reporter peptide.

    PubMed

    Duriez, Elodie; Trevisiol, Stephane; Domon, Bruno

    2015-02-06

    Peptide and protein quantification based on isotope dilution and mass spectrometry analysis are widely employed for the measurement of biomarkers and in system biology applications. The accuracy and reliability of such quantitative assays depend on the quality of the stable-isotope labeled standards. Although the quantification using stable-isotope labeled peptides is precise, the accuracy of the results can be severely biased by the purity of the internal standards, their stability and formulation, and the determination of their concentration. Here we describe a rapid and cost-efficient method to recalibrate stable isotope labeled peptides in a single LC-MS analysis. The method is based on the equimolar release of a protein reference peptide (used as surrogate for the protein of interest) and a universal reporter peptide during the trypsinization of a concatenated polypeptide standard. The quality and accuracy of data generated with such concatenated polypeptide standards are highlighted by the quantification of two clinically important proteins in urine samples and compared with results obtained with conventional stable isotope labeled reference peptides. Furthermore, the application of the UCRP standards in complex samples is described.

  11. DeuteRater: a tool for quantifying peptide isotope precision and kinetic proteomics.

    PubMed

    Naylor, Bradley C; Porter, Michael T; Wilson, Elise; Herring, Adam; Lofthouse, Spencer; Hannemann, Austin; Piccolo, Stephen R; Rockwood, Alan L; Price, John C

    2017-05-15

    Using mass spectrometry to measure the concentration and turnover of the individual proteins in a proteome, enables the calculation of individual synthesis and degradation rates for each protein. Software to analyze concentration is readily available, but software to analyze turnover is lacking. Data analysis workflows typically don't access the full breadth of information about instrument precision and accuracy that is present in each peptide isotopic envelope measurement. This method utilizes both isotope distribution and changes in neutromer spacing, which benefits the analysis of both concentration and turnover. We have developed a data analysis tool, DeuteRater, to measure protein turnover from metabolic D 2 O labeling. DeuteRater uses theoretical predictions for label-dependent change in isotope abundance and inter-peak (neutromer) spacing within the isotope envelope to calculate protein turnover rate. We have also used these metrics to evaluate the accuracy and precision of peptide measurements and thereby determined the optimal data acquisition parameters of different instruments, as well as the effect of data processing steps. We show that these combined measurements can be used to remove noise and increase confidence in the protein turnover measurement for each protein. Source code and ReadMe for Python 2 and 3 versions of DeuteRater are available at https://github.com/JC-Price/DeuteRater . Data is at https://chorusproject.org/pages/index.html project number 1147. Critical Intermediate calculation files provided as Tables S3 and S4. Software has only been tested on Windows machines. jcprice@chem.byu.edu. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

  12. Lead isotope determinations from sulfide mineral occurrences--Russian Far East

    USGS Publications Warehouse

    Church, Stan E.; Goryachev, Nikolai A.; Shpikerman, Vladimir I.

    2013-01-01

    The lead isotope database for sulfide deposits and occurrences in the Russian Far East was funded by the Mineral Resources Program, U.S. Geological Survey (USGS) in conjunction with the collaborative studies of mineral resources by the Russian Academy of Sciences and the U. S. Geological Survey (Nokleberg and others, 1996). Comparisons of these data with similar lead isotope data from Alaska published in Church, Delevaux, and others (1987) and Gaccetta and Church (1989) provide a basis for the following three-fold project objectives: 1. To utilize lead isotope signatures, in conjunction with regional mapping, to assess the relative ages and to categorize the types of mineral deposits studied, 2. To relate the lead isotope and trace-element geochemical signatures of specific deposits and occurrences to ore-forming processes, and 3. To use the lead isotope data to correlate lithotectonic terranes within the northern Cordillera (Alaska, Yukon Territories and British Columbia in Canada, and the western Cordillera of the United States). The report by Church, Gray, and others (1987) shows how this fingerprinting methodology can be applied to trace the offset of lithotectonic (or lithostratigraphic as labeled by some authors) terranes.The lead isotope data presented in table 1 represent the work completed on sulfide mineral deposits located in the Russian Far East from 1993 to 1995, when this study was terminated due to lack of funding. The lead isotope data are reported here for use by investigators who may find them of value in mineral exploration. No attempt is made to summarize the voluminous literature on these mineral deposits.

  13. 13 C dynamic nuclear polarization using isotopically enriched 4-oxo-TEMPO free radicals.

    PubMed

    Niedbalski, Peter; Parish, Christopher; Kiswandhi, Andhika; Lumata, Lloyd

    2016-12-01

    The nitroxide-based free radical 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) is a widely used polarizing agent in NMR signal amplification via dissolution dynamic nuclear polarization (DNP). In this study, we have thoroughly investigated the effects of 15 N and/or 2 H isotopic labeling of 4-oxo-TEMPO free radical on 13 C DNP of 3 M [1- 13 C] sodium acetate samples in 1 : 1 v/v glycerol : water at 3.35 T and 1.2 K. Four variants of this free radical were used for 13 C DNP: 4-oxo-TEMPO, 4-oxo-TEMPO- 15 N, 4-oxo-TEMPO-d 16 and 4-oxo-TEMPO- 15 N,d 16 . Our results indicate that, despite the striking differences seen in the electron spin resonance (ESR) spectral features, the 13 C DNP efficiency of these 15 N and/or 2 H-enriched 4-oxo-TEMPO free radicals are relatively the same compared with 13 C DNP performance of the regular 4-oxo-TEMPO. Furthermore, when fully deuterated glassing solvents were used, the 13 C DNP signals of these samples all doubled in the same manner, and the 13 C polarization buildup was faster by a factor of 2 for all samples. The data here suggest that the hyperfine coupling contributions of these isotopically enriched 4-oxo-TEMPO free radicals have negligible effects on the 13 C DNP efficiency at 3.35 T and 1.2 K. These results are discussed in light of the spin temperature model of DNP. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  14. Femtosecond Laser Ablation Multicollector ICPMS Analysis of Uranium Isotopes in NIST Glass

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Duffin, Andrew M.; Springer, Kellen WE; Ward, Jesse D.

    We have utilized femtosecond laser ablation coupled to multi-collector inductively couple plasma mass spectrometry to measure the uranium isotopic content of NIST 61x (x=0,2,4,6) glasses. The uranium content of these glasses is a linear two-component mixing between isotopically natural uranium and the isotopically depleted spike used in preparing the glasses. Laser ablation results match extremely well, generally within a few ppm, with solution analysis following sample dissolution and chemical separation. In addition to isotopic data, sample utilization efficiency measurements indicate that over 1% of ablated uranium atoms reach a mass spectrometer detector, making this technique extremely efficient. Laser sampling alsomore » allows for spatial analysis and our data indicate that rare uranium concentration inhomogeneities exist in NIST 616 glass.« less

  15. Development of Chemical Isotope Labeling LC-MS for Milk Metabolomics: Comprehensive and Quantitative Profiling of the Amine/Phenol Submetabolome.

    PubMed

    Mung, Dorothea; Li, Liang

    2017-04-18

    Milk is a complex sample containing a variety of proteins, lipids, and metabolites. Studying the milk metabolome represents an important application of metabolomics in the general area of nutritional research. However, comprehensive and quantitative analysis of milk metabolites is a challenging task due to the wide range of variations in chemical/physical properties and concentrations of these metabolites. We report an analytical workflow for in-depth profiling of the milk metabolome based on chemical isotope labeling (CIL) and liquid chromatography mass spectrometry (LC-MS) with a focus of using dansylation labeling to target the amine/phenol submetabolome. An optimal sample preparation method, including the use of methanol at a 3:1 ratio of solvent to milk for protein precipitation and dichloromethane for lipid removal, was developed to detect and quantify as many metabolites as possible. This workflow was found to be generally applicable to profile milk metabolomes of different species (cow, goat, and human) and types. Results from experimental replicate analysis (n = 5) of 1:1, 2:1, and 1:2 12 C-/ 13 C-labeled cow milk samples showed that 95.7%, 94.3%, and 93.2% of peak pairs, respectively, had ratio values within ±50% accuracy range and 90.7%, 92.6%, and 90.8% peak pairs had RSD values of less than 20%. In the metabolomic analysis of 36 samples from different categories of cow milk (brands, batches, and fat percentages) with experimental triplicates, a total of 7104 peak pairs or metabolites could be detected with an average of 4573 ± 505 (n = 108) pairs detected per LC-MS run. Among them, 3820 peak pairs were commonly detected in over 80% of the samples with 70 metabolites positively identified by mass and retention time matches to the dansyl standard library and 2988 pairs with their masses matched to the human metabolome libraries. This unprecedentedly high coverage of the amine/phenol submetabolome illustrates the complexity of the milk metabolome. Since

  16. In vitro labelling and detection of mesenchymal stromal cells: a comparison between magnetic resonance imaging of iron-labelled cells and magnetic resonance spectroscopy of fluorine-labelled cells.

    PubMed

    Rizzo, Stefania; Petrella, Francesco; Zucca, Ileana; Rinaldi, Elena; Barbaglia, Andrea; Padelli, Francesco; Baggi, Fulvio; Spaggiari, Lorenzo; Bellomi, Massimo; Bruzzone, Maria Grazia

    2017-01-01

    Among the various stem cell populations used for cell therapy, adult mesenchymal stromal cells (MSCs) have emerged as a major new cell technology. These cells must be tracked after transplantation to monitor their migration within the body and quantify their accumulation at the target site. This study assessed whether rat bone marrow MSCs can be labelled with superparamagnetic iron oxide (SPIO) nanoparticles and perfluorocarbon (PFC) nanoemulsion formulations without altering cell viability and compared magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) results from iron-labelled and fluorine-labelled MSCs, respectively. Of MSCs, 2 × 10 6 were labelled with Molday ION Rhodamine-B (MIRB) and 2 × 10 6 were labelled with Cell Sense. Cell viability was evaluated by trypan blue exclusion method. Labelled MSCs were divided into four samples containing increasing cell numbers (0.125 × 10 6 , 0.25 × 10 6 , 0.5 × 10 6 , 1 × 10 6 ) and scanned on a 7T MRI: for MIRB-labelled cells, phantoms and cells negative control, T1, T2 and T2* maps were acquired; for Cell Sense labelled cells, phantoms and unlabelled cells, a 19 F non-localised single-pulse MRS sequence was acquired. In total, 86.8% and 83.6% of MIRB-labelled cells and Cell Sense-labelled cells were viable, respectively. MIRB-labelled cells were visible in all samples with different cell numbers; pellets containing 0.5 × 10 6 and 1 × 10 6 of Cell Sense-labelled cells showed a detectable 19 F signal. Our data support the use of both types of contrast material (SPIO and PFC) for MSCs labelling, although further efforts should be dedicated to improve the efficiency of PFC labelling.

  17. High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

    PubMed Central

    Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

    2008-01-01

    Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

  18. The production of ultra-high purity single isotopes or tailored isotope mixtures by ICP-MS

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liezers, Martin; Farmer, Orville T.; Dion, Michael P.

    2015-01-01

    We report the development and testing of a simple collector arrangement for a commercial quadrupole ICP-MS that for the first time has been used to produce small quantities of highly enriched (>99.99%) single isotopes, with deposition rates >10 ng/hour. The collector assembly replaces the standard instrument detector allowing for implantation with simultaneous monitoring of the incident ion current. Even under zero bias implant conditions, low energy (<10 eV), ion collection efficiency was observed to be very high ~99%. 151Eu ion currents of 0.1-0.5 nA were collected on a simple, planar foil without resorting to any type of cup configuration. Recoverymore » of the enriched isotope from such foils is much simpler than from a more complex cup configuration. High rejection of adjacent mass isotopes was demonstrated by selectively implanting 167Er without any discernible co-implantation of 166Er and 168Er. The important analytical possibilities of the new approach to isotope ratio measurement, tracer purification and radiation measurements are discussed.« less

  19. RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro

    PubMed Central

    Herrmann, Elena; Young, Wayne; Rosendale, Douglas; Reichert-Grimm, Verena; Conrad, Ralf

    2017-01-01

    RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that “cross-feeding” occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system. PMID:28299315

  20. Stable carbon isotope ratios and intrinsic water-use efficiency of Miocene fossil leaves compared to modern congeners

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Marshall, J.D.; Zhang, J.; Rember, W.C.

    Miocene fossil leaves of forest trees were extracted from the Clarkia, Idaho fossil beds and their stable carbon isotope ratios were analyzed. Fossils had higher lignin concentrations and lower cellulose concentrations that modern leaves due to diagenesis and the HF used to extract the fossils. Therefore, [delta][sup 13]C of extracted fossil lignin was compared to that of modern lignin. Fossil lignin [delta][sup 13]C was significantly different from that of congeneric modern leaves (paired t-test, P<0.0001), but was 1.9% less negative. Gymnosperms (Metasequoia, Taxodium) were less negative than angiosperms (e.g., Magnolia, Quercus, Acer, Persea), but no difference between evergreen and deciduousmore » species was detected. Using published estimates of the concentration and [delta][sup 13]C of atmospheric CO[sub 2] during the Miocene was estimated the CO[sub 2] partial pressure gradient across the stomata (intrinsic water-use efficiency). Intrinsic water-use efficiency was at least 70% higher during this past [open quotes]greenhouse[close quotes] period than at present.« less

  1. Cellular Metabolic Activity and the Oxygen and Hydrogen Stable Isotope Composition of Intracellular Water and Metabolites

    NASA Astrophysics Data System (ADS)

    Kreuzer-Martin, H. W.; Hegg, E. L.

    2008-12-01

    Intracellular water is an important pool of oxygen and hydrogen atoms for biosynthesis. Intracellular water is usually assumed to be isotopically identical to extracellular water, but an unexpected experimental result caused us to question this assumption. Heme O isolated from Escherichia coli cells grown in 95% H218O contained only a fraction of the theoretical value of labeled oxygen at a position where the O atom was known to be derived from water. In fact, fewer than half of the oxygen atoms were labeled. In an effort to explain this surprising result, we developed a method to determine the isotope ratios of intracellular water in cultured cells. The results of our experiments showed that during active growth, up to 70% of the oxygen atoms and 50% of the hydrogen atoms in the intracellular water of E. coli are generated during metabolism and can be isotopically distinct from extracellular water. The fraction of isotopically distinct atoms was substantially less in stationary phase and chilled cells, consistent with our hypothesis that less metabolically-generated water would be present in cells with lower metabolic activity. Our results were consistent with and explained the result of the heme O labeling experiment. Only about 40% of the O atoms on the heme O molecule were labeled because, presumably, only about 40% of the water inside the cells was 18O water that had diffused in from the culture medium. The rest of the intracellular water contained 16O atoms derived from either nutrients or atmospheric oxygen. To test whether we could also detect metabolically-derived hydrogen atoms in cellular constituents, we isolated fatty acids from log-phase and stationary phase E. coli and determined the H isotope ratios of individual fatty acids. The results of these experiments showed that environmental water contributed more H atoms to fatty acids isolated in stationary phase than to the same fatty acids isolated from log-phase cells. Stable isotope analyses of

  2. Chloroformate derivatization for tracing the fate of Amino acids in cells and tissues by multiple stable isotope resolved metabolomics (mSIRM).

    PubMed

    Yang, Ye; Fan, Teresa W-M; Lane, Andrew N; Higashi, Richard M

    2017-07-11

    Amino acids have crucial roles in central metabolism, both anabolic and catabolic. To elucidate these roles, steady-state concentrations of amino acids alone are insufficient, as each amino acid participates in multiple pathways and functions in a complex network, which can also be compartmentalized. Stable Isotope-Resolved Metabolomics (SIRM) is an approach that uses atom-resolved tracking of metabolites through biochemical transformations in cells, tissues, or whole organisms. Using different elemental stable isotopes to label multiple metabolite precursors makes it possible to resolve simultaneously the utilization of these precursors in a single experiment. Conversely, a single precursor labeled with two (or more) different elemental isotopes can trace the allocation of e.g. C and N atoms through the network. Such dual-label experiments however challenge the resolution of conventional mass spectrometers, which must distinguish the neutron mass differences among different elemental isotopes. This requires ultrahigh resolution Fourier transform mass spectrometry (UHR-FTMS). When combined with direct infusion nano-electrospray ion source (nano-ESI), UHR-FTMS can provide rapid, global, and quantitative analysis of all possible mass isotopologues of metabolites. Unfortunately, very low mass polar metabolites such as amino acids can be difficult to analyze by current models of UHR-FTMS, plus the high salt content present in typical cell or tissue polar extracts may cause unacceptable ion suppression for sources such as nano-ESI. Here we describe a modified method of ethyl chloroformate (ECF) derivatization of amino acids to enable rapid quantitative analysis of stable isotope labeled amino acids using nano-ESI UHR-FTMS. This method showed excellent linearity with quantifiable limits in the low nanomolar range represented in microgram quantities of biological specimens, which results in extracts with total analyte abundances in the low to sub-femtomole range. We have

  3. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards & Labeling Programs for Copy Machines, External Power Supplies, LED Displays, Residential Gas Cooktops and Televisions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zheng, Nina; Zhou, Nan; Fridley, David

    2012-03-01

    This report presents a technical review of international minimum energy performance standards (MEPS), voluntary and mandatory energy efficiency labels and test procedures for five products being considered for new or revised MEPS in China: copy machines, external power supply, LED displays, residential gas cooktops and flat-screen televisions. For each product, an overview of the scope of existing international standards and labeling programs, energy values and energy performance metrics and description and detailed summary table of criteria and procedures in major test standards are presented.

  4. In Vivo Integrity and Biological Fate of Chelator-Free Zirconium-89-Labeled Mesoporous Silica Nanoparticles

    PubMed Central

    2015-01-01

    Traditional chelator-based radio-labeled nanoparticles and positron emission tomography (PET) imaging are playing vital roles in the field of nano-oncology. However, their long-term in vivo integrity and potential mismatch of the biodistribution patterns between nanoparticles and radio-isotopes are two major concerns for this approach. Here, we present a chelator-free zirconium-89 (89Zr, t1/2 = 78.4 h) labeling of mesoporous silica nanoparticle (MSN) with significantly enhanced in vivo long-term (>20 days) stability. Successful radio-labeling and in vivo stability are demonstrated to be highly dependent on both the concentration and location of deprotonated silanol groups (−Si–O–) from two types of silica nanoparticles investigated. This work reports 89Zr-labeled MSN with a detailed labeling mechanism investigation and long-term stability study. With its attractive radio-stability and the simplicity of chelator-free radio-labeling, 89Zr-MSN offers a novel, simple, and accurate way for studying the in vivo long-term fate and PET image-guided drug delivery of MSN in the near future. PMID:26213260

  5. In Vivo Integrity and Biological Fate of Chelator-Free Zirconium-89-Labeled Mesoporous Silica Nanoparticles.

    PubMed

    Chen, Feng; Goel, Shreya; Valdovinos, Hector F; Luo, Haiming; Hernandez, Reinier; Barnhart, Todd E; Cai, Weibo

    2015-08-25

    Traditional chelator-based radio-labeled nanoparticles and positron emission tomography (PET) imaging are playing vital roles in the field of nano-oncology. However, their long-term in vivo integrity and potential mismatch of the biodistribution patterns between nanoparticles and radio-isotopes are two major concerns for this approach. Here, we present a chelator-free zirconium-89 ((89)Zr, t1/2 = 78.4 h) labeling of mesoporous silica nanoparticle (MSN) with significantly enhanced in vivo long-term (>20 days) stability. Successful radio-labeling and in vivo stability are demonstrated to be highly dependent on both the concentration and location of deprotonated silanol groups (-Si-O(-)) from two types of silica nanoparticles investigated. This work reports (89)Zr-labeled MSN with a detailed labeling mechanism investigation and long-term stability study. With its attractive radio-stability and the simplicity of chelator-free radio-labeling, (89)Zr-MSN offers a novel, simple, and accurate way for studying the in vivo long-term fate and PET image-guided drug delivery of MSN in the near future.

  6. Characterization of labelling and de-labelling reagents for detection and recovery of tyrosine residue in peptide.

    PubMed

    Toyo'oka, Toshimasa; Mantani, Tomomi; Kato, Masaru

    2003-01-01

    This paper characterized the labelling and de-labelling reagents for reversible labelling of tyrosine (Tyr)-containing peptide, which involves detection and recovery. The phenolic hydroxyl group (-OH) in Tyr structure reacted with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F), and 1-fluoro-2,4-dinitrobenzene (DNFB) under mild conditions at room temperature at pH 9.3. The labels in the resulting derivatives were removed with the treatment of nucleophiles, such as thiols (cysteine, N-acetyl-L-cysteine and dithiothreitol) and amines (dimethylamine, methylamine, diethylamine, ethylamine and pyrrolidine). The de-labelling reactions of NBD-labelled N-acetyl-L-tyrosine (N-AcTyr) with the nucleophiles produced N-AcTyr, accompanied by NBD-nucleophile. Although DBD-F and DNFB also successfully labeled the -OH group in N-AcTyr, the efficiency of Cbond;O bond cleavage and recovery of N-AcTyr by the nucleophiles was relatively low compared with NBD-label. Among the de-labelling reagents, N-acetyl-L-cysteine and dimethylamine were recommended for the elimination of NBD moiety, with respect to the reaction rate, the side reaction, and the yield of recovery. The proposed procedure, which includes the labelling with NBD-F and the removal of NBD moiety by the nucleophiles, was successfully applied to the reversible labelling of N-terminal amine-blocked peptides, i.e. N-AcTyr-Val-Gly, Z-Glu-Tyr, Z-Phe-Tyr, N-Formyl-Met-Leu-Tyr, and N-AcArg-Pro-Pro-Gly-Phe-Ser-Pro-Tyr-Arg. Copyright 2003 John Wiley & Sons, Ltd.

  7. Dual-Labeled Near-Infrared/99mTc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells

    PubMed Central

    Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

    2016-01-01

    We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m (99mTc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with 99mTc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner. PMID:27399687

  8. Dual-Labeled Near-Infrared/(99m)Tc Imaging Probes Using PAMAM-Coated Silica Nanoparticles for the Imaging of HER2-Expressing Cancer Cells.

    PubMed

    Yamaguchi, Haruka; Tsuchimochi, Makoto; Hayama, Kazuhide; Kawase, Tomoyuki; Tsubokawa, Norio

    2016-07-07

    We sought to develop dual-modality imaging probes using functionalized silica nanoparticles to target human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer cells and achieve efficient target imaging of HER2-expressing tumors. Polyamidoamine-based functionalized silica nanoparticles (PCSNs) for multimodal imaging were synthesized with near-infrared (NIR) fluorescence (indocyanine green (ICG)) and technetium-99m ((99m)Tc) radioactivity. Anti-HER2 antibodies were bound to the labeled PCSNs. These dual-imaging probes were tested to image HER2-overexpressing breast carcinoma cells. In vivo imaging was also examined in breast tumor xenograft models in mice. SK-BR3 (HER2 positive) cells were imaged with stronger NIR fluorescent signals than that in MDA-MB231 (HER2 negative) cells. The increased radioactivity of the SK-BR3 cells was also confirmed by phosphor imaging. NIR images showed strong fluorescent signals in the SK-BR3 tumor model compared to muscle tissues and the MDA-MB231 tumor model. Automatic well counting results showed increased radioactivity in the SK-BR3 xenograft tumors. We developed functionalized silica nanoparticles loaded with (99m)Tc and ICG for the targeting and imaging of HER2-expressing cells. The dual-imaging probes efficiently imaged HER2-overexpressing cells. Although further studies are needed to produce efficient isotope labeling, the results suggest that the multifunctional silica nanoparticles are a promising vehicle for imaging specific components of the cell membrane in a dual-modality manner.

  9. A method to trace root-respired CO2 using a 13C label

    NASA Astrophysics Data System (ADS)

    Cooperdock, S.; Breecker, D.; Litvak, M. E.

    2014-12-01

    In order to partition total soil respiration into root respiration and decomposition under ambient conditions in desert soils, the following method was developed using 13C-labeled CO2 in a modern juniper savannah in central New Mexico. The labeled CO2 was mixed with ambient air and pumped into a small (2.5 m diameter and 1.4 m tall) juniper tree canopy . 10 L of the 13CO2 was sufficient to generate a stream of air at 20 L/min for 1 hour with a CO2 concentration of 540 ppm and a δ13C value of approximately 35,000‰. Plastic tarpaulins were used as a wind block. The 13CO2 -labeled air was applied to the canopy during peak photosynthesis between 10 and 11 am on June 30 2014 during which canopy air CO2 was elevated by approximately 10 ppm over ambient and had δ13C values ranging from 50 to 1000 ‰. Over the next three days, gas and tissue samples were collected in order to trace the 13C label through the juniper tree. Leaf and root samples collected from the labeled tree and from several control trees were loaded into exetainer vials, flushed with CO2-free air and incubated in the dark for 5 hours in order to measure the carbon isotope composition of respired CO2. Samples of soil pore space gas were collected from wells under the labeled tree and a control tree and were transported to the laboratory in He-flushed exetainer vials. The δ13C values of CO2 in the soil gas samples and in the headspace of incubation vials were measured using an isotope ratio mass spectrometer. The δ13C values of foliar respiration were significantly higher than those of the control (by 3.6‰, p < 0.01) one and two days after labeling and δ13C values of root-respired CO2 were significantly higher (by 0.7‰, p = 0.01) than those of the control three days after labeling. In addition, δ13C values of soil respired CO2, determined from measurements of soil pore space CO2 at 50 cm three days after labeling, were significantly higher (by 0.7‰, p < 0.03)) for the labeled tree than control

  10. Adapter reagents for protein site specific dye labeling.

    PubMed

    Thompson, Darren A; Evans, Eric G B; Kasza, Tomas; Millhauser, Glenn L; Dawson, Philip E

    2014-05-01

    Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this acetophenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. © 2014 Wiley Periodicals, Inc.

  11. Adapter Reagents for Protein Site Specific Dye Labeling

    PubMed Central

    Thompson, Darren A.; Evans, Eric G. B.; Kasza, Tomas; Millhauser, Glenn L.; Dawson, Philip E.

    2016-01-01

    Chemoselective protein labeling remains a significant challenge in chemical biology. Although many selective labeling chemistries have been reported, the practicalities of matching the reaction with appropriately functionalized proteins and labeling reagents is often a challenge. For example, we encountered the challenge of site specifically labeling the cellular form of the murine Prion protein with a fluorescent dye. To facilitate this labeling, a protein was expressed with site specific p-acetylphenylalanine. However, the utility of this aceto-phenone reactive group is hampered by the severe lack of commercially available aminooxy fluorophores. Here we outline a general strategy for the efficient solid phase synthesis of adapter reagents capable of converting maleimido-labels into aminooxy or azide functional groups that can be further tuned for desired length or solubility properties. The utility of the adapter strategy is demonstrated in the context of fluorescent labeling of the murine Prion protein through an adapted aminooxy-Alexa dye. PMID:24599728

  12. Category labels versus feature labels: category labels polarize inferential predictions.

    PubMed

    Yamauchi, Takashi; Yu, Na-Yung

    2008-04-01

    What makes category labels different from feature labels in predictive inference? This study suggests that category labels tend to make inductive reasoning polarized and homogeneous. In two experiments, participants were shown two schematic pictures of insects side by side and predicted the value of a hidden feature of one insect on the basis of the other insect. Arbitrary verbal labels were shown above the two pictures, and the meanings of the labels were manipulated in the instructions. In one condition, the labels represented the category membership of the insects, and in the other conditions, the same labels represented attributes of the insects. When the labels represented category membership, participants' responses became substantially polarized and homogeneous, indicating that the mere reference to category membership can modify reasoning processes.

  13. Relationship of leaf oxygen and carbon isotopic composition with transpiration efficiency in the C4 grasses Setaria viridis and Setaria italica.

    PubMed

    Ellsworth, Patrick Z; Ellsworth, Patrícia V; Cousins, Asaph B

    2017-06-15

    Leaf carbon and oxygen isotope ratios can potentially provide a time-integrated proxy for stomatal conductance (gs) and transpiration rate (E), and can be used to estimate transpiration efficiency (TE). In this study, we found significant relationships of bulk leaf carbon isotopic signature (δ13CBL) and bulk leaf oxygen enrichment above source water (Δ18OBL) with gas exchange and TE in the model C4 grasses Setaria viridis and S. italica. Leaf δ13C had strong relationships with E, gs, water use, biomass, and TE. Additionally, the consistent difference in δ13CBL between well-watered and water-limited plants suggests that δ13CBL is effective in separating C4 plants with different availability of water. Alternatively, the use of Δ18OBL as a proxy for E and TE in S. viridis and S. italica was problematic. First, the oxygen isotopic composition of source water, used to calculate leaf water enrichment (Δ18OLW), was variable with time and differed across water treatments. Second, water limitations changed leaf size and masked the relationship of Δ18OLW and Δ18OBL with E. Therefore, the data collected here suggest that δ13CBL but not Δ18OBL may be an effective proxy for TE in C4 grasses. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  14. Tracing nitrogenous disinfection byproducts after medium pressure UV water treatment by stable isotope labeling and high resolution mass spectrometry.

    PubMed

    Kolkman, Annemieke; Martijn, Bram J; Vughs, Dennis; Baken, Kirsten A; van Wezel, Annemarie P

    2015-04-07

    Advanced oxidation processes are important barriers for organic micropollutants (e.g., pharmaceuticals, pesticides) in (drinking) water treatment. Studies indicate that medium pressure (MP) UV/H2O2 treatment leads to a positive response in Ames mutagenicity tests, which is then removed after granulated activated carbon (GAC) filtration. The formed potentially mutagenic substances were hitherto not identified and may result from the reaction of photolysis products of nitrate with (photolysis products of) natural organic material (NOM). In this study we present an innovative approach to trace the formation of disinfection byproducts (DBPs) of MP UV water treatment, based on stable isotope labeled nitrate combined with high resolution mass spectrometry. It was shown that after MP UV treatment of artificial water containing NOM and nitrate, multiple nitrogen containing substances were formed. In total 84 N-DBPs were detected at individual concentrations between 1 to 135 ng/L bentazon-d6 equivalents, with a summed concentration of 1.2 μg/L bentazon-d6 equivalents. The chemical structures of three byproducts were confirmed. Screening for the 84 N-DBPs in water samples from a full-scale drinking water treatment plant based on MP UV/H2O2 treatment showed that 22 of the N-DBPs found in artificial water were also detected in real water samples.

  15. Protein Stable Isotope Fingerprinting (P-SIF): A New Tool to Understand Natural Isotopic Heterogeneity of Mixed Microbial Ecosystems

    NASA Astrophysics Data System (ADS)

    Pearson, A.; Mohr, W.; Tang, T.; Sattin, S.; Bovee, R.

    2014-12-01

    Protein stable isotope fingerprinting (P-SIF) is a method to measure the carbon isotope ratios of whole proteins separated from complex mixtures, including cultures and environmental samples. The goal of P-SIF is to expose the links between identity and function in microbial ecosystems by (i) determining the ratios of 13C/12C (values of δ13C) for different taxonomic divisions, and (ii) using those values as clues to the metabolic pathways employed by the respective organisms, while (iii) not perturbing the system, i.e., not adding exogenous substrates or isotope labels. To accomplish this, we employ two-dimensional HPLC to resolve a sample containing ca. 5-10 mg of mixed proteins into 960-1440 fractions. Each fraction then is split in two aliquots: The first is digested with trypsin for peptide sequencing, while the second is measured in triplicate using an isotope-ratio mass spectrometer interfaced with a spooling wire microcombustion device. Data from pure cultures show that bacteria have a narrow distribution of protein δ13C values within individual taxa (±0.7-1.2‰, 1σ). This is moderately larger than the mean precision of the triplicate isotope measurements (±0.5‰, 1σ) and may reflect heterogeneous distribution of 13C among the amino acids. When cells from different species are mixed together prior to protein extraction and separation, the results can predict accurately (to within ±1σ) the δ13C values of the original taxa. The number of data points required for this endmember prediction is ≥20/taxon, yielding a theoretical resolution of ca. 10 taxonomic units/sample. Initial tests on environmental samples suggest the approach will be useful to determine the overall trophic breadth of mixed microbial ecosystems.

  16. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    DOE PAGES

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; ...

    2015-08-28

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstratemore » stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this study, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.« less

  17. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstratemore » stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this study, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.« less

  18. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ostroumov, P. N., E-mail: ostroumov@anl.gov; Barcikowski, A.; Dickerson, C. A.

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstratemore » stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this paper, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.« less

  19. Highly tritiated water processing by isotopic exchange

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shu, W.M.; Willms, R.S.; Glugla, M.

    2015-03-15

    Highly tritiated water (HTW) is produced in fusion machines and one of the promising technologies to process it is isotopic exchange. 3 kinds of Pt-catalyzed zeolite (13X-APG, CBV-100-CY and HiSiv-1000) were tested as candidates for isotopic exchange of highly tritiated water (HTW), and CBV-100-CY (Na-Y type with a SiO{sub 2}/Al{sub 2}O{sub 3} ratio of ∼ 5.0) shows the best performance. Small-scale tritium testing indicates that this method is efficient for reaching an exchange factor (EF) of 100. Full-scale non-tritium testing implies that an EF of 300 can be achieved in 24 hours of operation if a temperature gradient is appliedmore » along the column. For the isotopic exchange, deuterium recycled from the Isotope Separation System (deuterium with 1% T and/or 200 ppm T) should be employed, and the tritiated water regenerated from the Pt-catalyzed zeolite bed after isotopic exchange should be transferred to Water Detritiation System (WDS) for further processing.« less

  20. Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

    PubMed

    Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

    2016-08-01

    Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Estimating the Efficiency of Phosphopeptide Identification by Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Hsu, Chuan-Chih; Xue, Liang; Arrington, Justine V.; Wang, Pengcheng; Paez Paez, Juan Sebastian; Zhou, Yuan; Zhu, Jian-Kang; Tao, W. Andy

    2017-06-01

    Mass spectrometry has played a significant role in the identification of unknown phosphoproteins and sites of phosphorylation in biological samples. Analyses of protein phosphorylation, particularly large scale phosphoproteomic experiments, have recently been enhanced by efficient enrichment, fast and accurate instrumentation, and better software, but challenges remain because of the low stoichiometry of phosphorylation and poor phosphopeptide ionization efficiency and fragmentation due to neutral loss. Phosphoproteomics has become an important dimension in systems biology studies, and it is essential to have efficient analytical tools to cover a broad range of signaling events. To evaluate current mass spectrometric performance, we present here a novel method to estimate the efficiency of phosphopeptide identification by tandem mass spectrometry. Phosphopeptides were directly isolated from whole plant cell extracts, dephosphorylated, and then incubated with one of three purified kinases—casein kinase II, mitogen-activated protein kinase 6, and SNF-related protein kinase 2.6—along with 16O4- and 18O4-ATP separately for in vitro kinase reactions. Phosphopeptides were enriched and analyzed by LC-MS. The phosphopeptide identification rate was estimated by comparing phosphopeptides identified by tandem mass spectrometry with phosphopeptide pairs generated by stable isotope labeled kinase reactions. Overall, we found that current high speed and high accuracy mass spectrometers can only identify 20%-40% of total phosphopeptides primarily due to relatively poor fragmentation, additional modifications, and low abundance, highlighting the urgent need for continuous efforts to improve phosphopeptide identification efficiency. [Figure not available: see fulltext.

  2. The reduction and oxidation of ceria: A natural abundance triple oxygen isotope perspective

    NASA Astrophysics Data System (ADS)

    Hayles, Justin; Bao, Huiming

    2015-06-01

    Ceria (CeO2) is a heavily studied material in catalytic chemistry for use as an oxygen storage medium, oxygen partial pressure regulator, fuel additive, and for the production of syngas, among other applications. Ceria powders are readily reduced and lose structural oxygen when subjected to low pO2 and/or high temperature conditions. Such dis-stoichiometric ceria can then re-oxidize under higher pO2 and/or lower temperature by incorporating new oxygen into the previously formed oxygen site vacancies. Despite extensive studies on ceria, the mechanisms for oxygen adsorption-desorption, dissociation-association, and diffusion of oxygen species on ceria surface and within the crystal structure are not well known. We predict that a large kinetic oxygen isotope effect should accompany the release and incorporation of ceria oxygen. As the first attempt to determine the existence and the degree of the isotope effect, this study focuses on a set of simple room-temperature re-oxidation experiments that are also relevant to a laboratory procedure using ceria to measure the triple oxygen isotope composition of CO2. Triple-oxygen-isotope labeled ceria powders are heated at 700 °C and cooled under vacuum prior to exposure to air. By combining results from independent experimental sets with different initial oxygen isotope labels and using a combined mass-balance and triangulation approach, we have determined the isotope fractionation factors for both high temperature reduction in vacuum (⩽10-4 mbar) and room temperature re-oxidation in air. Results indicate that there is a 1.5‰ ± 0.8‰ increase in the δ18O value of ceria after being heated in vacuum at 700 °C for 1 h. When the vacuum is broken at room temperature, the previously heated ceria incorporates 3-19% of its final structural oxygen from air, with a δ18O value of 2.1-4.1+7.7 ‰ for the incorporated oxygen. The substantial incorporation of oxygen from air supports that oxygen mobility is high in vacancy

  3. Isotope-labeling studies on the formation pathway of acrolein during heat processing of oils.

    PubMed

    Ewert, Alice; Granvogl, Michael; Schieberle, Peter

    2014-08-20

    Acrolein (2-propenal) is classified as a foodborne toxicant and was shown to be present in significant amounts in heated edible oils. Up to now, its formation was mainly suggested to be from the glycerol part of triacylglycerides, although a clear influence of the unsaturation of the fatty acid moiety was also obvious in previous studies. To unequivocally clarify the role of the glycerol and the fatty acid parts in acrolein formation, two series of labeled triacylglycerides were synthesized: [(13)C(3)]-triacylglycerides of stearic, oleic, linoleic, and linolenic acid and [(13)C(54)]-triacylglycerides with labeled stearic, oleic, and linoleic acid, but with unlabeled glycerol. Heating of each of the seven intermediates singly in silicon oil and measurement of the formed amounts of labeled and unlabeled acrolein clearly proved the fatty acid backbone as the key precursor structure. Enzymatically synthesized pure linoleic acid and linolenic acid hydroperoxides were shown to be the key intermediates in acrolein formation, thus allowing the discussion of a radical-induced reaction pathway leading to the formation of the aldehyde. Surprisingly, although several oils contained high amounts of acrolein after heating, deep-fried foods themselves, such as donuts or French fries, were low in the aldehyde.

  4. Labeling viral envelope lipids with quantum dots by harnessing the biotinylated lipid-self-inserted cellular membrane.

    PubMed

    Lv, Cheng; Lin, Yi; Liu, An-An; Hong, Zheng-Yuan; Wen, Li; Zhang, Zhenfeng; Zhang, Zhi-Ling; Wang, Hanzhong; Pang, Dai-Wen

    2016-11-01

    Highly efficient labeling of viruses with quantum dots (QDs) is the prerequisite for the long-term tracking of virus invasion at the single virus level to reveal mechanisms of virus infection. As one of the structural components of viruses, viral envelope lipids are hard to be labeled with QDs due to the lack of efficient methods to modify viral envelope lipids. Moreover, it is still a challenge to maintain the intactness and infectivity of labeled viruses. Herein, a mild method has been developed to label viral envelope lipids with QDs by harnessing the biotinylated lipid-self-inserted cellular membrane. Biotinylated lipids can spontaneously insert in cellular membranes of host cells during culture and then be naturally assembled on progeny Pseudorabies virus (PrV) via propagation. The biotinylated PrV can be labeled with streptavidin-conjugated QDs, with a labeling efficiency of ∼90%. Such a strategy to label lipids with QDs can retain the intactness and infectivity of labeled viruses to the largest extent, facilitating the study of mechanisms of virus infection at the single virus level. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Aircraft profile measurements of 18O/16O and D/H isotope ratios of cloud condensate and water vapor constrain precipitation efficiency and entrainment rates in tropical clouds

    NASA Astrophysics Data System (ADS)

    Noone, D. C.; Raudzens Bailey, A.; Toohey, D. W.; Twohy, C. H.; Heymsfield, A.; Rella, C.; Van Pelt, A. D.

    2011-12-01

    Convective clouds play a significant role in the moisture and heat balance of the tropics. The dynamics of organized and isolated convection are a function of the background thermodynamic profile and wind shear, buoyancy sources near the surface and the latent heating inside convective updrafts. The stable oxygen and hydrogen isotope ratios in water vapor and condensate can be used to identify dominant moisture exchanges and aspects of the cloud microphysics that are otherwise difficult to observe. Both the precipitation efficiency and the dilution of cloud updrafts by entrainment can be estimated since the isotopic composition outside the plume is distinct from inside. Measurements of the 18O/16O and D/H isotope ratios were made in July 2011 on 13 research flights of the NCAR C130 aircraft during the ICE-T (Ice in Clouds Experiment - Tropical) field campaign near St Croix. Measurements were made using an instrument based on the Picarro Wave-Length Scanning Cavity Ring Down platform that includes a number of optical, hardware and software modifications to allow measurements to be made at 5 Hz for deployment on aircraft. The measurement system was optimized to make precise measurements of the isotope ratio of liquid and ice cloud condensate by coupling the gas analyzer to the NCAR Counter flow Virtual Impactor inlet. The inlet system provides a particle enhancement while rejecting vapor. Sample air is vigorously heated before flowing into the gas phase analyzer. We present statistics that demonstrate the performance and calibration of the instrument. Measured profiles show that environmental air exhibits significant layering showing controls from boundary layer processes, large scale horizontal advection and regional subsidence. Condensate in clouds is consistent with generally low precipitation efficiency, although there is significant variability in the isotope ratios suggesting heterogeneity within plumes and the stochastic nature of detrainment processes

  6. Labeling and Functionalizing Amphipols for Biological Applications

    PubMed Central

    Bon, Christel Le; Popot, Jean-Luc; Giusti, Fabrice

    2014-01-01

    Amphipols (APols) are short amphipathic polymers developed as an alternative to detergents for handling membrane proteins (MPs) in aqueous solution. MPs are, as a rule, much more stable following trapping with APols than they are in detergent solutions. The best-characterized APol to date, called A8-35, is a mixture of short-chain sodium polyacrylates randomly derivatized with octylamine and isopropylamine. Its solution properties have been studied in detail, and it has been used extensively for biochemical and biophysical studies of MPs. One of the attractive characteristics of APols is that it is relatively easy to label them, isotopically or otherwise, without affecting their physical-chemical properties. Furthermore, several variously modified APols can be mixed, achieving multiple functionalization of MP/APol complexes in the easiest possible manner. Labeled or tagged APols are being used to study the solution properties of APols, their miscibility, their biodistribution upon injection into living organisms, their association with MPs and the composition, structure and dynamics of MP/APol complexes, examining the exchange of surfactants at the surface of MPs, labeling MPs to follow their distribution in fractionation experiments or to immobilize them, increasing the contrast between APols and solvent or MPs in biophysical experiments, improving NMR spectra, etc. Labeling or functionalization of APols can take various courses, each of which has its specific constraints and advantages regarding both synthesis and purification. The present review offers an overview of the various derivatives of A8-35 and its congeners that have been developed in our laboratory and discusses the pros and cons of various synthetic routes. PMID:24696186

  7. Closed flume inlet efficiency.

    DOT National Transportation Integrated Search

    2014-04-01

    The goal of the present study was to determine the efficiency of a specific culvert geometry, labeled as : Index 216 Closed Flume Inlet (CFI) in the FDOTs Design Standards, and to determine if geometric changes : affect the efficiency of the curre...

  8. Correction for isotopic interferences between analyte and internal standard in quantitative mass spectrometry by a nonlinear calibration function.

    PubMed

    Rule, Geoffrey S; Clark, Zlatuse D; Yue, Bingfang; Rockwood, Alan L

    2013-04-16

    Stable isotope-labeled internal standards are of great utility in providing accurate quantitation in mass spectrometry (MS). An implicit assumption has been that there is no "cross talk" between signals of the internal standard and the target analyte. In some cases, however, naturally occurring isotopes of the analyte do contribute to the signal of the internal standard. This phenomenon becomes more pronounced for isotopically rich compounds, such as those containing sulfur, chlorine, or bromine, higher molecular weight compounds, and those at high analyte/internal standard concentration ratio. This can create nonlinear calibration behavior that may bias quantitative results. Here, we propose the use of a nonlinear but more accurate fitting of data for these situations that incorporates one or two constants determined experimentally for each analyte/internal standard combination and an adjustable calibration parameter. This fitting provides more accurate quantitation in MS-based assays where contributions from analyte to stable labeled internal standard signal exist. It can also correct for the reverse situation where an analyte is present in the internal standard as an impurity. The practical utility of this approach is described, and by using experimental data, the approach is compared to alternative fits.

  9. Biosynthetic production of universally (13)C-labelled polyunsaturated fatty acids as reference materials for natural health product research.

    PubMed

    Le, Phuong Mai; Fraser, Catherine; Gardner, Graeme; Liang, Wei-Wan; Kralovec, Jaroslav A; Cunnane, Stephen C; Windust, Anthony J

    2007-09-01

    Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope (13)C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, (13)C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, (13)C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-(13)C-labelled fatty acids using U-(13)C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of (13)C-labelled DHA has been achieved with high chemical purity ( >99%) and high (13)C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-(13)C-DHA was enzymatically re-esterified to

  10. Gas chromatographic/mass spectrometric determination of carbon isotope composition in unpurified samples: methamphetamine example.

    PubMed

    Low, I A; Liu, R H; Legendre, M G; Piotrowski, E G; Furner, R L

    1986-10-01

    A gas chromatograph/quadrupole mass spectrometer system, operated in electron impact/selected ion monitoring mode, is used to determine the intensity ratio of the m/z 59 and the m/z 58 ions of the [C3H8N]+ fragment derived from methamphetamine samples synthesized with varying amounts of 13C-labeled methylamine. Crude products are introduced into the gas chromatograph without prior cleanup. The ratios measured were in excellent agreement with those calculated. A change in 0.25% use of 13C-methylamine is sufficient for product differentiation. The feasibility of using isotope labeling and subsequent mass spectrometric isotope ratio measurement as the basis of a compound tracing mechanism is discussed. Specifically, if methamphetamine samples manufactured from legal sources are asked to incorporate distinct 13C compositions, their sources can be traced when samples are diverted into illegal channels. Samples derived from illicit preparations can also be traced if the manufacturers of a precursor (methylamine in this case) incorporate distinct 13C compositions in their products.

  11. Taste Bud Labeling in Whole Tongue Epithelial Sheet in Adult Mice.

    PubMed

    Venkatesan, Nandakumar; Boggs, Kristin; Liu, Hong-Xiang

    2016-04-01

    Molecular labeling in whole-mount tissues provides an efficient way to obtain general information about the formation, maintenance, degeneration, and regeneration of many organs and tissues. However, labeling of lingual taste buds in whole tongue tissues in adult mice has been problematic because of the strong permeability barrier of the tongue epithelium. In this study, we present a simple method for labeling taste buds in the intact tongue epithelial sheet of an adult mouse. Following intralingual protease injection and incubation, immediate fixation of the tongue on mandible in 4% paraformaldehyde enabled the in situ shape of the tongue epithelium to be well maintained after peeling. The peeled epithelium was accessible to taste bud labeling with a pan-taste cell marker, keratin 8, and a type II taste cell marker, α-gustducin, in all three types of taste papillae, that is, fungiform, foliate, and circumvallate. Overnight incubation of tongue epithelial sheets with primary and secondary antibodies was sufficient for intense labeling of taste buds with both fluorescent and DAB visualizations. Labeled individual taste buds were easy to identify and quantify. This protocol provides an efficient way for phenotypic analyses of taste buds, especially regarding distribution pattern and number.

  12. Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

    PubMed

    Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

    2016-05-01

    Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction

  13. Determination of depleted uranium in urine via isotope ratio measurements using large-bore direct injection high efficiency nebulizer-inductively coupled plasma mass spectrometry.

    PubMed

    Westphal, Craig S; McLean, John A; Hakspiel, Shelly J; Jackson, William E; McClain, David E; Montaser, Akbar

    2004-09-01

    Inductively coupled plasma mass spectrometry (ICP-MS), coupled with a large-bore direct injection high efficiency nebulizer (LB-DIHEN), was utilized to determine the concentration and isotopic ratio of uranium in 11 samples of synthetic urine spiked with varying concentrations and ratios of uranium isotopes. Total U concentrations and (235)U/(238)U isotopic ratios ranged from 0.1 to 10 microg/L and 0.0011 and 0.00725, respectively. The results are compared with data from other laboratories that used either alpha-spectrometry or quadrupole-based ICP-MS with a conventional nebulizer-spray chamber arrangement. Severe matrix effects due to the high total dissolved solid content of the samples resulted in a 60 to 80% loss of signal intensity, but were compensated for by using (233)U as an internal standard. Accurate results were obtained with LB-DIHEN-ICP-MS, allowing for the positive identification of depleted uranium based on the (235)U/(238)U ratio. Precision for the (235)U/(238)U ratio is typically better than 5% and 15% for ICP-MS and alpha-spectrometry, respectively, determined over the concentrations and ratios investigated in this study, with the LB-DIHEN-ICP-MS system providing the most accurate results. Short-term precision (6 min) for the individual (235)U and (238)U isotopes in synthetic urine is better than 2% (N = 7), compared to approximately 5% for conventional nebulizer-spray chamber arrangements and >10% for alpha-spectrometry. The significance of these measurements is discussed for uranium exposure assessment of Persian Gulf War veterans affected by depleted uranium ammunitions.

  14. Liquid chromatography/electrospray ionization/isotopic dilution mass spectrometry analysis of n-(phosphonomethyl) glycine and mass spectrometry analysis of aminomethyl phosphonic acid in environmental water and vegetation matrixes.

    PubMed

    Grey, L; Nguyen, B; Yang, P

    2001-01-01

    A liquid chromatography/electrospray/mass spectrometry (LC/ES/MS) method was developed for the analysis of glyphosate (n-phosphonomethyl glycine) and its metabolite, aminomethyl phosphonic acid (AMPA) using isotope-labelled glyphosate as a method surrogate. Optimized parameters were achieved to derivatize glyphosate and AMPA using 9-fluorenylmethyl chloroformate (FMOC-Cl) in borate buffer prior to a reversed-phase LC analysis. Method spike recovery data obtained using laboratory and real world sample matrixes indicated an excellent correlation between the recovery of the native and isotope-labelled glyphosate. Hence, the first performance-based, isotope dilution MS method with superior precision, accuracy, and data quality was developed for the analysis of glyphosate. There was, however, no observable correlation between the isotope-labelled glyphosate and AMPA. Thus, the use of this procedure for the accurate analysis of AMPA was not supported. Method detection limits established using standard U.S. Environmental Protection Agency protocol were 0.06 and 0.30 microg/L, respectively, for glyphosate and AMPA in water matrixes and 0.11 and 0.53 microg/g, respectively, in vegetation matrixes. Problems, solutions, and the method performance data related to the analysis of chlorine-treated drinking water samples are discussed. Applying this method to other environmental matrixes, e.g., soil, with minimum modifications is possible, assuring accurate, multimedia studies of glyphosate concentration in the environment and the delivery of useful multimedia information for regulatory applications.

  15. Site-Specific Carbon Isotopes in Organics

    NASA Astrophysics Data System (ADS)

    Piasecki, A.; Eiler, J. M.

    2012-12-01

    propane and labeled propane (13CH3-12CH2-12CH3). Results are consistent with the expected relative fractionations between the two fragments, indicating limited 'scrambling' of carbon positions of less than 2% in the source. The limits of precision of this method are currently ~0.5 ‰, sufficient to resolve known or suspected position-specific isotope effects in propane. We have explored the expected temperature-dependent equilibrium isotopic distributions of propane using density functional theory and quantum mechanical models of vibrational isotope effects. These models predict the homogeneous isotope exchange equilibria among the various isotopologues of propane, which include several of a wide range of effects that should be measurable by our methods. At 300 K we predict that the central carbon site is 15‰ higher in δ13C and 95 ‰ higher in δD than the terminal carbon site; similarly the molecule containing both a 13C and D in the central site is enriched by ~120 ‰ relative to a random isotopic distribution at 300 K. These predictions present targets for future experimental and empirical studies of the temperature dependence of isotopic ordering in propane. More generally, the methods we are developing for the study of intramolecular isotopic distributions in propane will serve as a model for future study of similar effects in other organic compounds. [1]DeNiro, Epstein (1977) Science Volume 197, 261-263.

  16. Testing plant use of mobile vs immobile soil water sources using stable isotope experiments.

    PubMed

    Vargas, Ana I; Schaffer, Bruce; Yuhong, Li; Sternberg, Leonel da Silveira Lobo

    2017-07-01

    We tested for isotope exchange between bound (immobile) and mobile soil water, and whether there is isotope fractionation during plant water uptake. These are critical assumptions to the formulation of the 'two water worlds' hypothesis based on isotope profiles of soil water. In two different soil types, soil-bound water in two sets of 19-l pots, each with a 2-yr-old avocado plant (Persea americana), were identically labeled with tap water. After which, one set received isotopically enriched water whereas the other set received tap water as the mobile phase water. After a dry down period, we analyzed plant stem water as a proxy for soil-bound water as well as total soil water by cryogenic distillation. Seventy-five to 95% of the bound water isotopically exchanged with the mobile water phase. In addition, plants discriminated against 18 O and 2 H during water uptake, and this discrimination is a function of the soil water loss and soil type. The present experiment shows that the assumptions for the 'two water worlds' hypothesis are not supported. We propose a novel explanation for the discrepancy between isotope ratios of the soil water profile and other water compartments in the hydrological cycle. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  17. Development and Interlaboratory Study of a Liquid Chromatography Tandem Mass Spectrometric Methodfor the Determination of Multiple Mycotoxins inCereals Using Stable Isotope Dilution.

    PubMed

    Ye, Jin; Wu, Yu; Guo, Qilei; Lu, Meiling; Wang, Songshan; Xin, Yuanyuan; Xie, Gang; Zhang, Yan; Mariappan, Meena; Wang, Songxue

    2018-05-01

    An efficient, rapid, accurate, and cost-effective method based on stable isotope dilution and LC tandem MS was developed for the determination of multimycotoxins in cereals. The samples were extracted using acetonitrile-water-acetic acid (70 + 29 + 1, v/v/v), followed by dilution and centrifugation without any further cleanup. The mycotoxins were separated on a C18 column. Interference due to matrix effects was efficiently compensated for with [13C]-labeled stable isotope internal standards. The method demonstrated excellent linear relations, with regression coefficients above 0.999. Spiked recoveries at three different concentrations ranged from 80.9 to 115.9%, and RSDs were below 14% for all mycotoxins. The trueness of the method was also verified by participating in two proficiency tests, and satisfactory z-scores (|z| < 1.1) were obtained. In addition, an international interlaboratory study was organized to evaluate the methods. Eight laboratories characterized recovery, repeatability, and reproducibility studies in wheat, maize, and barley. The interlaboratory results were analyzed according to ISO 5725-2. Cochran and Grubbs tests were used to remove outliers. The mean recoveries of all 16 mycotoxins ranged from 87 to 111%. Repeatability, reproducibility, and Horwitz ratio values were 3.5-16.2, 5.4-33.6, and 0.16-1.65%, respectively. The results demonstrate that the method is reliable to determine multimycotoxins in cereals.

  18. Tracing subducted crustal materials in the mantle by using magnesium isotopes

    NASA Astrophysics Data System (ADS)

    Teng, F. Z.

    2016-12-01

    Recent studies show that some continental basalt, mantle-metasomatised peridotite and cratonic eclogite have heterogeneous Mg isotopic compositions. These isotopically distinct Mg isotopic compositions have been explained by the incorporation of subducted materials in their mantle sources though the detailed mechanisms are still not well understood. In particular, how Mg-poor crustal materials can modify Mg isotopic systematics of Mg-rich mantle is unknown. Subduction zones are the most efficient sites for crust and mantle interactions, hence should be where the most prominent Mg isotopic variation occurs. However, to date, little is known on Mg isotope systematics in the subduction factory. Here I first review and report new Mg isotopic data for arc lava, subarc peridotite and the subducted slab (marine sediment, altered basalt and abyssal peridotite), then use them to constrain the origins of mantle Mg isotopic heterogeneity and lay the foundation for using Mg isotopes as new tools for tracing crust-mantle interactions. The main conclusions are 1) fluid-rock interactions can modify Mg isotopic systematics of abyssal peridotites; 2) island arc lavas have non-MORB Mg isotopic compositions, reflecting distinct surbarc mantle Mg isotopic signature; 3) continental arcs have non-MORB Mg isotopic compositions, likely resulting from crustal contamination and 4) the isotopically heterogeneous continental basalts are mainly produced by mixing of isotopically distinct magmas instead of being partial melting products of metasomatised mantle peridotites.

  19. Highly abundant defense proteins in human sweat as revealed by targeted proteomics and label-free quantification mass spectrometry.

    PubMed

    Csősz, É; Emri, G; Kalló, G; Tsaprailis, G; Tőzsér, J

    2015-10-01

    The healthy human skin with its effective antimicrobial defense system forms an efficient barrier against invading pathogens. There is evidence suggesting that the composition of this chemical barrier varies between diseases, making the easily collected sweat an ideal candidate for biomarker discoveries. Our aim was to provide information about the normal composition of the sweat, and to study the chemical barrier found at the surface of skin. Sweat samples from healthy individuals were collected during sauna bathing, and the global protein panel was analysed by label-free mass spectrometry. SRM-based targeted proteomic methods were designed and stable isotope labelled reference peptides were used for method validation. Ninety-five sweat proteins were identified, 20 of them were novel proteins. It was shown that dermcidin is the most abundant sweat protein, and along with apolipoprotein D, clusterin, prolactin-inducible protein and serum albumin, they make up 91% of secreted sweat proteins. The roles of these highly abundant proteins were reviewed; all of which have protective functions, highlighting the importance of sweat glands in composing the first line of innate immune defense system, and maintaining the epidermal barrier integrity. Our findings with regard to the proteins forming the chemical barrier of the skin as determined by label-free quantification and targeted proteomics methods are in accordance with previous studies, and can be further used as a starting point for non-invasive sweat biomarker research. © 2015 European Academy of Dermatology and Venereology.

  20. Recent developments in application of stable isotope analysis on agro-product authenticity and traceability.

    PubMed

    Zhao, Yan; Zhang, Bin; Chen, Gang; Chen, Ailiang; Yang, Shuming; Ye, Zhihua

    2014-02-15

    With the globalisation of agro-product markets and convenient transportation of food across countries and continents, the potential for distribution of mis-labelled products increases accordingly, highlighting the need for measures to identify the origin of food. High quality food with identified geographic origin is a concern not only for consumers, but also for agriculture farmers, retailers and administrative authorities. Currently, stable isotope ratio analysis in combination with other chemical methods gradually becomes a promising approach for agro-product authenticity and traceability. In the last five years, a growing number of research papers have been published on tracing agro-products by stable isotope ratio analysis and techniques combining with other instruments. In these reports, the global variety of stable isotope compositions has been investigated, including light elements such as C, N, H, O and S, and heavy isotopes variation such as Sr and B. Several factors also have been considered, including the latitude, altitude, evaporation and climate conditions. In the present paper, an overview is provided on the authenticity and traceability of the agro-products from both animal and plant sources by stable isotope ratio analysis. Copyright © 2013 Elsevier Ltd. All rights reserved.