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1

Efficient synthesis of D-branched-chain amino acids and their labeled compounds with stable isotopes using D-amino acid dehydrogenase.  

PubMed

D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (>99 %) and optical purity (>99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives. PMID:23661083

Akita, Hironaga; Suzuki, Hirokazu; Doi, Katsumi; Ohshima, Toshihisa

2014-02-01

2

Isotope-labeled immunoassays without radiation waste  

E-print Network

Isotope-labeled immunoassays without radiation waste Guomin Shan*, Wei Huang*, Shirley J. Gee with radioactive materials, and (iii) short shelf-life of the labeled re- agents. The advantage of isotopic with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay

Hammock, Bruce D.

3

Glutathione specifically labeled with isotopes.  

PubMed

A procedure for synthesis of glutathione selectivity labeled with isotopes is described. A strain of Escherichia coli enriched in its content of gamma-glutamylcysteine synthetase and glutathione synthetase by recombinant DNA techniques is immobilized in a carrageenan matrix and treated with toluene to render the cells more permeable to the substrates. The immobilized cell matrix is incubated with a mixture containing the appropriately labeled amino acid, the other amino acid constituents of glutathione, ATP, and acetylphosphate. The radiolabeled product is isolated by column chromatography. PMID:2867714

Murata, K; Abbott, W A; Bridges, R J; Meister, A

1985-10-01

4

Isotope-coded protein label.  

PubMed

A great variety of technologies using stable isotope labeling in combination with mass spectrometry have been described being tools to identify and relatively quantify proteins within complex mixtures. Here, we present a method, termed isotope-coded protein label (ICPL), which is capable of high-throughput quantitative proteome profiling on a global scale. Since ICPL is based on tagging stable isotope derivatives at the free amino groups of intact proteins, the method is applicable to any protein sample, including extracts from tissues or body fluids. All separation methods currently employed in proteome studies can be used to reduce complexity on the protein level. After enzymatic cleavage of the protein fractions, the ratios of peptides from different proteome states can be calculated by simple MS-based mass spectrometric analyses. Only peptides representing different expression levels in the different proteomic states are further analyzed by tandem-mass spectrometry to identify respective proteins. For quantification of proteins from multiplexed ICPL experiments, ICPLQuant was developed, a software package especially designed to cover the whole ICPL workflow. The ICPL method results in accurate and reproducible quantification of proteins and high sequence coverage, indispensable for a comprehensive detection of posttranslational modifications and discrimination of protein isoforms. PMID:22665300

Kellermann, Josef; Lottspeich, Friedrich

2012-01-01

5

Synthesis of isotopically labeled epothilones  

PubMed Central

The epothilones, including epothilones B and D, are macrocyclic lactones which have potent cytotoxicities and which promote the polymerization of tubulin to mictotubules by binding to and stabilizing the tubulin polymer. They have a very similar mechanism of action to paclitaxel (Taxol®). The determination of the microtubule-binding conformation of the epothilones is an important piece of information in designing improved analogs for possible clinical use, and internuclear distance information that will assist the determination of this conformation can be obtained by REDOR NMR studies of microtubule-bound epothilones with appropriate stable isotope labels. Analogs of epothilone B and epothilone D with [2H3] and [19F] labels were prepared from an advanced precursor for potential use in REDOR NMR studies to determine internuclear distances in tubulin-bound ligand. PMID:24307484

Ganesh, Thota; Brodie, Peggy J.; Banerjee, Abhijit; Bane, Susan; Kingston, David G. I.

2014-01-01

6

Multiple isotopic labels for quantitative mass spectrometry  

PubMed Central

Quantitative mass spectrometry is often performed using isotopically-labeled samples. While the 4-trimethylammoniumbutyryl (TMAB) labels have many advantages over other isotopic tags, only two forms have previously been synthesized (i.e. a heavy form containing 9 deuteriums and a light form without deuterium). In the present report, two additional forms containing 3 and 6 deuteriums have been synthesized and tested. These additional isotopic tags perform identically to the previously reported tags; peptides labeled with the new TMAB reagents co-elute from reverse phase HPLC columns with peptides labeled with the lighter and heavier TMAB reagents. Altogether, these 4 tags allow for multivariate analysis in a single liquid chromatography/mass spectrometry analysis, with each isotopically tagged peptide differing in mass by 3 Da per tag incorporated. The synthetic scheme is described in simple terms so that a biochemist without specific training in organic chemistry can perform the synthesis. The interpretation of tandem mass spectrometry data for the TMAB-labeled peptides is also described in more detail. The additional TMAB isotopic reagents described here, together with the additional description of the synthesis and analysis should allow these labels to be more widely used for proteomics and peptidomics analyses. PMID:19551992

Morano, Cain; Zhang, Xin; Fricker, Lloyd D.

2009-01-01

7

Intrinsic isotopic 13C labelling of polyphenols.  

PubMed

The intrinsic isotopic labelling of plants with (13)CO2 is an effective method to generate highly labelled compounds using photosynthesis and avoiding labour-intensive complex organic syntheses. In this study, the intrinsic isotopic labelling of polyphenols in parsley, spinach and peppermint is shown for the first time. The plants were grown in an atmosphere where (12)CO2 was replaced by (13)CO2, in order to generate highly labelled compounds. The total content of (13)C as well as the individual polyphenols were analysed by Isotopic Ratio-MS and HPLC-Iontrap-MS(n). After 34 days of plant growth under (13)CO2, degree of labelling was found to be higher than 90 atom% (13)C for most polyphenols, predominantly consisting of highly and fully labelled isotopomers; the total plant material contained more than 88 atom% (13)C. Such highly labelled compounds can be used in future studies to dissect both metabolism and bioavailability of polyphenols in humans. PMID:23870998

Gleichenhagen, Maike; Zimmermann, Benno F; Herzig, Birgit; Janzik, Ingar; Jahnke, Siegfried; Boner, Markus; Stehle, Peter; Galensa, Rudolf

2013-12-01

8

Stereospecific multiple isotopic labeling of benzyl alcohol.  

PubMed

Isotopically labeled enzymatic substrates and biological metabolites are useful for many mechanistic analyses, particularly the study of kinetic and equilibrium isotope effects, determining the stereospecificity of enzymes, and resolving metabolic pathways. Here, we present the one-pot synthesis, purification, and kinetic analysis of 7R-[(2) H]-phenyl-[(14) C]-benzyl alcohol. The procedure involves a chemoenzymatic synthesis that couples formate dehydrogenase to alcohol dehydrogenase with a catalytic amount of nicotinamide cofactor. The reaction goes to completion overnight, and the measurement of a competitive kinetic isotope effect on the enzymatic oxidation of the purified product identified no (1) H contamination. This measurement is very sensitive to such isotopic contamination and verified the high level of isotopic and enantiomeric purity yielded by the new synthetic procedure. PMID:24327376

Roston, Daniel; Kohen, Amnon

2014-02-01

9

Stereospecific Multiple Isotopic Labeling of Benzyl Alcohol  

PubMed Central

Isotopically labeled enzymatic substrates and biological metabolites are useful for many mechanistic analyses, particularly the study of kinetic and equilibrium isotope effects, determining the stereospecificity of enzymes, and resolving metabolic pathways. Here we present the 1-pot synthesis, purification, and kinetic analysis of 7R-[2H]-phenyl-[14C]-benzyl alcohol. The procedure involves a chemoenzymatic synthesis that couples formate dehydrogenase to alcohol dehydrogenase with a catalytic amount of nicotinamide cofactor. The reaction goes to completion overnight, and the measurement of a competitive kinetic isotope effect on the enzymatic oxidation of the purified product identified no 1H contamination. This measurement is very sensitive to such isotopic contamination and verified the high level of isotopic and enantiomeric purity yielded by the new synthetic procedure. PMID:24327376

Roston, Daniel; Kohen, Amnon

2015-01-01

10

Cyclic enzymatic solid phase synthesis of isotopically labeled DNA oligonucleotides.  

PubMed

Isotopic labeling of DNA using standard solid phase synthesis requires expensive phosphoramidites that are used in large excess. We have developed a protocol where enzymatic, cyclic, solid phase synthesis of DNA facilitates a more economical use of the less expensive labeled DNA triphosphates (dNTP). In this approach, the DNA template is immobilized on an epoxy-activated solid support. Both the support and the linkage between DNA and resin are inert to high pH conditions which are required for product release in this scheme. Efficient covalent attachment of the DNA to the resin was achieved when the reaction was carried out in MgCl2/CAPS. The enzymatic fill in reaction as well as product release and recycling conditions were optimized for efficient reuse of dNTPs without any purification. The developed protocol was used to generate a selectively [(13)C, (15)N] G labeled 10-mer duplex. PMID:20183571

Khan, Ahmed M; Mishra, Subrata H; Germann, Markus W

2009-11-01

11

Quantitative Analysis of Snake Venoms Using Soluble Polymer-based Isotope Labeling*S?  

PubMed Central

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-containing peptides, and the subsequent tagged peptides are released and analyzed using nanoflow liquid chromatography-tandem mass spectrometry. The SoPIL strategy was used to quantify venom proteins from two pairs of venomous snakes: Crotalus scutulatus scutulatus type A, C. scutulatus scutulatus type B, Crotalus oreganus helleri, and Bothrops colombiensis. The hemorrhagic, hemolytic, clotting ability, and fibrinogenolytic activities of crude venoms were measured and correlated with difference in protein abundance determined by the SoPIL analysis. The SoPIL approach could provide an efficient and widely applicable tool for quantitative proteomics. PMID:18089550

Galan, Jacob A.; Guo, Minjie; Sanchez, Elda E.; Cantu, Esteban; Rodriguez-Acosta, Alexis; Perez, John C.; Tao, W. Andy

2008-01-01

12

Raman spectroscopic and mass spectrometric investigations of the hydrogen isotopes and isotopically labelled methane  

Microsoft Academic Search

Suitable analytical methods must be tested and developed for monitoring the individual process steps within the fuel cycle of a fusion reactor and for tritium accountability. The utility of laser-Raman spectroscopy accompanied by mass spectrometry with an Omegatron was investigated using the analysis of all hydrogen isotopes and isotopically labeled methanes as an example. The Omegatron is useful for analyzing

Fluor Daniel Hanford

1997-01-01

13

Synthesis of stable isotope labelled internal standards for drug-drug interaction (DDI) studies.  

PubMed

The syntheses of stable isotope labelled internal standards of important CYP-isoform selective probes, like testosterone 1, diclofenac 3, midazolam 5, and dextromethorphan 7, as well as their corresponding hydroxylated metabolites 6?-hydroxytestosterone 2, 4'-hydroxydiclofenac 4, 1'-hydroxymidazolam 6 and dextrorphan 8 are reported. Microwave-enhanced H/D-exchange reactions applying either acid, base, or homogeneous and heterogeneous transition metal catalysis, or combinations thereof proved to be highly efficient for direct deuterium labelling of the above mentioned probes. Compared to conventional stepwise synthetic approaches, the combination of H/D exchange and biotransformation provides the potential for considerable time- and cost savings, in particular for the synthesis of the stable isotope labelled internal standards of 4'-hydroxydiclofenac 4 and 1'-hydroxymidazolam 6. PMID:22890009

Atzrodt, J; Blankenstein, J; Brasseur, D; Calvo-Vicente, S; Denoux, M; Derdau, V; Lavisse, M; Perard, S; Roy, S; Sandvoss, M; Schofield, J; Zimmermann, J

2012-09-15

14

From isotope labeled CH3CN to N2 inside single-walled carbon nanotubes  

E-print Network

From isotope labeled CH3CN to N2 inside single-walled carbon nanotubes Christian Kramberger to this peculiar place? We have used N15 and C13 isotope labeled acetonitrile during the synthesis of single-walled carbon nanotubes to investigate this process. The isotope shifts of phonons and vibrons are observed

Maruyama, Shigeo

15

Quantitating isotopic molecular labels with accelerator mass spectrometry.  

PubMed

Accelerator mass spectrometry (AMS) traces isotopically labeled biochemicals and provides significant new directions for understanding molecular kinetics and dynamics in biological systems. AMS traces low-abundance radioisotopes for high specificity but detects them with MS for high sensitivity. AMS reduces radiation exposure doses to levels safe for use in human volunteers of all ages. Total radiation exposures are equivalent to those obtained in very short airplane flights, a commonly accepted radiation risk. Waste products seldom reach the Nuclear Regulatory Commission (NRC) definition of radioactive waste material for (14)C and (3)H. Attomoles of labeled compounds are quantified in milligram-sized samples, such as 20 microl of blood. AMS is available from several facilities that offer services and new spectrometers that are affordable. Detailed examples of designing AMS studies are provided, and the methods of analyzing AMS data are outlined. PMID:16401517

Vogel, John S; Love, Adam H

2005-01-01

16

Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture  

Microsoft Academic Search

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling\\u000a of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling\\u000a of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as\\u000a a source of high-quality, non-toxic yeastolates with the major

T. A. Egorova-Zachernyuk; G. J. C. G. M. Bosman; A. M. A. Pistorius; W. J. DeGrip

2009-01-01

17

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

PubMed Central

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-13C]-compound to generate [U-13C]-volatile standards for use in subsequent experimental profiling studies. [U-13C]-?-linolenic acid (C18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-13C]-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. Using 250 ?L starting sample, labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-13C]-labeled standards, limits of detection comparable to or better than previous HS-SPME reports were achieved, 0.010–1.04 ng/g. The performance of the [U-13C]-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-13C]-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-13C]-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-13C]-sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard. PMID:22662968

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

2012-01-01

18

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling  

PubMed Central

Tracing rare 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 or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration, and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling. PMID:24457314

Soong, Jennifer L; Reuss, Dan; Pinney, Colin; Boyack, Ty; Haddix, Michelle L; Stewart, Catherine E; Cotrufo, M. Francesca

2014-01-01

19

Triple isotopic labeling and kinetic isotope effects: Exposing H-transfer steps in enzymatic systems†  

PubMed Central

Kinetic isotope effect (KIE) studies can provide insight into the mechanism and kinetics of specific chemical steps in complex catalytic cascades. Recent results from hydrogen KIE measurements have examined correlations between enzyme dynamics and catalytic function, leading to a surge of studies in this area. Unfortunately, most enzymatic H-transfer reactions are not rate-limiting and the observed KIEs do not reliably reflect the intrinsic KIEs on the chemical step of interest. Given their importance to understanding the chemical step under study, accurate determination of the intrinsic KIE from the observed data is essential. In 1975, Northrop developed an elegant method to assess intrinsic KIEs from their observed values [Northrop, D. B. (1975) Steady-state analysis of kinetic isotope effects in enzymic reactions, Biochemistry, 14, 2644–2651]. The Northrop method involves KIE measurements using all three hydrogen isotopes, where one of them serves as the reference isotope. This method has been successfully used with different combinations of observed KIEs over the years but criteria for a rational choice of reference isotope have never before been experimentally determined. Here we compare different reference isotopes (and hence distinct experimental designs) using the reduction of dihydrofolate and dihydrobiopterin by two dissimilar enzymes as model reactions. A number of isotopic labeling patterns have been applied to facilitate the comparative study of reference isotopes. The results demonstrate the versatility of the Northrop method, and that such experiments are limited only by synthetic techniques, availability of starting materials, and the experimental error associated with the use of distinct combinations of isotopologues. PMID:21688781

Sen, Arundhuti; Yahashiri, Atsushi; Kohen, Amnon

2011-01-01

20

Two Efficient Label-Equivalence-Based Connected-Component Labeling Algorithms for 3-D Binary Images  

PubMed Central

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. PMID:21324785

He, Lifeng; Chao, Yuyan; Suzuki, Kenji

2014-01-01

21

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

DOEpatents

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).

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

2014-12-09

22

A Simple and Efficient Connected Components Labeling Algorithm  

Microsoft Academic Search

We describe a two-scan algorithm for labeling connected components in binary images in raster format. Unlike the classical two-scan approach, our algorithm processes equivalences during the first scan by merging equivalence classes as soon as a new equivalence is found. We show that this significantly improves the efficiency of the labeling process with respect to the classical approach. The data

Luigi Di Stefano; Andrea Bulgarelli

1999-01-01

23

N-glycosylamine-mediated isotope labeling for mass spectrometry-based quantitative analysis of N-linked glycans.  

PubMed

N-linked glycosylation is a major protein modification involved in many essential cellular functions. Methods capable of quantitative glycan analysis are highly valuable and have been actively pursued. Here we describe a novel N-glycosylamine-based strategy for isotopic labeling of N-linked glycans for quantitative analysis by use of mass spectrometry (MS). This strategy relies on the primary amine group on the reducing end of freshly released N-linked glycans for labeling, and eliminates the need for the harsh labeling reaction conditions and/or tedious cleanup procedures required by existing methods. By using NHS-ester amine chemistry we used this strategy to label N-linked glycans from a monoclonal antibody with commercially available tandem mass tags (TMT). Only duplex experiments can be performed with currently available TMT reagents, because quantification is based on the intensity of intact labeled glycans. Under mild reaction conditions, greater than 95% derivatization was achieved in 30 min and the labeled glycans, when kept at -20 °C, were stable for more than 10 days. By performing glycan release, TMT labeling, and LC-MS analysis continuously in a single volatile aqueous buffer without cleanup steps, we were able to complete the entire analysis in less than 2 h. Quantification was highly accurate and the dynamic range was large. Compared with previously established methods, N-glycosylamine-mediated labeling has the advantages of experimental simplicity, efficient labeling, and preserving glycan integrity. PMID:23670280

Gong, Bing; Hoyt, Erik; Lynaugh, Heather; Burnina, Irina; Moore, Renee; Thompson, Alissa; Li, Huijuan

2013-07-01

24

Uniform sup 13 C isotope labeling of proteins with sodium acetate for NMR studies: Application to human carbonic anhydrase II  

SciTech Connect

Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of ({sup 13}C{sub 6}, 99%)glucose. The authors demonstrate here that uniformly (>95%) {sup 13}C and {sup 15}N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium (1,2-{sup 13}C{sub 2},99%)acetate as the sole carbon source and ({sup 15}N, 99%)ammonium chloride as the sole nitrogen source. In addition, the authors demonstrate that this labeling scheme can be extended to include uniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium (1-{sup 13}C, 99%)acetate and sodium (2-{sup 13}C, 99%)acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labeling using isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the {sup 1}H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium (1,2-{sup 13}C{sub 2}, 99%)acetate growth media is considerably less than the cost of the ({sup 13}C{sub 6}, 99%)glucose growth media. They report here the first published {sup 13}C and {sup 15}N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.

Venters, R.S.; Calderone, T.L.; Spicer, L.D.; Fierke, C.A. (Duke Univ., Durham, NC (USA))

1991-05-07

25

Multisubstrate Isotope Labeling and Metagenomic Analysis of Active Soil Bacterial Communities  

PubMed Central

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

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

26

Autotrophic Production of Stable-Isotope-Labeled Arginine in Ralstonia eutropha Strain H16  

PubMed Central

With the aim of improving industrial-scale production of stable-isotope (SI)-labeled arginine, we have developed a system for the heterologous production of the arginine-containing polymer cyanophycin in recombinant strains of Ralstonia eutropha under lithoautotrophic growth conditions. We constructed an expression plasmid based on the cyanophycin synthetase gene (cphA) of Synechocystis sp. strain PCC6308 under the control of the strong PcbbL promoter of the R. eutropha H16 cbbc operon (coding for autotrophic CO2 fixation). In batch cultures growing on H2 and CO2 as sole sources of energy and carbon, respectively, the cyanophycin content of cells reached 5.5% of cell dry weight (CDW). However, in the absence of selection (i.e., in antibiotic-free medium), plasmid loss led to a substantial reduction in yield. We therefore designed a novel addiction system suitable for use under lithoautotrophic conditions. Based on the hydrogenase transcription factor HoxA, this system mediated stabilized expression of cphA during lithoautotrophic cultivation without the need for antibiotics. The maximum yield of cyanophycin was 7.1% of CDW. To test the labeling efficiency of our expression system under actual production conditions, cells were grown in 10-liter-scale fermentations fed with 13CO2 and 15NH4Cl, and the 13C/15N-labeled cyanophycin was subsequently extracted by treatment with 0.1 M HCl; 2.5 to 5 g of [13C/15N]arginine was obtained per fed-batch fermentation, corresponding to isotope enrichments of 98.8% to 99.4%. PMID:22941075

Lütte, Steffen; Pohlmann, Anne; Zaychikov, Evgeny; Becher, Johannes R.; Heumann, Hermann; Friedrich, Bärbel

2012-01-01

27

Production, purification and detergent exchange of isotopically labeled Bacillussubtilis cytochrome b??? (SdhC).  

PubMed

Cytochrome b??? 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 b???. In this work we present an efficient procedure for the isolation of preparative amounts of isotopically labeled B. subtilis cytochrome b??? 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. PMID:21641999

Baureder, Michael; Hederstedt, Lars

2011-11-01

28

Preparation of heteroelement-incorporated and stable isotope-labeled protein standards for quantitative proteomics.  

PubMed

A major obstacle for further development of quantitative proteomics is the lack of accurately quantified protein standards. The following protocol describes innovative methods for the production of stable isotope-labeled protein standards. Their production is achieved by cell-free protein synthesis, which enables simultaneous incorporation of selenomethionine and stable isotope-labeled amino acids. The selenium tag allows sensitive and accurate quantification by inductively coupled plasma mass spectrometry (ICP-MS). The stable isotope label allows internal standardization in mass spectrometry-based proteomics by electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Both label types can be placed within a single protein RISQ standard (recombinant isotope-labeled and selenium quantified) or can be distributed over two types of related RSQ and RIQ standards for the same target protein (recombinant selenium quantified and recombinant isotope-labeled and quantified). The combination of cell-free synthesis as production method with ICP-MS and ESI-MS/MS as detection methods results in protein standards, which are quantified at an outstanding level of accuracy. PMID:24792000

Konopka, Anna; Zinn, Nico; Wild, Christina; Lehmann, Wolf D

2014-01-01

29

Stable isotope-labeling studies in metabolomics: new insights into structure and dynamics of metabolic networks  

PubMed Central

The rapid emergence of metabolomics has enabled system-wide measurements of metabolites in various organisms. However, advances in the mechanistic understanding of metabolic networks remain limited, as most metabolomics studies cannot routinely provide accurate metabolite identification, absolute quantification and flux measurement. Stable isotope labeling offers opportunities to overcome these limitations. Here we describe some current approaches to stable isotope-labeled metabolomics and provide examples of the significant impact that these studies have had on our understanding of cellular metabolism. Furthermore, we discuss recently developed software solutions for the analysis of stable isotope-labeled metabolomics data and propose the bioinformatics solutions that will pave the way for the broader application and optimal interpretation of system-scale labeling studies in metabolomics. PMID:24568354

Chokkathukalam, Achuthanunni; Kim, Dong-Hyun; Barrett, Michael P; Breitling, Rainer; Creek, Darren J

2014-01-01

30

Stable isotope N-phosphorylation labeling for Peptide de novo sequencing and protein quantification based on organic phosphorus chemistry.  

PubMed

In this paper, we describe the development of a novel stable isotope N-phosphorylation labeling (SIPL) strategy for peptide de novo sequencing and protein quantification based on organic phosphorus chemistry. The labeling reaction could be performed easily and completed within 40 min in a one-pot reaction without additional cleanup procedures. It was found that N-phosphorylation labeling reagents were activated in situ to form labeling intermediates with high reactivity targeting on N-terminus and ?-amino groups of lysine under mild reaction conditions. The introduction of N-terminal-labeled phosphoryl group not only improved the ionization efficiency of peptides and increased the protein sequence coverage for peptide mass fingerprints but also greatly enhanced the intensities of b ions, suppressed the internal fragments, and reduced the complexity of the tandem mass spectrometry (MS/MS) fragmentation patterns of peptides. By using nano liquid chromatography chip/time-of-flight mass spectrometry (nano LC-chip/TOF MS) for the protein quantification, the obtained results showed excellent correlation of the measured ratios to theoretical ratios with relative errors ranging from 0.5% to 6.7% and relative standard deviation of less than 10.6%, indicating that the developed method was reproducible and precise. The isotope effect was negligible because of the deuterium atoms were placed adjacent to the neutral phosphoryl group with high electrophilicity and moderately small size. Moreover, the SIPL approach used inexpensive reagents and was amenable to samples from various sources, including cell culture, biological fluids, and tissues. The method development based on organic phosphorus chemistry offered a new approach for quantitative proteomics by using novel stable isotope labeling reagents. PMID:23134482

Gao, Xiang; Wu, Hanzhi; Lee, Kim-Chung; Liu, Hongxia; Zhao, Yufen; Cai, Zongwei; Jiang, Yuyang

2012-12-01

31

Energy-efficient appliance labeling in China: Lessons for successful labeling programs in varied markets  

SciTech Connect

Appliance ownership and production has increased dramatically in China in the past two decades. From extremely low levels in 1980, China's appliance industry has become one of the largest in the world, with sales topping U.S. $14.4 billion in 2000. In 1981, less than 1 percent of urban Chinese households owned a refrigerator; by 1998, that number had increased to over 75 percent. This dramatic increase in sales and ownership leads to an excellent opportunity to impact energy consumption in China by affecting the energy efficiency of appliances being bought and sold. In general, Chinese consumers value energy efficiency and are knowledgeable about the operating costs of major appliances. However, the Chinese marketplace does not provide information that consumers trust about the energy consumption of specific products. Thus, several interdependent organizations have emerged in China to provide information and market supports for energy efficiency. This paper describes the appliance market in China and the evolution of its standards and labeling programs and the agencies that implement them. It discusses the authors' work with these organizations in developing energy efficiency criteria and supporting an energy efficiency endorsement labeling program in China. It describes how the authors have used their experience with ENERGY STAR{reg_sign} and other programs in the U.S. to work with China to develop a successful program specific to Chinese conditions, with a particular emphasis on refrigerators. It then gives the author's market assessment of the Chinese refrigerator market and recommendations for a successful labeling program and transferable lessons for developing energy efficiency labeling programs in varied markets. This paper is based on the authors' market research, their support in setting energy efficiency criteria in China, interviews with Chinese manufacturers, retailers, and sales staff, and the development and implementation of labeling strategies and promotion in China.

Lin, Jiang; Townend, Jeanne; Fridley, David; McNeil, Gary; Silva, Tony; Clark, Robin

2002-08-20

32

Multisubstrate isotope labeling and metagenomic analysis of active soil bacterial communities.  

PubMed

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 ((12)C) or stable-isotope-labeled ((13)C) 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 (13)C-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. Importance: The ability to identify genes based on function, instead of sequence homology, allows the discovery of genes that would not be identified through sequence alone. This is arguably the most powerful application of metagenomics for the recovery of novel genes and a natural partner of the stable-isotope-probing approach for targeting active-yet-uncultured microorganisms. We expanded on previous efforts to combine stable-isotope probing and metagenomics, enriching microorganisms from multiple soils that were active in degrading plant-derived carbohydrates, followed by construction of a cellulose-based metagenomic library and recovery of glycoside hydrolases through functional metagenomics. The major advance of our study was the discovery of active-yet-uncultivated soil microorganisms and enrichment of their glycoside hydrolases. We recovered positive cosmid clones in a higher frequency than would be expected with direct metagenomic analysis of soil DNA. This study has generated an invaluable metagenomic resource that future research will exploit for genetic and enzymatic potential. PMID:25028422

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; Charles, T C; Neufeld, J D

2014-01-01

33

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans.  

PubMed

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with (15)N,(13)C by providing them with isotopically labeled bacteria. (2)H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the "test" GPCR to demonstrate the viability of this approach. Although the worms' cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization. PMID:25461480

Salom, David; Cao, Pengxiu; Yuan, Yiyuan; Miyagi, Masaru; Feng, Zhaoyang; Palczewski, Krzysztof

2015-03-01

34

Preparation of amino-acid-type selective isotope labeling of protein expressed in Pichia pastoris.  

PubMed

We report the culture conditions for successful amino-acid-type selective (AATS) isotope labeling of protein expressed in Pichia pastoris (P. pastoris). Rhodostomin (Rho), a six disulfide-bonded protein expressed in P. pastoris with the correct fold, was used to optimize the culture conditions. The concentrations of [alpha-15N] selective amino acid, nonlabeled amino acids, and ammonium chloride, as well as induction time, were optimized to avoid scrambling and to increase the incorporation rate and protein yield. The optimized protocol was successfully applied to produce AATS isotope-labeled Rho. The labeling of [alpha-15N]Cys has a 50% incorporation rate, and all 12 cysteine resonances were observed in HSQC spectrum. The labeling of [alpha-15N]Leu, -Lys, and -Met amino acids has an incorporation rate greater than 65%, and the expected number of resonances in the HSQC spectra were observed. In contrast, the labeling of [alpha-15N]Asp and -Gly amino acids has a low incorporation rate and the scrambling problem. In addition, the culture condition was successfully applied to label dendroaspin (Den), a four disulfide-bonded protein expressed in P. pastoris. Therefore, the described condition should be generally applicable to other proteins produced in the P. pastoris expression system. This is the first report to present a protocol for AATS isotope labeling of protein expressed in P. pastoris for NMR study. PMID:16283643

Chen, Chiu-Yueh; Cheng, Chun-Ho; Chen, Yi-Chun; Lee, Jenq-Chang; Chou, Shan-Ho; Huang, Wenya; Chuang, Woei-Jer

2006-01-01

35

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

PubMed Central

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

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

2013-01-01

36

An experimental evaluation of transgenerational isotope labelling in a coral reef grouper  

Microsoft Academic Search

Transgenerational isotope labelling (TRAIL) using enriched stable isotopes provides a novel means of mass-marking marine fish\\u000a larvae and estimating larval dispersal. The technique, therefore, provides a new way of addressing questions about demographic\\u000a population connectivity and larval export from no-take marine protected areas. However, successful field applications must\\u000a be preceded by larval rearing studies that validate the geochemical marking technique,

David H. Williamson; Geoffrey P. Jones; Simon R. Thorrold

2009-01-01

37

Proteome scale turnover analysis in live animals using stable isotope metabolic labeling.  

PubMed

At present most quantitative proteomics investigations are focused on the analysis of protein expression differences between two or more sample specimens. With each analysis a static snapshot of a cellular state is captured with regard to protein expression. However, any information on protein turnover cannot be obtained using classic methodologies. Protein turnover, the result of protein synthesis and degradation, represents a dynamic process, which is of equal importance to understanding physiological processes. Methods employing isotopic tracers have been developed to measure protein turnover. However, applying these methods to live animals is often complicated by the fact that an assessment of precursor pool relative isotope abundance is required. Also, data analysis becomes difficult in case of low label incorporation, which results in a complex convolution of labeled and unlabeled peptide mass spectrometry signals. Here we present a protein turnover analysis method that circumvents this problem using a (15)N-labeled diet as an isotopic tracer. Mice were fed with the labeled diet for limited time periods and the resulting partially labeled proteins digested and subjected to tandem mass spectrometry. For the interpretation of the mass spectrometry data, we have developed the ProTurnyzer software that allows the determination of protein fractional synthesis rates without the need of precursor relative isotope abundance information. We present results validating ProTurnyzer with Escherichia coli protein data and apply the method to mouse brain and plasma proteomes for automated turnover studies. PMID:21314131

Zhang, Yaoyang; Reckow, Stefan; Webhofer, Christian; Boehme, Michael; Gormanns, Philipp; Egge-Jacobsen, Wolfgang M; Turck, Christoph W

2011-03-01

38

Radiation oxidation of polypropylene: A solid-state 13C NMR study using selective isotopic labeling  

Microsoft Academic Search

Polypropylene samples, in which the three different carbon atoms along the chain were selectively labeled with carbon-13, were subjected to radiation under inert and air atmospheres, and to post-irradiation exposure in air at various temperatures. By using solid-state 13C NMR measurements at room temperature, we have been able to identify and quantify the oxidation products. The isotopic labeling provides insight

Daniel M. Mowery; Roger A. Assink; Dora K. Derzon; Sara B. Klamo; Robert Bernstein; Roger L. Clough

2007-01-01

39

Precursor ion scans for the targeted detection of stable-isotope-labeled peptides.  

PubMed

Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large-scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse-labeled with a stable isotope by metabolic incorporation of 'heavy' amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple-quadrupole ion trap mass spectrometer. PIS-based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several-fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique. PMID:19844962

Colzani, Mara; Bienvenut, Willy V; Faes, Eveline; Quadroni, Manfredo

2009-11-01

40

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...

41

Ion-Surface Reactions Involving Isotopically Labeled Langmuir-Blodgett Films  

E-print Network

(i.e., secondary ion mass spectrometry, SIMS11 ). Various mechanisms for alkyl and/or hydrogen for investigating ion-surface reactions is a custom tandem mass spectrometer equipped with two Extrel (PittsburghIon-Surface Reactions Involving Isotopically Labeled Langmuir-Blodgett Films Chungang Gu and Vicki

Wysocki, Vicki H.

42

Isotopic labelling studies on far-infrared spectra of nickel-histamine complexes  

NASA Astrophysics Data System (ADS)

Nickel-histamine (hm) complexes type [Ni(hm)Cl 2] and [Ni(hm) 3] X2 (Where X=Cl, Br, I, ClO 4) were investigated in the far-infrared region. Metal isotope labelling and deuteration effects were employed for observed band assignments. Metal-ligand vibrations were discussed and correlated with the structures of the complexes.

Dro?d?ewski, Piotr; Kordon, Ewa

2000-11-01

43

Analysis of Isotopic Labeling in Peptide Fragments by Tandem Mass Spectrometry  

PubMed Central

Phenotype in multicellular organisms is the consequence of dynamic metabolic events that occur in a spatially dependent fashion. This spatial and temporal complexity presents challenges for investigating metabolism; creating a need for improved methods that effectively probe biochemical events such as amino acid biosynthesis. Isotopic labeling can provide a temporal-spatial recording of metabolic events through, for example, the description of enriched amino acids in the protein pool. Proteins are therefore an important readout of metabolism and can be assessed with modern mass spectrometers. We compared the measurement of isotopic labeling in MS2 spectra obtained from tandem mass spectrometry under either higher energy collision dissociation (HCD) or collision induced dissociation (CID) at varied energy levels. Developing soybean embryos cultured with or without 13C-labeled substrates, and Escherichia coli MG1655 enriched by feeding 7% uniformly labeled glucose served as a source of biological material for protein evaluation. CID with low energies resulted in a disproportionate amount of heavier isotopologues remaining in the precursor isotopic distribution. HCD resulted in fewer quantifiable products; however deviation from predicted distributions were small relative to the CID-based comparisons. Fragment ions have the potential to provide information on the labeling of amino acids in peptides, but our results indicate that without further development the use of this readout in quantitative methods such as metabolic flux analysis is limited. PMID:24626471

Allen, Doug K.; Evans, Bradley S.; Libourel, Igor G. L.

2014-01-01

44

Isotope labeling pattern study of central carbon metabolites using GC/MS.  

PubMed

Determination of fluxes by (13)C tracer experiments depends on monitoring the (13)C labeling pattern of metabolites during isotope experiments. In metabolome-based (13)C metabolic flux analysis, liquid chromatography combined with mass spectrometry or tandem mass spectrometry (LC/MS or LC/MS/MS, respectively) has been mainly used as an analytical platform for isotope pattern studies of central carbon metabolites. However, gas chromatography with mass spectrometry (GC/MS) has several advantages over LC/MS, such as high sensitivity, low cost, ease of operation, and availability of mass spectra databases for comparison. In this study, analysis of isotope pattern for central carbon metabolites using GC/MS was demonstrated. First, a proper set of mass ions for central carbon metabolites was selected based on carbon backbone information and structural isomers of mass fragment ions. A total of 34 mass fragment ions was selected and used for the quantification of 25 central carbon metabolites. Then, to quantify isotope fractions, a natural mass isotopomer library for selected mass fragment ions was constructed and subtracted from isotopomer mass spectra data. The results revealed a surprisingly high abundance of partially labeled (13)C intermediates, such as 56.4% of fructose 6-phosphate and 47.6% of dihydroxyacetone phosphate at isotopic steady state, which were generated in the pentose phosphate pathway. Finally, dynamic changes of isotope fragments of central metabolites were monitored with a U-(13)C glucose stimulus response experiment in Kluyveromyces marxianus. With a comprehensive study of isotope patterns of central carbon metabolites using GC/MS, 25 central carbon metabolites and their isotopic fractions were successfully quantified. Dynamic and precise acquisition of isotope pattern can then be used in combination with proper kinetic models to calculate metabolic fluxes. PMID:25463204

Jung, Joon-Young; Oh, Min-Kyu

2015-01-01

45

Utilization of organophosphate:phosphate antiporter for isotope-labeling experiments in E. coli.  

PubMed

The transport of organophosphates across the cytoplasma membrane is mediated by organophosphate:phosphate antiporter proteins. In this work, we present the application of a recombinant phosphoenolpyruvate:phosphate antiporter for isotopic labeling experiments in E. coli strains. The antiporters UhpT, UhpT-D388C, and PgtP were investigated regarding transport activity and growth on phosphoenolpyruvate as sole carbon source. The expression of the protein variant UhpT-D388C in a shikimic acid producing E. coli strain was used to show the successful isotopic labeling of shikimic acid from extracellular phosphoenolpyruvate. The results demonstrate the possibility of a direct incorporation of exogenously applicated glycolysis intermediates into E. coli cells for (13) C-labeling experiments. PMID:25273627

Albermann, Christoph; Weiner, Michael; Tröndle, Julia; Weuster-Botz, Dirk; Sprenger, Georg A

2014-10-01

46

Extensive backbone dynamics in the GCAA RNA tetraloop analyzed using 13C NMR spin relaxation and specific isotope labeling  

PubMed Central

Conformational dynamics play a key role in the properties and functions of proteins and nucleic acids. Heteronuclear NMR spin relaxation is a uniquely powerful site-specific probe of dynamics in proteins and has found increasing applications to nucleotide base side chains and anomeric sites in RNA. Applications to the nucleic acid ribose backbone, however, have been hampered by strong magnetic coupling among ring carbons in uniformly 13C-labeled samples. In this work, we apply a recently-developed, metabolically-directed isotope labeling scheme that places 13C with high efficiency and specificity at the nucleotide ribose C2’ and C4’ sites. We take advantage of this scheme to explore backbone dynamics in the well-studied GCAA RNA tetraloop. Using a combination of CPMG (Carr-Purcell-Meiboom-Gill) and R1? relaxation dispersion spectroscopy to explore exchange processes on the microsecond to millisecond timescale, we find an extensive pattern of dynamic transitions connecting a set of relatively well-defined conformations. In many cases, the observed transitions appear to be linked to C3’-endo/C2’-endo sugar pucker transitions of the corresponding nucleotides, and may also be correlated across multiple nucleotides within the tetraloop. These results demonstrate the power of NMR spin relaxation based on alternate-site isotope labeling to open a new window into the dynamic properties of ribose backbone groups in RNA. PMID:19049467

Johnson, James E.; Hoogstraten, Charles G.

2009-01-01

47

Unambiguous assignment of short- and long-range structural restraints by solid-state NMR spectroscopy with segmental isotope labeling.  

PubMed

We present an efficient method for the reduction of spectral complexity in the solid-state NMR spectra of insoluble protein assemblies, without loss of signal intensity. The approach is based on segmental isotope labeling by using the split intein DnaE from Nostoc punctiforme. We show that the segmentally (13) C,(15) N-labeled prion domain of HET-s exhibits significantly reduced spectral overlap while retaining the wild-type structure and spectral quality. A large number of unambiguous distance restraints were thus collected from a single two-dimensional (13) C,(13) C cross-correlation spectrum. The observed resonances could be unambiguously identified as intramolecular without the need for preparing a dilute, less sensitive sample. PMID:25394265

Schubeis, Tobias; Lührs, Thorsten; Ritter, Christiane

2015-01-01

48

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

NASA Astrophysics Data System (ADS)

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.

Chandra, Subhash

2004-06-01

49

Use of (13)C stable isotope labelling for pathway and metabolic flux analysis in Leishmania parasites.  

PubMed

This protocol describes the combined use of metabolite profiling and stable isotope labelling to define pathways of central carbon metabolism in the protozoa parasite, Leishmania mexicana. Parasite stages are cultivated in standard or completely defined media and then rapidly transferred to chemically equivalent media containing a single (13)C-labelled nutrient. The incorporation of label can be followed over time or after establishment of isotopic equilibrium by harvesting parasites with rapid metabolic quenching. (13)C enrichment of multiple intracellular polar and apolar (lipidic) metabolites can be quantified using gas chromatography-mass spectrometry (GC-MS), while the uptake and secretion of (13)C-labelled metabolites can be measured by (13)C-NMR. Analysis of the mass isotopomer distribution of key metabolites provides information on pathway structure, while analysis of labelling kinetics can be used to infer metabolic fluxes. This protocol is exemplified using L. mexicana labelled with (13)C-U-glucose. The method can be used to measure perturbations in parasite metabolism induced by drug inhibition or genetic manipulation of enzyme levels and is broadly applicable to any cultured parasite stages. PMID:25388122

Saunders, Eleanor C; de Souza, David P; Chambers, Jennifer M; Ng, Milica; Pyke, James; McConville, Malcolm J

2015-01-01

50

Preparation of uniformly isotope labeled KcsA for solid state NMR: Expression, purification, reconstitution into liposomes and functional assay  

PubMed Central

We report the expression, purification, liposome reconstitution and functional validation of uniformly 13C and 15N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ~ 35–40 milligrams per liter of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-Vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR. PMID:23916531

Bhate, Manasi P.; Wylie, Benjamin J.; Thompson, Ameer; Tian, Lin; Nimigean, Crina; McDermott, Ann E.

2013-01-01

51

Efficient Site-Specific Labeling of Proteins via Cysteines  

PubMed Central

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

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

2011-01-01

52

Raman spectroscopic investigation of polycrystalline structures of CVD-grown graphene by isotope labeling  

NASA Astrophysics Data System (ADS)

Topological defects, such as point defects, dislocations and grain boundaries, have a dramatic influence on the chemical and physical properties of large-scale graphene grown by chemical vapor deposition (CVD) method. Here we demonstrate the Raman visualization of polycrystalline structures in an isotopically modified CVD graphene. By means of the reversible reaction of methane on a copper catalyst, the etching of 12C-lattice and surface deposition of 13C-atoms occur in CVD graphene by sequentially introducing hydrogen and isotopic methane after standard growth of graphene with full monolayer coverage. Spatial Raman spectroscopic mapping on labeled graphene reveals pronounced network-like 13C-rich regions, which are further identified to exist along the grain boundaries of graphene by low-energy electron microscopy. The structural defects inside the graphene grains are also targeted in the isotope labeling process. Our work opens a new way to investigate multiple grain structures in CVD graphene with a simple spectroscopic technique.Topological defects, such as point defects, dislocations and grain boundaries, have a dramatic influence on the chemical and physical properties of large-scale graphene grown by chemical vapor deposition (CVD) method. Here we demonstrate the Raman visualization of polycrystalline structures in an isotopically modified CVD graphene. By means of the reversible reaction of methane on a copper catalyst, the etching of 12C-lattice and surface deposition of 13C-atoms occur in CVD graphene by sequentially introducing hydrogen and isotopic methane after standard growth of graphene with full monolayer coverage. Spatial Raman spectroscopic mapping on labeled graphene reveals pronounced network-like 13C-rich regions, which are further identified to exist along the grain boundaries of graphene by low-energy electron microscopy. The structural defects inside the graphene grains are also targeted in the isotope labeling process. Our work opens a new way to investigate multiple grain structures in CVD graphene with a simple spectroscopic technique. Electronic supplementary information (ESI) available: Fig. S1, experimental scheme; Fig. S2-3, scanning electron microscopy analysis and optical images; Fig. S4-5, additional LEEM analysis of labeled graphene and hydrogen-etched graphene; Fig. S6-10, additional Raman spectra of labeled graphene. See DOI: 10.1039/c4nr03824j

Wang, Shengnan; Suzuki, Satoru; Hibino, Hiroki

2014-10-01

53

Selective isotopic labeling of a nitroxide spin label to enhance sensitivity for T2 oxymetry  

NASA Astrophysics Data System (ADS)

The synthesis and application of a novel compound, a variant of a standard spin label which has been used for T2-based oxymetry, is described. The compound is 4-hydro-3carbamoyl-2,2,5,5-tetraperdeuteromethylpyrrolin-1-yloxy- d12 (mHCTPO). It was developed to optimize both the sensitivity of the T 2 method to oxygen tension and the spectrometer signal for a given concentration of spin label. The compound is used with a very low frequency (250 MHz) electron paramagnetic resonance spectrometer for in vivo application and for imaging, but has application at X band. The compound provides a convenient spectral feature distinguishing broadening associated with self-interaction from that due to environmental oxygen.

Halpern, Howard J.; Peril, Miroslav; Nguyen, Thanh-D.; Spencer, David P.; Teicher, Beverly A.; Lin, Yawares J.; Bowman, Michael K.

54

Secondary isotope effects in liquid chromatography behaviour of 2H and 3H labelled solutes and solvents.  

PubMed

The separation of solutes that differ only in the extent of isotopic substitution of their hydrogen atoms, using either mixtures of isotopically non-modified or perdeuterated solvents as mobile phases, is described. The occurrence of a secondary isotope effect is demonstrated in reversed-phase liquid chromatography, which is independent of the nature of the stationary phase (different octadecyl-bonded silicas, an embedded alkylamide-bonded silica, as well as one polymeric stationary phase were tested), and the water content and the nature of organic modifier of the mobile phase. The separation of 24 structurally different isotopologue pairs (apolar compounds and polar compounds with exchangeable or non-exchangeable hydrogen atoms) is examined using reversed-phase liquid chromatography. It is found that the greater the number of isotopically substituted hydrogen atoms in a given organic solute, the better is the separation of a particular isotopologue pair. The single secondary isotope effect is shown to be dependent on the number of isotopic substitutions. The greater the number of these substitutions, the smaller is the single isotope effect. The single secondary isotope effect is higher for aromatic hydrocarbons than for aliphatic hydrocarbons. A secondary isotope effect is also observed in chiral chromatography and normal-phase liquid chromatography, as well as on changing the nature of the substituting isotope, i.e.: tritium instead of deuterium. Thus, we have demonstrated that the total secondary isotopic effect for hydrogen/tritium is higher than for hydrogen/deuterium. This isotope effect involves only the consequences of changes in interactions due to nuclear motions. Overall this study confirms the predominance of hydrophobic effects in retention processes in reversed-phase liquid chromatography. In reversed-phase liquid chromatography, a secondary isotope effect related to mobile phase composition is also observed. The behaviour of deuterium oxide and water in mobile phases of the same composition (%, w/w) is compared. Independent of the nature of the organic modifier (methanol, acetonitrile or ethanol), the effect of replacing H2O with 2H2O in the mobile phase, is an increase in the retention factors and an improvement in the chromatographic resolution of isotopologue pairs. This increase in the resolution is not accompanied by a change in the chromatographic selectivity. The measurement of liquid-liquid extraction coefficients proves that the effect is mainly due to the modification of the phase ratio. In general the effect of 2H-labelled solvents (2H2O and C2H3CN) as mobile phase components, compared to their isotopically non-modified isomers, can be rationalized on the basis of their lower polarisabilities. Overall the use of perdeuterated rather than isotopically non-modified solvents as mobile phase components leads to the most efficient separation systems. PMID:16631181

Valleix, Alain; Carrat, Sandrine; Caussignac, Céline; Léonce, Estelle; Tchapla, Alain

2006-05-26

55

Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study.  

PubMed

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as (15)N. By utilising hydroponic media that contain (15)N inorganic salts as the sole nitrogen source, near to 100% (15)N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled (14)N- and (15)N-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of (14)N/(15)N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the (14)N and (15)N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct (14)N and (15)N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). PMID:18538804

Bindschedler, Laurence V; Palmblad, Magnus; Cramer, Rainer

2008-07-01

56

An Efficient Method to Estimate Labelled Sample Size for Transductive LDA(QDA/MDA)  

E-print Network

An Efficient Method to Estimate Labelled Sample Size for Transductive LDA(QDA/MDA) Based on Bayes to Estimate Labelled Sample Size 275 kernel, and by maximizing the margin based on the unlabelled data labelled sample size becomes a necessity. Moreover, a detailed analysis of labelled sample size under

57

IMPACT OF DURATION OF INFUSION OF CHOICE ISOTOPE LABEL ON ISOTOPE RECYCLING IN GLUCOSE HOMEOSTASIS  

Technology Transfer Automated Retrieval System (TEKTRAN)

The purposes of this study were to quantitate the impact of the duration of infusion and choice of stable isotope of glucose on measures of glucose rate of appearance (glucose Ra) and to determine whether the differences observed were due to tracer recycling via the glycogen pool (direct pathway) or...

58

Mass-related inversion symmetry breaking and phonon self-energy renormalization in isotopically labeled AB-stacked bilayer graphene  

E-print Network

A mass-related symmetry breaking in isotopically labeled bilayer graphene (2LG) was investigated during in-situ electrochemical charging of AB stacked (AB-2LG) and turbostratic (t-2LG) layers. The overlap of the two ...

Araujo, Paulo Antonio Trinidade

59

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

PubMed Central

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

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

60

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

PubMed

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. PMID:21158445

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

2011-03-01

61

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

PubMed

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. PMID:24874385

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

2014-11-01

62

Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes  

Microsoft Academic Search

Abstact  A reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium-\\u000a and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5,1-imine ([G-3H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-3H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci\\/mol, respectively. The isotopomeric distribution of\\u000a deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal

Yu. A. Zolotarev; Yu. Yu. Firsova; A. Abaimov; A. K. Dadayan; V. S. Kosik; A. V. Novikov; N. V. Krasnov; B. V. Vaskovskii; I. V. Nazimov; G. I. Kovalev; N. F. Myasoedov

2009-01-01

63

A novel stable isotope labelling assisted workflow for improved untargeted LC-HRMS based metabolomics research.  

PubMed

Many untargeted LC-ESI-HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on non-labelled and highly (13)C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U-(13)C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC-HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87-135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U-(13)C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples. PMID:25057268

Bueschl, Christoph; Kluger, Bernhard; Lemmens, Marc; Adam, Gerhard; Wiesenberger, Gerlinde; Maschietto, Valentina; Marocco, Adriano; Strauss, Joseph; Bödi, Stephan; Thallinger, Gerhard G; Krska, Rudolf; Schuhmacher, Rainer

2014-01-01

64

[Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes].  

PubMed

A reaction of high-temperature solid-phase catalytic isotope exchange (HSCIE) was studied for the preparation of tritium- and deuterium-labeled ligands of glutamate and dopamine receptors. Tritium-labeled (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclopenten-5,10-imine ([G-(3)H]MK-801) and R(+)-7-hydroxy-N,N-di-n-propyl-2-aminotetraline ([G-(3)H]-7-OH-DPAT) were obtained with a specific activity of 210 and 120 Ci/mol, respectively. The isotopomeric distribution of deuterium-labeled ligands was studied using time-of-flight mass-spectrometer MX 5310 (ESI-o-TOF) with electrospray and orthogonal ion injection. Mean deuterium incorporation per ligand molecule was 11.09 and 3.21 atoms for [G-(2)H]MK-801 and [G-(2)H]-7-OH-DPAT, respectively. The isotope label was shown to be distributed all over the ligand molecule. The radioreceptor binding of tritium-labeled ligands [G-(3)H]MK-801 and [G-(3)H]-7-OH-DPAT was analyzed using the brain structure of Vistar rats. It was demonstrated that [G-(3)H]MK-801 specifically binds to hippocampus membranes with K(d) 8.3 +/- 1.4 nM, B(max) being 3345 +/- 300 fmol/mg protein. The [G-(3)H]-7-OH-DPAT ligand specifically binds to rat striatum membranes with K(d) 10.01 +/- 0.91 nM and B(max) 125 +/- 4.5 fmol/mg protein. It was concluded that the HSCIE reaction can be used for the preparation of highly tritium-labeled (+)-MK-801 and 7-OH-DPAT with retention of their physiological activities. PMID:19621047

Zolotarev, Iu A; Firstova, Iu Iu; Abaimov, D A; Dadaian, A K; Kosik, V S; Novikov, A V; Krasnov, N V; Vas'kovski?, B V; Nazimov, I V; Kovalev, G I; Miasoedov, N F

2009-01-01

65

Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry.  

PubMed

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis. PMID:25427836

Chen, Yi-Ting; Tuan, Li-Ping; Chen, Hsiao-Wei; Wei, I-An; Chou, Min-Yuan; Chen, Han-Min; Tyan, Yu-Chang; Chen, Sung-Fang

2015-01-01

66

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

PubMed

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. PMID:19345114

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

2009-07-01

67

Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics  

Microsoft Academic Search

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultane- ous and automated identification and quantitation of complex protein mixtures. Here we describe a method, termed SILAC, for stable isotope labeling by amino acids in cell culture, for the in vivo incorporation of

Shao-En Ong; Blagoy Blagoev; Irina Kratchmarova; Dan Bach Kristensen; Hanno Steen; Akhilesh Pandey; Matthias Mann

2002-01-01

68

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

PubMed

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 (53)Cr, (65)Cu and (106)Cd was fed for 2h 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 (53)Cr occurred after 5h of depuration (GPT), whereas 90% of the ingested (53)Cr was recovered in the feces after 22.5h of depuration (GRT). (53)Chromium was not significantly accumulated in the soft tissues upon exposure. In contrast, (65)Cu and (106)Cd assimilation was detectable for most experimental snails, i.e., (65/63)Cu and (106/114)Cd 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 (53)Cr egested in the feces during depuration and the concentration of (53)Cr 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. PMID:17467071

Croteau, Marie-Noële; Luoma, Samuel N; Pellet, Bastien

2007-06-15

69

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

USGS Publications Warehouse

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.

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

2007-01-01

70

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

PubMed

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. PMID:24228619

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

2013-11-27

71

Site-specific Orientation of an ?-helical Peptide Ovispirin-1 from Isotope Labeled SFG Spectroscopy  

PubMed Central

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

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

2013-01-01

72

Recombinant isotope labeled and selenium quantified proteins for absolute protein quantification.  

PubMed

A novel, widely applicable method for the production of absolutely quantified proteins is described, which can be used as internal standards for quantitative proteomic studies based on mass spectrometry. These standards are recombinant proteins containing an isotope label and selenomethionine. For recombinant protein expression, assembly of expression vectors fitted to cell-free protein synthesis was conducted using the gateway technology which offers fast access to a variety of genes via open reading frame libraries and an easy shuttling of genes between vectors. The proteins are generated by cell-free expression in a medium in which methionine is exchanged against selenomethionine and at least one amino acid is exchanged by a highly stable isotope labeled analogue. After protein synthesis and purification, selenium is used for absolute quantification by element mass spectrometry, while the heavy amino acids in the protein serve as reference in subsequent analyses by LC-ESI-MS or MALDI-MS. Accordingly, these standards are denominated RISQ (for recombinant isotope labeled and selenium quantified) proteins. In this study, a protein was generated containing Lys+6 ([(13)C(6)]-lysine) and Arg+10 ([(13)C(6),(15)N(4)]-arginine) so that each standard tryptic peptide contains a labeled amino acid. Apolipoprotein A1 was synthesized as RISQ protein, and its use as internal standard led to quantification of a reference material within the specified value. Owing to their cell-free expression, RISQ proteins do not contain posttranslational modifications. Thus, correct quantitative data by ESI- or MALDI-MS are restricted to quantifications based on peptides derived from unmodified regions of the analyte protein. Therefore, besides serving as internal standards, RISQ proteins stand out as new tools for quantitative analysis of covalent protein modifications. PMID:20163147

Zinn, Nico; Winter, Dominic; Lehmann, Wolf D

2010-03-15

73

METHOD TO TEST ISOTOPIC SEPARATION EFFICIENCY OF PALLADIUM PACKED COLUMNS  

SciTech Connect

The isotopic effect of palladium has been applied in different ways to separate hydrogen isotopes for many years. At Savannah River Site palladium deposited on kieselguhr (Pd/k) is used in a thermal cycling absorption process (TCAP) to purify tritium for over ten years. The need to design columns for different throughputs and the desire to advance the performance of TCAP created the need to evaluate different column designs and packing materials for their separation efficiency. In this work, columns with variations in length, diameter and metal foam use, were tested using an isotope displacement method. A simple computer model was also developed to calculate the number of theoretical separation stages using the test results. The effects of column diameter, metal foam and gas flow rate were identified.

Heung, L; Gregory Staack, G; James Klein, J; William Jacobs, W

2007-06-27

74

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

PubMed Central

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 given metabolites on development-dependent changes in the 56 identified 13C-HSQC signals, we have determined the changes in metabolic networks that are associated with energy and nitrogen metabolism. PMID:19030231

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

2008-01-01

75

Selectively Dispersed Isotope Labeling for Protein Structure Determination by Magic Angle Spinning NMR  

PubMed Central

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-13C] acetate does not label ? carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-13C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA-CO coupling in the backbone should improve the resolution of NCACX spectra. PMID:23990199

Eddy, Matthew T.; Belenky, Marina; Sivertsen, Astrid; Griffin, Robert G.; Herzfeld, Judith

2013-01-01

76

Chemical imaging of biological materials by NanoSIMS using isotopic and elemental labels  

SciTech Connect

The NanoSIMS 50 combines unprecedented spatial resolution (as good as 50 nm) with ultra-high sensitivity (minimum detection limit of {approx}200 atoms). The NanoSIMS 50 incorporates an array of detectors, enabling simultaneous collection of 5 species originating from the same sputtered volume of a sample. The primary ion beam (Cs{sup +} or O{sup -}) can be scanned across the sample to produce quantitative secondary ion images. This capability for multiple isotope imaging with high spatial resolution provides a novel new approach to the study of biological materials. Studies can be made of sub-regions of tissues, mammalian cells, and bacteria. Major, minor and trace element distributions can be mapped on a submicron scale, growth and metabolism can be tracked using stable isotope labels, and biogenic origin can be determined based on composition. We have applied this technique extensively to mammalian and prokaryotic cells and bacterial spores. The NanoSIMS technology enables the researcher to interrogate the fate of molecules of interest within cells and organs through elemental and isotopic labeling. Biological applications at LLNL will be discussed.

Weber, P K; Fallon, S J; Pett-Ridge, J; Ghosal, S; Hutcheon, I D

2006-04-10

77

Ultrafast liquid water transport through graphene-based nanochannels measured by isotope labelling.  

PubMed

Based on isotope labelling, we found that liquid water can afford an ultrafast permeation through graphene-based nanochannels with a diffusion coefficient 4-5 orders of magnitude greater than in the bulk case. When dissolving ions in sources, the diffusion coefficient of ions through graphene channels lies in the same order of magnitude as water, while the ion diffusion is slightly faster than water, indicating that the ions are mainly transported by water flows and the delicate interactions between ions and nanocapillary walls also take effect in the accelerated ion transportation. PMID:25608844

Sun, Pengzhan; Liu, He; Wang, Kunlin; Zhong, Minlin; Wu, Dehai; Zhu, Hongwei

2015-02-01

78

Multiplexed Analysis of Cage and Cage Free Chicken Egg Fatty Acids Using Stable Isotope Labeling and Mass Spectrometry  

PubMed Central

Binary stable isotope labeling couple with LC-ESI-MS has been used as a powerful non-targeted approach for the relative quantification of lipids, amino acids, and many other important metabolite classes. A multiplexed approach using three or more isotopic labeling reagents greatly reduces analytical run-time while maintaining excellent sensitivity and reproducibility. Three isotopic cholamine labeling reagents have been developed to take advantage of the pre-ionized character of cholamine, for ESI, and the ease by which stable isotopes can be incorporated into the cholamine structure. These three cholamine labeling reagents have been used to relatively quantify three fatty acid samples simultaneously. The quantification resulted in the observation of 12 fatty acids that had an average absolute error of 0.9% and an average coefficient of variation of 6.1%. Caged versus cage-free isotope labeling experiments showed that cage-free eggs have an increased level of omega-3 fatty acids as compared to caged eggs. This multiplexed fatty acid analysis provides an inexpensive and expedited tool for broad-based lipid profiling that will further aid discoveries in the mechanisms of fatty acid action in cells. PMID:24317525

Torde, Richard G.; Therrien, Andrew J.; Shortreed, Michael R.; Smith, Lloyd M.; Lamos, Shane M.

2014-01-01

79

Isotope effect in negative ion chemical ionization mass spectrometry of deuterium labelled lormetazepam.  

PubMed

This study identified the reason for the poor quantification of lormetazepam-TMS (1) using negative ion chemical ionization with lormetazepam-1,1,1-2H3-TMS (2) as an internal standard. Mass spectra of lormetazepam and its deuterium labelled compounds determined at various ion source temperatures (100-250 degrees C) gave almost the same behaviour for 1 and lormetazepam-3',4',5',6'-2H4-TMS (3) but a different one for 2, suggesting that the poor quantification was due to an isotope effect. This was confirmed by the findings that the ratios of ion currents of the base peaks of 1 and 3 were independent of the ion source temperature but those of 1 and 2 varied markedly with it. This phenomenon was also observed in the mass fragmentography of the molecular ion, although to a lesser degree than that of the above fragment. In both positive ion chemical ionization and electron impact ionization modes, no isotope effect arose because there was no corresponding fragment to cause the isotope effect. PMID:2955830

Takahashi, S

1987-06-01

80

Software for quantitative proteomic analysis using stable isotope labeling and data independent acquisition.  

PubMed

Many software tools have been developed for analyzing stable isotope labeling (SIL)-based quantitative proteomic data using data dependent acquisition (DDA). However, programs for analyzing SIL-based quantitative proteomics data obtained with data independent acquisition (DIA) have yet to be reported. Here, we demonstrated the development of a new software for analyzing SIL data using the DIA method. Performance of the DIA on SYNAPT G2MS was evaluated using SIL-labeled complex proteome mixtures with known heavy/light ratios (H/L = 1:1, 1:5, and 1:10) and compared with the DDA on linear ion trap (LTQ)-Orbitrap MS. The DIA displays relatively high quantitation accuracy for peptides cross all intensity regions, while the DDA shows an intensity dependent distribution of H/L ratios. For the three proteome mixtures, the number of detected SIL-peptide pairs and dynamic range of protein intensities using DIA drop stepwise, whereas no significant changes in these aspects using DDA were observed. The new software was applied to investigate the proteome difference between mouse embryonic fibroblasts (MEFs) and MEF-derived induced pluripotent stem cells (iPSCs) using (16)O/(18)O labeling. Our study expanded the capacities of our UNiquant software pipeline and provided valuable insight into the performance of the two cutting-edge MS platforms for SIL-based quantitative proteomic analysis today. PMID:21834580

Huang, Xin; Liu, Miao; Nold, Michael J; Tian, Changhai; Fu, Kai; Zheng, Jialin; Geromanos, Scott J; Ding, Shi-Jian

2011-09-15

81

Differential 12C-/13C-isotope dansylation labeling and fast liquid chromatography/mass spectrometry for absolute and relative quantification of the metabolome.  

PubMed

We report a new quantitative metabolome profiling technique based on differential (12)C-/(13)C-isotope dansylation labeling of metabolites, fast liquid chromatography (LC) separation and electrospray ionization Fourier-transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) detection. An isotope reagent, (13)C-dansyl chloride, can be readily synthesized. This reagent, along with (12)C-dansyl chloride, provides a simple and robust means of labeling metabolites containing primary amine, secondary amine, or phenolic hydroxyl group(s). It is shown that dansylation labeling offers 1-3 orders of magnitude ESI signal enhancement over the underivatized counterparts. Dansylation alters the chromatographic behaviors of polar and ionic metabolites normally not retainable on a reversed phase (RP) column to an extent that they can be retained and separated by RPLC with high efficiency. There is no isotopic effect on RPLC separation of the differential isotope labeled metabolites, and (12)C-/(13)C-labeled isoforms of metabolites are coeluted and detected by MS for precise and accurate quantification and confident metabolite identification. It is demonstrated that, in the analysis of 20 amino acids, a linear response of over 2 orders of magnitude is achieved for relative metabolite quantification with an average relative standard deviation (RSD) of about 5.3% from replicate experiments. A dansylation standard compound library consisting of 121 known amines and phenols has been constructed and is proven to be useful for absolute metabolite quantification and MS-based metabolite identification in biological samples. As an example, the absolute concentrations of 93 metabolites, ranging from 30 nM to 2510 microM, can be determined from a pooled sample of human urines collected in 5 consecutive days labeled with (12)C-dansylation and spiked with the 121 (13)C-dansylated standards. Relative concentration variations of these metabolites in individual urine samples can also be monitored by mixing the (13)C-dansylated pooled urine sample with the (12)C-dansylated individual sample. With a 12 min fast LC separation combined with FTICR MS, 672 metabolites were detected in a human urine sample with each metabolite peak having a signal-to-noise ratio of greater than 20; the identities of most of the metabolites remain to be determined. This work illustrates that dansylation labeling and fast LC/FTICR MS can be a powerful technique for quantitative profiling of at least 672 metabolites in urine samples in 12 min. PMID:19309105

Guo, Kevin; Li, Liang

2009-05-15

82

Cost-effective production of 13C, 15N stable isotope-labelled biomass from phototrophic microalgae for various biotechnological applications.  

PubMed

The present study outlines a process for the cost-effective production of 13C/15N-labelled biomass of microalgae on a commercial scale. The core of the process is a bubble column photobioreactor with exhaust gas recirculation by means of a low-pressure compressor. To avoid accumulation of dissolved oxygen in the culture, the exhaust gas is bubbled through a sodium sulphite solution prior to its return to the reactor. The engineered system can be used for the production of 13C, 15N, and 13C-15N stable isotope-labelled biomass as required. To produce 13C-labelled biomass, 13CO2 is injected on demand for pH control and carbon supply, whereas for 15N-labelled biomass Na15NO3 is supplied as nitrogen source at the stochiometric concentration. The reactor is operated in semicontinuous mode at different biomass concentrations, yielding a maximum mean biomass productivity of 0.3 gL(-1) day(-1). In order to maximize the uptake efficiency of the labelled substrates, the inorganic carbon is recovered from the supernatant by acidification/desorption processes, while the nitrate is delivered at stochiometric concentration and the harvesting of biomass is performed when the 15NO3- is depleted. In these conditions, elemental analysis of both biomass and supernatant shows that 89.2% of the injected carbon is assimilated into the biomass and 6.9% remains in the supernatant. Based on elemental analysis, 97.8% of the supplied nitrogen is assimilated into the biomass and 1.3% remains in the supernatant. Stable isotope-labelling enrichment has been analysed by GC-MS results showing that the biomass is highly labelled. All the fatty acids are labelled; more than 96% of the carbon present in these fatty acids is 13C. The engineered system was stably operated for 3 months, producing over 160 g of 13C and/or 15N-labelled biomass. The engineered bioreactor can be applied for the labelling of various microalgae. PMID:16257578

Acién Fernández, F G; Fernández Sevilla, J M; Egorova-Zachernyuk, T A; Molina Grima, E

2005-12-01

83

Convenient procedures for the biosynthesis, isolation, and isotope labeling of cytochalasins.  

PubMed Central

Efforts to improve small-scale yields of useful cytochalasins by fermentation resulted in selection of an enriched aflatoxin medium which increased yields by fivefold over those reported in the literature. With Helminthosporium dematoideum and Zygosporium masonii in stationary culture for 3 weeks, cytochalasins B and D were obtained in quantities approaching 700 and 500 mg/liter, respectively. It appears that the critical component in this growth medium is factors associated with whole wheat. By using these procedures, coupled with improvements in isolation, supplementation with two radioactive phenylalanine species readily produced [14C]- or [3H]cytochalasin B. Oxidation of carrier-free radioactive cytochalasin B to cytochasasin A readily provided this labeled congener as well. The isotopic ocnversion of precursor to crystalline products that met analytical criteria ranged from 2 to 4% of the administered radioactivity. PMID:571262

Zabel, R A; Miller, C A; Tanenbaum, S W

1979-01-01

84

Ultrafast, Unimpeded Liquid Water Transport Through Graphene-Based Nanochannels Measured by Isotope Labelling  

E-print Network

Graphene-based laminates, with ultralong and tortuous nanocapillaries formed by simply stacking graphene flakes together, have great promises in filtration and separation. However, the information on liquid water trans-membrane permeation is lacking, which is the most fundamental problem and of crucial importance in solution-based mass transport. Here, based on isotope labelling, we investigate the liquid water transportation through graphene-based nanocapillaries under no external hydrostatic pressures. Liquid water can afford an unimpeded permeation through graphene-based nanochannels with a diffusion coefficient 4~5 orders of magnitude larger than through sub-micrometer-sized polymeric channels. When dissolving ions in sources, the diffusion coefficient of ions through graphene channels lies in the same order of magnitude as water, while the ion diffusion is faster than water, indicating that the ions are mainly transported by fast water flows and the delicate interactions between ions and nanocapillary wa...

Sun, Pengzhan; Wang, Kunlin; Zhong, Minlin; Wu, Dehai; Zhu, Hongwei

2014-01-01

85

Split-Field Drift Tube/Mass Spectrometry and Isotopic Labeling Techniques for Determination of Single Amino Acid Polymorphisms  

E-print Network

of Single Amino Acid Polymorphisms Stephen J. Valentine, S. Sevugarajan, Ruwan T. Kurulugama, Stormy L/mass spectrometry and isotopic labeling techniques is evaluated as a means of identifying single amino acid, these species have identical mobility distributions. Peptides having sequences that differ by one amino acid

Clemmer, David E.

86

Growth of single wall carbon nanotubes from 13C isotope labelled organic solvents inside single wall carbon nanotube hosts  

Microsoft Academic Search

We present the growth of single wall carbon nanotubes (SWCNTs) from organic solvents such as benzene and toluene inside a host SWCNT. The solvents encapsulated in SWCNTs are transformed to an inner tube when subject to a heat treatment under dynamic vacuum at 1270°C. We used isotope labeling of the different carbon sources to prove that the source of the

Ferenc Simon; Hans Kuzmany

2006-01-01

87

Quantifying instantaneous regeneration rates of plant leaf waxes using stable hydrogen isotope labeling.  

PubMed

Leaf waxes protect terrestrial plants from biotic and abiotic stresses and are important sedimentary biomarkers for terrestrial plants. Thus, understanding the production and ablation of leaf waxes is critical in plant physiology and for geochemical studies. However, there have been no accurate approaches to quantify leaf wax production at different time scales. In this study, we demonstrate a novel approach to study leaf wax regeneration by irrigating plants with a pulse of deuterium-enriched water, followed by measurements of leaf wax D/H ratios by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). We demonstrate the efficacy of this approach using the grass species Phleum pratense in a greenhouse environment. Using a binary isotope mass balance model, we are able to quantify the regeneration rates of the C(16), C(18) acids and leaf waxes (C(23)-C(31) n-alkanes; C(22)-C(30) n-acids) over a diurnal cycle. Our results show that within one day 33-47% of C(16) and C(18) acids are regenerated, and thus the recycling time for these compounds is 2-3 days. For C(22)-C(26) n-alkyl lipids, 7-21% are regenerated within one day and thus they require 5-16 days to recycle. In comparison, the recycling time for long-chain n-alkyl lipids (C(27)-C(31)) is as long as 71-128 days. Our approach can be applied to different plants at shorter or longer time scales by adjusting the degree of isotopic labeling, sampling intervals and the amount of irrigation water. PMID:22173799

Gao, Li; Burnier, Andre; Huang, Yongsong

2012-01-30

88

Location of Structural Transitions in an Isotopically Labeled Lung Surfactant SP-B Peptide by IRRAS  

PubMed Central

Pulmonary surfactant, a lipid/protein complex that lines the air/water interface in the mammalian lung, functions to reduce the work of breathing. Surfactant protein B (SP-B) is a small, hydrophobic protein that is an essential component of this mixture. Structure-function relationships of SP-B are currently under investigation as the protein and its peptide analogs are being incorporated into surfactant replacement therapies. Knowledge of the structure of SP-B and its related peptides in bulk and monolayer phases will facilitate the design of later generation therapeutic agents. Prior infrared reflection-absorption spectroscopic studies reported notable, reversible surface pressure-induced antiparallel ?-sheet formation in a synthetic peptide derived from human SP-B, residues 9–36 (SP-B9–36). In the current work, infrared reflection-absorption spectroscopy is applied in conjunction with isotopic labeling to detect the site and pressure dependence of the conformational change. SP-B9–36, synthesized with 13C=O-labeled Ala residues in positions 26, 28, 30, and 32, shifted the ?-sheet marker band to ?1600 cm?1 and thus immediately identified this structural element within the labeled region. Surface pressure-induced alterations in the relative intensities of Amide I band constituents are interpreted using a semiempirical transition dipole coupling model. In addition, electron micrographs reveal the formation of tubular myelin structures from in vitro preparations using SP-B9–36 in place of porcine SP-B indicating that the peptide has the potential to mimic this property of the native protein. PMID:12829488

Flach, Carol R.; Cai, Peng; Dieudonné, Darline; Brauner, Joseph W.; Keough, Kevin M. W.; Stewart, June; Mendelsohn, Richard

2003-01-01

89

Radiation oxidation of polypropylene: A solid-state 13C NMR study using selective isotopic labeling  

NASA Astrophysics Data System (ADS)

Polypropylene samples, in which the three different carbon atoms along the chain were selectively labeled with carbon-13, were subjected to radiation under inert and air atmospheres, and to post-irradiation exposure in air at various temperatures. By using solid-state 13C NMR measurements at room temperature, we have been able to identify and quantify the oxidation products. The isotopic labeling provides insight into chemical reaction mechanisms, since oxidation products can be traced back to their positions of origin on the macromolecule. The major products include peroxides and alcohols, both formed at tertiary carbon sites along the chain. Other products include methyl ketones, acids, esters, peresters, and hemiketals formed from reaction at the tertiary carbon, together with in-chain ketones and esters from reaction at the secondary chain carbon. No evidence is found of products arising from reactions at the methyl side chain. Significant temperature-dependent differences are apparent; for example much higher yields of chain-end methyl ketones, which are the indicator product of chain scission, are generated for both elevated temperature irradiation and for post-irradiation treatment at elevated temperatures. Time-dependent plots of yields of the various oxidation products have been obtained under a wide range of conditions, including the post-irradiation oxidation of a sample at room temperature in air that has been monitored for 2 years. Radiation-oxidation products of polypropylene are contrasted to products measured for 13C-labeled polyethylene in an earlier investigation: the peroxides formed in irradiated polypropylene are remarkably longer lived, the non-peroxidic products are significantly different, and the overall ratios of oxidation products in polypropylene change relatively little as a function of the extent of oxidation.

Mowery, Daniel M.; Assink, Roger A.; Derzon, Dora K.; Klamo, Sara B.; Bernstein, Robert; Clough, Roger L.

2007-05-01

90

A Method for Sporulating Budding Yeast Cells That Allows for Unbiased Identification of Kinase Substrates Using Stable Isotope Labeling by Amino Acids in Cell Culture  

PubMed Central

Quantitative proteomics has been widely used to elucidate many cellular processes. In particular, stable isotope labeling by amino acids in cell culture (SILAC) has been instrumental in improving the quality of data generated from quantitative high-throughput proteomic studies. SILAC uses the cell’s natural metabolic pathways to label proteins with isotopically heavy amino acids. Incorporation of these heavy amino acids effectively labels a cell’s proteome, allowing the comparison of cell cultures treated under different conditions. SILAC has been successfully applied to a variety of model organisms including yeast, fruit flies, plants, and mice to look for kinase substrates as well as protein–protein interactions. In budding yeast, several kinases are known to play critical roles in different aspects of meiosis. Therefore, the use of SILAC to identify potential kinase substrates would be helpful in the understanding the specific mechanisms by which these kinases act. Previously, it has not been possible to use SILAC to quantitatively study the phosphoproteome of meiotic Saccharomyces cerevisiae cells, because yeast cells sporulate inefficiently after pregrowth in standard synthetic medium. In this study we report the development of a synthetic, SILAC-compatible, pre-sporulation medium (RPS) that allows for efficient sporulation of S. cerevisiae SK1 diploids. Pre-growth in RPS supplemented with heavy amino acids efficiently labels the proteome, after which cells proceed relatively synchronously through meiosis, producing highly viable spores. As proof of principle, SILAC experiments were able to identify known targets of the meiosis-specific kinase Mek1. PMID:25168012

Suhandynata, Ray; Liang, Jason; Albuquerque, Claudio. P.; Zhou, Huilin; Hollingsworth, Nancy M.

2014-01-01

91

NMR Study of 100 kDa HCV IRES RNA Using Segmental Isotope Labeling Insil Kim, Peter J. Lukavsky, and Joseph D. Puglisi*  

E-print Network

labeling of RNA, in which part of the RNA is labeled with stable isotope, reduces the complexity of spectra, and could potentially allow NMR studies of very large RNAs and their complexes. Unambiguous resonance and hammerhead ribozyme. Segmental labeling allowed the NMR characterization of domain II of the 100 kDa internal

Puglisi, Joseph

92

Isotope Coded Protein Labeling Coupled Immunoprecipitation (ICPL-IP): A Novel Approach for Quantitative Protein Complex Analysis From Native Tissue*  

PubMed Central

High confidence definition of protein interactions is an important objective toward the understanding of biological systems. Isotope labeling in combination with affinity-based isolation of protein complexes has increased in accuracy and reproducibility, yet, larger organisms—including humans—are hardly accessible to metabolic labeling and thus, a major limitation has been its restriction to small animals, cell lines, and yeast. As composition as well as the stoichiometry of protein complexes can significantly differ in primary tissues, there is a great demand for methods capable to combine the selectivity of affinity-based isolation as well as the accuracy and reproducibility of isotope-based labeling with its application toward analysis of protein interactions from intact tissue. Toward this goal, we combined isotope coded protein labeling (ICPL)1 with immunoprecipitation (IP) and quantitative mass spectrometry (MS). ICPL-IP allows sensitive and accurate analysis of protein interactions from primary tissue. We applied ICPL-IP to immuno-isolate protein complexes from bovine retinal tissue. Protein complexes of immunoprecipitated ?-tubulin, a highly abundant protein with known interactors as well as the lowly expressed small GTPase RhoA were analyzed. The results of both analyses demonstrate sensitive and selective identification of known as well as new protein interactions by our method. PMID:23268931

Vogt, Andreas; Fuerholzner, Bettina; Kinkl, Norbert; Boldt, Karsten; Ueffing, Marius

2013-01-01

93

Rapid speciation and quantification of selenium compounds by HPLC-ICP MS using multiple standards labelled with different isotopes.  

PubMed

Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP MS) is now commonly used to investigate metabolic and toxicological aspects of some metals and metalloids. We have developed a rapid method for simultaneous identification and quantification of metabolites of selenium (Se) compounds using multiple standards labelled with different isotopes. A mixture of the labelled standards was spiked in a selenised garlic extract and the sample was subjected to speciation analysis by HPLC-ICP MS. The selenised garlic contains ?-glutamyl-methylselenocysteine, methylselenocysteine, and selenomethionine and the concentrations of those Se compounds were 723.8, 414.8, and 310.7 ng Se ml(-1), respectively. The isotopically labelled standards were also applied to the speciation of Se in rat urine. Selenate, methylselenonic acid, selenosugar, and trimethyselenium ions were found to be excreted by the present speciation procedure. Multiple standards labelled with different stable isotopes enable high-throughput identification and quantitative measurements of Se metabolites. PMID:21756199

Ohta, Yuki; Suzuki, Noriyuki; Kobayashi, Yayoi; Hirano, Seishiro

2011-09-01

94

Investigation of the mechanism of n-butane oxidation on vanadium phosphorus oxide catalysts: evidence from isotopic labeling studies.  

PubMed

The selective oxidation of n-butane to maleic acid catalyzed by vanadium phosphates (VPO) is one of the most complex partial oxidation reactions used in industry today. Numerous reaction mechanisms have been proposed in the literature, many of which have butenes, butadiene, and furan as reaction intermediates. We have developed an experimental protocol to study the mechanism of this reaction in which (13)C-isotopically labeled n-butane is flowed over a catalyst bed and the reaction products are analyzed using (13)C NMR spectroscopy. This protocol approximates the conditions found in an industrial reactor without requiring an exorbitant amount of isotopically labeled material. When [1,4-(13)C]n-butane reacted on VPO catalysts to produce maleic acid and butadiene, the isotopic labels were observed in both the 1,4 and 2,3 positions of butadiene and maleic acid. The ratio of label scrambling was typically 1:20 for the 2,3:1,4 positions in maleic acid. For butadiene, the ratio of label scrambling was consistently much higher, at 2:3 for the 2,3:1,4 positions. Because of the discrepancy in the amount of label scrambling between maleic acid and butadiene, butadiene is unlikely to be the primary reaction intermediate for the conversion of n-butane to maleic anhydride under typical industrial conditions. Ethylene was always observed as a side product for n-butane oxidation on VPO catalysts. Fully (13)C-labeled butane produced about 5-13 times as much isotopically labeled ethylene as did [1,4-(13)C]butane, indicating that ethylene was produced mainly from the two methylene carbons of n-butane. When the reaction was run under conditions which minimize total oxidation products such as CO and CO(2), the amounts of ethylene and carbon oxides produced from fully (13)C-labeled butane were almost equal. This strongly suggests that the total oxidation of n-butane on VPO catalysts involves the oxidation and abstraction of the two methyl groups of n-butane, and the two methylene groups of n-butane form ethylene. An organometallic mechanism is proposed to explain these results. PMID:11853438

Chen, Bin; Munson, Eric J

2002-02-27

95

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

PubMed Central

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

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

2012-01-01

96

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

PubMed

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. PMID:24766305

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

2014-05-20

97

Measuring supply chain carbon efficiency : a carbon label framework  

E-print Network

In the near term, efficiency improvements represent a key option for reducing the impacts of climate change. The growing awareness of climate change has increased the attention regarding the carbon emissions "embedded" in ...

Craig, Anthony (Anthony J.)

2012-01-01

98

Isotope labeling-based quantitative proteomics of developing seeds of castor oil seed (Ricinus communis L.).  

PubMed

In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed-storage proteins (SSPs), toxins, and allergens. Additionally, we have used off-line hydrophilic interaction chromatography (HILIC) as a step of peptide fractionation preceding the reverse-phase nanoLC coupled to a LTQ Orbitrap. We were able to identify a total of 1875 proteins, and from these 1748 could be mapped to extant castor gene models, considerably expanding the number of proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism, and catabolism of fatty acid and the pattern of deposition of SSPs, toxins, and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of SSP that are differentially expressed during seed development. PMID:24090105

Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit; Soares, Emanuela L; Soares, Arlete A; Roepstorff, Peter; Domont, Gilberto B; Campos, Francisco A P

2013-11-01

99

Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS  

NASA Astrophysics Data System (ADS)

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.

Zhu, Zhikai; Go, Eden P.; Desaire, Heather

2014-06-01

100

Tracking down sulphate-reducing microorganisms by molecular and isotope-labelling techniques  

NASA Astrophysics Data System (ADS)

Sulphate-reducing microorganisms (SRM) are of great ecological importance for carbon compound degradation and sulphur cycling in many anoxic ecosystems, including marine sediments, peatlands, and oil reservoirs. However, the activity of SRM can result in oil souring and pipeline corrosion and thus is also an economic burden for the oil industry. Molecular diversity surveys based on rRNA genes and dsrAB, genes that encode major subunits of the dissimilatory sulfite reductase, indicate that our view of the natural diversity of SRM (as we know it from cultivation) is far from being complete. This enormous phylogenetic diversity complicates unbiased identification and quantification of SRM by molecular methods such as fluorescence in situ hybridization, real-time PCR or DNA microarrays. Combining these 16S rRNA and dsrAB-based molecular methods with substrate-mediated isotope labelling techniques is a potential solution for identification and functional characterization of yet uncultivated SRM. Using SRM in peatlands as an example, the problems and opportunities of these techniques for diagnosing and monitoring SRM in the environment will be discussed in this talk.

Loy, Alexander

2010-05-01

101

Investigation of bn-44 Peptide Fragments Using High Resolution Mass Spectrometry and Isotope Labeling  

NASA Astrophysics Data System (ADS)

An N-terminal deuterohemin-containing hexapeptide (DhHP-6) was designed as a short peptide cytochrome c (Cyt c) mimetic to study the effect of N-terminal charge on peptide fragmentation pathways. This peptide gave different dissociation patterns than normal tryptic peptides. Upon collision-induced dissociation (CID) with an ion trap mass spectrometer, the singly charged peptide ion containing no added proton generated abundant and characteristic bn-44 ions instead of bn-28 (an) ions. Studies by high resolution mass spectrometry (HRMS) and isotope labeling indicate that elimination of 44 Da fragments from b ions occurs via two different pathways: (1) loss of CH3CHO (44.0262) from a Thr side chain; (2) loss of CO2 (43.9898) from the oxazolone structure in the C-terminus. A series of analogues were designed and analyzed. The experimental results combined with Density Functional Theory (DFT) calculations on the proton affinity of the deuteroporphyrin demonstrate that the production of these novel bn-44 ions is related to the N-terminal charge via a charge-remote rather than radical-directed fragmentation pathway.

Wang, Bing; Yu, Jiayi; Wang, Huixin; Wei, Zhonglin; Guo, Xinhua; Xiao, Zhaohui; Zeng, Zhoufang; Kong, Wei

2014-12-01

102

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

Microsoft Academic Search

BackgroundMetabolic 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

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

2008-01-01

103

A device for single leaf labelling with CO2 isotopes to study carbon allocation and partitioning in Arabidopsis thaliana  

PubMed Central

Background Plant biomass consists primarily of carbohydrates derived from photosynthesis. Monitoring the assimilation of carbon via the Calvin-Benson cycle and its subsequent utilisation is fundamental to understanding plant growth. The use of stable and radioactive carbon isotopes, supplied to plants as CO2, allows the measurement of fluxes through the intermediates of primary photosynthetic metabolism, long-distance transport of sugars in the vasculature, and the synthesis of structural and storage components. Results Here we describe the design of a system for supplying isotopically labelled CO2 to single leaves of Arabidopsis thaliana. We demonstrate that the system works well using short pulses of 14CO2 and that it can be used to produce robust qualitative and quantitative data about carbon export from source leaves to the sink tissues, such as the developing leaves and the roots. Time course experiments show the dynamics of carbon partitioning between storage as starch, local production of biomass, and export of carbon to sink tissues. Conclusion This isotope labelling method is relatively simple to establish and inexpensive to perform. Our use of 14CO2 helps establish the temporal and spatial allocation of assimilated carbon during plant growth, delivering data complementary to those obtained in recent studies using 13CO2 and MS-based metabolomics techniques. However, we emphasise that this labelling device could also be used effectively in combination with 13CO2 and MS-based techniques. PMID:24252607

2013-01-01

104

Isotope labeled internal standards (ILIS) as a basis for quality control in clinical studies using plasma samples.  

PubMed

For clinical proteomic studies, the quality of the biofluid samples such as human blood plasma is extremely important. In this study we have investigated the stability of human plasma samples by spiking stable isotope-labeled peptides into the plasma and monitoring their degradation under different storage conditions. FPA-1, C4A and C3f were synthesized with isotopically labeled amino acids, and used as reference peptides. The mixture of internal calibrants was spiked into plasma at the starting point of investigation, mimicking the time of collection for future biobanking efforts, and their qualitative and quantitative changes were analyzed over time by using both MALDI-MS (LTQ Orbitrap XL) and nanoLC-ESI-MS (LTQ XL ETD). We have found that all three synthetic peptides were stable in plasma at -20 and -80 degrees C during the examined 2-month period. However, different proteolytic degradation profiles of the peptides were observed at room temperature. We anticipate that the use of these isotope-labeled peptides as internal standards (ILIS) provides a quality control for long-term storage and proteomic plasma analysis. PMID:20176153

Rezeli, Melinda; Végvári, Akos; Marko-Varga, György; Laurell, Thomas

2010-04-18

105

Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis.  

PubMed

A method was developed for the precise and accurate determination of ovalbumin labelled with p-hydroxy-mercuribenzoic acid (pHMB) using polyacrylamide gel electrophoresis with ns-laser ablation-inductively coupled plasma mass spectrometry. Following systematic optimisation of the ablation process in terms of detection sensitivity, two different quantification strategies were applied: external calibration using standards of the derivatized protein after (13)C(+) normalization and, as a proof of concept, label-specific isotope dilution analysis (IDA) using pHMB enriched in the isotope (199)Hg. Due to the inhomogeneous distribution of the protein within the gel bands, it could be demonstrated that the IDA approach was superior in terms of precision and accuracy. Furthermore, it permits a reliable quantification, if more complex separation protocols are applied, as typically occurring analyte loss and degradation can be compensated for as soon as complete mixture of spike and sample is achieved. The estimated limit of detection was 160 fmol in the case of ovalbumin. In contrast to earlier studies using metals naturally present in proteins, no loss of mercury was observed during separation under denaturing conditions and other sample preparation steps. Using label-specific IDA, the measured isotope ratios in the gel corresponded to recoveries between 95% and 103%. PMID:21773737

Kutscher, Daniel J; Fricker, Mattias B; Hattendorf, Bodo; Bettmer, Jörg; Günther, Detlef

2011-11-01

106

Development of a New Extended Motor Product Label for Industrial Energy Efficiency  

E-print Network

Labeling programs • Working with supply chain • Vendor and customer education ESL-IE-14-05-11 Proceedings of the Thrity-Sixth Industrial Energy Technology Conference New Orleans, LA. May 20-23, 2014 Extended Motor Products: A New Opportunity • Opportunities...Development of a New Extended Motor Product Label for Industrial Energy Efficiency Ethan A. Rogers, Senior Program Manager, Industry Robert Boteler, Visiting Fellow R. Neal Elliott, Associate Director for Research 2014 Industrial Energy Technology...

Rogers, E.; Boteler, R.; Elliot, R. N.

2014-01-01

107

Seasonal liver protein differences in a hibernator revealed by quantitative proteomics using whole animal isotopic labeling  

PubMed Central

Hibernation is an energy-saving strategy used by diverse species of mammals to survive winter. It is characterized by cycles between multi-day periods of torpor with low body temperature (Tb), and short periods of rapid, spontaneous rewarming. The ability to retain cellular integrity and function throughout torpor and rewarming is a key attribute of hibernation. Livers from winter hibernators are resistant to cellular damage induced by cold storage followed by warm reperfusion. Identifying proteins that differ between the summer-sensitive and winter-protected phenotypic states is one useful approach that may elucidate the molecular mechanisms that underlie this protection. Here we employ a novel quantitative proteomics screening strategy whereby a newly-weaned 13-lined ground squirrel was metabolically labeled by ingesting heavy-isotope substituted (15N) Spirulina. The liver protein extract from this animal provided a common reference for quantitative evaluation of protein differences by its addition to extracts from pooled samples of summer active (SA) or winter entrance (Ent) phase hibernating ground squirrels. We identified 61 significantly different proteins between the two groups and compared them to proteins identified previously in the same samples using 2D gels. Of the 20 proteins common to the two datasets, the direction and magnitude of their differences were perfectly concordant for 18, providing confidence that both sets of altered proteins reflect bona fide differences between the two physiological states. Furthermore, the 41 novel proteins recovered in this study included many new enzymes in pathways identified previously: specifically, additional enzymes belonging to the urea cycle, amino acid and carbohydrate degradation, and lipid biosynthetic pathways were decreased, whereas enzymes involved in ketone body synthesis, fatty acid utilization, protein synthesis and gluconeogenesis were increased in the samples from entrance hibernators compared to summer active animals, providing additional specific evidence for the importance of these pathways in the hibernating phenotype. PMID:21481655

Rose, J. Cameron; Epperson, L. Elaine; Carey, Hannah V.; Martin, Sandra L.

2011-01-01

108

Stable isotope labeled n-alkanes to assess digesta passage kinetics through the digestive tract of ruminants.  

PubMed

We describe the use of carbon stable isotope ((13)C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically (13)C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha(-1)) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the ?(13)C (i.e. the ratio (13)C:(12)C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C27-C36) and passage kinetics were determined for the most abundant C29, C31 and C33 n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K 1) among individual n-alkanes (3.71-3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p?0.002) increase with carbon chain length. K 1 estimates were comparable to those of the (13)C labeled digestible dry matter fraction (3.38%/h; r?=?0.61 to 0.71; p?0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of (13)C versus (12)C. Our results suggest that (13)C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the ?(13)C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics. PMID:24124493

Warner, Daniel; Ferreira, Luis M M; Breuer, Michel J H; Dijkstra, Jan; Pellikaan, Wilbert F

2013-01-01

109

Stable Isotope Labeled n-Alkanes to Assess Digesta Passage Kinetics through the Digestive Tract of Ruminants  

PubMed Central

We describe the use of carbon stable isotope (13C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically 13C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha?1) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the ?13C (i.e. the ratio 13C:12C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C27–C36) and passage kinetics were determined for the most abundant C29, C31 and C33 n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K1) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p?0.002) increase with carbon chain length. K1 estimates were comparable to those of the 13C labeled digestible dry matter fraction (3.38%/h; r?=?0.61 to 0.71; p?0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of 13C versus 12C. Our results suggest that 13C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the ?13C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics. PMID:24124493

Warner, Daniel; Ferreira, Luis M. M.; Breuer, Michel J. H.; Dijkstra, Jan; Pellikaan, Wilbert F.

2013-01-01

110

Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe*  

PubMed Central

Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, 13C615N4-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable 13C615N4-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. PMID:25368411

Carpy, Alejandro; Patel, Avinash; Tay, Ye Dee; Hagan, Iain M.; Macek, Boris

2015-01-01

111

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

SciTech Connect

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 novel 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).

Mark E. Fuller; Tullis C. Onstott

2003-12-17

112

Investigation of non-segregation graphene growth on Ni via isotope-labeled alcohol catalytic chemical vapor deposition  

NASA Astrophysics Data System (ADS)

Here we present CVD growth of graphene on Ni and investigate the growth mechanism using isotopically labeled 13C-ethanol as the precursor. Results show that during low-pressure alcohol catalytic CVD (LP-ACCVD), a growth time of less than 30 s yields graphene films with high surface coverage (>80%). Moreover, when isotopically labeled ethanol precursors were sequentially introduced, Raman mapping revealed that both 12C and 13C graphene flakes exist. This shows that even at high temperature (~900 °C) the graphene flakes form independently, suggesting a different growth mechanism for ethanol-derived graphene on Ni from the segregation process for methane-derived graphene. We interpret this growth mechanism using a direct surface-adsorptive growth model in which small carbon fragments catalyzed from ethanol decomposition products first nucleate at metal step edges or grain boundaries to initiate graphene growth, and then expand over the entire metal surface.Here we present CVD growth of graphene on Ni and investigate the growth mechanism using isotopically labeled 13C-ethanol as the precursor. Results show that during low-pressure alcohol catalytic CVD (LP-ACCVD), a growth time of less than 30 s yields graphene films with high surface coverage (>80%). Moreover, when isotopically labeled ethanol precursors were sequentially introduced, Raman mapping revealed that both 12C and 13C graphene flakes exist. This shows that even at high temperature (~900 °C) the graphene flakes form independently, suggesting a different growth mechanism for ethanol-derived graphene on Ni from the segregation process for methane-derived graphene. We interpret this growth mechanism using a direct surface-adsorptive growth model in which small carbon fragments catalyzed from ethanol decomposition products first nucleate at metal step edges or grain boundaries to initiate graphene growth, and then expand over the entire metal surface. Electronic supplementary information (ESI) available: Additional Raman imaging maps from as-grown (isotopic) graphene samples, as well as AFM images of Ni surfaces before and after H2 annealing. See DOI: 10.1039/c3nr01080e

Zhao, Pei; Hou, Bo; Chen, Xiao; Kim, Sungjin; Chiashi, Shohei; Einarsson, Erik; Maruyama, Shigeo

2013-06-01

113

Efficient isotope separation by single-photon atomic sorting  

SciTech Connect

We propose a general and scalable approach to isotope separation. The method is based on an irreversible change of the mass-to-magnetic moment ratio of a particular isotope in an atomic beam, followed by a magnetic multipole whose gradients deflect and guide the atoms. The underlying mechanism is a reduction of the entropy of the beam by the information of a single scattered photon for each atom that is separated. We numerically simulate isotope separation for a range of examples, which demonstrate this technique's general applicability to almost the entire periodic table. The practical importance of the proposed method is that large-scale isotope separation should be possible, using ordinary inexpensive magnets and the existing technologies of supersonic beams and lasers.

Jerkins, M.; Chavez, I.; Raizen, M. G. [Center for Nonlinear Dynamics and Department of Physics, University of Texas at Austin, Austin, Texas 78712 (United States); Even, U. [Sackler School of Chemistry, Tel-Aviv University, Tel-Aviv (Israel)

2010-09-15

114

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

NASA Astrophysics Data System (ADS)

The Individuality Normalization when Labeling with Isotopic Glycan Hydrazide Tags (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 attributable to isotopic overlap and a post-acquisition normalization of the data to account for any systematic bias, a plot of the experimental H:L ratio versus 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.

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

2013-09-01

115

Phosphorus use efficiency by cotton measured through 32P isotope technique  

NASA Astrophysics Data System (ADS)

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.

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

2012-04-01

116

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

SciTech Connect

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 in 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 the development of a monitoring system to track compliance with standards and labeling, CLASP, with support from Japan's Ministry of Economy, Trade and Industry (METI), has expanded its ongoing collaboration with the China National Institute of Standards (CNIS) to include enforcement and monitoring. CNIS has already begun working on the issue of compliance. CNIS has conducted modest sample testing in 2006 for refrigerators, freezers and room air-conditioners, and repeated the same task in 2007 with a similar sample size for three products (refrigerators, freezers, air-conditioners and clothes washers). And, CNIS, with technical support from LBNL, has analyzed the data collected through testing. At the same time, parallel effort has also been paid to look at the potential impact of the label to 2020. In conjunction with CNIS, CLASP technical experts reviewed the standards development timeline of the four products currently subject to the mandatory energy information label. CLASP, with the support of METI/IEEJ, collaborated with CNIS to develop the efficiency grades, providing: technical input to the process; comment and advice on particular technical issues; as well as evaluation of the results. In addition, in order to effectively evaluate the impact of the label on China's market, CLASP further provided assistance to CNIS to collect data on both the efficiency distribution and product volume distribution of refrigerators on the market. This short report summarizes the status of Standards and Labeling program, current enforcement and monitoring mechanism in China, and states the importance of international collaborations.

Zhou, Nan

2008-03-01

117

Metabolic labeling with stable isotope nitrogen (15N) to follow amino acid and protein turnover of three plastid proteins in Chlamydomonas reinhardtii  

PubMed Central

Background The length of time that a protein remains available to perform its function is significantly influenced by its turnover rate. Knowing the turnover rate of proteins involved in different processes is important to determining how long a function might progress even when the stimulus has been removed and no further synthesis of the particular proteins occurs. In this article, we describe the use of 15N-metabolic labeling coupled to GC-MS to follow the turnover of free amino acids and LC-MS/MS to identify and LC-MS to follow the turnover of specific proteins in Chlamydomonas reinhardtii. Results To achieve the metabolic labeling, the growth medium was formulated with standard Tris acetate phosphate medium (TAP) in which14NH4Cl was replaced with 15NH415NO3 and (14NH4)6Mo7O24.4H2O was replaced with Na2MoO4.2H2O. This medium designated 15N-TAP allowed CC-125 algal cells to grow normally. Mass isotopic distribution revealed successful 15N incorporation into 13 amino acids with approximately 98% labeling efficiency. Tryptic digestion of the 55 kDa SDS-PAGE bands from 14N- and 15N-labeled crude algal protein extracts followed by LC-MS/MS resulted in the identification of 27 proteins. Of these, five displayed peptide sequence confidence levels greater than 95% and protein sequence coverage greater than 25%. These proteins were the RuBisCo large subunit, ATP synthase CF1 alpha and beta subunits, the mitochondrial protein (F1F0 ATP synthase) and the cytosolic protein (S-adenosyl homocysteine hydroxylase). These proteins were present in both labeled and unlabeled samples. Once the newly synthesized 15N-labeled free amino acids and proteins obtained maximum incorporation of the 15N-label, turnover rates were determined after transfer of cells into 14N-TAP medium. The t½ values were determined for the three plastid proteins (RuBisCo, ATP synthase CF1 alpha and beta) by following the reduction of the 15N-fractional abundance over time. Conclusion We describe a more rapid and non-radioactive method to measure free amino acid and protein turnover. Our approach is applicable for determination of protein turnover for various proteins, which will lead to a better understanding of the relationship between protein lifetime and functionality. PMID:24580857

2014-01-01

118

Biosynthesis of 15N3-labeled enniatins and beauvericin and their application to stable isotope dilution assays.  

PubMed

The first stable isotope dilution assay for the determination of enniatins A, A1, B, and B1 and beauvericin was developed. The (15)N(3)-labeled enniatins and beauvericin were biosynthesized by feeding two Fusarium strains Na(15)NO(3) and subsequently isolated from the fungal culture. The chemical structures of the biosynthesized products were characterized by LC-MS/MS and (1)H NMR. Standard solutions of (15)N(3)-labeled beauvericin, enniatin A, and enniatin A1 were accurately quantitated by quantitative NMR. On the basis of the use of the labeled products as internal standards, stable isotope dilution assays were developed and applied to various food samples using LC-MS/MS. The sample extracts were directly injected without any tedious cleanup procedures. The limits of detection were 3.9, 2.6, 3.7, 1.9, and 4.4 ?g/kg for enniatins A, A1, B, and B1 and beauvericin, respectively. Limits of quantitation were 11.5 (enniatin A), 7.6 (enniatin A1), 10.9 (enniatin B), 5.8 (enniatin B1), and 13.1 ?g/kg (beauvericin). Recoveries were within the range between 90 and 120%, and good intraday and interday precisions with coefficients of variation between 1.35 and 8.61% were obtained. Thus, the stable isotope dilution assay presented here is similarly sensitive and precise but more accurate than assays reported before. Analyses of cereals and cereal products revealed frequent contaminations of barley, wheat, rye, and oats with enniatins B and B1, whereas beauvericin was not quantifiable. PMID:22734473

Hu, Ling; Rychlik, Michael

2012-07-25

119

Glycation Isotopic Labeling with 13C-Reducing Sugars for Quantitative Analysis of Glycated Proteins in Human Plasma*  

PubMed Central

Non-enzymatic glycation of proteins is a post-translational modification produced by a reaction between reducing sugars and amino groups located in lysine and arginine residues or in the N-terminal position. This modification plays a relevant role in medicine and food industry. In the clinical field, this undesired role is directly linked to blood glucose concentration and therefore to pathological conditions derived from hyperglycemia (>11 mm glucose) such as diabetes mellitus or renal failure. An approach for qualitative and quantitative analysis of glycated proteins is here proposed to achieve the three information levels for their complete characterization. These are: 1) identification of glycated proteins, 2) elucidation of sugar attachment sites, and 3) quantitative analysis to compare glycemic states. Qualitative analysis was carried out by tandem mass spectrometry after endoproteinase Glu-C digestion and boronate affinity chromatography for isolation of glycated peptides. For this purpose, two MS operational modes were used: higher energy collisional dissociation-MS2 and CID-MS3 by neutral loss scan monitoring of two selective neutral losses (162.05 and 84.04 Da for the glucose cleavage and an intermediate rearrangement of the glucose moiety). On the other hand, quantitative analysis was based on labeling of proteins with [13C6]glucose incubation to evaluate the native glycated proteins labeled with [12C6]glucose. As glycation is chemoselective, it is exclusively occurring in potential targets for in vivo modifications. This approach, named glycation isotopic labeling, enabled differentiation of glycated peptides labeled with both isotopic forms resulting from enzymatic digestion by mass spectrometry (6-Da mass shift/glycation site). The strategy was then applied to a reference plasma sample, revealing the detection of 50 glycated proteins and 161 sugar attachment positions with identification of preferential glycation sites for each protein. A predictive approach was also tested to detect potential glycation sites under high glucose concentration. PMID:19955080

Priego-Capote, Feliciano; Scherl, Alexander; Müller, Markus; Waridel, Patrice; Lisacek, Frédérique; Sanchez, Jean-Charles

2010-01-01

120

Reductive carbonylation of aryl halides employing a two-chamber reactor: a protocol for the synthesis of aryl aldehydes including 13C- and D-isotope labeling.  

PubMed

A protocol has been developed for conducting the palladium-catalyzed reductive carbonylation of aryl iodides and bromides using 9-methylfluorene-9-carbonyl chloride (COgen) as a source of externally delivered carbon monoxide in a sealed two-chamber system (COware), and potassium formate as the in situ hydride source. The method is tolerant to a wide number of functional groups positioned on the aromatic ring, and it can be exploited for the isotope labeling of the aldehyde group. Hence, reductive carbonylations run with (13)COgen provide a facile access to (13)C-labeled aromatic aldehydes, whereas with DCO2K, the aldehyde is specifically labeled with deuterium. Two examples of double isotopic labeling are also demonstrated. Finally, the method was applied to the specific carbon-13 labeling of the ?-amyloid binding compound, florbetaben. PMID:23692554

Korsager, Signe; Taaning, Rolf H; Lindhardt, Anders T; Skrydstrup, Troels

2013-06-21

121

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 ...

122

Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation  

SciTech Connect

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.

Korzekwa, K.R.; Swinney, D.C.; Trager, W.F. (National Heart, Lung and Blood Institute, Bethesda, MD (USA))

1989-11-14

123

Anaerobic Central Metabolic Pathways in Shewanella oneidensis MR-1 Reinterpreted in the Light of Isotopic Metabolite Labeling?  

PubMed Central

It has been proposed that during growth under anaerobic or oxygen-limited conditions, Shewanella oneidensis MR-1 uses the serine-isocitrate lyase pathway common to many methylotrophic anaerobes, in which formaldehyde produced from pyruvate is condensed with glycine to form serine. The serine is then transformed through hydroxypyruvate and glycerate to enter central metabolism at phosphoglycerate. To examine its use of the serine-isocitrate lyase pathway under anaerobic conditions, we grew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source, with either trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor. Analysis of cellular metabolites indicated that a large percentage (>70%) of lactate was partially oxidized to either acetate or pyruvate. The 13C isotope distributions in amino acids and other key metabolites indicate that under anaerobic conditions, although glyoxylate synthesized from the isocitrate lyase reaction can be converted to glycine, a complete serine-isocitrate pathway is not present and serine/glycine is, in fact, oxidized via a highly reversible degradation pathway. The labeling data also suggest significant activity in the anapleurotic (malic enzyme and phosphoenolpyruvate carboxylase) reactions. Although the tricarboxylic acid (TCA) cycle is often observed to be incomplete in many other anaerobes (absence of 2-oxoglutarate dehydrogenase activity), isotopic labeling supports the existence of a complete TCA cycle in S. oneidensis MR-1 under certain anaerobic conditions, e.g., TMAO-reducing conditions. PMID:17114268

Tang, Yinjie J.; Meadows, Adam L.; Kirby, James; Keasling, Jay D.

2007-01-01

124

Untargeted Profiling of Tracer-Derived Metabolites Using Stable Isotopic Labeling and Fast Polarity-Switching LC–ESI-HRMS  

PubMed Central

An untargeted metabolomics workflow for the detection of metabolites derived from endogenous or exogenous tracer substances is presented. To this end, a recently developed stable isotope-assisted LC–HRMS-based metabolomics workflow for the global annotation of biological samples has been further developed and extended. For untargeted detection of metabolites arising from labeled tracer substances, isotope pattern recognition has been adjusted to account for nonlabeled moieties conjugated to the native and labeled tracer molecules. Furthermore, the workflow has been extended by (i) an optional ion intensity ratio check, (ii) the automated combination of positive and negative ionization mode mass spectra derived from fast polarity switching, and (iii) metabolic feature annotation. These extensions enable the automated, unbiased, and global detection of tracer-derived metabolites in complex biological samples. The workflow is demonstrated with the metabolism of 13C9-phenylalanine in wheat cell suspension cultures in the presence of the mycotoxin deoxynivalenol (DON). In total, 341 metabolic features (150 in positive and 191 in negative ionization mode) corresponding to 139 metabolites were detected. The benefit of fast polarity switching was evident, with 32 and 58 of these metabolites having exclusively been detected in the positive and negative modes, respectively. Moreover, for 19 of the remaining 49 phenylalanine-derived metabolites, the assignment of ion species and, thus, molecular weight was possible only by the use of complementary features of the two ion polarity modes. Statistical evaluation showed that treatment with DON increased or decreased the abundances of many detected metabolites. PMID:25372979

2014-01-01

125

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

SciTech Connect

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, activities 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%, possibly due to the implementation of newly strengthened efficiency standards in 2009. Areas for improvement include: Greater awareness at the local level to ensure that all manufacturers register their products with the label certification project and to minimize their resistance to inspections; improvement of the product sampling methodology to include representative testing of both large and small manufacturers and greater standardization of testing tools and procedures; and continued improvement in local enforcement efforts.

Zhou, Nan; Zheng, Nina; Fino-Chen, Cecilia; Fridley, David; Ning, Cao

2011-09-26

126

An atypical naturally split intein engineered for highly efficient protein labeling.  

PubMed

Protein trans-splicing catalyzed by split inteins is a powerful technique for assembling a polypeptide backbone from two separate parts. However, split inteins with robust efficiencies and short fragments suitable for peptide synthesis are rare and have mostly been artificially created. The novel split intein AceL-TerL was identified from metagenomic data and characterized. It represents the first naturally occurring, atypically split intein. The N-terminal fragment of only 25 amino acids is the shortest natural intein fragment to date and was easily amenable to chemical synthesis with a fluorescent label. Optimal protein trans-splicing activity was observed at low temperatures. Further improved mutants were selected by directed protein evolution. The engineered intein variants with up to 50-fold increased rates showed unprecedented efficiency in chemically labeling of a diverse set of proteins. These inteins should prove valuable tools for protein semi-synthesis and other intein-related biotechnological applications. PMID:24382817

Thiel, Ilka V; Volkmann, Gerrit; Pietrokovski, Shmuel; Mootz, Henning D

2014-01-27

127

Kinetic isotope effects significantly influence intracellular metabolite [superscript 13]C labeling patterns and flux determination  

E-print Network

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide ...

Stephanopoulos, Gregory

128

Synthesis of isotopically labeled R- or S-[.sup.13C, .sup.2H] glycerols  

DOEpatents

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.

Martinez, Rodolfo A. (Santa Fe, NM); Unkefer, Clifford J. (Los Alamos, NM); Alvarez, Marc A. (Santa Fe, NM)

2008-01-22

129

Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments  

PubMed Central

The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state 13C labelling and analysed labelling in protein amino acids from plastid and cytosol. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol, respectively. Developing Brassica napus embryos were cultured in the presence of [U-13C]-sucrose, [U-13C]-glucose, [U-13C]-glutamine or [U-13C]-alanine to generate proteins. The large subunits (LSU) and small subunits (SSU) of Rubisco were isolated and the labelling in their constituent amino acids was analysed by gas chromatography-mass spectrometry. Amino acids including alanine, glycine and serine exhibited different 13C enrichment in the LSU and SSU, demonstrating that these pools have different metabolic origins and are not isotopically equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of this novel approach to other macromolecules, organelles and cell types of eukaryotes are discussed. PMID:22292468

Allen, Doug K; Laclair, Russell W; Ohlrogge, John B; Shachar-Hill, Yair

2012-01-01

130

Automated LC-HRMS(/MS) approach for the annotation of fragment ions derived from stable isotope labeling-assisted untargeted metabolomics.  

PubMed

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 (12)C- and uniformly (13)C (U-(13)C)-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-(13)C-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

Neumann, Nora K N; Lehner, Sylvia M; Kluger, Bernhard; Bueschl, Christoph; Sedelmaier, Karoline; Lemmens, Marc; Krska, Rudolf; Schuhmacher, Rainer

2014-08-01

131

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

PubMed Central

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

2014-01-01

132

Regional cooperation in energy efficiency standard-setting and labeling in North America  

SciTech Connect

The North American Energy Working Group (NAEWG) was established in 2001 by the governments of Canada, Mexico, and the United States. The goals of NAEWG are to foster communication and cooperation on energy-related matters of common interest, and to enhance North American energy trade and interconnections consistent with the goal of sustainable development, for the benefit of all three countries. At its outset, NAEWG established teams to address different aspects of the energy sector. One, the Energy Efficiency Expert Group, undertook activity in three areas: (1) analyzing commonalities and differences in the test procedures of Canada, Mexico, and the United States, and identifying specific products for which the three countries might consider harmonization; (2) exploring possibilities for increased mutual recognition of laboratory test results; and (3) looking at possibilities for enhanced cooperation in the Energy Star voluntary endorsement labeling program. To support NAEWG's Expert Group on Energy Efficiency (NAEWG-EE), USDOE commissioned Lawrence Berkeley National Laboratory, representing the Collaborative Labeling and Appliance Standards Program (CLASP), to prepare a resource document comparing current standards, labels, and test procedure regulations in Canada, Mexico, and the United States. The resulting document identified 46 energy-using products for which at least one of the three countries has energy efficiency regulations. Three products--refrigerators/freezers, room air conditioners, and integral horsepower three-phase electric motors--have identical minimum energy performance standards (MEPS) and test procedures in the three countries. Ten other products have different MEPS and test procedures, but have the near-term potential for harmonization. NAEWG-EE is currently working to identify mechanisms for mutual recognition of test results. With consultative support from the United States and Canada through NAEWG-EE, Mexico is exploring possibilities for extending the Energy Star endorsement label to Mexico.

Wiel, Stephen; Van Wie McGrory, Laura

2003-08-04

133

Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters†  

PubMed Central

Deuterated styrene ([2H8]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 [2H8]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 [2H8]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. PMID:11571187

Alexandrino, Maria; Knief, Claudia; Lipski, André

2001-01-01

134

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

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, -MS(3), 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(CH(3))(2)) and 'heavy' (S(CD(3))(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-MS(3) 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

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

2012-04-01

135

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

NASA Astrophysics Data System (ADS)

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.

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

2012-04-01

136

Highly efficient cellular labeling of mesoporous nanoparticles in human mesenchymal stem cells: implication for stem cell tracking  

Microsoft Academic Search

Tracking the distribution of stem cells is crucial to their therapeutic use. However, the usage of current vectors in cellular labeling is restricted by their low internalizing efficiency. Here, we reported a cellular labeling approach with a novel vector composed of mesoporous silica nanoparticles (MSNs) conjugated with fluorescein isothiocyanate in human bone marrow mesenchymal stem cells and 3T3-L1 cells, and

Dong-Ming Huang; Yann Hung; Bor-Sheng Ko; Szu-Chun Hsu; Wei-Hsuan Chen; Chung-Liang Chien; Chih-Pin Tsai; Chieh-Ti Kuo; Ju-Chiun Kang; Chung-Shi Yang; Chung-Yuan Mou; Yao-Chang Chen

2005-01-01

137

Lewis Acid-Base, Molecular Modeling, and Isotopic Labeling in a Sophomore Inorganic Chemistry Laboratory  

ERIC Educational Resources Information Center

An experiment to prepare a deuterium labeled adduct of a Lewis acid and Lewis base, to use computational methods allowing students to visualize the LUMO of Lewis acids, the HOMO of Lewis bases and the molecular orbitals of the adduct that is formed is developed. This allows students to see the interplay between calculated and experimental results.

Nataro, Chip; Ferguson, Michelle A.; Bocage, Katherine M.; Hess, Brian J.; Ross, Vincent J.; Swarr, Daniel T.

2004-01-01

138

Tetrairon(III) single-molecule magnet monolayers on gold: insights from ToF-SIMS and isotopic labeling.  

PubMed

To work as magnetic components in molecular electronics and spintronics, single-molecule magnets (SMMs) must be reliably interfaced with metals. The organization on gold of a Fe4 SMM carrying two acetyl-protected thiol groups has been studied by exploiting the surface sensitivity of time-of-flight secondary ion mass spectrometry (ToF-SIMS), additionally powered by the use of an isotopic labeling strategy. Deposition from millimolar dichloromethane solutions results in a higher surface coverage and better packed monolayers as compared with previous protocols based on more diluted solutions. Fe4 complexes are chemically tethered to the surface via a single Au-S bond while they still contain an intact SAc group. PMID:25000391

Totaro, Pasquale; Poggini, Lorenzo; Favre, Annaick; Mannini, Matteo; Sainctavit, Philippe; Cornia, Andrea; Magnani, Agnese; Sessoli, Roberta

2014-07-29

139

A flexible fluorescence correlation spectroscopy based method for quantification of the DNA double labeling efficiency with precision control  

NASA Astrophysics Data System (ADS)

We developed a laser-based method to quantify the double labeling efficiency of double-stranded DNA (dsDNA) in a fluorescent dsDNA pool with fluorescence correlation spectroscopy (FCS). Though, for quantitative biochemistry, accurate measurement of this parameter is of critical importance, before our work it was almost impossible to quantify what percentage of DNA is doubly labeled with the same dye. The dsDNA is produced by annealing complementary single-stranded DNA (ssDNA) labeled with the same dye at 5? end. Due to imperfect ssDNA labeling, the resulting dsDNA is a mixture of doubly labeled dsDNA, singly labeled dsDNA and unlabeled dsDNA. Our method allows the percentage of doubly labeled dsDNA in the total fluorescent dsDNA pool to be measured. In this method, we excite the imperfectly labeled dsDNA sample in a focal volume of <1?fL with a laser beam and correlate the fluctuations of the fluorescence signal to get the FCS autocorrelation curves; we express the amplitudes of the autocorrelation function as a function of the DNA labeling efficiency; we perform a comparative analysis of a dsDNA sample and a reference dsDNA sample, which is prepared by increasing the total dsDNA concentration c (c > 1) times by adding unlabeled ssDNA during the annealing process. The method is flexible in that it allows for the selection of the reference sample and the c value can be adjusted as needed for a specific study. We express the precision of the method as a function of the ssDNA labeling efficiency or the dsDNA double labeling efficiency. The measurement precision can be controlled by changing the c value.

Hou, Sen; Tabaka, Marcin; Sun, Lili; Trochimczyk, Piotr; Kaminski, Tomasz S.; Kalwarczyk, Tomasz; Zhang, Xuzhu; Holyst, Robert

2014-08-01

140

Analysis of SRC Oncogenic Signaling in Colorectal Cancer by Stable Isotope Labeling with Heavy Amino Acids in Mouse Xenografts*  

PubMed Central

The non-receptor tyrosine kinase SRC is frequently deregulated in human colorectal cancer (CRC), and SRC increased activity has been associated with poor clinical outcomes. In nude mice engrafted with human CRC cells, SRC over-expression favors tumor growth and is accompanied by a robust increase in tyrosine phosphorylation in tumor cells. How SRC contributes to this tumorigenic process is largely unknown. We analyzed SRC oncogenic signaling in these tumors by means of a novel quantitative proteomic analysis. This method is based on stable isotope labeling with amino acids of xenograft tumors by the addition of [13C6]-lysine into mouse food. An incorporation level greater than 88% was obtained in xenograft tumors after 30 days of the heavy lysine diet. Quantitative phosphoproteomic analysis of these tumors allowed the identification of 61 proteins that exhibited a significant increase in tyrosine phosphorylation and/or association with tyrosine phosphorylated proteins upon SRC expression. These mainly included molecules implicated in vesicular trafficking and signaling and RNA binding proteins. Most of these proteins were specific targets of SRC signaling in vivo, as they were not identified by analysis via stable isotope labeling by amino acids in cell culture (SILAC) of the same CRC cells in culture. This suggests that oncogenic signaling induced by SRC in tumors significantly differs from that induced by SRC in cell culture. We next confirmed this notion experimentally with the example of the vesicular trafficking protein and SRC substrate TOM1L1. We found that whereas TOM1L1 depletion only slightly affected SRC-induced proliferation of CRC cells in vitro, it drastically decreased tumor growth in xenografted nude mice. We thus concluded that this vesicular trafficking protein plays an important role in SRC-induced tumor growth. Overall, these data show that SILAC analysis in mouse xenografts is a valuable approach for deciphering tyrosine kinase oncogenic signaling in vivo. PMID:23023324

Sirvent, Audrey; Vigy, Oana; Orsetti, Beatrice; Urbach, Serge; Roche, Serge

2012-01-01

141

Highly accurate quantification of hydroxyproline-containing peptides in blood using a protease digest of stable isotope-labeled collagen.  

PubMed

Collagen-derived hydroxyproline (Hyp)-containing dipeptides and tripeptides, which are known to possess physiological functions, appear in blood at high concentrations after oral ingestion of gelatin hydrolysate. However, highly accurate and sensitive quantification of the Hyp-containing peptides in blood has been challenging because of the analytical interference from numerous other blood components. We recently developed a stable isotope-labeled collagen named "SI-collagen" that can be used as an internal standard in various types of collagen analyses employing liquid chromatography-mass spectrometry (LC-MS). Here we prepared stable isotope-labeled Hyp-containing peptides from SI-collagen using trypsin/chymotrypsin and plasma proteases by mimicking the protein degradation pathways in the body. With the protease digest of SI-collagen used as an internal standard mixture, we achieved highly accurate simultaneous quantification of Hyp and 13 Hyp-containing peptides in human blood by LC-MS. The area under the plasma concentration-time curve of Hyp-containing peptides ranged from 0.663 ± 0.022 nmol/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate. PMID:25417748

Taga, Yuki; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

2014-12-17

142

Deciphering systemic wound responses of the pumpkin extrafascicular phloem by metabolomics and stable isotope-coded protein labeling.  

PubMed

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

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

2012-12-01

143

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

NASA Astrophysics Data System (ADS)

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.

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

2012-09-01

144

Characterization of volatile nylon 6.6 thermal-oxidative degradation products by selective isotopic labeling and cryo-GC/MS.  

PubMed

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. PMID:22711515

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

2012-09-01

145

High-throughput synthesis of stable isotope-labeled transmembrane proteins for targeted transmembrane proteomics using a wheat germ cell-free protein synthesis system.  

PubMed

Using a wheat germ cell-free protein synthesis system, we developed a high-throughput method for the synthesis of stable isotope-labeled full-length transmembrane proteins as proteoliposomes to mimic the in vivo environment, and we successfully constructed an internal standard library for targeted transmembrane proteomics by using mass spectrometry. PMID:25431973

Takemori, Nobuaki; Takemori, Ayako; Matsuoka, Kazuhiro; Morishita, Ryo; Matsushita, Natsuki; Aoshima, Masato; Takeda, Hiroyuki; Sawasaki, Tatsuya; Endo, Yaeta; Higashiyama, Shigeki

2015-02-20

146

Comparison of faecal monitoring and area under the curve techniques to determine iron absorption in humans using stable isotope labelling.  

PubMed

The objective of the study was to compare faecal monitoring and area under the curve (AUC) techniques for measuring iron absorption from a stable isotope label. Nine healthy female subjects (age: 33+/-8 yr) were recruited to take part in the study. After an overnight fast, each subject received a 5 mg dose of highly enriched Fe-57 and all faecal samples were collected for 10 days post-dose to allow for estimation of iron absorption by the faecal monitoring method (geometric mean: 25%). Serial blood samples (12 in total) were also collected from each volunteer for 6h post-dose to estimate iron absorption by the well-validated area under the plasma iron concentration curve (AUC) method (geometric mean: 18%). The faecal monitoring method yields higher iron absorption results compared to AUC. This discrepancy may be due to the steps involved in faecal processing where systematic errors are likely to take place and also to the lack of complete faecal recovery associated with issues of cleanliness and hygiene when passing stools. Both of these lead to losses of the unabsorbed isotope and to an overestimate of iron absorption. PMID:20569926

Sarria, Beatriz; Dainty, Jack R

2010-07-01

147

Isomerization of stable isotopically labeled elaidic acid to cis and trans monoenes by ruminal microbes  

Microsoft Academic Search

A previous study showed that oleic acid was con- verted by mixed ruminal microbes to stearic acid and also converted to a multitude of trans octadecenoic acid iso- mers. This study traced the metabolism of one of these trans C18:1 isomers upon its incubation with mixed ruminal microbes. Unlabeled and labeled (18-( 13 C) trans -9 C18:1) elaidic acid were

Julie M. Proell; Erin E. Mosley; Gary L. Powell; Thomas C. Jenkins

2002-01-01

148

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

PubMed Central

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. PMID:19196281

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

2009-01-01

149

Human lactation: maternal transfer of dietary triglycerides labeled with stable isotopes  

SciTech Connect

A stable isotope tracer method was utilized to measure quantitatively the secretion of diet-derived fatty acids (FA) into human milk. A mixture of (/sup 2/H6)tripalmitin, (/sup 2/H18)-triolein, and (/sup 2/H12)trilinolein was administered to three healthy, lactating women 22 to 30 years of age. Milk and blood samples were collected sequentially for 72 hr. The FA composition and concentration of total plasma, lipoprotein, and milk triglycerides were determined by gas-liquid chromatography (GLC) and the isotopic enrichment was determined by gas-liquid chromatography-mass spectrometry (GLC-MS). There were no statistically significant differences in mammary secretion of the individual fats, either by a single individual or between subjects. The mean secretion of fat by one breast was 5.11 +/- 1.26% of the dose (CV = 25%). There was a significant 6.0-hr delay between peak occurrence of the tracer in plasma and its occurrence in milk. The lipids are transported to the mammary gland primarily by the chylomicron and very low density lipoprotein triglycerides.

Hachey, D.L.; Thomas, M.R.; Emken, E.A.; Garza, C.; Brown-Booth, L.; Adlof, R.O.; Klein, P.D.

1987-10-01

150

Competition for phosphorus: differential uptake from dual-isotope--labeled soil interspaces between shrub and grass.  

PubMed

Two species of Agropyron grass differed strikingly in their capacity to compete for phosphate in soil interspaces shared with a common competitor, the sagebrush Artemisia tridentata. Of the total phosphorus-32 and -33 absorbed by Artemisia, 86 percent was from the interspace shared with Agropyron spicatum and only 14 percent from that shared with Agropyron desertorum. Actively absorbing mycorrhizal roots of Agropyron and Artemisia were present in both interspaces, where competition for the labeled phosphate occurred. The results have important implications about the way in which plants compete for resources below ground in both natural plant communities and agricultural intercropping systems. PMID:17795898

Caldwell, M M; Eissenstat, D M; Richards, J H; Allen, M F

1985-07-26

151

Competition for phosphorus: differential uptake from dual-isotope-labeled soil interspaces between shrub and grass  

SciTech Connect

Two species of Agropyron grass differed strikingly in their capacity to compete for phosphate in soil interspaces shared with a common competitor, the sagebrush Artemisia tridentata. Of the total phosphorus-32 and -33 absorbed by Artemisia, 86% was from the interspace shared with Agropyron spicatum and only 14% from that shared with Agropyron desertorum. Actively absorbing mycorrhizal roots of Agropyron and Artemisia were present in both interspaces, where competition for the labeled phosphate occurred. The results have important implications about the way in which plants compete for resources below ground in both natural plant communities and agricultural intercropping systems.

Caldwell, M.M.; Eissenstat, D.M.; Richards, J.H.; Allen, M.F.

1985-07-26

152

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

NASA Astrophysics Data System (ADS)

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.

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.; Sivá?ek, I.; Stepantsov, S. V.; Vanin, D. V.; Vedeneev, V. Yu.; Yukhimchuk, S. A.; Granja, C.; Pospisil, S.

2014-06-01

153

Isotope-labelled urea to test colon drug delivery devices in vivo: principles, calculations and interpretations.  

PubMed

This paper describes various methodological aspects that were encountered during the development of a system to monitor the in vivo behaviour of a newly developed colon delivery device that enables oral drug treatment of inflammatory bowel diseases. [(13)C]urea was chosen as the marker substance. Release of [(13)C]urea in the ileocolonic region is proven by the exhalation of (13)CO2 in breath due to bacterial fermentation of [(13)C]urea. The (13)CO2 exhalation kinetics allows the calculation of a lag time as marker for delay of release, a pulse time as marker for the speed of drug release and the fraction of the dose that is fermented. To determine the total bioavailability, also the fraction of the dose absorbed from the intestine must be quantified. Initially, this was done by calculating the time-dependent [(13)C]urea appearance in the body urea pool via measurement of (13)C abundance and concentration of plasma urea. Thereafter, a new methodology was successfully developed to obtain the bioavailability data by measurement of the urinary excretion rate of [(13)C]urea. These techniques required two experimental days, one to test the coated device, another to test the uncoated device to obtain reference values for the situation that 100 % of [(13)C]urea is absorbed. This is hampered by large day-to-day variations in urea metabolism. Finally, a completely non-invasive, one-day test was worked out based on a dual isotope approach applying a simultaneous administration of [(13)C]urea in a coated device and [(15)N2]urea in an uncoated device. All aspects of isotope-related analytical methodologies and required calculation and correction systems are described. PMID:24313370

Maurer, Marina J M; Schellekens, Reinout C A; Wutzke, Klaus D; Stellaard, Frans

2013-01-01

154

A novel dual-isotope labelling method for distinguishing between soil sources of N2O.  

PubMed

We present a novel 18O-15N-enrichment method for the distinction between nitrous oxide (N2O) from nitrification, nitrifier denitrification and denitrification based on a method with single- and double-15N-labelled ammonium nitrate. We added a new treatment with 18O-labelled water to quantify N2O from nitrifier denitrification. The theory behind this is that ammonia oxidisers use oxygen (O2) from soil air for the oxidation of ammonia (NH3), but use H2O for the oxidation of the resulting hydroxylamine (NH2OH) to nitrite (NO2-). Thus, N2O from nitrification would therefore be expected to reflect the 18O signature of soil O2, whereas the 18O signature of N2O from nitrifier denitrification would reflect that of both soil O2 and H2O. It was assumed that (a) there would be no preferential removal of 18O or 16O during nitrifier denitrification or denitrification, (b) the 18O signature of the applied 18O-labelled water would remain constant over the experimental period, and (c) any O exchange between H(2)18O and NO3- would be negligible under the chosen experimental conditions. These assumptions were tested and validated for a silt loam soil at 50% water-filled pore space (WFPS) following application of 400 mg N kg-1 dry soil. We compared the results of our new method with those of a conventional inhibition method using 0.02% v/v acetylene (C2H2) and 80% v/v O2 in helium. Both the 18O-15N-enrichment and inhibitor methods identified nitrifier denitrification to be a major source of N2O, accounting for 44 and 40%, respectively, of N2O production over 24 h. However, compared to our 18O-15N-method, the inhibitor method overestimated the contribution from nitrification at the expense of denitrification, probably due to incomplete inhibition of nitrifier denitrification and denitrification by large concentrations of O2 and a negative effect of C2H2 on denitrification. We consider our new 18O-15N-enrichment method to be more reliable than the use of inhibitors; it enables the distinction between more soil sources of N2O than was previously possible and has provided the first direct evidence of the significance of nitrifier denitrification as a source of N2O in fertilised arable soil. PMID:16220527

Wrage, N; van Groenigen, J W; Oenema, O; Baggs, E M

2005-01-01

155

The use of stable-isotopically labeled oleic acid to interrogate lipid assembly in vivo: assessing pharmacological effects in preclinical species  

PubMed Central

The use of stable isotopically labeled substrates and analysis by mass spectrometry have provided substantial insight into rates of synthesis, disposition, and utilization of lipids in vivo. The information to be gained from such studies is of particular benefit to therapeutic research where the underlying causes of disease may be related to the production and utilization of lipids. When studying biology through the use of isotope tracers, care must be exercised in interpreting the data to ensure that any response observed can truly be interpreted as biological and not as an artifact of the experimental design or a dilutional effect on the isotope. We studied the effects of dosing route and tracer concentration on the mass isotopomer distribution profile as well as the action of selective inhibitors of microsomal tri­glyceride transfer protein (MTP) in mice and diacylglycerol acyltransferase 1 (DGAT1) in nonhuman primates, using a stable-isotopically labeled approach. Subjects were treated with inhibitor and subsequently given a dose of uniformly 13C-labeled oleic acid. Samples were analyzed using a rapid LC-MS technique, allowing the effects of the intervention on the assembly and disposition of triglycerides, cholesteryl esters, and phospholipids to be determined in a single 3 min run from just 10 ?l of plasma. PMID:21415123

McLaren, David G.; He, Timothy; Wang, Sheng-Ping; Mendoza, Vivienne; Rosa, Raymond; Gagen, Karen; Bhat, Gowri; Herath, Kithsiri; Miller, Paul L.; Stribling, Sloan; Taggart, Andrew; Imbriglio, Jason; Liu, Jinqi; Chen, Dunlu; Pinto, Shirly; Balkovec, James M.; DeVita, Robert J.; Marsh, Donald J.; Castro-Perez, Jose M.; Strack, Alison; Johns, Douglas G.; Previs, Stephen F.; Hubbard, Brian K.; Roddy, Thomas P.

2011-01-01

156

A review on test procedure, energy efficiency standards and energy labels for room air conditioners and refrigerator–freezers  

Microsoft Academic Search

Air conditioners and refrigerator–freezers are major energy users in a household environment and hence efficiency improvement of these appliances can be considered as an important step to reduce their energy consumption along with environmental pollution prevention. Energy efficiency standards and labels are commonly used tools to reduce the energy uses for household appliances for many countries around the world. The

T. M. I. Mahlia; R. Saidur

2010-01-01

157

Stable isotope labeling method for the investigation of protein haptenation by electrophilic skin sensitizers.  

PubMed

The risk of contact sensitization is a major consideration in the development of new formulations for personal care products. However, developing a mechanistic approach for non-animal risk assessment requires further understanding of haptenation of skin proteins by sensitizing chemicals, which is the molecular initiating event causative of skin sensitization. The non-stoichiometric nature of protein haptenation results in relatively low levels of modification, often of low abundant proteins, presenting a major challenge for their assignment in complex biological matrices such as skin. Instrumental advances over the last few years have led to a considerable increase in sensitivity of mass spectrometry (MS) techniques. We have combined these advancements with a novel dual-labeling/LC-MS(E) approach to provide an in-depth direct comparison of human serum albumin (HSA), 2,4-dinitro-1-chlorobenzene (DNCB), 5-chloro-2-methyl-4-isothiazolin-3-one (MCI), trans-cinnamaldehyde, and 6-methyl coumarin. These data have revealed novel insights into the differences in protein haptenation between sensitizers with different reaction mechanisms and sensitizing potency; the extreme sensitizers DNCB and MCI were shown to modify a greater number of nucleophilic sites than the moderate sensitizer cinnamaldehyde; and the weak/non-sensitizer 6-methyl coumarin was restricted to only a single nucleophilic residue within HSA. The evaluation of this dual labeling/LC-MS(E) approach using HSA as a model protein has also demonstrated that this strategy could be applied to studying global haptenation in complex mixtures of skin-related proteins by different chemicals. PMID:25145658

Parkinson, Erika; Boyd, Pete; Aleksic, Maja; Cubberley, Richard; O'Connor, David; Skipp, Paul

2014-11-01

158

An efficient simulator for pinhole imaging of PET isotopes  

NASA Astrophysics Data System (ADS)

Today, small-animal multi-pinhole single photon emission computed tomography (SPECT) can reach sub-half-millimeter image resolution. Recently we have shown that dedicated multi-pinhole collimators can also image PET tracers at sub-mm level. Simulations play a vital role in the design and optimization of such collimators. Here we propose and validate an efficient simulator that models the whole imaging chain from emitted positron to detector signal. This analytical simulator for pinhole positron emission computed tomography (ASPECT) combines analytical models for pinhole and detector response with Monte Carlo (MC)-generated kernels for positron range. Accuracy of ASPECT was validated by means of a MC simulator (MCS) that uses a kernel-based step for detector response with an angle-dependent detector kernel based on experiments. Digital phantom simulations with ASPECT and MCS converge to almost identical images. However, ASPECT converges to an equal image noise level three to four orders of magnitude faster than MCS. We conclude that ASPECT could serve as a practical tool in collimator design and iterative image reconstruction for novel multi-pinhole PET.

Goorden, M. C.; van der Have, F.; Kreuger, R.; Beekman, F. J.

2011-03-01

159

The simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards.  

PubMed

An LC/MS/MS assay we published for tenofovir (TFV) plasma levels is a useful tool for monitoring the pharmacotherapy of HIV-positive individuals [T. Delahunty, L. Bushman, C.V. Fletcher, J. Chromatogr. B 830 (2006) 6-12]. A new combination therapy consisting of the TFV pro-drug (300 mg) and another reverse transcriptase inhibitor, emtricitabine (FTC, 200 mg) has become available in a convenient once-daily dosage form (Truvada). This widely used medication has prompted us to develop and validate a convenient assay to determine simultaneously TFV and FTC plasma concentrations. In view of their chemical similarity to the analytes, stable isotope internal standards (IS) were chosen. These consisted of TFV labeled uniformly with (13)C in the adenine moiety (Iso-TFV) and FTC labeled with 13C and 15N in the cytosine moiety (Iso-FTC). Trifluoroacetic acid was added to the patient's EDTA plasma (containing the IS) to produce a de-proteinated extract after high speed centrifugation. The extracts were directly injected into the mobile phase (3% acetonitrile/1% acetic acid, aq.) stream flowing at 200 microL/min. A Synergi Polar-RP, 2.0 mm x 150 mm, reversed-phase analytical column was used to achieve the chromatographic separation. Detection of the analytes was achieved by ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) in the positive ion mode were 288/176 and 293/181 ions for TFV and Iso-TFV, respectively and the precursor/product transitions (m/z) were 248/130 and 251/133 ions for FTC and Iso-FTC, respectively. When the analyte/IS abundance ratios were plotted against the specified concentrations, the linearity of the concentration curves were in the range 10 ng/mL to 1500 ng/mL for both analytes (250 microL plasma extracted), with a minimum quantifiable limit of 10 ng/mL for both analytes. The inter- and intra-day accuracy and precision for both TFV and FTC were within +/-20% at the LLOQ and +/-15% at the other QC levels. We have expanded the method originally designed for the assay of TFV alone to incorporate the simultaneous determination of the latter and FTC using stable isotope IS. This assay has been successfully used for the periodic monitoring of 678 HIV-positive patients being treated with the combination therapy. PMID:19493710

Delahunty, Tom; Bushman, Lane; Robbins, Brian; Fletcher, Courtney V

2009-07-01

160

Structure and vibrational dynamics of isotopically labeled lithium borohydride using neutron diffraction and spectroscopy  

SciTech Connect

The crystalline structure of a {sup 7}Li and {sup 11}B labeled lithium borohydride has been investigated using neutron powder diffraction at 3.5, 360, and 400 K. The B-H bond lengths and H-B-H angles for the [BH{sub 4}]{sup -} tetrahedra indicated that the tetrahedra maintained a nearly ideal configuration throughout the temperature range investigated. The atomic displacement parameters at 360 K suggest that the [BH{sub 4}]{sup -} tetrahedra become increasingly disordered as a result of large amplitude librational and reorientational motions as the orthorhombic to hexagonal phase transition (T=384 K) is approached. In the high-temperature hexagonal phase, the [BH{sub 4}]{sup -} tetrahedra displayed extreme disorder about the trigonal axis along which they are aligned. Neutron vibrational spectroscopy data were collected at 5 K over an energy range of 10-170 meV, and were found to be in good agreement with prior Raman and low-resolution neutron spectroscopy studies. - Graphical abstract: The structure of {sup 7}Li{sup 11}BH{sub 4} in the low-temperature Pnma phase, including atomic displacement ellipsoids, at 3.5 K.

Hartman, Michael R. [NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899-8562 (United States)], E-mail: mike.hartman@oregonstate.edu; Rush, John J. [NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899-8562 (United States); Udovic, Terrence J. [NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, MD 20899-8562 (United States); Bowman, Robert C. [Jet Propulsion Laboratory, California Institute of Technology, Pasadena, CA 91109 (United States); Hwang, Son-Jong [Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125 (United States)

2007-04-15

161

Metabolic cartography: experimental quantification of metabolic fluxes from isotopic labelling studies  

SciTech Connect

For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on {sup 13}CO{sub 2} dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

O'Grady J.; Schwender J.; Shachar-Hill, Y.; Morgan, J. A.

2012-03-01

162

Magic angle sample spinning sup 13 C nuclear magnetic resonance of isotopically labeled bacteriorhodopsin  

SciTech Connect

Bacteriorhodopsin (bR), the light-driven proton pump protein from Halobacterium halobium, was biosynthetically labeled with (4-{sup 13}C)Asp. The incorporation yield was 48%. The magic angle sample spinning (MASS) {sup 13}C nuclear magnetic resonance (NMR) spectrum of this sample revealed six different peaks superimposed on a broad band of naturally abundant peptide-bond {sup 13}C. Two of the six carbonyl signals can be attributed to internal-protonated Asp carboxyl groups, one of which might be Asp115. An additional resonance at 110 ppm can be associated with the C-11 carbon of Trp, indicating an unusual biosynthetic pathway of this amino acid in Halobacterium halobium. Similar measurements performed on papain-treated purple membrane which lacks the C-terminal tail display two new intense signals at 178 and 178.9 ppm. If the same spectrum is taken without cross-polarization, these signals do not decrease or disappear. On the basis of their intensities and their chemical shifts, one can assign in addition to the C-terminal Asp four Asp residues facing the cytoplasmic phase. In native bR, at least two of these form a salt-bridge-like bond which also might include the C-terminal tail. These experiments not only provide data about the chemical environment of the Asp residues within the hydrophobic core of bacteriorhodopsin but also yield information about the interactions between surface components.

Engelhard, M.; Hess, B.; Emeis, D.; Metz, G.; Kreutz, W.; Siebert, F. (Max-Planck-Institut fuer Ernaehrungsphysiologie, Dortmund (Germany, F.R.))

1989-05-02

163

Relationship between stable carbon isotope discrimination and water use efficiency under regulated deficit irrigation of pear-jujube tree  

Microsoft Academic Search

To investigate the relationship between stable carbon isotope discrimination (?) of different organs and water use efficiency (WUE) under different water deficit levels, severe, moderate and low water deficit levels were treated at bud burst to leafing, flowering to fruit set, fruit growth and fruit maturation stages of field grown pear-jujube tree, and leaf stable carbon isotope discrimination (?L) at

Ningbo Cui; Taisheng Du; Shaozhong Kang; Fusheng Li; Xiaotao Hu; Mixia Wang; Zhijun Li

2009-01-01

164

ISOTOPES  

E-print Network

over ammonia-hydrogen exchange include higher separationAmmonia-hydrogen Exchange This reaction has both a larger separationammonia) fraction with which it is in isotopic equilibrium, necessitating the separation

Lederer, C. Michael

2013-01-01

165

The utility of isotope-coded protein labeling for prioritization of proteins found in ovarian cancer patient urine.  

PubMed

Urine offers a number of attractive features as a sample type for biomarker discovery, including noninvasive sampling, quantity and availability, stability, and a narrow dynamic range. In this study we report the first application of isotope coded protein labeling (ICPL), coupled with in-solution isoelectric fractionation and LC-MALDI-TOF/TOF, to examine and prioritize urinary proteins from ovarian cancer patients. Following the definition of stringent exclusion criteria a total of 579 proteins were identified with 43% providing quantitation data. Protein abundance changes were validated for selected proteins by ESI-Qq-TOF MS, following which Western blot and immunohistochemical analysis by tissue microarray was used to explore the biological relevance of the proteins identified. Several established markers (e.g., HE4, osteopontin) were identified at increased levels in ovarian cancer patient urine, validating the approach used; we also identified a number of potential marker candidates (e.g., phosphatidylethanolamine binding protein 1, cell-adhesion molecule 1) previously unreported in the context of ovarian cancer. We conclude that the ICPL strategy for identification and relative quantitation of urine proteins is an appropriate tool for biomarker discovery studies, and can be applied for the selection of potential biomarker candidates for further characterization. PMID:23952987

Rainczuk, Adam; Condina, Mark; Pelzing, Matthias; Dolman, Sebastiaan; Rao, Jyothsna; Fairweather, Nicole; Jobling, Tom; Stephens, Andrew N

2013-09-01

166

Quantification of protein deposits on silicone hydrogel materials using stable-isotopic labeling and multiple reaction monitoring.  

PubMed

This study was designed to use multiple reaction monitoring (MRM) for accurate quantification of contact lens protein deposits. Worn lenses used with a multipurpose disinfecting solution were collected after wear. Individual contact lenses were extracted and then digested with trypsin. MRM in conjunction with stable-isotope-labeled peptide standards was used for protein quantification. The results show that lysozyme was the major protein detected from both lens types. The amount of protein extracted from contact lenses was affected by the lens material. Except for keratin-1 (0.83 ± 0.61 vs 0.77 ± 0.20, p = 0.81) or proline rich protein-4 (0.11 ± 0.04 vs 0.15 ± 0.12, p = 0.97), the amounts of lysozyme, lactoferrin, or lipocalin-1 extracted from balafilcon A lenses (12.9 ± 9.01, 0.84 ± 0.50 or 2.06 ± 1.6, respectively) were significantly higher than that extracted from senofilcon A lenses (0.88 ± 0.13, 0.50 ± 0.10 or 0.27 ± 0.23, respectively) (p < 0.05). The amount of protein extracted from contact lenses was dependent on both the individual wearer and the contact lens material. This may have implications for the development of clinical responses during lens wear for different people and with different types of contact lenses. The use of MRM-MS is a powerful analytical tool for the quantification of specific proteins from single contact lenses after wear. PMID:22784025

Omali, Negar Babaei; Zhao, Zhenjun; Zhong, Ling; Raftery, Mark J; Zhu, Hua; Ozkan, Jerome; Willcox, Mark

2012-01-01

167

A novel method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies  

NASA Astrophysics Data System (ADS)

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.

Wu, Dianming; Kampf, Christopher; Pöschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Sörgel, Matthias

2014-05-01

168

Advanced Identification of Proteins in Uncharacterized Proteomes by Pulsed in Vivo Stable Isotope Labeling-based Mass Spectrometry*  

PubMed Central

Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. This problem is aggravated by the presence of large numbers of expressed sequence tag clones that lack homologues in other species, which makes it difficult to identify new proteins irrespective of whether such molecules are involved in species-specific biological processes. We have used a pulsed stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry method, which is based on the detection of paired peptides after [13C6]lysine incorporation into proteins in vivo, to greatly increase the confidence of protein identification in cross-species database searches. The method was applied to identify nearly 3000 proteins in regenerating tails of the urodele amphibian Notophthalmus viridescens, which possesses outstanding capabilities in the regeneration of complex tissues. We reason that pulsed in vivo SILAC represents a versatile tool to identify new proteins in species for which only limited sequence information exists. PMID:20139370

Looso, Mario; Borchardt, Thilo; Krüger, Marcus; Braun, Thomas

2010-01-01

169

Automated resonance assignment of the 21 kDa stereo-array isotope labeled thioldisulfide oxidoreductase DsbA  

NASA Astrophysics Data System (ADS)

The automated chemical shift assignment algorithm FLYA has been extended for use with stereo-array isotope labeled (SAIL) proteins to determine the sequence-specific resonance assignments of large proteins. Here we present the assignment of the backbone and sidechain chemical shifts of the 21 kDa thioldisulfide oxidoreductase DsbA from Escherichia coli that were determined with the SAIL-FLYA algorithm in conjunction with automated peak picking. No manual corrections of peak lists or assignments were applied. The assignments agreed with manually determined reference assignments in 95.4% of the cases if 16 input spectra were used, 94.1% if only 3D 13C/15N-resolved NOESY, CBCA(CO)NH, and 2D [13C/15N,1H]-HSQC were used, and 86.8% if exclusively 3D 13C/15N-resolved NOESY spectra were used. Considering only the assignments that are classified as reliable by the SAIL-FLYA algorithm, the degrees of agreement increased to 97.5%, 96.5%, and 94.2%, respectively. With our approach it is thus possible to automatically obtain almost complete and correct assignments of proteins larger than 20 kDa.

Schmidt, Elena; Ikeya, Teppei; Takeda, Mitsuhiro; Löhr, Frank; Buchner, Lena; Ito, Yutaka; Kainosho, Masatsune; Güntert, Peter

2014-12-01

170

Isotopically Labeled Expression in E. coli, Purification, and Refolding of the Full Ectodomain of the Influenza Virus Membrane Fusion Protein  

PubMed Central

This paper describes methods to produce an isotopically labeled 23 kDa viral membrane protein with purified yield of 20 mg/L of E. coli shake flask culture. This yield is sufficient for NMR structural studies and the protein production methods are simple, straightforward, and rapid and likely applicable to other recombinant membrane proteins expressed in E. coli. The target FHA2 protein is the full ectodomain construct of the influenza virus hemagglutinin protein which catalyzes fusion between the viral and the cellular endosomal membranes during infection. The high yield of FHA2 was achieved by: (1) initial growth in rich medium to A600 ~ 8 followed by a switch to minimal medium and induction of protein expression; and (2) obtaining protein both from purification of the detergent-soluble lysate and from solubilization, purification, and refolding of inclusion bodies. The high cell density was achieved after optimization of pH, oxygenation, and carbon source and concentration, and the refolding protocol was optimized using circular dichroism spectroscopy. For a single residue of membrane-associated FHA2 that was obtained from purification and refolding of inclusion bodies, native conformation was verified by the 13CO chemical shift measured using solid-state nuclear magnetic resonance spectroscopy. PMID:18640277

Curtis-Fisk, Jaime; Spencer, Ryan M.; Weliky, David P.

2008-01-01

171

Stable isotope labeling confirms mixotrophic nature of streamer biofilm communities at alkaline hot springs  

PubMed Central

Streamer biofilm communities (SBC) are often observed within chemosynthetic zones of Yellowstone hot spring outflow channels, where temperatures exceed those conducive to photosynthesis. Nearest the hydrothermal source (75–88°C) SBC comprise thermophilic Archaea and Bacteria, often mixed communities including Desulfurococcales and uncultured Crenarchaeota, as well as Aquificae and Thermus, each carrying diagnostic membrane lipid biomarkers. We tested the hypothesis that SBC can alternate their metabolism between autotrophy and heterotrophy depending on substrate availability. Feeding experiments were performed at two alkaline hot springs in Yellowstone National Park: Octopus Spring and “Bison Pool,” using various 13C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest 13C uptake, at both sites, was from acetate into almost all bacterial fatty acids, particularly into methyl-branched C15, C17 and C19 fatty acids that are diagnostic for Thermus/Meiothermus, and some Firmicutes as well as into universally common C16:0 and C18:0 fatty acids. 13C-glucose showed a similar, but a 10–30 times lower uptake across most fatty acids. 13C-bicarbonate uptake, signifying the presence of autotrophic communities was only significant at “Bison Pool” and was observed predominantly in non-specific saturated C16, C18, C20, and C22 fatty acids. Incorporation of 13C-formate occurred only at very low rates at “Bison Pool” and was almost undetectable at Octopus Spring, suggesting that formate is not an important carbon source for SBC. 13C-uptake into archaeal lipids occurred predominantly with 13C-acetate, suggesting also that archaeal communities at both springs have primarily heterotrophic carbon assimilation pathways. We hypothesize that these communities are energy-limited and predominantly nurtured by input of exogenous organic material, with only a small fraction being sustained by autotrophic growth.

Schubotz, Florence; Hays, Lindsay E.; Meyer-Dombard, D'Arcy R.; Gillespie, Aimee; Shock, Everett L.; Summons, Roger E.

2015-01-01

172

Residue-Specific Structural Kinetics of Proteins through the Union of Isotope Labeling, Mid-IR Pulse Shaping, and Coherent 2D IR Spectroscopy  

PubMed Central

We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for 13C=18O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide form type 2 diabetes. PMID:20472067

Middleton, Chris T.; Woys, Ann Marie; Mukherjee, Sudipta S.; Zanni, Martin T.

2010-01-01

173

Reproducibility of an HPLC-ESI-MS/MS Method for the Measurement of Stable-Isotope Enrichment of in Vivo-Labeled Muscle ATP Synthase Beta Subunit  

PubMed Central

We sought to evaluate the reproducibility of a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach to measure the stable-isotope enrichment of in vivo-labeled muscle ATP synthase ? subunit (?-F1-ATPase), a protein most directly involved in ATP production, and whose abundance is reduced under a variety of circumstances. Muscle was obtained from a rat infused with stable-isotope-labeled leucine. The muscle was homogenized, ?-F1-ATPase immunoprecipitated, and the protein was resolved using 1D-SDS PAGE. Following trypsin digestion of the isolated protein, the resultant peptide mixtures were subjected to analysis by HPLC-ESI-MS/MS, which resulted in the detection of multiple ?-F1-ATPase peptides. There were three ?-F1-ATPase unique peptides with a leucine residue in the amino acid sequence, and which were detected with high intensity relative to other peptides and assigned with >95% probability to ?-F1-ATPase. These peptides were specifically targeted for fragmentation to access their stable-isotope enrichment based on MS/MS peak areas calculated from extracted ion chromatographs for selected labeled and unlabeled fragment ions. Results showed best linearity (R2?=?0.99) in the detection of MS/MS peak areas for both labeled and unlabeled fragment ions, over a wide range of amounts of injected protein, specifically for the ?-F1-ATPase134-143 peptide. Measured stable-isotope enrichment was highly reproducible for the ?-F1-ATPase134-143 peptide (CV?=?2.9%). Further, using mixtures of synthetic labeled and unlabeled peptides we determined that there is an excellent linear relationship (R2?=?0.99) between measured and predicted enrichment for percent enrichments ranging between 0.009% and 8.185% for the ?-F1-ATPase134-143 peptide. The described approach provides a reliable approach to measure the stable-isotope enrichment of in-vivo-labeled muscle ?-F1-ATPase based on the determination of the enrichment of the ?-F1-ATPase134-143 peptide. PMID:22022551

Everman, Sarah; Yi, Zhengping; Langlais, Paul; Mandarino, Lawrence J.; Luo, Moulun; Roberts, Christine; Katsanos, Christos S.

2011-01-01

174

Amino acid-specific isotopic labeling and active site NMR studies of iron(II)- and iron(III)-superoxide dismutase from Escherichia coli  

Microsoft Academic Search

We have developed and employed multiple amino acid-specific isotopic labeling schemes to obtain definitive assignments for active site 1H NMR resonances of iron(II)- and iron(III)-superoxide dismutase (Fe(II)SOD and Fe(III)SOD) from Escherichia coli. Despite the severe relaxivity of high-spin Fe(III), we have been able to assign resonances to ligand His' d1 protons near 100 ppm, and ß and a protons collectively

David L. Sorkin; Anne-Frances Miller

2000-01-01

175

NanoLC Coupled to MALDI-TOF\\/TOF as an Alternative to LC-ESI-MS for Peptide Quantitation by Stable Isotope Labeling  

Microsoft Academic Search

In the last years several techniques have been developed to achieve protein quantitation using MALDI- TOF\\/TOF systems, e.g . ICAT TM , SILAC TM and iTRAQ TM , most recently. Here, we present nano-LC coupled offline to MALDI-TOF\\/TOF mass spectrometry as an alternative to commonly used online LC-ESI-MS to quantify target proteins by stable isotopic labeling, SIL (2). A mixture

J. Mateos; P. Fernández; C. Ruiz-Romero; C. Fernández; I. Rodriguez; F. J. Blanco

176

Simultaneous determination of glucose turnover, alanine turnover, and gluconeogenesis in human using a double stable-isotope-labeled tracer infusion and gas chromatography-mass spectrometry analysis  

SciTech Connect

We have developed and validated a new method to measure simultaneously glucose turnover, alanine turnover, and gluconeogenesis in human, in steady and non-steady states, using a double stable-isotope-labeled tracer infusion and GC-MS analysis. The method is based on the concomitant infusion and dilution of D-(2,3,4,6,6-2H5)glucose and L-(1,2,3-13C3)alanine. The choice of the tracers was done on the basis of a minimal overlap between the ions of interest and those arising from natural isotopic abundances. Alanine was chosen as the gluconeogenic substrate because it is the major gluconeogenic amino acid extracted by the liver and, with lactate, constitutes the bulk of the gluconeogenic precursors. The method was validated by comparing the results obtained during simultaneous infusion of trace amounts of both stable isotope labeled compounds with the radioactive tracers (D-(3-3H)glucose and L-(1,2,3-14C3)alanine) in a normal and a diabetic subject; the radiolabeled tracers were used as the accepted reference procedure. A slight overestimation of glucose turnover (7.3 versus 6.8 in normal and 10.8 versus 9.2 mumol/kg min in diabetic subject) was noticed when the stable isotope-labeled tracers were used. For the basal turnover rate of alanine, similar values were obtained with both methods (6.2 mumol/kg min). For gluconeogenesis, higher values were observed in the basal state with the stable isotopes (0.42 versus 0.21 mumol/kg min); however, these differences disappeared in the postprandial period after the ingestion of a mixed meal. Despite those minor differences, the overall correlation with the reference method was excellent for glucose turnover (r = 0.87) and gluconeogenesis (r = 0.86).

Martineau, A.; Lecavalier, L.; Falardeau, P.; Chiasson, J.L.

1985-12-01

177

Efficient packing Fourier-transform approach for ultrahigh resolution isotopic distribution calculations.  

PubMed

Fine isotopic structure patterns resolvable by ultrahigh-resolution mass spectrometers are diagnostic of the elemental composition of moderately large compounds. Despite the proven performance of Fourier transforms algorithms to calculate accurate high resolution isotopic distribution, its application to finer ultrahigh resolving power exhibits limited performance. Fast Fourier transforms algorithm requires sampling the relevant range at equally spaced mass values, but ultrahigh resolution mass spectrum displays highly localized complex patterns (peaks) separated in between by relatively large unstructured intervals. Computational efforts consumed on those uninformative intervals are a waste of resources. A fast and memory efficient procedure is introduced in this paper to calculate the isotopic distribution of a single relatively high-mass molecule at ultrahigh resolution by Fourier transforms approaches. The whole isotopic distribution is packed closer to the monoisotopic peak without distorting the actual scale of the peak fine structure. This packing procedure reduced 8 to 32 times the computation resources in comparison to the same calculation performed without packing. The procedure can be readily implemented in existing software. PMID:20112948

Fernandez-de-Cossio, Jorge

2010-03-01

178

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

NASA Astrophysics Data System (ADS)

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 ?13CH4-signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) 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 (?trans = 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 ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox, = 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 rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged organic rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost affected ecosystems and their potential strengths in response to global warming.

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

2012-12-01

179

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

NASA Astrophysics Data System (ADS)

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 ?13CH4 signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) 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 (?trans = 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 ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox = 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 potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged, organic-rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost-affected ecosystems and their potential strengths in response to global warming.

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

2013-04-01

180

Simultaneous measurements of the accumulation of isotope-labelled protein and erythrocytes in skin reactions of allergic inflammation in the guinea-pig  

PubMed Central

This paper describes a method for simultaneous measurement of the accumulation of plasma protein and erythrocytes in skin reactions of hypersensitivity to bovine ?-globulin (BGG) and tuberculin PPD in the guinea-pig. The procedure consists in giving 125I-labelled plasma albumin and 51Cr-labelled autologous erythrocytes together by intravenous injection into guinea-pigs bearing skin lesions of allergic inflammation at different times and for different periods during the development of the skin reactions. Isotope accumulation in excised skin reactions is measured by scintillation counting at different stages during the evolution of hypersensitivity responses. Skin reactions of combined anaphylactic and Arthus hypersensitivity to BGG were characterized by pronounced increased vascular permeability principally in the first hour. In established 24-hour hypersensitivity reactions to both antigens (BGG and PPD) there was continuing accumulation of both plasma albumin and erythrocytes. During the development of the tuberculin reaction, an intermediate phase of isotope accumulation occurred between 6 and 9 hours after skin testing; serum transfer studies showed that this intermediate peak was not attributable to circulating antibody alone. These isotope tracer techniques were also applied to study vascular permeability in systemically transferred reactions of delayed hypersensitivity and in the vascular response to the intradermal injection of an inflammatory factor generated by antigen-activation of sensitized lymphocytes. It was concluded that isotope tracing provided objective and sensitive methods for analysing microvascular responses in allergic inflammation. PMID:4262654

Morley, J.; Williams, T. J.; Slater, A. J.; Cubitt, D.; Dumonde, D. C.

1972-01-01

181

An optimal defense strategy for phenolic glycoside production in Populus trichocarpa--isotope labeling demonstrates secondary metabolite production in growing leaves.  

PubMed

Large amounts of carbon are required for plant growth, but young, growing tissues often also have high concentrations of defensive secondary metabolites. Plants' capacity to allocate resources to growth and defense is addressed by the growth-differentiation balance hypothesis and the optimal defense hypothesis, which make contrasting predictions. Isotope labeling can demonstrate whether defense compounds are synthesized from stored or newly fixed carbon, allowing a detailed examination of these hypotheses. Populus trichocarpa saplings were pulse-labeled with 13CO2 at the beginning and end of a growing season, and the 13C signatures of phenolic glycosides (salicinoids), sugars, bulk tissue, and respired CO2 were traced over time. Half of the saplings were also subjected to mechanical damage. Populus trichocarpa followed an optimal defense strategy, investing 13C in salicinoids in expanding leaves directly after labeling. Salicinoids turned over quickly, and their production continued throughout the season. Salicin was induced by early-season damage, further demonstrating optimal defense. Salicinoids appear to be of great value to P. trichocarpa, as they command new C both early and late in the growing season, but their fitness benefits require further study. Export of salicinoids between tissues and biochemical pathways enabling induction also needs research. Nonetheless, the investigation of defense production afforded by isotope labeling lends new insights into plants' ability to grow and defend simultaneously. PMID:24739022

Massad, Tara Joy; Trumbore, Susan E; Ganbat, Gantsetseg; Reichelt, Michael; Unsicker, Sybille; Boeckler, Andreas; Gleixner, Gerd; Gershenzon, Jonathan; Ruehlow, Steffen

2014-07-01

182

ABRF Research Group Development and Characterization of a Proteomics Normalization Standard Consisting of 1,000 Stable Isotope Labeled Peptides  

PubMed Central

The ABRF Proteomics Standards Research Group (sPRG) is reporting the progress of a two-year study (2012–2014) which focuses on the generation of interassay, interspecies, and interlaboratory peptide standard that can be used for normalization of protein abundance measurements in mass spectrometry based quantitative proteomics analyses. The standard has been formulated as two mixtures: 1,000 stable isotope 13C/15N-labeled (SIL) synthetic peptides alone, and peptides mixed with a tryptic digest of a HEK 293 cell lysate. The sequences of the synthetic peptides were derived from 552 proteins conserved across proteomes of commonly analyzed species: Homo sapiens, Mus musculus and Rattus norvegicus. The selected peptides represent a full range of hydrophobicities and isoelectric points, typical of tryptic peptides derived from complex proteomic samples. The standard was designed to represent proteins of various concentrations, spanning three orders of magnitude. First year efforts were focused on selection of appropriate protein and peptide candidates, peptide synthesis, quality assessment and LC-MS/MS evaluation conducted in laboratories of sPRG members. Using a variety of instrumental configurations and bioinformatics approaches, a thorough characterization of all 1,000 peptides was established. In the second year, the group launched the study to the entire proteomics community. A lyophilized mixture of HEK 293 tryptic digest cell lysate spiked with the 1,000 SIL peptide standards was provided to each participant. Also provided were a Skyline tutorial, tutorial datasets, three MS/MS spectral libraries generated from linear ion-trap (CID), Q-TOF/QQQ (CID), or Orbitrap (HCD) instrumentation, and a Panorama data repository. Participants were asked to analyze the sample in triplicate and calculate ratios of the spiked SIL to endogenous peptides and coefficients of variance for each peptide. Over 40 datasets were returned, and results following thorough characterization of the standard using various instrumental configurations will be reported.

Dufresne, Craig; Hawke, David; Ivanov, Alexander R.; Koller, Antonius; MacLean, Brendan; Phinney, Brett; Rose, Kristie; Rudnick, Paul; Searle, Brian; Shaffer, Scott; Colangelo, Christopher M.

2014-01-01

183

Pathway of oxygen incorporation from O2 in TiO2 photocatalytic hydroxylation of aromatics: oxygen isotope labeling studies.  

PubMed

The hydroxylation process is the primary, and even the rate-determining step of the photocatalytic degradation of aromatic compounds. To make clear the hydroxylation pathway of aromatics, the TiO(2) photocatalytic hydroxylation of several model substrates, such as benzoic acid, benzene, nitrobenzene, and benzonitrile, has been studied by an oxygen-isotope-labeling method, which can definitively assign the origin of the O atoms (from oxidant O(2) or solvent H(2)O) in the hydroxyl groups of the hydroxylated products. It is found that the oxygen source of the hydroxylated products depends markedly on the reaction conditions. The percentage of the products with O(2)-derived hydroxyl O atoms increases with the irradiation time, while it decreases with the increase of substrate concentration. More intriguingly, when photogenerated valence-band holes (h(vb)(+)) are removed, nearly all the O atoms (>97?%) in the hydroxyl groups of the hydroxylated products of benzoic acid come from O(2), whereas the scavenging of conduction-band electrons (e(cb)(-)) makes almost all the hydroxyl O atoms (>95?%) originate from solvent H(2)O. In the photocatalytic oxidation system with benzoic acid and benzene coexisting in the same dispersion, the percentage of O(2)-derived hydroxyl O atoms in the hydroxylated products of strongly adsorbed benzoic acid (ca. 30?%) is much less than in that of weakly adsorbed benzene (phenol) (>60?%). Such dependences provide unique clues to uncover the photocatalytic hydroxylation pathway. Our experiments show that the main O(2)-incorporation pathway involves the reduction of O(2) by e(cb)(-) and the subsequent formation of free (?)OH via H(2)O(2), which was usually overlooked in the past photocatalytic studies. Moreover, in the hydroxylation initiated by h(vb)(+), unlike the conventional mechanism, the O atom in O(2) cannot incorporate into the product through the direct coupling between molecular O(2) and the substrate-based radicals. PMID:22266774

Li, Yue; Wen, Bo; Yu, Cailan; Chen, Chuncheng; Ji, Hongwei; Ma, Wanhong; Zhao, Jincai

2012-02-13

184

Quantitative determination of free and total bisphenol A in human urine using labeled BPA glucuronide and isotope dilution mass spectrometry.  

PubMed

Bisphenol A (BPA) is a widely used industrial chemical in the manufacturing of polycarbonate plastic bottles, food and beverage can linings, thermal receipts, and dental sealants. Animal and human studies suggest that BPA may disrupt normal hormonal function and hence, potentially, have negative effects on the human health. While total BPA is frequently reported, it is recognized that free BPA is the biologically active form and is rarely reported in the literature. The objective of this study was to develop a sensitive and improved method for the measurement of free and total BPA in human urine. Use of a labeled conjugated BPA (bisphenol A-d6 ?-D-glucuronide) allowed for the optimization of the enzymatic reaction and permitted an accurate determination of the conjugated BPA concentration in urine samples. In addition, a (13)C12-BPA internal standard was used to account for the analytical recoveries and performance of the isotope dilution method. Solid-phase extraction (SPE) combined with derivatization and analysis using a triple quadrupole GC-EI/MS/MS system achieved very low method detection limit of 0.027 ng/mL. BPA concentrations were measured in urine samples collected during the second and third trimesters of pregnancy in 36 Canadian women. Total maternal BPA concentrations in urine samples ranged from not detected to 9.40 ng/mL (median, 1.21 ng/mL), and free BPA concentrations ranged from not detected to 0.950 ng/mL (median, 0.185 ng/mL). Eighty-six percent of the women had detectable levels of conjugated BPA, whereas only 22 % had detectable levels of free BPA in their urine. BPA levels measured in this study agreed well with data reported internationally. PMID:24817354

Kubwabo, Cariton; Kosarac, Ivana; Lalonde, Kaela; Foster, Warren G

2014-07-01

185

Stable isotope labeling by amino acids in cell culture-based liquid chromatography-mass spectrometry assay to measure microtubule dynamics in neuronal cell cultures.  

PubMed

Microtubules (MTs) are highly dynamic polymers composed of ?- and ?-tubulin heterodimers. Dysregulation of MT dynamics in neurons may be a contributing factor in the progression of various neurodegenerative diseases. We developed a stable isotope labeling by amino acids in cell culture (SILAC)-based liquid chromatography-mass spectrometry (LC-MS) method to measure the fraction of [(13)C6]leucine-labeled ?-tubulin-derived surrogate peptides. Using this approach, we measured the time course of incorporation of [(13)C6]leucine label into the MT and dimer pools isolated from cycling cells and rat primary hippocampal neurons. We found that the MT pool is in rapid equilibrium with the dimer pool in the cycling cells, consistent with rapid MT polymerization/depolymerization during cell proliferation. Conversely, in neurons, we found that labeling of the MT pool was rapid, whereas the dimer pool was delayed. These results suggest that newly synthesized ?-tubulin is first incorporated into MTs or complexes that co-sediment with MTs and that appearance of labeled ?-tubulin in the dimer pool may be a consequence of MT depolymerization or breakdown. Our results demonstrate that a SILAC-based approach can be used to measure MT dynamics and may have utility for exploring MT dysregulation in various models of neurodegenerative disease. PMID:25175011

Polson, Craig; Cantone, Joseph L; Wei, Cong; Drexler, Dieter M; Meredith, Jere E

2014-12-01

186

Use of an oral stable isotope label to confirm variation in red blood cell mean age that influences HbA1c interpretation.  

PubMed

HbA1c is commonly used to monitor glycemic control. However, there is growing evidence that the relationship between HbA1c and mean blood glucose (MBG) is influenced by variation in red blood cell (RBC) lifespan in hematologically normal individuals. Correction of HbA1c for mean RBC age (MRBC ) requires a noninvasive, accurate, and affordable method to measure RBC survival. In this study, we evaluated whether a stable isotope approach would satisfy these requirements. RBC lifespan and MRBC were determined in a group of nine hematologically normal diabetic and nondiabetic subjects using oral (15) N-glycine to label heme in an age cohort of RBC. The MRBC was 58.7 ± 9.1 (2SD) days and RBC lifespan was 106 ± 21 (2SD) days. This degree of variation (±15-20%) is consistent with previous studies using other techniques. In a subset of seven subjects, MRBC determined with the biotin label technique were available from approximately five years prior, and strongly correlated with the stable isotope values (R(2) = 0.79). This study suggests that the MRBC is stable over time but varies substantially among individuals, and supports the importance of its variation in HbA1c interpretation. The characteristics of the stable isotope method support its suitability for studies to directly evaluate the impact of variation in MRBC on the interpretation of HbA1c. Am. J. Hematol. 90:50-55, 2015. © 2014 Wiley Periodicals, Inc. PMID:25293624

Khera, Paramjit K; Smith, Eric P; Lindsell, Christopher J; Rogge, Mary Colleen; Haggerty, Shannon; Wagner, David A; Palascak, Mary B; Mehta, Shilpa; Hibbert, Jacqueline M; Joiner, Clinton H; Franco, Robert S; Cohen, Robert M

2015-01-01

187

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors  

PubMed Central

Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family. PMID:24223875

Mehlhorn, Jennifer; Steinocher, Helena; Beck, Sebastian; Kennis, John T. M.; Hegemann, Peter; Mathes, Tilo

2013-01-01

188

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

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. PMID:25638265

Michael, Claudia; Rizzi, Andreas M

2015-02-27

189

Insights into nitrogen allocation and recycling from nitrogen elemental analysis and 15N isotope labelling in 14 genotypes of willow.  

PubMed

Minimizing nitrogen (N) fertilization inputs during cultivation is essential for sustainable production of bioenergy and biofuels. The biomass crop willow (Salix spp.) is considered to have low N fertilizer requirements due to efficient recycling of nutrients during the perennial cycle. To investigate how successfully different willow genotypes assimilate and allocate N during growth, and remobilize and consequently recycle N before the onset of winter dormancy, N allocation and N remobilization (to and between different organs) were examined in 14 genotypes of a genetic family using elemental analysis and (15)N as a label. Cuttings were established in pots in April and sampled in June, August and at onset of senescence in October. Biomass yield of the trees correlated well with yields recorded in the field. Genotype-specific variation was observed for all traits measured and general trends spanning these sampling points were identified when trees were grouped by biomass yield. Nitrogen reserves in the cutting fuelled the entirety of the canopy establishment, yet earlier cessation of this dependency was linked to higher biomass yields. The stem was found to be the major N reserve by autumn, which constitutes a major source of N loss at harvest, typically every 2-3 years. These data contribute to understanding N remobilization in short rotation coppice willow and to the identification of traits that could potentially be selected for in breeding programmes to further improve the sustainability of biomass production. PMID:24186940

Brereton, Nicholas J B; Pitre, Frederic E; Shield, Ian; Hanley, Steven J; Ray, Michael J; Murphy, Richard J; Karp, Angela

2014-11-01

190

Recent developments in solid-state magic-angle spinning, nuclear magnetic resonance of fully and significantly isotopically labelled peptides and proteins.  

PubMed Central

In recent years, a large number of solid-state nuclear magnetic resonance (NMR) techniques have been developed and applied to the study of fully or significantly isotopically labelled ((13)C, (15)N or (13)C/(15)N) biomolecules. In the past few years, the first structures of (13)C/(15)N-labelled peptides, Gly-Ile and Met-Leu-Phe, and a protein, Src-homology 3 domain, were solved using magic-angle spinning NMR, without recourse to any structural information obtained from other methods. This progress has been made possible by the development of NMR experiments to assign solid-state spectra and experiments to extract distance and orientational information. Another key aspect to the success of solid-state NMR is the advances made in sample preparation. These improvements will be reviewed in this contribution. Future prospects for the application of solid-state NMR to interesting biological questions will also briefly be discussed. PMID:15306412

Straus, Suzana K

2004-01-01

191

Stimulating carbon efficient supply chains : carbon labels and voluntary public private partnerships  

E-print Network

This thesis looks at the potential of labeling products with life cycle greenhouse gas emission information as a bottom-up, complementary alternative to carbon cap and trade systems. By improving the transparency of product ...

Tan, Kwan Chong

2009-01-01

192

Quantitative analysis of 3-alkyl-2-methoxypyrazines in juice and wine using stable isotope labelled internal standard assay.  

PubMed

A solid phase microextraction (HS-SPME)-GC-MS methodology was established for the analysis of 3-alkyl-2-methoxypyrazines (MPs) in wine using a stable isotope dilution assay. The compounds analysed were 3-isobutyl-2-methoxypyrazine (IBMP), 3-sec-butyl-2-methoxypyrazine (SBMP), and 3-isopropyl-2-methoxypyrazine (IPMP) using their respective deuterated analogues ([2H3]-IBMP, [2H3]-SBMP, [2H3]-IPMP) as internal standards, synthesised during this work. A divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) fibre was selected for isolation of MPs and the effects of matrix parameters such as pH and ethanol concentration were examined in the development of the method. Best results were obtained at a pH of approximately 6 and with a wine dilution factor of 1:2.5, resulting in an ethanol concentration of approximately 5% (v/v). Relative standard deviations (RSDs) of replicate samples were 5.6-7% for all MPs at 5 ng L(-1) and <5% for 15 and 30 ng L(-1) samples. The limit of detection was <0.5 ng L(-1) in juice and 1-2 ng L(-1) in wine. The recovery efficiencies for spiked wine samples were between 99 and 102% for all three MPs. Using this method, we investigated the impact of the Multicoloured Asian Lady Beetle (MALB) on MPs in wine. In red wine fermented with live MALB, IPMP is the most prevalent MP detected, although SBMP concentrations are also increased and IBMP is unchanged from background levels. MALB that have been dead for 1 day before addition to juice can still contribute to elevated SBMP concentrations in wine, but not if they have been dead for 3 days or longer. Clarifying juice prior to fermentation leads to substantially lower IPMP concentration in the subsequent wine when compared with unclarified juice. PMID:18377916

Kotseridis, Y S; Spink, M; Brindle, I D; Blake, A J; Sears, M; Chen, X; Soleas, G; Inglis, D; Pickering, G J

2008-05-01

193

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

PubMed

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 ?L of urine as the starting material. The workflow involves aliquoting 10 ?L of an individual urine sample for ¹²C-dansylation labeling that target amines and phenols. Another 10 ?L 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 (A?). 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 metabolomics studies in human cerebrospinal fluid or blood samples. This work illustrates the utility of this isotope labeling LC-MS method for biomarker discovery using mouse urine metabolomics. PMID:25164377

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

2014-10-01

194

Large-scale synthesis of isotopically labeled 13C2-tenuazonic acid and development of a rapid HPLC-MS/MS method for the analysis of tenuazonic acid in tomato and pepper products.  

PubMed

Tenuazonic acid is a fungal secondary metabolite that is produced by a number of Alternaria species and is therefore a natural contaminant of food and feed samples. This paper describes a new strategy for the efficient and economical large-scale synthesis of the isotopically labeled internal standard (13)C(2)-tenuazonic acid via a three-step procedure. Furthermore, a new reliable and quick method based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) cleanup is presented for the determination of tenuazonic acid in food and feed samples utilizing high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) by application of the stable isotope dilution analysis. This new method has a limit of detection (LOD) of 0.86 ?g/kg and a limit of quantitation (LOQ) of 2.89 ?g/kg. In total 26 tomato samples and 4 bell pepper samples from the German market were analyzed. Tenuazonic acid was found in each sample with levels from 3 to 2330 ?g/kg. PMID:23230907

Lohrey, Lilia; Marschik, Stefanie; Cramer, Benedikt; Humpf, Hans-Ulrich

2013-01-01

195

Determination of bovine lactoferrin in dairy products by ultra-high performance liquid chromatography-tandem mass spectrometry based on tryptic signature peptides employing an isotope-labeled winged peptide as internal standard.  

PubMed

A new and sensitive determination method was developed for bovine lactoferrin in dairy products including infant formulas based on the signature peptide by ultra high-performance liquid chromatography and triple-quadrupole tandem mass spectrometry under the multiple reaction monitoring mode. The simple pretreatment procedures included the addition of a winged peptide containing the isotope-labeled signature peptide as internal standard, followed by an enzymatic digestion with trypsin. The signature peptide was chosen and identified from the tryptic hydrolyzates of bovine lactoferrin by ultra high-performance liquid chromatography and quadrupole-time-of-flight tandem mass spectrometry based on sequence database search. Analytes were separated on an ACQUITY UPLC BEH 300 C18 column and monitored by MS/MS in seven minutes. Quantitative result bias due to matrix effect and tryptic efficiency was corrected through the use of synthetic isotope-labeled standards. The limit of detection and limit of quantification were 0.3 mg/100 g and 1.0 mg/100 g, respectively. Bovine lactoferrin within the concentration range of 10-1000 nmol L(-1) showed a strong linear relationship with a linear correlation coefficient (r) of >0.998. The intra- and inter-day precision of the method were RSD<6.5% and RSD<7.1%, respectively. Excellent repeatability (RSD<6.4%) substantially supported the application of this method for the determination of bovine lactoferrin in dairy samples. The present method was successfully validated and applied to determination of bovine lactoferrin in dairy products including infant formulas. PMID:24856400

Zhang, Jingshun; Lai, Shiyun; Cai, Zengxuan; Chen, Qi; Huang, Baifen; Ren, Yiping

2014-06-01

196

Validation of methane measurement using headspace-GC-MS and quantification by a stable isotope-labeled internal standard generated in situ.  

PubMed

A previous study has shown the possibility to identify methane (CH4 ) using headspace-GC-MS and quantify it with a stable isotope as internal standard. The main drawback of the GC-MS methods discussed in literature for CH4 measurement is the absence of a specific internal standard necessary to perform quantification. However, it becomes essential to develop a safer method to limit the manipulation of gaseous CH4 and to precisely control the injected amount of gas for spiking and calibration by comparison with external calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate a labeled gas as an internal standard in a vial on the basis of the formation of CH4 by the reaction of Grignard reagent methylmagnesium chloride with deuterated water. This method allows precise measurement of CH4 concentrations in gaseous sample as well as in a solid or a liquid sample after a thermodesorption step in a headspace vial. A full accuracy profile validation of this method is then presented. PMID:23585415

Varlet, Vincent; Smith, Fiona; Augsburger, Marc

2013-06-01

197

A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green.  

PubMed

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains. PMID:24671497

Prieto, Daniel; Aparicio, Gonzalo; Morande, Pablo E; Zolessi, Flavio R

2014-09-01

198

Plasma nitrogen isotopic fractionation and feed efficiency in growing beef heifers.  

PubMed

Fractionation of N isotopes occurs in many metabolic reactions which causes tissue proteins to become enriched in ¹?N while urea (urine) is depleted in ¹?N relative to the diet. We investigated ¹?N enrichment of whole plasma and its relationship with feed conversion efficiency (FCE) in growing beef heifers (n 84) offered 2 kg/d of concentrates with grass silage ad libitum. Heifers were on average 299 (SD 48·3) d old and weighed 311 (SD 48·8) kg. Plasma was obtained on day 79 (n 84) of the experiment and from a subset of animals (n 20) on four occasions between days 16 and 79. Silage DM intake (DMI) averaged 4·1 (SD 0·74) kg/d and concentrate DMI was 1·72 kg/d. Mean mid-test live weight was 333 (SD 47·6) kg, daily gain was 0·53 (SD 0·183) kg, FCE (g live-weight gain/g DMI) was 0·09 (SD 0·028) and residual feed intake (RFI) was 0 (SD 0·428). N isotopic fractionation (?¹?N; plasma ?¹?N - diet ?¹?N) averaged 3·58 ‰ on day 79 (n 84) and 3·90 ‰ for the subset of heifers. There was no relationship between ?¹?N and RFI. Plasma ?¹?N and ?¹?N were related to both FCE (negative) and animal weight (positive) for the whole population, and repeatable for the subset of animals over four time points. These relationships of ?¹?N with FCE and animal weight are consistent with the anticipated negative relationship with N-use efficiency. There is potential to use ?¹?N to provide rapid, low-cost estimates of FCE in cattle. PMID:24387820

Wheadon, N M; McGee, M; Edwards, G R; Dewhurst, R J

2014-05-01

199

Importance of bacterivory and preferential selection toward diatoms in larvae of Crepidula fornicata (L.) assessed by a dual stable isotope (13C, 15N) labeling approach  

NASA Astrophysics Data System (ADS)

In Europe, the gastropod Crepidula fornicata is an invasive species characterized by a long reproductive period (from February to November). Thus, its larvae are exposed to variations in available food sources (in terms of quantity and quality). We aimed to investigate if bacteria could contribute to larval food both in presence or absence of phytoplankton, and to compare these results to seasonal variations of bacteria and phytoplankton abundances at a coastal site in the English Channel. First, ingestion of fluorescent beads of 0.5 to 2 ?m diameter, showed that larvae were able to ingest particles of typical bacterial size. Then we used a dual stable isotope labeling approach which consisted in labeling a bacterial pelagic community with 15N and a diatom (Chaetoceros gracilis) culture with 13C, and supplying larvae with 15N-labeled bacteria, 13C-labeled diatoms, and both labeled sources. This technique has, to our knowledge, never been applied to invertebrate larvae. After 24 h of experiment, larvae were significantly enriched in all treatments: + 21.5‰ (??13C) when supplied with diatoms, + 1364‰ (??15N) when supplied with bacteria, and + 24‰ (??13C) and + 135‰ (??15N) when supplied with the two mixed sources. These results indicated that bacteria can contribute to the larval nutrition in C. fornicata, even in the presence of phytoplankton. Our results however suggested that larvae of C. fornicata preferentially used diatoms and showed that the supply of free bacteria did not alter the uptake of diatoms. Considering the seasonal variations of bacteria and phytoplankton abundances at the study site, these results suggested that bacteria may constitute a complementary resource for the larvae of C. fornicata when phytoplankton is abundant and may become a substitute resource when phytoplankton is less available. This approach offers promising perspectives to trace food sources and assess nitrogen and carbon fluxes between planktotrophic larvae and their preys.

Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frédérique; Leroux, Cédric; Comtet, Thierry

2012-05-01

200

Efficient Access to 3?-Terminal Azide-Modified RNA for Inverse Click-Labeling Patterns  

PubMed Central

Labeled RNA becomes increasingly important for molecular diagnostics and biophysical studies on RNA with its diverse interaction partners, which range from small metabolites to large macromolecular assemblies, such as the ribosome. Here, we introduce a fast synthesis path to 3?-terminal 2?-O-(2-azidoethyl) modified oligoribonucleotides for subsequent bioconjugation, as exemplified by fluorescent labeling via Click chemistry for an siRNA targeting the brain acid-soluble protein 1 gene (BASP1). Importantly, the functional group pattern is inverse to commonly encountered alkyne-functionalized “click”-able RNA and offers increased flexibility with respect to multiple and stepwise labeling of the same RNA molecule. Additionally, our route opens up a minimal step synthesis of 2?-O-(2-aminoethyl) modified pyrimidine nucleoside phosphoramidites which are of widespread use to generate amino-modified RNA for N-hydroxysuccinimide (NHS) ester-based conjugations. PMID:24358989

2013-01-01

201

Phosphorylcholine-coated semiconducting polymer nanoparticles as rapid and efficient labeling agents for in vivo cell tracking.  

PubMed

Despite the pressing need to noninvasively monitor transplanted cells in vivo with fluorescence imaging, desirable fluorescent agents with rapid labeling capability, durable brightness, and ideal biocompatibility remain lacking. Here, phosphorylcholine-coated near-infrared (NIR) fluorescent semiconducting polymer nanoparticles (SPNs) are reported as a new class of rapid, efficient, and cytocompatible labeling nanoagents for in vivo cell tracking. The phosphorylcholine coating results in efficient and rapid endocytosis and allows the SPN to enter cells within 0.5 h in complete culture medium apparently independent of the cell type, while its NIR fluorescence leads to a tissue penetration depth of 0.5 cm. In comparison to quantum dots and Cy5.5, the SPN is tolerant to physiologically ubiquitous reactive oxygen species (ROS), resulting in durable fluorescence both in vitro and in vivo. These desirable physical and physiological properties of the SPN permit cell tracking of human renal cell carcinoma (RCC) cells in living mice at a lower limit of detection of 10 000 cells with no obvious alteration of cell phenotype after 12 d. SPNs thus can provide unique opportunities for optimizing cellular therapy and deciphering pathological processes as a cell tracking label. PMID:24668903

Pu, Kanyi; Shuhendler, Adam J; Valta, Maija P; Cui, Lina; Saar, Matthias; Peehl, Donna M; Rao, Jianghong

2014-08-01

202

Ultrascale and microscale growth dynamics of the cidaroid spine of Phyllacanthus imperialis revealed by ²?Mg labeling and NanoSIMS isotopic imaging.  

PubMed

Growth dynamics of the primary spine of the cidaroid sea urchin Phyllacanthus imperialis was assessed for the first time using pulsed (26) Mg-labeling and NanoSIMS isotopic imaging. The sea urchin was incubated twice (for 48 h) in artificial seawater with elevated level of (26) Mg. After each labeling event, the sea urchin was returned for 72 h to seawater with natural isotopic abundance of (26) Mg. NanoSIMS ion microprobe was subsequently used to visualize the labeled regions of the spine with submicrometer lateral resolution. The growth of the new skeleton was restricted to the distalmost and peripheral portions of the spine. Skeletogenesis involved mostly the deposition of continuous thickening layers and lateral growth involving bridges between previously formed trabeculae. The timescale of formation of individual thickening layers (ca. 1 µm in width) on the stereom trabeculae was on the order of 1 day. Longitudinal growth occurred mainly at the periphery in the form of small portions of the thickening deposits or more massive microspines that appeared to branch and fuse with those above and below. These microspines were found to grow at about 10 µm/day. These results reveal that the skeletal growth of a juvenile cidaroid spine is complex and highly heterogeneous, with different extension rates depending on the stage of the stereom development and/or direction of the growth fronts. The growth pattern observed here at the submicrometer scale provides direct evidence supporting the earlier suggestions that the lamellar structure of echinoderm stereom is formed by periodic deposition of continuous mineral layers. PMID:24595980

Gorzelak, Przemys?aw; Stolarski, Jaros?aw; Dery, Aurélie; Dubois, Philippe; Escrig, Stéphane; Meibom, Anders

2014-07-01

203

A stable isotope-labeled internal standard is essential for correcting for the interindividual variability in the recovery of lapatinib from cancer patient plasma in quantitative LC-MS/MS analysis  

PubMed Central

The development and validation of a LC-MS/MS method is often performed using pooled human plasma, which may fail to account for variations in interindividual matrices. Since calibrator standards and quality control samples are routinely prepared in pooled human plasma, variations in the extraction recovery and/or matrix effect between pooled plasma and individual patient plasma can cause erroneous measurements. Using both pooled human plasma as well as individual healthy donor and cancer patient plasma samples, we evaluated the analytical performance of two classes of internal standards (i.e., non-isotope-labeled and isotope-labeled) in the quantitative LC-MS/MS analysis of lapatinib. After exhaustive extraction with organic solvent, the recovery of lapatinib, a highly plasma protein-bound drug, varied up to 2.4-fold (range, 29 – 70%) in 6 different donors of plasma and varied up to 3.5-fold (range, 16 – 56%) in the pretreatment plasma samples from 6 cancer patients. No apparent matrix effects were observed for lapatinib in both pooled and individual donor or patient plasma samples. The calibration curve range was 5 – 5000 ng/ml of lapatinib in plasma. Both the non-isotope-labeled (zileuton) and isotope-labeled (lapatinib-d3) internal standard methods showed acceptable specificity, accuracy (within 100 ± 10%), and precision (< 11%) in the determination of lapatinib in pooled human plasma. Nevertheless, only the isotope-labeled internal standard could correct for the interindividual variability in the recovery of lapatinib from patient plasma samples. As inter- and intra-patient matrix variability is commonly presented in the clinical setting, this study provides an example underscoring the importance of using a stable isotope-labeled internal standard in quantitative LC-MS/MS analysis for therapeutic drug monitoring or pharmacokinetic evaluation. PMID:24189203

Wu, Jianmei; Wiegand, Richard; LoRusso, Patricia; Li, Jing

2013-01-01

204

Transpiration efficiency over an annual cycle, leaf gas exchange and wood carbon isotope ratio of three tropical tree species  

E-print Network

Transpiration efficiency over an annual cycle, leaf gas exchange and wood carbon isotope ratio. Cumulative transpiration was determined by repeatedly weighing the pots with a pallet truck scale. Dry matter that leaf-level processes largely determined variation among the three tropical tree species in whole

Bermingham, Eldredge

205

Insights into oxidation mechanisms in gamma-irradiated polypropylene, utilizing selective isotopic labeling with analysis by GC/MS, NMR and FTIR  

NASA Astrophysics Data System (ADS)

In an effort to shed additional light on the chemical mechanisms underlying the radiation-oxidation of polypropylene (PP), we are using samples having selective 13C isotopic labeling at the three unique sites within the macromolecular structure. After radiation exposure, we applied GC/MS, solid-state 13C NMR, and FTIR to evaluate the applicability of each technique in identifying the molecular labeling of the oxidation products, with the goal of determining the site of origin of the products with respect to the macromolecule. Using GC/MS, we have identified the position of origin of CO 2 and CO from the polymer. Most of the CO 2 (60%) and CO (>90%) come from the C(1) (methylene) position of PP, with (30%) of the CO 2 originating from the C(3) (methyl) position, and 10% coming from the C(2) (tertiary) position. By GC/MS we have also identified the labeling patterns in four volatile oxidation products (acetone, methylisobutylketone, isobutane, and methyl acetate), and have used this information to map each compound onto the macromolecular framework. Using NMR we have quantified the time-dependent formation of solid-phase degradation products occurring from post-irradiation aging of PP samples held for 28 months at room temperature in air. Most of the solid oxidation products occur at the C(2) (tertiary) site; the predominant species, C(2) peroxides, increase linearly during the first 2 years, after which they plateau at a relatively high concentration.

Bernstein, Robert; Thornberg, Steven M.; Assink, Roger A.; Mowery, Daniel M.; Kathleen Alam, M.; Irwin, Adriane N.; Hochrein, James M.; Derzon, Dora K.; Klamo, Sara B.; Clough, Roger L.

2007-12-01

206

Preparation of stable isotope-labeled 2-nitrobenzaldehyde derivatives of four metabolites of nitrofuran antibiotics and their comprehensive characterization by UV, MS, and NMR techniques.  

PubMed

A convenient method is presented for the preparation of the carbon-13-labeled 2-nitrobenzaldehyde derivatives of the nitrofuran metabolites 3-amino-2-oxazolidinone (AOZ), semicarbazide (SC), 1-aminohydantoin (AH), and 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), with the purpose of using them as internal standards for the quantification of trace levels of nitrofuran residues by liquid chromatography-tandem mass spectrometry in foods of animal origin. The synthesis encompasses the nitration of [1,2,3,4,5,6-(13)C(6)]toluene prior to chromyl compound-mediated oxidation of the methyl group into the corresponding aldehyde. The four metabolites of nitrofuran antibiotics were derivatized independently with the resulting ring-labeled 2-nitrobenzaldehyde (NBA) to obtain the target compounds. Both the isotopically enriched and native substances were used to perform a comprehensive fragmentation study by electrospray ionization (ESI) collision-induced dissociation (CID) mass spectrometry (MS). Full characterization of the nitrofuran derivatives was accomplished with ultraviolet (UV) and exhaustive nuclear magnetic resonance (NMR) analysis. A major advantage of the described procedure is that it can be extended to the preparation of other carbon-13-labeled derivatives of metabolites of nitrofuran antibiotics. PMID:14558749

Delatour, Thierry; Gremaud, Eric; Mottier, Pascal; Richoz, Janique; Arce Vera, Francia; Stadler, Richard H

2003-10-22

207

A low-toxic artificial fluorescent glycoprotein can serve as an efficient cytoplasmic labeling in living cell.  

PubMed

To maintain the virtue of good optical property and discard the dross of conventional fluorescent staining dyes, we provide a strategy for designing new fluorescent scaffolds. In this study, a novel fluorescent labeling glycoprotein (chitosan-poly-l-cysteine, CPC) was synthesized through graft copolymerization. CPC gives emission peak at 465-470nm when excited at 386nm. The submicro-scale CPC microspheres could be localized and persisted specifically in the cytoplasm of living cells, with strong blue fluorescence. Moreover, CPC was highly resistant to photo bleaching, the fluorescence was remained stable for up to 72h as the cells grew and developed. The glycoprotein CPC was bio-compatible and in zero grade cytotoxicity as quantified by MTT assay. The fluorescent labeling process with our newly designed glycoprotein CPC is exceptionally efficient. PMID:25498627

Si, Jiangju; Liang, Dawei; Kong, Dan; Wu, Sufang; Yuan, Lan; Xiang, Yan; Jiang, Lei

2015-03-01

208

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...

209

Does an energy efficiency label alter consumers' purchasing decisions? A latent class approach based on a stated choice experiment in Shanghai.  

PubMed

In this paper we conducted a hypothetical choice experiment in Shanghai, China, to examine whether the China Energy Efficiency Label influences consumers' choices of air conditioners and refrigerators. A latent class approach was applied to observe both heterogeneities among the respondents and product brands. Our results suggested that consumers in Shanghai were well aware of the China Energy Efficiency Label and tended to pay more attention to products with such labels. In addition, air conditioners and refrigerators affixed with a hypothetical label that indicates saving in electricity bills compared with a standard model received significant preferences, which suggested that the more information manufacturers provide, the more their products would be preferred by consumers. Finally, weighted by class probability, the willingness to pay values for more energy efficient refrigerators were higher than those for more energy efficient air conditioners, implying that Shanghai consumers have greater incentive to pay more for appliances they use more frequently. PMID:19595499

Shen, Junyi; Saijo, Tatsuyoshi

2009-08-01

210

Can isotopes in nocturnal air drainage revewl water-use efficiency of vegetation in mountain watersheds?  

NASA Astrophysics Data System (ADS)

For ecologists interested in measuring the exchange of water and carbon in the soil-plant-atmosphere continuum, mountainous terrain is largely off limits; airflow patterns in uneven terrain can wreak havoc in the interpretation of vertical fluxes. However, well-developed air drainage systems might be used to advantage. In particular, we postulate that the isotopic composition of CO2 in nocturnal air drainage could be used to monitor vegetation water-use efficiency (WUE) of whole basins. Recent studies show that the carbon isotope composition of ecosystem-respired CO2 (d13CR ) is strongly influenced by WUE in the recent past. Therefore, frequent measurements of d13CR, which may be accomplished through Keeling Plot analyses, could provide a powerful way to monitor and study WUE, which in turn would improve understanding of connections between climate, vegetation, and basin hydrology. In a pilot study, we are examining the feasibility of sampling for Keeling Plot analyses in nocturnal air drainage in two small watersheds in the western Cascade Mountains of Oregon, USA. This feasibility depends on uncoupling between the air drainage and the bulk atmosphere, trapping respired CO2 in the airflow, and on defining the land surface of the drainage system. We found that downhill drainage predominates at night in our systems, and often during the day as well. The drainage layer is typically deep (often more than 10 m), swiftly moving, and well mixed. CO2 concentrations are typically well above ambient (400-450 ppm), indicating some degree of uncoupling. The land surface contributing to respired CO2 at the mouth of the drainage system appears to be smaller than the entire watershed, but the area and consistency of this footprint requires further investigation. At present we are cautiously optimistic about the approach.

Bond, B.; Unsworth, M.; Mix, A.; Alstad, K.; Mahrt, L.; Pypker, T.; Ocheltree, T.

2003-04-01

211

Spatially tracking 13C labeled substrate (bicarbonate) accumulation in microbial communities using laser ablation isotope ratio mass spectrometry  

SciTech Connect

This is a manuscript we would like to submit for publication in Environmental Microbiology Reports. This manuscript contains a description of a laser ablation isotope ratio mass spectrometry methodology developed at PNNL and applied to a microbial system at a PNNL project location – Hot Lake, Washington. I will submit a word document containing the entire manuscript with this Erica input request form.

Moran, James J.; Doll, Charles G.; Bernstein, Hans C.; Renslow, Ryan S.; Cory, Alexandra B.; Hutchison, Janine R.; Lindemann, Stephen R.; Fredrickson, Jim K.

2014-08-25

212

Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children  

Technology Transfer Automated Retrieval System (TEKTRAN)

BACKGROUND: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. OBJECTIVE: Our objectives were to produce heme iron enriched in (58)Fe and to use it to study the absorption of heme iron and the effect of iron and ...

213

Comparative analysis of monoclonal antibody N-glycosylation using stable isotope labelling and UPLC-fluorescence-MS.  

PubMed

A twoplex method using (12)C6 and (13)C6 stable isotope analogues (?mass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection. PMID:25623139

Millán Martín, Silvia; Delporte, Cédric; Farrell, Amy; Navas Iglesias, Natalia; McLoughlin, Niaobh; Bones, Jonathan

2015-03-01

214

Development of Isotope Labeling LC-MS for Human Salivary Metabolomics and Application to Profiling Metabolome Changes Associated with Mild Cognitive Impairment  

PubMed Central

Saliva is a readily available biofluid that may contain metabolites of interest for diagnosis and prognosis of diseases. In this work, a differential 13C-/12C-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 ?L 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, 12C-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 13C-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 one 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. PMID:23150892

Zheng, Jiamin; Dixon, Roger A.; Li, Liang

2012-01-01

215

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)

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.

Guo, Kevin; Bamforth, Fiona; Li, Liang

2011-02-01

216

99mTc-labeling kinetics of four thiol-containing chelators and 2-hydrazinopyridine: Factors influencing their radiolabeling efficiency  

Microsoft Academic Search

The relative 99mTc-labeling efficiency of several potentially tetradentate thiol-containing chelators was studied by competing them with glucoheptonate in the [99mTc]glucoheptonate complex, and was compared to that of 2-hydrazinopyridine (HYPY), a model compound for HYNIC (hydrazinonicotinamide). The thiol-containing chelators in their unprotected forms include 4,5-bis(mercaptoacetamido)pentanoic acid (H4L1), N-[2-(mercapto)propionyl]glycylglycylglycine (H4L2), 2-(mercapto)ethylaminoacetyl-l-cysteine (H3L3), and N,N?-ethylenediyl-bis-l-cysteine diethyl ester (H3L4). There are several factors that

S. Liu; D. Scott Edwards; A. R. Harris; P. R. Singh

1997-01-01

217

Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard.  

PubMed

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/?L). 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. PMID:25466018

Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda-Yamamoto, Mari

2015-04-15

218

IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation.  

PubMed

In this study, we present a fully automated tool, called IDEAL-Q, for label-free quantitation analysis. It accepts raw data in the standard mzXML format as well as search results from major search engines, including Mascot, SEQUEST, and X!Tandem, as input data. To quantify as many identified peptides as possible, IDEAL-Q uses an efficient algorithm to predict the elution time of a peptide unidentified in a specific LC-MS/MS run but identified in other runs. Then, the predicted elution time is used to detect peak clusters of the assigned peptide. Detected peptide peaks are processed by statistical and computational methods and further validated by signal-to-noise ratio, charge state, and isotopic distribution criteria (SCI validation) to filter out noisy data. The performance of IDEAL-Q has been evaluated by several experiments. First, a serially diluted protein mixed with Escherichia coli lysate showed a high correlation with expected ratios and demonstrated good linearity (R(2) = 0.996). Second, in a biological replicate experiment on the THP-1 cell lysate, IDEAL-Q quantified 87% (1,672 peptides) of all identified peptides, surpassing the 45.7% (909 peptides) achieved by the conventional identity-based approach, which only quantifies peptides identified in all LC-MS/MS runs. Manual validation on all 11,940 peptide ions in six replicate LC-MS/MS runs revealed that 97.8% of the peptide ions were correctly aligned, and 93.3% were correctly validated by SCI. Thus, the mean of the protein ratio, 1.00 +/- 0.05, demonstrates the high accuracy of IDEAL-Q without human intervention. Finally, IDEAL-Q was applied again to the biological replicate experiment but with an additional SDS-PAGE step to show its compatibility for label-free experiments with fractionation. For flexible workflow design, IDEAL-Q supports different fractionation strategies and various normalization schemes, including multiple spiked internal standards. User-friendly interfaces are provided to facilitate convenient inspection, validation, and modification of quantitation results. In summary, IDEAL-Q is an efficient, user-friendly, and robust quantitation tool. It is available for download. PMID:19752006

Tsou, Chih-Chiang; Tsai, Chia-Feng; Tsui, Ying-Hao; Sudhir, Putty-Reddy; Wang, Yi-Ting; Chen, Yu-Ju; Chen, Jeou-Yuan; Sung, Ting-Yi; Hsu, Wen-Lian

2010-01-01

219

Using stable isotopes to reconcile differences in nitrogen uptake efficiency relative to late season fertilization of northern red oak seedlings in Wisconsin bare-root nurseries  

NASA Astrophysics Data System (ADS)

Cultural applications (e.g., timing, amount) of nitrogen (N) fertilizer in bareroot tree nurseries have been assessed for some time. However, the use of different metrologies to quantify the efficient use of fertilizer N and its allocation within biomass has confounded comparisons between fertilization regimes. This inconsistency is especially problematic when quantifying N fertilizer uptake efficiency (NFUE) of late season N fertilization in northern red oak (Quercus rubra L.) (NRO) seedlings characterized by episodic flushes in growth and N storage in perennial tissue to support spring growth. The use of isotopic tracers could help elucidate these differences. We therefore hypothesized that: 1) calculations of NFUE using isotopically enriched fertilizer would yield lower, more precise estimates of NFUE relative to traditional methods due to differences in the accounting of mineralized and reabsorbed N, and 2) a significant fraction of leaf N in older leaves (early flushes) would be reabsorbed into root and shoot tissue before abscission relative to leaves produced toward the end of the growing season (late flushes). To test these hypotheses, we conducted an experiment in two-year old NRO seedlings at two bare-root nurseries in Wisconsin. We applied a total of 147 mg N seedling-1 in pulses from early July after the seedlings completed their second leaf flush until late August. The treatments consisted of three replicated plots of 15N enriched (1.000 atom%) ammonium sulfate, three non-enriched plots, and three unfertilized plots (controls) at each nursery. Subsequent changes in plant N uptake and N allocation were quantified from destructively harvested samples taken at 40, 60, and 120 days after the fertilization began. We evaluated three common methods currently used to estimate NFUE (total N without control, total N with control, and isotopic difference). The total N without control method overestimated mean NFUE by 3.2 times relative to the isotope method, because mineralized N uptake and reabsorption of leaf N was unaccounted for. The total N with control method also overestimated mean NFUE, but only by 20% relative to the isotope method; variation associated with the effects of N fertilization on mineralization and immobilization was large enough to preclude significant difference between these methods. The difference of non-labeled N between day 60 and day 120 revealed that the roots and shoots absorbed 95% and 5%, respectively, of initial leaf N. However, isotopic mass balance between day 60 and day 120 indicated that the NRO seedlings did not reabsorb leaf fertilized N from the youngest leaves before abscission. This study shows that using stable isotopes to understand plant-soil interactions in response to fertilization will help elucidate the contribution of additional N fluxes (e.g., N reabsorption) within perennial plants and thus improve fertility management of production systems.

Fujinuma, R.; Balster, N. J.

2009-12-01

220

Use of Differential Isotopic Labeling and Mass Spectrometry To Analyze Capacitation-Associated Changes in the Phosphorylation Status of Mouse Sperm Proteins  

PubMed Central

Mammalian sperm need to reside in the female reproductive tract for a finite period of time before acquiring fertilizing competence. The biochemical changes associated with this process are collectively known as “capacitation”. With the use of the mouse as an experimental model, we have previously demonstrated that capacitation is associated with a cAMP-dependent increase in protein tyrosine phosphorylation. However, little is known about the identity and function of the protein targets of this phosphorylation cascade. In the present work, we have used differential isotopic labeling coupled with immobilized metal affinity chromatography (IMAC)-based phosphopeptide enrichment and analysis on a hybrid linear ion trap/FT-ICR mass spectrometer to measure the changes in protein phosphorylation resulting from the capacitation process. As no kinase activators and/or phosphatase inhibitors were used in the preparation of the sperm samples, phosphorylated residues identified in this study represent in vivo sites of phosphorylation. Also, in contrast to other methods which rely on the incorporation of isotopically labeled amino acids at the protein level (e.g., SILAC), the present technique is based on the Fisher esterification of protein digests, allowing for the comparison of phosphorylation status in the absence of protein synthesis. This approach resulted in the identification of 55 unique, in vivo sites of phosphorylation and permitted the relative extent of phosphorylation, as a consequence of capacitation, to be calculated for 42 different phosphopeptides. This work represents the first effort to determine which specific protein phosphorylation sites change their phosphorylation status in vivo as a result of the mammalian capacitation process. PMID:19186949

Platt, Mark D.; Salicioni, Ana M.; Hunt, Donald F.; Visconti, Pablo E.

2010-01-01

221

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

PubMed Central

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

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

2012-01-01

222

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

SciTech Connect

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. Application 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.

Merkley, Eric D.; Baker, Erin Shammel; Crowell, Kevin L.; Orton, Daniel J.; Taverner, Thomas; Ansong, Charles; Ibrahim, Yehia M.; Burnet, Meagan C.; Cort, John R.; Anderson, Gordon A.; Smith, Richard D.; Adkins, Joshua N.

2013-02-20

223

Iron uptake and ferrokinetics in healthy male subjects of an iron-based oral phosphate binder (SBR759) labeled with the stable isotope (58)Fe.  

PubMed

SBR759 is a novel polynuclear iron(III) oxide-hydroxide starch·sucrose·carbonate complex being developed for oral use in chronic kidney disease (CKD) patients with hyperphosphatemia on hemodialysis. SBR759 binds inorganic phosphate released by food uptake and digestion in the gastro-intestinal tract increasing the fecal excretion of phosphate with concomitant reduction of serum phosphate concentrations. Considering the high content of ?20% w/w covalently bound iron in SBR759 and expected chronic administration to patients, absorption of small amounts of iron released from the drug substance could result in potential iron overload and toxicity. In a mechanistic iron uptake study, 12 healthy male subjects (receiving comparable low phosphorus-containing meal typical for CKD patients: ?1000 mg phosphate per day) were treated with 12 g (divided in 3 × 4 g) of stable (58)Fe isotope-labeled SBR759. The ferrokinetics of [(58)Fe]SBR759-related total iron was followed in blood (over 3 weeks) and in plasma (over 26 hours) by analyzing with high precision the isotope ratios of the natural iron isotopes (58)Fe, (57)Fe, (56)Fe and (54)Fe by multi-collector inductively coupled mass spectrometry (MC-ICP-MS). Three weeks following dosing, the subjects cumulatively absorbed on average 7.8 ± 3.2 mg (3.8-13.9 mg) iron corresponding to 0.30 ± 0.12% (0.15-0.54%) SBR759-related iron which amounts to approx. 5-fold the basal daily iron absorption of 1-2 mg in humans. SBR759 was well-tolerated and there was no serious adverse event and no clinically significant changes in the iron indices hemoglobin, hematocrit, ferritin concentration and transferrin saturation. PMID:25017110

Gschwind, Hans-Peter; Schmid, Dietmar G; von Blanckenburg, Friedhelm; Oelze, Marcus; van Zuilen, Kirsten; Slade, Alan J; Stitah, Sylvie; Kaufmann, Daniel; Swart, Piet

2014-11-01

224

Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis  

Microsoft Academic Search

A method was developed for the precise and accurate determination of ovalbumin labelled with p-hydroxy-mercuribenzoic acid (pHMB) using polyacrylamide gel electrophoresis with ns-laser ablation–inductively coupled plasma\\u000a mass spectrometry. Following systematic optimisation of the ablation process in terms of detection sensitivity, two different\\u000a quantification strategies were applied: external calibration using standards of the derivatized protein after 13C+ normalization and, as a

Daniel J. Kutscher; Mattias B. Fricker; Bodo Hattendorf; Jörg Bettmer; Detlef Günther

225

Quantitation of methadone enantiomers in humans using stable isotope-labeled (2H3)-, (2H5)-, and (2H8)Methadone  

SciTech Connect

A new technique for simultaneous stereoselective kinetic studies of methadone enantiomers was developed using three deuterium-labeled forms of methadone and GLC-chemical-ionization mass spectrometry. A racemic mixture (1:1) of (R)-(-)-(2H5)methadone (l-form) and (S)-(R)-(2H3)methadone (d-form) was administered orally in place of a single daily dose of unlabeled (+/-)-(2H0)methadone in long-term maintenance patients. Racemic (+/-)-(2H8)methadone was used as an internal standard for the simultaneous quantitation of (2H0)-, (2H3)-, and (2H5)methadone in plasma and urine. A newly developed extraction procedure, using a short, disposable C18 reversed-phase cartridge and improved chemical-ionization procedures employing ammonia gas, resulted in significant reduction of the background impurities contributing to the ions used for isotopic abundance measurements. These improvements enabled the measurement of labeled plasma methadone levels for 120 hr following a single dose. This methodology was applied to the study of methadone kinetics in two patients; in both patients, the analgesically active l-enantiomer of the drug had a longer plasma elimination half-life and a smaller area under the plasma disappearance curve than did the inactive d-form.

Nakamura, K.; Hachey, D.L.; Kreek, M.J.; Irving, C.S.; Klein, P.D.

1982-01-01

226

Iron absorption from a malted cocoa drink fortified with ferric orthophosphate using the stable isotope 58Fe as an extrinsic label.  

PubMed

The potential use of an extrinsic label to measure iron absorption from a ferric orthophosphate-fortified malted cocoa drink was examined by measuring the solubility of the FePO4 in 0.1 M-hydrochloric acid. The validity of using the stable isotope 58Fe as an extrinsic label was tested by comparing Fe absorption by rats from wheat flour extrinsically-labelled with 58Fe or 59Fe. Fe absorption from a malted cocoa drink (20 g powder made up with hot water) fortified with FePO4 (0.5 mg Fe/g powder) was measured in human subjects using 58Fe as an extrinsic label. Absorption was calculated by measuring unabsorbed 58Fe in faeces. Absorptions of Fe from the drink fortified with either FePO4 or ferrous sulphate were compared. The effect of the addition of ascorbic acid to the drink (1 mg/g powder) on Fe availability was also examined. The effect of fasting on Fe absorption from the drink was determined in rats by giving the drink to fasting animals or shortly after they had consumed a small meal. The FePO4 was totally soluble in 0.1 M-HCl and there were no differences in absorption between 58Fe- and 59Fe-labelled wheat flour. In the human experiment the proportion of Fe absorbed from the drink plus FePO4 and ascorbic acid was (mean with SE) 0.25 (0.02), from the drink plus FePO4 0.24 (0.02) and from the drink plus FeSO4 0.23 (0.03). Fasting had a significant effect on Fe availability; rats given the drink shortly after a small meal absorbed less than half as much Fe as those given the drink on a fasted stomach. It was concluded that the FePO4 used to fortify the malted cocoa drink was as well absorbed as FeSO4 but that the high levels of absorption were a reflection of the fasting condition of the subjects. The level of ascorbic acid was not great enough to enhance the availability of the FePO4 any further. PMID:6882732

Fairweather-Tait, S J; Minski, M J; Richardson, D P

1983-07-01

227

Efficiency of monolaurin in mitigating ruminal methanogenesis and modifying C-isotope fractionation when incubating diets composed of either C3 or C4  

E-print Network

Efficiency of monolaurin in mitigating ruminal methanogenesis and modifying C-isotope fractionation in ruminants has been an important goal for several decades. Free lauric acid, known to suppress ruminal metha disappearance. The influence on 13 C-isotope abundance and fractionation supports the hypothesis that ruminal

Gilli, Adrian

228

ACCESSING OVERSEAS MARKETS ENERGY EFFICIENCY STANDARDS AND APPLIANCE LABELING IN ASIA AND LATIN AMERICA  

EPA Science Inventory

The purpose of the project is to reduce pollution and environmental degradation by increasing the efficiency of energy end-uses in the industrial and household sectors of key Asian and Latin American countries. This will be accomplished by encouraging the adoption and harmo...

229

99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency  

PubMed Central

99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, 99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a 99mTc-labeled HYNIC-DAPI compound with that of 99mTc pertechnetate (99mTcO4?). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by 99mTcO4? (0.51), and the number of DSBs increased fivefold in the 99mTc-HYNIC-DAPI-treated sample compared with the 99mTcO4? treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the 99mTcO4– treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the 99mTc-HYNIC-DAPI-treated samples. These results indicated that 99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the 99mTc-labeled compound with DNA. In contrast to these results, 99mTcO4? induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of 99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately 10-fold in terms of inducing DSBs. PMID:25098953

Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

2014-01-01

230

Highly efficient click labeling using 2-[18F]fluoroethyl azide and synthesis of an 18F N-hydroxysuccinimide ester as conjugation agent  

PubMed Central

Introduction Click labeling using 2-[18F]fluoroethyl azide has been proven to be promising methods of radiolabeling small molecules and peptides, some of which are undergoing clinical evaluations. However, the previously reported method afforded low yield, poor purities and under desirable reproducibility. Methods A vacuum distillation method was used to isolate 2-[18F]fluoroethyl azide, and the solvent effect of acetonitrile (ACN) and dimethylformamide (DMF) on the click labeling using Cu(I) from copper sulfate/sodium ascorbate was studied. The labeling conditions were optimized to radiosynthesize a hydroxysuccinimide ester (NHS). Results 2-[18F]fluoroethyl azide was isolated by the vacuum distillation method with > 80% yield within 10 min in a “pure” and click-ready form. It was found that the amount of DMF was critical for maintaining high levels of Cu(I) from copper sulfate/sodium ascorbate in order to rapidly complete the click labeling reaction. The addition of bathophenanthrolinedisulfonic acid disodium salt (BPDS) to the mixture of copper sulfate/sodium ascorbate also greatly improved the click labeling efficiency. Through exploiting these optimizations, a base-labile N-hydroxysuccinimide (NHS) ester was rapidly radiosynthesized in 90% isolated yield with good chemical and radiochemical purities. Conclusions We have developed a general method to click-label small molecules efficiently using [18F]2 for research and clinical use. This NHS ester can be used for conjugation chemistry to label antibodies, peptides and small molecules as PET tracers. PMID:22770647

Zhou, Dong; Chu, Wenhua; Dence, Carmen S.; Mach, Robert H.; Welch, Michael J.

2012-01-01

231

Use of photopatterned porous polymer monoliths as passive micromixers to enhance mixing efficiency of on-chip labeling reactions  

PubMed Central

In order to increase the extent of reaction for on-chip fluorescent labeling of proteins, a passive mixer has been prepared by using UV light to photopattern a periodic arrangement of porous polymer monolith structures directly within the channel of a plastic microfluidic chip. By optimizing the composition of the polymerization solution and irradiation time we demonstrated the ability to photopattern monoliths in regularly repeating 100 ?m segments at the tee-junction of the disposable device. To evaluate the efficiency of this dual functional mixer-reactor, fluorescamine and lysine were introduced in separate channels upstream of the tee-junction and the intensity of laser-induced fluorescence resulting from the fluorogenic labeling reaction was monitored. The fluorescence level after passing the photopatterned periodic monolith configuration was better than both an equivalent 1 cm long continuous monolithic segment and an open channel. These results indicate that the periodic arrangement of monoliths, with regularly spaced open areas between 100 ?m plugs, is responsible for enhancing the mixing performance and overall rate of chemical reaction carried out in the system. In addition to facilitating preparation of a dual functional mixer-reactor, the ability to accurately photopattern monoliths in a channel is an enabling technology for seamlessly integrating multiple monoliths into a single microdevice. PMID:19294297

Mair, Dieudonne A.; Schwei, Thomas R.; Dinio, Theresa S.; Fréchet, Jean M. J.; Svec, Frantisek

2009-01-01

232

Efficient In Vitro Labeling Rabbit Bone Marrow-Derived Mesenchymal Stem Cells with SPIO and Differentiating into Neural-Like Cells  

PubMed Central

Mesenchymal stem cells (MSCs) can differentiate into neural cells to treat nervous system diseases. Magnetic resonance is an ideal means for cell tracking through labeling cells with superparamagnetic iron oxide (SPIO). However, no studies have described the neural differentiation ability of SPIO-labeled MSCs, which is the foundation for cell therapy and cell tracking in vivo. Our results showed that bone marrow-derived mesenchymal stem cells (BM-MSCs) labeled in vitro with SPIO can be induced into neural-like cells without affecting the viability and labeling efficiency. The cellular uptake of SPIO was maintained after labeled BM-MSCs differentiated into neural-like cells, which were the basis for transplanted cells that can be dynamically and non-invasively tracked in vivo by MRI. Moreover, the SPIO-labeled induced neural-like cells showed neural cell morphology and expressed related markers such as NSE, MAP-2. Furthermore, whole-cell patch clamp recording demonstrated that these neural-like cells exhibited electrophysiological properties of neurons. More importantly, there was no significant difference in the cellular viability and [Ca2+]i between the induced labeled and unlabeled neural-like cells. In this study, we show for the first time that SPIO-labeled MSCs retained their differentiation capacity and could differentiate into neural-like cells with high cell viability and a good cellular state in vitro. PMID:25234466

Zhang, Ruiping; Li, Jing; Li, Jianding; Xie, Jun

2014-01-01

233

Characterization of TATP gas phase product ion chemistry via isotope labeling experiments using ion mobility spectrometry interfaced with a triple quadrupole mass spectrometer.  

PubMed

Identification of the fragment ion species associated with the ion reaction mechanism of triacetone triperoxide (TATP), a homemade peroxide-based explosive, is presented. Ion mobility spectrometry (IMS) has proven to be a key analytical technique in the detection of trace explosive material. Unfortunately, IMS alone does not provide chemical identification of the ions detected; therefore, it is unknown what ion species are actually formed and separated by the IMS. In IMS, ions are primarily characterized by their drift time, which is dependent on the ion?s mass and molecular cross-section; thus, IMS as a standalone technique does not provide structural signatures, which is in sharp contrast to the chemical and molecular information that is generally obtained from other customary analytical techniques, such as NMR, Raman and IR spectroscopy and mass spectrometry. To help study the ion chemistry that gives rise to the peaks observed in IMS, the hardware of two different commercial IMS instruments has been directly coupled to triple quadrupole (QQQ) mass spectrometers, in order to ascertain each ion?s corresponding mass/charge (m/z) ratios with different dopants at two temperatures. Isotope labeling was then used to help identify and confirm the molecular identity of the explosive fragment and adduct ions of TATP. The m/z values and isotope labeling experiments were used to help propose probable molecular formulas for the ion fragments. In this report, the fragment and adduct ions m/z 58 and 240 of TATP have been confirmed to be [C3H6NH·H](+) and [TATP·NH4](+), respectively; while the fragment ions m/z 73 and 89 of TATP are identified as having the molecular formulas [C4H9NH2](+) and [C4H9O2](+), respectively. It is anticipated that the work in this area will not only help to facilitate improvements in mobility-based detection (IMS and MS), but also aid in the development and optimization of MS-based detection algorithms for TATP. PMID:24913870

Tomlinson-Phillips, Jill; Wooten, Alfred; Kozole, Joseph; Deline, James; Beresford, Pamela; Stairs, Jason

2014-09-01

234

New development in the tritium labelling of peptides and proteins using solid catalytic isotopic exchange with spillover-tritium.  

PubMed

The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140-180 degrees C was analyzed. This reaction was used for preparing [(3)H]enkephalins such as [(3)H]DALG with specific activity of 138 Ci/mmol and [(3)H]LENK with specific activity of 120 Ci/mmol at 180 degrees C. The analogues of [(3)H]ACTG(4-10) with specific activity of 80 Ci/mmol, [(3)H]zervamicin IIB with specific activity of 70 Ci/mmol and [(3)H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [(3)H]Peptide analysis using (3)H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the beta-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [(3)H] beta-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity. PMID:12707815

Zolotarev, Yu A; Dadayan, A K; Bocharov, E V; Borisov, Yu A; Vaskovsky, B V; Dorokhova, E M; Myasoedov, N F

2003-04-01

235

Efficient MRI labeling of endothelial progenitor cells: design of thiolated surface stabilized superparamagnetic iron oxide nanoparticles.  

PubMed

The aim of this study was to design thiolated surface stabilized superparamagnetic iron oxide nanoparticles (TSS-SPIONs) for efficient internalization with high MRI sensitivity. TSS-SPIONs were developed by chelation between thiolated chitosan-thioglycolic acid (chitosan-TGA) hydrogel and iron ions (Fe(2+)/Fe(3+)). Likely, unmodified chitosan hydrogel SPIONs (UC-SPIONs) and uncoated SPIONs were used as control. Moreover, TSS-SPIONs were investigated regarding to their iron core size, hydrodynamic diameter, zeta potential, iron contents, molar relaxivities (r1 and r2), and cellular internalization. TSS-SPIONs demonstrated an iron oxide core diameter (crystallite size by XRD) of 3.1 ± 0.02 nm, a hydrodynamic diameter of 94 ± 20 nm, a zeta potential of +21 ± 5 mV, and an iron content of 3.6 ± 0.9 mg/mL. In addition, internalization of TSS-SPIONs into human endothelial progenitor cells (EPC) from umbilical cord blood was more than threefold and 17-fold higher in contrast to UC-SPIONs and SPIONs, respectively. With twofold lower incubation iron concentration of TSS-SPIONs, more than threefold higher internalization was achieved as compared to Resovist®. Also, cell viability of more than 90% was observed in the presence of TSS-SPIONs after 24h. The molar MR relaxivities (r2) value at 1.5 T was threefold higher than that of Resovist® and demonstrated that TSS-SPIONs have the potential as very effective T2 contrast-enhancement agent. According to these findings, TSS-SPIONs with efficient internalization, lower cytotoxicity, and high MRI sensitivity seem to be promising for cell tracking. PMID:23481176

Shahnaz, Gul; Kremser, Christian; Reinisch, Andreas; Vetter, Anja; Laffleur, Flavia; Rahmat, Deni; Iqbal, Javed; Dünnhaupt, Sarah; Salvenmoser, Willi; Tessadri, Richard; Griesser, Ulrich; Bernkop-Schnürch, Andreas

2013-11-01

236

Alzheimer's disease biomarkers detection in human samples by efficient capturing through porous magnetic microspheres and labelling with electrocatalytic gold nanoparticles.  

PubMed

A nanobiosensor based on the use of porous magnetic microspheres (PMM) as efficient capturing/pre-concentrating platform is presented for detection of Alzheimer's disease (AD) biomarkers. These PMMs prepared by a multistep swelling polymerization combined with iron oxide precipitation afford carboxyl functional groups suitable for immobilization of antibodies on the particle surface allowing an enhanced efficiency in the capturing of AD biomarkers from human serum samples. The AD biomarkers signaling is produced by gold nanoparticle (AuNP) tags monitored through their electrocatalytic effect towards hydrogen evolution reaction (HER). Novel properties of PMMs in terms of high functionality and high active area available for enhanced catalytic activity of the captured AuNPs electrocatalytic tags are exploited for the first time. A thorough characterization by scanning transmission electron microscope in high angle annular dark field mode (STEM-HAADF) demonstrates the enhanced ability of PMMs to capture a higher quantity of analyte and consequently of electrocatalytic label, when compared with commercially available microspheres. The optimized and characterized PMMs are also applied for the first time for the detection of beta amyloid and ApoE at clinical relevant levels in cerebrospinal fluid (CSF), serum and plasma samples of patients suffering from AD. PMID:25153932

de la Escosura-Muñiz, Alfredo; Plichta, Zden?k; Horák, Daniel; Merkoçi, Arben

2015-05-15

237

Acetylation and glycation of fibrinogen in vitro occur at specific lysine residues in a concentration dependent manner: A mass spectrometric and isotope labeling study  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer Fibrinogen was incubated in vitro with glucose or aspirin. Black-Right-Pointing-Pointer Acetylations and glycations were found at twelve lysine sites by mass spectrometry. Black-Right-Pointing-Pointer The labeling by aspirin and glucose occurred dose-dependently. Black-Right-Pointing-Pointer No competition between glucose and aspirin for binding to fibrinogen was found. -- Abstract: Aspirin may exert part of its antithrombotic effects through platelet-independent mechanisms. Diabetes is a condition in which the beneficial effects of aspirin are less prominent or absent - a phenomenon called 'aspirin resistance'. We investigated whether acetylation and glycation occur at specific sites in fibrinogen and if competition between glucose and aspirin in binding to fibrinogen occurs. Our hypothesis was that such competition might be one explanation to 'aspirin resistance' in diabetes. After incubation of fibrinogen in vitro with aspirin (0.8 mM, 24 h) or glucose (100 mM, 5-10 days), we found 12 modified sites with mass spectrometric techniques. Acetylations in the {alpha}-chain: {alpha}K191, {alpha}K208, {alpha}K224, {alpha}K429, {alpha}K457, {alpha}K539, {alpha}K562, in the {beta}-chain: {beta}K233, and in the {gamma}-chain: {gamma}K170 and {gamma}K273. Glycations were found at {beta}K133 and {gamma}K75, alternatively {gamma}K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [{sup 14}C-acetyl]salicylic acid and [{sup 14}C]glucose, a labeling of 0.013-0.084 and 0.12-0.5 mol of acetylated and glycated adduct/mol fibrinogen, respectively, was found for clinically (12.9-100 {mu}M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen.

Svensson, Jan, E-mail: jan.svensson@ki.se [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden) [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Bergman, Ann-Charlotte [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden)] [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Adamson, Ulf [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)] [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden); Blombaeck, Margareta [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden)] [Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital (Solna), SE-171 76 Stockholm (Sweden); Wallen, Hakan; Joerneskog, Gun [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)] [Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm (Sweden)

2012-05-04

238

Rapid detection and characterization of reactive drug metabolites in vitro using several isotope-labeled trapping agents and ultra-performance liquid chromatography/time-of-flight mass spectrometry.  

PubMed

Reactive metabolites are believed to be one of the main reasons for unexpected drug-induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione-trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide-trapped metabolites were found for eight and semicarbazide-trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup. PMID:19224530

Rousu, Timo; Pelkonen, Olavi; Tolonen, Ari

2009-03-01

239

Identification of a novel neurotrophic factor from primary retinal Müller cells using stable isotope labeling by amino acids in cell culture (SILAC).  

PubMed

Retinal Müller glial cells (RMGs) have a primary role in maintaining the homeostasis of the retina. In pathological situations, RMGs execute protective and regenerative effects, but they can also contribute to neurodegeneration. It has recently been recognized that cultured primary RMGs secrete pro-survival factors for retinal neurons for up to 2 weeks in culture, but this ability is lost when RMGs are cultivated for longer durations. In our study, we investigated RMG supernatants for novel neuroprotective factors using a quantitative proteomic approach. Stable isotope labeling by amino acids in cell culture (SILAC) was used on primary porcine RMGs. Supernatants of RMGs cultivated for 2 weeks were compared with supernatants from cells that had already lost their protective capacity. Using this approach, we detected established neurotrophic factors such as transferrin, osteopontin, and leukemia inhibitory factor and identified C-X-C motif chemokine 10 (CXCL10) as a novel candidate neuroprotective factor. All factors prolonged photoreceptor survival in vitro. Ex vivo treatment of retinal explants with leukemia inhibitory factor or CXCL10 demonstrated a neuroprotective effect on photoreceptors. Western blots on CXCL10- and leukemia inhibitory factor-stimulated explanted retina and photoreceptor lysates indicated activation of pro-survival signal transducer and activator of transcription signaling and B-cell lymphoma pathways. These findings suggest that CXCL10 contributes to the supportive potential of RMGs toward retinal neurons. PMID:24925906

von Toerne, Christine; Menzler, Jacob; Ly, Alice; Senninger, Nicole; Ueffing, Marius; Hauck, Stefanie M

2014-09-01

240

Characterization of L-phenylalanine metabolism to acetophenone and 1-phenylethanol in the flowers of Camellia sinensis using stable isotope labeling.  

PubMed

Acetophenone (AP) and 1-phenylethanol (1PE) are the two major endogenous volatile compounds in flowers of Camellia sinensis var. Yabukita. Until now no information has been available on the biosynthesis of AP and 1PE in plants. Here we propose that AP and 1PE are derived from L-phenylalanine (L-Phe), based on feeding experiments using stable isotope-labeled precursors L-[(2)H(8)]Phe and L-[(13)C(9)]Phe. The subacid conditions in the flowers result in more hydrogenation than dehydrogenation in the transformation between AP and 1PE. Due to the action of some enzyme(s) responsible for the formation of (R)-1PE from AP in the flowers, (R)-1PE is the dominant endogenous steroisomer of 1PE. The modification of 1PE into nonvolatile glycosidic forms is one of the reasons for why only a little 1PE is released from the flowers. The levels of AP, 1PE, and glycosides of 1PE increase during floral development, whereas the level of L-Phe decreases. These metabolites occur mostly in the anthers. PMID:22209218

Dong, Fang; Yang, Ziyin; Baldermann, Susanne; Kajitani, Yutaka; Ota, Shogo; Kasuga, Hisae; Imazeki, Yumi; Ohnishi, Toshiyuki; Watanabe, Naoharu

2012-02-15

241

Preliminary Quantitative Profile of Differential Expression between Rat L6 Myoblasts and Myotubes by Stable Isotope Labeling by Amino acids in Cell Culture  

PubMed Central

Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muslce myogenesis in vitro. When mononucleate L6 myoblast cells reach confluent in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracelluar matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In sum, our results provide new clues for understanding the mechanism of myogenesis. PMID:19253283

Cui, Ziyou; Chen, Xiulan; Lu, Bingwen; Park, Sung Kyu; Xu, Tao; Xie, Zhensheng; Xue, Peng; Hou, Junjie; Hang, Haiying; Yates, John R.; Yang, Fuquan

2010-01-01

242

Comparative cytochrome P450 proteomics in the livers of immunodeficient mice using 18O stable isotope labeling.  

PubMed

Quantitative changes in cytochrome P450 (CYP) proteins involved in drug metabolism as a consequence of drug treatment are important parameters in predicting the fates and pharmacological consequences of xenobiotics and drugs. In this study we undertook comparative P450 proteomics using liver from control and 1,4-bis-2-(3,5-dichloropyridyloxybenzene) (TCPOBOP)-dosed mice. The method involved separation of microsomal proteins by SDS-PAGE, trypsin digestion, and postdigest 18O/16O labeling followed by nano-LC-MS/MS for peptide identification and LC-MS for relative quantification. Seventeen P450 proteins were identified from mouse liver of which 16 yielded data sufficient for relative quantification. All the P450s detected were unambiguously identified except the highly homologous CYP2A4/2A5. With the exception of CYP2A12, -2D10, and -2F2, the levels of all the P450s quantified were affected by treatment with TCPOBOP (3 mg/kg). CYP1A2, -2A4/5, -2B10, -2B20, -2C29, -2C37, -2C38, -3A11, and -39A1 were up-regulated, and CYP2C40, -2E1, -3A41, and -27A1 down-regulated. The response of CYP2B20 to stimulation has not been distinguished previously from that of CYP2B10 because of the poor discrimination between these two proteins (they share 87% sequence identity). Differential response to chemical stimulation by closely related members of the CYP2C subfamily was also observed. PMID:17296599

Lane, Catherine S; Wang, Yuqin; Betts, Richard; Griffiths, William J; Patterson, Laurence H

2007-06-01

243

The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2.  

PubMed

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol. PMID:22493088

Qi, Jenson; Lang, Wensheng; Geisler, John G; Wang, Ping; Petrounia, Ioanna; Mai, Selyna; Smith, Charles; Askari, Hossein; Struble, Geoffrey T; Williams, Robyn; Bhanot, Sanjay; Monia, Brett P; Bayoumy, Shariff; Grant, Eugene; Caldwell, Gary W; Todd, Matthew J; Liang, Yin; Gaul, Micheal D; Demarest, Keith T; Connelly, Margery A

2012-06-01

244

The use of stable isotope-labeled glycerol and oleic acid to differentiate the hepatic functions of DGAT1 and -2  

PubMed Central

Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with 13C3-D5-glycerol or 13C18-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that 13C3-D5-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, 13C18-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D5-glycerol to mice and measured plasma levels of D5-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D5-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered 13C18-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol. PMID:22493088

Qi, Jenson; Lang, Wensheng; Geisler, John G.; Wang, Ping; Petrounia, Ioanna; Mai, Selyna; Smith, Charles; Askari, Hossein; Struble, Geoffrey T.; Williams, Robyn; Bhanot, Sanjay; Monia, Brett P.; Bayoumy, Shariff; Grant, Eugene; Caldwell, Gary W.; Todd, Matthew J.; Liang, Yin; Gaul, Micheal D.; Demarest, Keith T.; Connelly, Margery A.

2012-01-01

245

Cytotoxicity, cytocompatibility, cell-labeling efficiency, and in vitro cellular magnetic resonance imaging of gadolinium-catalyzed single-walled carbon nanotubes  

PubMed Central

Cell tracking by magnetic resonance imaging (MRI) is an emerging technique that typically requires the use of MRI contrast agents (CAs). A MRI CA for cellular imaging should label cells efficiently at potentially safe concentrations, have high relaxivity, and not affect the cellular machinery. In this article, we report the cytotoxicity, cytocompatibility, and cell labeling efficiency in NIH/3T3 fibroblasts of novel, single-walled carbon nanotubes synthesized using gadolinium nano-particles as catalysts (Gd-SWCNTs). Cells incubated with the Gd-SWCNT showed a dose- (50–100 ?g/mL nanotube concentration) and time- (12–48 h) dependent decrease in viability. 30% cell death was observed for cells incubated with Gd-SWCNTs at the maximum dose of 100 ?g/mL for 48 h. Cells incubated with the Gd-SWCNTs at concentrations between 1–10 ?g/mL for 48 h showed no change in viability or proliferation compared to untreated controls. Additionally, at these potentially safe concentrations, up to 48 h, the cells showed no phosphatidyl serine externalization (pre-apoptotic condition), caspase-3 activity (point of no return for apoptosis), genetic damage, or changes in their division cycle. Localization of Gd-SWCNTs within the cells was confirmed by transmission electron microscopy (TEM) and Raman microscopy, and these results show 100% cell labeling efficiency. Elemental analysis also indicates significant uptake of Gd-SWCNTs by the cells (108–109 Gd3+ ions per cell). Finally, T1-weighted MRI at 3 T of Gd-SWCNT-labelled cells show up to a four-fold increase in MR signal intensities as compared to untreated cells. These results indicate that Gd-SWCNTs label cells efficiently at potentially safe concentrations, and enhance MRI contrast without any structural damage to the cells. PMID:23686792

Avti, Pramod K.; Caparelli, Elisabeth D.; Sitharaman, Balaji

2013-01-01

246

Isotope ratio analysis of actinides, fission products, and geolocators by high-efficiency multi-collector thermal ionization mass spectrometry  

NASA Astrophysics Data System (ADS)

A ThermoFisher "Triton" multi-collector thermal ionization mass spectrometer (MC-TIMS) was evaluated for trace and ultra-trace level isotope ratio analysis of actinides (uranium, plutonium, and americium), fission products and geolocators (strontium, cesium, and neodymium). Total efficiencies (atoms loaded to ions detected) of up to 0.5-2% for U, Pu, and Am, and 1-30% for Sr, Cs, and Nd can be reported employing resin bead load techniques onto flat ribbon Re filaments or resin beads loaded into a millimeter-sized cavity drilled into a Re rod. This results in detection limits of <0.1 fg (104 atoms to 105 atoms) for 239-242+244Pu, 233+236U, 241-243Am, 89,90Sr, and 134,135,137Cs, and <=1 pg for natural Nd isotopes (limited by the chemical processing blank) using a secondary electron multiplier (SEM) or multiple-ion counters (MICs). Relative standard deviations (RSD) as small as 0.1% and abundance sensitivities of 1 × 106 or better using a SEM are reported here. Precisions of RSD [approximate]0.01-0.001% using a multi-collector Faraday cup array can be achieved at sub-nanogram concentrations for strontium and neodymium and are suitable to gain crucial geolocation information. The analytical protocols reported herein are of particular value for nuclear forensic and nuclear safeguard applications.

Bürger, S.; Riciputi, L. R.; Bostick, D. A.; Turgeon, S.; McBay, E. H.; Lavelle, M.

2009-09-01

247

Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics Study of a Thyroid Hormone-regulated Secretome in Human Hepatoma Cells*  

PubMed Central

The thyroid hormone, 3, 3?,5-triiodo-l-thyronine (T3), regulates cell growth, development, differentiation, and metabolism via interactions with thyroid hormone receptors (TRs). However, the secreted proteins that are regulated by T3 are yet to be characterized. In this study, we used the quantitative proteomic approach of stable isotope labeling with amino acids in cell culture coupled with nano-liquid chromatography-tandem MS performed on a LTQ-Orbitrap instrument to identify and characterize the T3-regulated proteins secreted in human hepatocellular carcinoma cell lines overexpressing TR?1 (HepG2-TR?1). In total, 1742 and 1714 proteins were identified and quantified, respectively, in three independent experiments. Among these, 61 up-regulated twofold and 11 down-regulated twofold proteins were identified. Eight proteins displaying increased expression and one with decreased expression in conditioned media were validated using Western blotting. Real-time quantitative RT-PCR further disclosed induction of plasminogen activator inhibitor-1 (PAI-1), a T3 target, in a time-course and dose-dependent manner. Serial deletions of the PAI-1 promoter region and subsequent chromatin immunoprecipitation assays revealed that the thyroid hormone response element on the promoter is localized at positions –327/–312. PAI-1 overexpression enhanced tumor growth and migration in a manner similar to what was seen when T3 induced PAI-1 expression in J7-TR?1 cells, both in vitro and in vivo. An in vitro neutralizing assay further supported a crucial role of secreted PAI-1 in T3/TR-regulated cell migration. To our knowledge, these results demonstrate for the first time that proteins involved in the urokinase plasminogen activator system, including PAI-1, uPAR, and BSSP4, are augmented in the extra- and intracellular space of T3-treated HepG2-TR?1 cells. The T3-regulated secretome generated in the current study may provide an opportunity to establish the mechanisms underlying T3-associated tumor progression and prognosis. PMID:22171322

Chen, Cheng-Yi; Chi, Lang-Ming; Chi, Hsiang-Cheng; Tsai, Ming-Ming; Tsai, Chung-Ying; Tseng, Yi-Hsin; Lin, Yang-Hsiang; Chen, Wei-Jan; Huang, Ya-Hui; Lin, Kwang-Huei

2012-01-01

248

New insights into the coordination of Cu(II) by the amyloid-B 16 peptide from Fourier transform IR spectroscopy and isotopic labeling.  

PubMed

Alzheimer's disease is a neurodegenerative disorder in which the formation of amyloid-? (A?) aggregates plays a causative role. There is ample evidence that Cu(II) can bind to A? and modulate its aggregation. Moreover, Cu(II) bound to A? might be involved in the production of reactive oxygen species, a process supposed to be involved in the Alzheimer's disease. The native A?40 contains a high affinity binding site for Cu(II), which is comprised in the N-terminal portion. Thus, A?16 (amino acid 1-16 of A?) has often been used as a model for Cu(II)-binding to monomeric A?. The Cu(II)-binding to A? is pH dependent and at pH 7.4, two different type of Cu(II) coordinations exist in equilibrium. These two forms are predominant at pH 6.5 and pH 9.0. In either form, a variety of studies show that the N-terminal Asp and the three His play a key role in the coordination, although the exact binding of these amino acids has not been addressed. Therefore, we studied the coordination modes of Cu(II) at pH 6.5 and 9.0 with the help of Fourier transform infrared (FTIR) spectroscopy. Combined with isotopic labeling of the amino acids involved in the coordination sphere, the data points toward the coordination of Cu(II) via the carboxylate of Asp1 at both pH values in a pseudobridging monovalent fashion. At low pH, His6 binds copper via N?, while His13 and His14 are bound via N?. At high pH, direct evidence is given on the coordination of Cu(II) via the N? atom of His6. Additionally, this study clearly shows the effect of Cu(II) binding on the protonation state of the His residues where a proton displacement takes places on the nitrogen atoms of the imidazole ring. PMID:22026330

El Khoury, Youssef; Dorlet, Pierre; Faller, Peter; Hellwig, Petra

2011-12-15

249

Passage of stable isotope-labeled grass silage fiber and fiber-bound protein through the gastrointestinal tract of dairy cows.  

PubMed

Fractional passage rates are required to predict nutrient absorption in ruminants but data on nutrient-specific passage kinetics are largely lacking. With the use of the stable isotope ratio (?) as an internal marker, we assessed passage kinetics of fiber and fiber-bound nitrogen (N) of intrinsically labeled grass silage from fecal and omasal excretion patterns of ?(13)C and ?(15)N. In a 6×6 Latin square, lactating dairy cows received grass silages [455 g/kg of total diet dry matter (DM) ] in a 2×3 factorial arrangement from ryegrass swards fertilized at low (45 kg of N/ha) or high (90 kg of N/ha) levels of N and harvested at 3 maturity stages. Feed intake (16.7±0.48 kg of DM/d; mean ± standard error of the mean) and milk yield (26.7±0.92 kg/d) increased at the high level of N fertilization and at decreasing maturity. Nutrient digestibility decreased with increasing plant maturity, particularly at the high level of N fertilization, essentially reflecting dietary treatment effects on the nutritional composition of the grass silage. Fractional rumen passage rates (K1) were highest and total mean retention time in the gastrointestinal tract (TMRT) was lowest when based on the external marker chromium mordanted fiber (Cr-NDF; 0.047/h and 38.0 h, respectively). Fecal ?(13)C in the acid detergent fiber fraction ((13)CADF) provided the lowest K1 (0.023/h) and the highest TMRT (61.1 h) and highest peak concentration time (PCT; 24.3h) among markers. In comparison, fecal fiber-bound N ((15)NADF) had a considerably higher K1 (0.032/h) and lower TMRT (46.4 h) than (13)CADF. Total N (measured with (15)NDM) had a comparable K1 (0.034/h) to that of (15)NADF but provided the highest fractional passage rates from the proximal colon-cecum (K2; 0.37/h) and lowest PCT (17.4 h) among markers. A literature review indicated unclear effects of grass silage maturity on K1 and unknown effects of N fertilization on K1. Our study indicated no effect of advancing maturity on fecal K1 and a trend for K1 to increase with the high level of N fertilization. Parameter K2 increased, whereas PCT and TMRT generally decreased with the high level of N fertilization. Omasal digesta sampling largely confirmed results based on fecal sampling. Results indicate that the use of ?(13)C and ?(15)N can describe fiber-specific passage kinetics of forage. PMID:24119806

Warner, D; Dijkstra, J; Hendriks, W H; Pellikaan, W F

2013-12-01

250

Identification of Subunit-Subunit Interaction Sites in ?A-WT Crystallin and Mutant ?A-G98R Crystallin Using Isotope-Labeled Cross-Linker and Mass Spectrometry  

PubMed Central

Cataract is characterized by progressive protein aggregation and loss of vision. ?-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in ?-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit–subunit interaction sites in cataract-causing mutant ?A-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker–containing peptides. In this study, we cross-linked wild-type (?A-WT) and mutant (?A-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d0 and d4) cross-linker–BS2G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88 — K99 in the ?5 strand of the mutant ?A-G98R crystallin that is not found in wild-type ?A-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly. PMID:23755258

Kannan, Rama; Santhoshkumar, Puttur; Mooney, Brian P.; Sharma, K. Krishna

2013-01-01

251

Absolute quantification of UGT1A1 in various tissues and cell lines using isotope label-free UPLC-MS/MS method determines its turnover number and correlates with its glucuronidation activities.  

PubMed

Uridine 5'-diphosphate-glucuronosyltransferase (UGT)1A1 is a major phase II metabolism enzyme responsible for glucuronidation of drugs and endogenous compounds. The purpose of this study was to determine the expression level of UGT1A1 in human liver microsomes and human cell lines by using an isotope label-free LC-MS/MS method. A Waters Ultra performance liquid chromatography (UPLC) system coupled with an API 5500Qtrap mass spectrometer was used for the analysis. Two signature peptides (Pep-1, and Pep-2) were employed to quantify UGT1A1 by multiple reaction monitoring (MRM) approach. Standard addition method was used to validate the assay to account for the matrix effect. 17?-Estradiol was used as the marker substrate to determine UGT1A1 activities. The validated method has a linear range of 200-0.0195nM for both signature peptides. The precision, accuracy, and matrix effect were in acceptable ranges. UGT1A1 expression levels were then determined using 8 individual human liver microsomes, a pooled human liver microsomes, three UGT1A1 genotyped human liver microsomes, and four cell lines (Caco-2, MCF-7, Hela, and HepG2). The correlations study showed that the UGT1A1 protein levels were strongly correlated with its glucuronidation activities in human liver microsomes (R(2)=0.85) and in microsomes prepared from cell lines (R(2)=0.95). Isotope-labeled peptides were not necessary for LC-MS/MS quantitation of proteins. The isotope label-free absolute quantification method used here had good accuracy, sensitivity, linear range, and reproducibility, and were used successfully for the accurate determination of UGT1A1 from tissues and cell lines. PMID:24055854

Xu, Beibei; Gao, Song; Wu, Baojian; Yin, Taijun; Hu, Ming

2014-01-01

252

Ash, Carbon Isotope Discrimination, and Silicon as Estimators of Transpiration Efficiency in Crested Wheatgrass  

Microsoft Academic Search

Breeding and selection for higher transpiration efficiency (W) has been hampered by tedious and costly methodology. Rapid and less costly methods are needed for screening W in plant improvement programmes. We report the relationship of ash, silicon (Si) concentration, and Si uptake to W in crested wheatgrass (Agropyron desertorum (Fischer ex Link) Schultes), an important C3 range grass in western

H. F. Mayland; D. A. Johnson; K. H. Asay; A USDA-ARS; B USDA-ARS

253

RESULTS OF THE STUDY OF POLPAK PACKING FOR HIGH EFFICIENCY COLUMNS FOR SEPARATION OF ISOTOPES  

Microsoft Academic Search

A description is given of a new polpak packing for laboratory and ; commercial scale coiumns. Resistance of flow, separation capacity, and hold-up ; were studied in dependence on the characteristic size of packing elements, feed ; rate, and pressure. The packing examined is characterized by high efficiency; ; the pressure drop per theoretical plate is very low. Therefore the

A. Selecki; B. Tyminski

1963-01-01

254

Fluorescence energy transfer efficiency in labeled yeast cytochrome c: a rapid screen for ion biocompatibility in aqueous ionic liquids  

SciTech Connect

A fluorescence energy transfer de-quenching assay was implemented to follow the equilibrium unfolding behaviour of site-specific tetramethylrhodamine-labelled yeast cytochrome c in aqueous ionic liquid solutions; additionally, this approach offers the prospect of naked eye screening for biocompatible ion combinations in hydrated ionic liquids.

Baker, Sheila N [ORNL; Zhao, Hua [Savannah State University; Pandey, Siddharth [Indian Institute of Technology, Delhi; Heller, William T [ORNL; Bright, Frank [University of Buffalo, The State University of New York; Baker, Gary A [ORNL

2011-01-01

255

Mechanisms of trichloramine removal with activated carbon: Stoichiometric analysis with isotopically labeled trichloramine and theoretical analysis with a diffusion-reaction model.  

PubMed

This study investigated the mechanism by which activated carbon removes trichloramine, a byproduct of water treatment that has a strongly offensive chlorinous odor. A stoichiometrical mass balance for (15)N before and after activated carbon treatment of laboratory-prepared (15)N-labeled trichloramine solutions clearly revealed that the mechanism of trichloramine removal with activated carbon was not adsorption but rather reductive decomposition to nitrogen gas. There was a weak positive correlation between the surface decomposition rate constant of trichloramine and the concentration of basic functional groups on the surface of the carbon particles, the suggestion being that the trichloramine may have been reduced by sulfhydryl groups (-SH) on the activated carbon surface. Efficient decomposition of trichloramine was achieved with super powdered activated carbon (SPAC), which was prepared by pulverization of commercially available PAC into very fine particles less than 1 ?m in diameter. SPAC could decompose trichloramine selectively, even when trichloramine and free chlorine were present simultaneously in water, the indication being that the strong disinfection capability of residual free chlorine could be retained even after trichloramine was effectively decomposed. The residual ratio of trichloramine after carbon contact increased somewhat at low water temperatures of 1-5 °C. At these low temperatures, biological treatment, the traditional method for control of a major trichloramine precursor (ammonium nitrogen), is inefficient. Even at these low temperatures, SPAC could reduce the trichloramine concentration to an acceptable level. A theoretical analysis with a diffusion-reaction model developed in the present study revealed that the increase in the trichloramine residual with decreasing water temperature was attributable to the temperature dependence of the rate of the reductive reaction rather than to the temperature dependence of the diffusive mass transfer rate. PMID:25466640

Sakuma, Miki; Matsushita, Taku; Matsui, Yoshihiko; Aki, Tomoko; Isaka, Masahito; Shirasaki, Nobutaka

2014-11-13

256

Evaluation of a method for nitrotyrosine site identification and relative quantitation using a stable isotope-labeled nitrated spike-in standard and high resolution fourier transform MS and MS/MS analysis.  

PubMed

The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO-), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z=181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards. PMID:24736779

Seeley, Kent W; Fertig, Alison R; Dufresne, Craig P; Pinho, Joao P C; Stevens, Stanley M

2014-01-01

257

Evaluation of a Method for Nitrotyrosine Site Identification and Relative Quantitation Using a Stable Isotope-Labeled Nitrated Spike-In Standard and High Resolution Fourier Transform MS and MS/MS Analysis  

PubMed Central

The overproduction of reactive oxygen and nitrogen species (ROS and RNS) can have deleterious effects in the cell, including structural and possible activity-altering modifications to proteins. Peroxynitrite is one such RNS that can result in a specific protein modification, nitration of tyrosine residues to form nitrotyrosine, and to date, the identification of nitrotyrosine sites in proteins continues to be a major analytical challenge. We have developed a method by which 15N-labeled nitrotyrosine groups are generated on peptide or protein standards using stable isotope-labeled peroxynitrite (O15NOO?), and the resulting standard is mixed with representative samples in which nitrotyrosine formation is to be measured by mass spectrometry (MS). Nitropeptide MS/MS spectra are filtered using high mass accuracy Fourier transform MS (FTMS) detection of the nitrotyrosine immonium ion. Given that the nitropeptide pair is co-isolated for MS/MS fragmentation, the nitrotyrosine immonium ions (at m/z = 181 or 182) can be used for relative quantitation with negligible isotopic interference at a mass resolution of greater than 50,000 (FWHM, full width at half-maximum). Furthermore, the standard potentially allows for the increased signal of nitrotyrosine-containing peptides, thus facilitating selection for MS/MS in a data-dependent mode of acquisition. We have evaluated the methodology in terms of nitrotyrosine site identification and relative quantitation using nitrated peptide and protein standards. PMID:24736779

Seeley, Kent W.; Fertig, Alison R.; Dufresne, Craig P.; Pinho, Joao P. C.; Stevens, Stanley M.

2014-01-01

258

Carbon transfer from photosynthesis to below ground fine root/hyphae respiration in Quercus serrata using stable carbon isotope pulse labeling  

NASA Astrophysics Data System (ADS)

Studying carbon allocation in trees is a key to better understand belowground carbon cycle and its response to climate change. Tracing 13C in tree and soil compartments after pulse labeling is one of powerful tool to study the fate of carbon in forest ecosystems. This experiment was conducted in Yamashiro experimental forest, Kyoto, Japan. Annual mean temperature and precipitation from 1994 to 2009 are 15.5 ° C and 1,388 mm respectively. The branch pulse labeling were done 7 times in 2011 using same branch of Quercus serrata (H:11.7 m, DBH; 33.7 cm) to see seasonal variations of carbon velocity. Whole crown labeling of Quercus serrata (H:9 m, DBH; 13.7 cm) was done in 2012 to study carbon allocation and to especially focus on belowground carbon flux until to the hyphae respiration. Pure 13CO2 (99.9%) was injected to the labeling chamber which was set to branch or crown. Then, after one hour of branch labeling and 3.5 hour for crown labeling, the chamber was opened. Trunk respiration chambers, fine root chambers and hyphae chambers were set to the target tree to trace labeled carbon in the CO2 efflux. 41 ?m mesh was used to exclude ingrowth of roots into hyphae chambers. The results show that the velocity of carbon through the tree varied seasonally, with higher velocity in summer than autumn, averaging 0.47 m h-1. Half-lives of labeled carbon in autotrophic respiration were similar above and below ground during the growing season, but they were twice longer in trunk than in root in autumn. From the whole crown labeling done end of growing season, the 13CO2 signal was observed 25 hours after labeling in trunk chamber and 34-37.7 hours after labeling in fine root and hyphae respiration almost simultaneously. Half-lives of 13 was longer in trunk than below ground. Trunk respiration was still using labelled carbon during winter suggesting that winter trunk respiration is partly fueled by carbon stored in the trunk at the end of the growing season.

Dannoura, M.; Kominami, Y.; Takanashi, S.; Takahashi, K.

2013-12-01

259

High-efficiency preparative-scale reversed-phase high-performance liquid chromatographic purification of 14C-labelled antibiotics.  

PubMed

The 14C-labelled antibiotics [2-14C]mupirocin, and [thienyl-3-14C]temocillin cannot be satisfactorily purified on a small scale by conventional methods of chromatography or recrystallisation. Their purification was successfully achieved by high-efficiency preparative-scale reversed-phase high-performance liquid chromatography. The purifications employed 250 mm X 10 mm I.D. or 22 mm I.D. stainless-steel columns packed with Merck LiChrosorb RP-18 (10 microns) stationary phase which were eluted with aqueous buffer solutions at flow-rates of 10-25 ml min-1 using conventional analytical instrumentation. PMID:3106386

Morecombe, D J

1987-03-13

260

Isotope ratio analysis of actinides, fission products, and geolocators by high-efficiency multi-collector thermal ionization mass spectrometry  

SciTech Connect

A ThermoFisher 'Triton' multi-collector thermal ionization mass spectrometer (MC-TIMS) was evaluated for trace and ultra-trace level isotoperatioanalysis of actinides (uranium, plutonium, and americium), fission products and geolocators (strontium, cesium, and neodymium). Total efficiencies (atoms loaded to ions detected) of up to 0.5-2% for U, Pu, and Am, and 1-30% for Sr, Cs, and Nd can be reported employing resin bead load techniques onto flat ribbon Re filaments or resin beads loaded into a millimeter-sized cavity drilled into a Re rod. This results in detection limits of <0.1 fg (10{sup 4} atoms to 10{sup 5} atoms) for {sup 239-242+244}Pu, {sup 233+236}U, {sup 241-243}Am, {sup 89,90}Sr, and {sup 134,135,137}Cs, and {le} 1 pg for natural Nd isotopes (limited by the chemical processing blank) using a secondary electron multiplier (SEM) or multiple-ion counters (MICs). Relative standard deviations (RSD) as small as 0.1% and abundance sensitivities of 1 x 10{sup 6} or better using a SEM are reported here. Precisions of RSD {approx} 0.01-0.001% using a multi-collector Faraday cup array can be achieved at sub-nanogram concentrations for strontium and neodymium and are suitable to gain crucial geolocation information. The analytical protocols reported herein are of particular value for nuclear forensic and nuclear safeguard applications.

Bürger, Stefan [New Brunswick Laboratory, Argonne, IL; Riciputi, Lee R [Los Alamos National Laboratory (LANL); Bostick, Debra A [ORNL; Turgeon, Steven [University of Alberta, Edmondton, Canada; McBay, Eddie H [ORNL; Lavelle, Mark [ORNL

2009-01-01

261

Lectin intravital perfusion studies in tumor-bearing mice: micrometer-resolution, wide-area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor.  

PubMed

Intravital lectin perfusion was combined with computer-guided scanning digital microscopy to map the perfused elements of the vasculature in tumor-bearing mice. High-precision composite images (spatial precision 1.3 micron and optical resolution 1.5 micron) were generated to permit exact positioning, reconstruction, analysis, and mapping of entire tumor cross-sections (c. 1 cm in diameter). Collation of these mosaics with nuclear magnetic resonance maps in the same tumor plane identified sites of rapid contrast medium uptake as tumor blood vessels. Digitized imaging after intravital double labeling allowed polychromatic visualization of two different types of mismatched staining. First, simultaneous application of two lectins, each bearing a different fluorochrome, revealed organ-specific differential processing in the microvascular wall. Second, sequential application of two boluses of one lectin, bearing different fluorochromes successively, distinguished between double-labeled microvessels, representing efficiently perfused vascular segments, and single-labeled microvessels, with inefficient or intermittent perfusion. Intravital lectin perfusion images of blood vessels in the vital functional state thus highlighted biologically significant differences in vessel function and served as high-resolution adjuncts to MR imaging. PMID:9562571

Debbage, P L; Griebel, J; Ried, M; Gneiting, T; DeVries, A; Hutzler, P

1998-05-01

262

Production of stable-isotope-labeled bovine heme and its use to measure heme-iron absorption in children1-4  

Microsoft Academic Search

Background: The use of stable isotopes has provided valuable insights into iron absorption in humans, but the data have been limited to nonheme iron. Objective: Our objectives were to produce heme iron enriched in 58 Fe and to use it to study the absorption of heme iron and the effect of iron and zinc intakes on heme-iron absorption in children.

Paz Etcheverry; Gordon E Carstens; Erin Brown; Keli M Hawthorne; Zhensheng Chen; Ian J Griffin

263

Simultaneous detection of stable isotope-labeled and unlabeled L-tryptophan and of its main metabolites, L-kynurenine, serotonin and quinolinic acid, by gas chromatography/negative ion chemical ionization mass spectrometry.  

PubMed

A method for the detection of unlabeled and (15)N2 -labeled L-tryptophan (L-Trp), L-kynurenine (L-Kyn), serotonin (5-HT) and quinolinic acid (QA) in human and rat plasma by GC/MS is described. Labeled and unlabeled versions of these four products were analyzed as their acyl substitution derivatives using pentafluoropropionic anhydride and 2,2,3,3,3-pentafluoro-1-propanol. Products were then separated by GC and analyzed by selected ion monitoring using negative ion chemical ionization mass spectrometry. L-[(13)C11, (15)N2]-Trp, methyl-serotonin and 3,5-pyridinedicarboxylic acid were used as internal standards for this method. The coefficients of variation for inter-assay repeatability were found to be approximately 5.2% for L-Trp and (15)N2-Trp, 17.1% for L-Kyn, 16.9% for 5-HT and 5.8% for QA (n?=?2). We used this method to determine isotope enrichments in plasma L-Trp over the course of a continuous, intravenous infusion of L-[(15) N2 ]Trp in pregnant rat in the fasting state. Plasma (15)N2-Trp enrichment reached a plateau at 120?min. The free Trp appearance rate (Ra) into plasma was 49.5?±?3.35?µmol/kg/h. The GC/MS method was applied to determine the enrichment of (15)N-labeled L-Trp, L-Kyn, 5-HT and QA concurrently with the concentration of non-labeled L-Trp, L-Kyn, 5-HT and QA in plasma. This method may help improve our understanding on L-Trp metabolism in vivo in animals and humans and potentially reveal the relative contribution of the four pathways of L-Trp metabolism. PMID:24677305

Sano, Mitsue; Ferchaud-Roucher, Véronique; Nael, Charlotte; Aguesse, Audrey; Poupeau, Guillaume; Castellano, Blandine; Darmaun, Dominique

2014-02-01

264

Efficient Blind Spectral Unmixing of Fluorescently Labeled Samples Using Multi-Layer Non-Negative Matrix Factorization  

PubMed Central

The ample variety of labeling dyes and staining methods available in fluorescence microscopy has enabled biologists to advance in the understanding of living organisms at cellular and molecular level. When two or more fluorescent dyes are used in the same preparation, or one dye is used in the presence of autofluorescence, the separation of the fluorescent emissions can become problematic. Various approaches have been recently proposed to solve this problem. Among them, blind non-negative matrix factorization is gaining interest since it requires little assumptions about the spectra and concentration of the fluorochromes. In this paper, we propose a novel algorithm for blind spectral separation that addresses some of the shortcomings of existing solutions: namely, their dependency on the initialization and their slow convergence. We apply this new algorithm to two relevant problems in fluorescence microscopy: autofluorescence elimination and spectral unmixing of multi-labeled samples. Our results show that our new algorithm performs well when compared with the state-of-the-art approaches for a much faster implementation. PMID:24260120

Zudaire, Isabel; Ortiz-de-Solorzano, Carlos

2013-01-01

265

Use of an automated chromium reduction system for hydrogen isotope ratio analysis of physiological fluids applied to doubly labeled water analysis.  

PubMed

The doubly labeled water method is commonly used to measure total energy expenditure in free-living subjects. The method, however, requires accurate and precise deuterium abundance determinations, which can be laborious. The aim of this study was to evaluate a fully automated, high-throughput, chromium reduction technique for the measurement of deuterium abundances in physiological fluids. The chromium technique was compared with an off-line zinc bomb reduction technique and also subjected to test-retest analysis. Analysis of international water standards demonstrated that the chromium technique was accurate and had a within-day precision of <1 per thousand. Addition of organic matter to water samples demonstrated that the technique was sensitive to interference at levels between 2 and 5 g l(-1). Physiological samples could be analyzed without this interference, plasma by 10000 Da exclusion filtration, saliva by sedimentation and urine by decolorizing with carbon black. Chromium reduction of urine specimens from doubly labeled water studies indicated no bias relative to zinc reduction with a mean difference in calculated energy expenditure of -0.2 +/- 3.9%. Blinded reanalysis of urine specimens from a second doubly labeled water study demonstrated a test-retest coefficient of variation of 4%. The chromium reduction method was found to be a rapid, accurate and precise method for the analysis of urine specimens from doubly labeled water. PMID:11006607

Schoeller, D A; Colligan, A S; Shriver, T; Avak, H; Bartok-Olson, C

2000-09-01

266

Method development for the redox speciation analysis of iron by ion chromatography-inductively coupled plasma mass spectrometry and carryover assessment using isotopically labeled analyte analogues.  

PubMed

An ion chromatography-inductively coupled plasma mass spectrometry (IC-ICP-MS) method was developed for the redox speciation analysis of iron (Fe) based on in-column complexation of Fe(2+) and Fe(3+) by dipicolinic acid (DPA). The effects of column type, mobile phase composition and molecular ion interference were studied in the method optimization. The carryover of the target species in the IC-ICP-MS method was uniquely and effectively evaluated using isotopically enriched analogues of the analytes ((54)Fe(2+) and (57)Fe(3+)). Standard solutions of the enriched standards were injected into the system following analysis of a sample, and the ratios of the isotopes of iron in the enriched standards were calculated based on the chromatographic peak areas. The concentrations of the analytes carried over from the sample to the enriched standards were determined using the quantitative relationship in isotope dilution mass spectrometry (IDMS). In contrast to the routine way of evaluating carryover effect by injecting a blank solution after sample analysis, the use of isotopically enriched standards identified significant analyte carryover in the present method. Extensive experiments were carried out to systematically identify the source of the carryover and to eliminate the problem; the separation column was found to be the exclusive source. More than 95% of the analyte carryover was eliminated by reducing the length of the column. The detection limit of the IC-ICP-MS method (MDL) for the iron species was 2ngg(-1). The method was used to determine Fe(2+) and Fe(3+) in synthetic aqueous standard solutions and a beverage sample. PMID:24819017

Wolle, Mesay Mulugeta; Fahrenholz, Timothy; Rahman, G M Mizanur; Pamuku, Matt; Kingston, H M 'Skip'; Browne, Damien

2014-06-20

267

New Multidimensional Editing Experiments for Measurement of Amide Deuterium Isotope Effects on C ?Chemical Shifts in 13C, 15N-Labeled Proteins  

Microsoft Academic Search

Novel multidimensional NMR pulse sequences for measurement of the three- and four-bond amide deuterium isotope effect on the chemical shifts of13C?in proteins are presented. The sequences result in editing into two subspectra of a heteronuclear triple resonance spectrum {?(N), ?(C?), ?(H?)} according to there being a deuterium or a proton attached to15N for the pertinent correlations. The new experiments are

Axel Meissner; Ole Winneche Sørensen

1998-01-01

268

Transformations and availability of phosphorus in three contrasting soil types from native and farming systems: A study using fractionation and isotopic labeling techniques  

Microsoft Academic Search

Background, aim, and scope  Despite the contribution of many sequential P fractionation schemes to the study of P transformations in agricultural soils,\\u000a the nature of P in each fraction remains qualitative rather than mechanistic. This study used the sequential extraction and\\u000a isotopic dilution techniques to assess the recovery of a tracer (32P) in soil P fractions and to elucidate the transformation

D. T. Vu; C. Tang; R. D. Armstrong

2010-01-01

269

Estimation of the efficiency of hydrocarbon mineralization in soil by measuring CO2-emission and variations in the isotope composition of carbon dioxide  

NASA Astrophysics Data System (ADS)

Estimation of the efficiency of hydrocarbon mineralization in soil by measuring CO2-emission and variations in the isotope composition of carbon dioxide E. Dubrovskaya1, O. Turkovskaya1, A. Tiunov2, N. Pozdnyakova1, A. Muratova1 1 - Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, Saratov, 2 - A.N. Severtsov Institute of Ecology and Evolution, RAS, Moscow, Russian Federation Hydrocarbon mineralization in soil undergoing phytoremediation was investigated in a laboratory experiment by estimating the variation in the 13?/12? ratio in the respired ??2. Hexadecane (HD) was used as a model hydrocarbon pollutant. The polluted soil was planted with winter rye (Secale cereale) inoculated with Azospirillum brasilense strain SR80, which combines the abilities to promote plant growth and to degrade oil hydrocarbon. Each vegetated treatment was accompanied with a corresponding nonvegetated one, and uncontaminated treatments were used as controls. Emission of carbon dioxide, its isotopic composition, and the residual concentration of HD in the soil were examined after two and four weeks. At the beginning of the experiment, the CO2-emission level was higher in the uncontaminated than in the contaminated soil. After two weeks, the quantity of emitted carbon dioxide decreased by about three times and did not change significantly in all uncontaminated treatments. The presence of HD in the soil initially increased CO2 emission, but later the respiration was reduced. During the first two weeks, nonvegetated soil had the highest CO2-emission level. Subsequently, the maximum increase in respiration was recorded in the vegetated contaminated treatments. The isotope composition of plant material determines the isotope composition of soil. The soil used in our experiment had an isotopic signature typical of soils formed by C3 plants (?13C,-22.4‰). Generally, there was no significant fractionation of the carbon isotopes of the substrates metabolized by the soil microbiota. The plants and microorganisms used had the isotopic signatures similar to that of the soil, whereas the ?13C of HD was -47.9‰. The HD mineralization level was assessed by determining the difference between the isotopic compositions of soil CO2 immediately after pollution and during remediation. In the unvegetated soil, about 13% of initially added HD was mineralized, the phytoremediation increased the total decomposition of the contaminant to 19%, and an additional plant inoculation with strain SR80 raised it to 33%. The GC analysis of soil demonstrated that contaminant loss in the plant treatments and in the inoculated plant treatment was 71 and 72%, respectively, whereas in the nonvegetated treatments, it was 64 and 66%, respectively. Thus, the elimination of the contaminant resulted from its total mineralization (CO2 emission) and partial chemical transformation.

Dubrovskaya, Ekaterina; Turkovskaya, Olga

2010-05-01

270

Label-Free Efficient and Accurate Detection of Cystic Fibrosis Causing Mutations Using an Azimuthally Rotated GC-SPR Platform.  

PubMed

Plasmonic nanosensors are candidates for the development of new sensors with low detection limits, high sensitivity, and specificity for target detection: these characteristics are of critical importance in the screening of mutations responsible for inherited diseases. In this work, we focused our study on the detection of some of the most frequent mutations responsible for cystic fibrosis (CF) among the Italian population. For the detection of the CF mutations we adopted a recently developed and highly sensitive Grating Coupled-Surface Plasmon Resonance (GC-SPR) enhanced spectroscopy method for label-free molecular identification exploiting a conical illumination configuration. Gold sinusoidal gratings functionalized with heterobifunctional PEG were used as sensing surfaces, and the specific biodetection was achieved through the coupling with DNA hairpin probes designed for single nucleotide discrimination. Such substrates were used to test unlabeled PCR amplified homozygous wild type (wt) and heterozygous samples, deriving from clinical samples, for the screened mutations. Hybridization conditions were optimized to obtain the maximum discrimination ratio (DR) between the homozygous wild type and the heterozygous samples. SPR signals obtained from hybridizing wild type and heterozygous samples show DRs able to identify univocally the correct genotypes, as confirmed by fluorescence microarray experiments run in parallel. Furthermore, SPR genotyping was not impaired in samples containing unrelated DNA, allowing the platform to be used for the concomitant discrimination of several alleles also scalable for a high throughput screening setting. PMID:25359284

Meneghello, Anna; Antognoli, Agnese; Sonato, Agnese; Zacco, Gabriele; Ruffato, Gianluca; Cretaio, Erica; Romanato, Filippo

2014-12-01

271

Determination of extremely low (236)U/(238)U isotope ratios in environmental samples by sector-field inductively coupled plasma mass spectrometry using high-efficiency sample introduction.  

PubMed

A method by inductively coupled plasma mass spectrometry (ICP-MS) was developed which allows the measurement of (236)U at concentration ranges down to 3 x 10(-14)g g(-1) and extremely low (236)U/(238)U isotope ratios in soil samples of 10(-7). By using the high-efficiency solution introduction system APEX in connection with a sector-field ICP-MS a sensitivity of more than 5,000 counts fg(-1) uranium was achieved. The use of an aerosol desolvating unit reduced the formation rate of uranium hydride ions UH(+)/U(+) down to a level of 10(-6). An abundance sensitivity of 3 x 10(-7) was observed for (236)U/(238)U isotope ratio measurements at mass resolution 4000. The detection limit for (236)U and the lowest detectable (236)U/(238)U isotope ratio were improved by more than two orders of magnitude compared with corresponding values by alpha spectrometry. Determination of uranium in soil samples collected in the vicinity of Chernobyl nuclear power plant (NPP) resulted in that the (236)U/(238)U isotope ratio is a much more sensitive and accurate marker for environmental contamination by spent uranium in comparison to the (235)U/(238)U isotope ratio. The ICP-MS technique allowed for the first time detection of irradiated uranium in soil samples even at distances more than 200 km to the north of Chernobyl NPP (Mogilev region). The concentration of (236)U in the upper 0-10 cm soil layers varied from 2 x 10(-9)g g(-1) within radioactive spots close to the Chernobyl NPP to 3 x 10(-13)g g(-1) on a sampling site located by >200 km from Chernobyl. PMID:16504353

Boulyga, Sergei F; Heumann, Klaus G

2006-01-01

272

Efficient Estimators for Quantum Instanton Evaluation of theKinetic Isotope Effects: Application to the Intramolecular HydrogenTransfer in Pentadiene  

SciTech Connect

The quantum instanton approximation is used to compute kinetic isotope effects for intramolecular hydrogen transfer in cis-1,3-pentadiene. Due to the importance of skeleton motions, this system with 13 atoms is a simple prototype for hydrogen transfer in enzymatic reactions. The calculation is carried out using thermodynamic integration with respect to the mass of the isotopes and a path integral Monte Carlo evaluation of relevant thermodynamic quantities. Efficient 'virial' estimators are derived for the logarithmic derivatives of the partition function and the delta-delta correlation functions. These estimators require significantly fewer Monte Carlo samples since their statistical error does not increase with the number of discrete time slices in the path integral. The calculation treats all 39 degrees of freedom quantum-mechanically and uses an empirical valence bond potential based on a modified general AMBER force field.

Vanicek, Jiri; Miller, William H.

2007-06-13

273

An efficient and compact difference-frequency-generation spectrometer and its application to (12)CH(3)D/(12)CH(4) isotope ratio measurements.  

PubMed

We have developed an efficient and compact 3.4 ?m difference-frequency-generation spectrometer using a 1.55 ?m distributed feedback (DFB) laser diode, a 1.06 ?m DFB laser diode, and a ridge-waveguide periodically poled lithium niobate. It is continuously tunable in the 30 cm(-1) span and is applied to (12)CH(3)D/(12)CH(4) isotope ratio measurements. The suitable pair of (12)CH(3)D ?(4) (p)P(7,6) and (12)CH(4) ?(2)+?(4) R(6) F(1)((1)) lines enabled us to determine their isotope ratio with a precision repeatability of 0.8‰ using a sample and a working standard of pure methane with an effective signal averaging time of 100 ms. PMID:22163569

Tsuji, Kiyoshi; Teshima, Hiroaki; Sasada, Hiroyuki; Yoshida, Naohiro

2010-01-01

274

An Efficient and Compact Difference-Frequency-Generation Spectrometer and Its Application to 12CH3D/12CH4 Isotope Ratio Measurements  

PubMed Central

We have developed an efficient and compact 3.4 ?m difference-frequency-generation spectrometer using a 1.55 ?m distributed feedback (DFB) laser diode, a 1.06 ?m DFB laser diode, and a ridge-waveguide periodically poled lithium niobate. It is continuously tunable in the 30 cm?1 span and is applied to 12CH3D/12CH4 isotope ratio measurements. The suitable pair of 12CH3D ?4 pP(7,6) and 12CH4 ? 2+?4 R(6) F1(1) lines enabled us to determine their isotope ratio with a precision repeatability of 0.8‰ using a sample and a working standard of pure methane with an effective signal averaging time of 100 ms. PMID:22163569

Tsuji, Kiyoshi; Teshima, Hiroaki; Sasada, Hiroyuki; Yoshida, Naohiro

2010-01-01

275

[18F]SiFA-isothiocyanate: a new highly effective radioactive labeling agent for lysine-containing proteins.  

PubMed

A highly efficient (18)F-labeling synthon for universal protein labeling is reported. Diverse (18)F-labeled proteins of 66-144 kDa were prepared with [(18)F]SiFA-isothiocyanate synthesized by an isotopic (19)F for (18)F exchange at the silicon atom. Overall preparative radiochemical yields were 20-40 % after 40-50 min. No bone uptake of (18)F radioactivity was detected until 90 min post-injection of (18)F-SiFA-RSA; this demonstrates the metabolic stability of the [(18)F]SiFA moiety. PMID:19422010

Rosa-Neto, Pedro; Wängler, Björn; Iovkova, Ljuba; Boening, Guido; Reader, Andrew; Jurkschat, Klaus; Schirrmacher, Esther

2009-05-25

276

Highly Efficient Circulating Tumor Cell Isolation from Whole Blood and Label-Free Enumeration Using Polymer-Based Microfluidics with an Integrated Conductivity Sensor  

PubMed Central

A novel microfluidic device that can selectively and specifically isolate exceedingly small numbers of circulating tumor cells (CTCs) through a monoclonal antibody (mAB) mediated process by sampling large input volumes (?1 mL) of whole blood directly in short time periods (<37 min) was demonstrated. The CTCs were concentrated into small volumes (190 nL), and the number of cells captured was read without labeling using an integrated conductivity sensor following release from the capture surface. The microfluidic device contained a series (51) of high-aspect ratio microchannels (35 ?m width × 150 ?m depth) that were replicated in poly(methyl methacrylate), PMMA, from a metal mold master. The microchannel walls were covalently decorated with mABs directed against breast cancer cells overexpressing the epithelial cell adhesion molecule (EpCAM). This microfluidic device could accept inputs of whole blood, and its CTC capture efficiency was made highly quantitative (>97%) by designing capture channels with the appropriate widths and heights. The isolated CTCs were readily released from the mAB capturing surface using trypsin. The released CTCs were then enumerated on-device using a novel, label-free solution conductivity route capable of detecting single tumor cells traveling through the detection electrodes. The conductivity readout provided near 100% detection efficiency and exquisite specificity for CTCs due to scaling factors and the nonoptimal electrical properties of potential interferences (erythrocytes or leukocytes). The simplicity in manufacturing the device and its ease of operation make it attractive for clinical applications requiring one-time use operation. PMID:18557614

Adams, André A.; Okagbare, Paul I.; Feng, Juan; Hupert, Matuesz L.; Patterson, Don; Göttert, Jost; McCarley, Robin L.; Nikitopoulos, Dimitris; Murphy, Michael C.; Soper, Steven A.

2008-01-01

277

Evaluation of the efficiency of Pd/H2 -catalyzed benzylic H/D exchange of dehydroabietinal with D(2) O and synthesis of a tritium-labeled analogue.  

PubMed

Dehydroabietinal (DA) has been identified as an important signaling molecule in systemic acquired resistance in plants. Deuterium and tritium-labeled DA were synthesized to confirm its role in signaling and to further elucidate the mechanism by which DA induces systemic acquired resistance. Pd/H2 -catalyzed exchange of benzylic hydrogen atoms of DA with (2) H-H2 O or (3) H-H2 O was conducted with >97% label incorporation for (2) H-DA and a specific activity of 12.6?mCi/mmol for (3) H-DA synthesized from 90?mCi/mmol (3) H-H2 O. The extent of deuterium labeling at each benzylic position was determined via an inverse-gated (13) C NMR experiment. C7 and C15 were 87% and 81% labeled, respectively. Isotope-induced chemical shift changes at C6 were used to approximate the amount of singly (66%) and doubly (17%) labeled (2) H-DA at C7. Results also indicated that two of the three benzylic protons in DA underwent facile exchange. Exchange at the remaining position was likely hampered by steric interactions of nearby methyl groups at the surface of the Pd catalyst. PMID:24448746

Petros, Robby A; Shah, Jyoti

2014-01-01

278

Subcellular Flux Analysis of Central Metabolism in a Heterotrophic Arabidopsis Cell Suspension Using Steady-State Stable Isotope Labeling1[W][OA  

PubMed Central

The presence of cytosolic and plastidic pathways of carbohydrate oxidation is a characteristic feature of plant cell metabolism. Ideally, steady-state metabolic flux analysis, an emerging tool for creating flux maps of heterotrophic plant metabolism, would capture this feature of the metabolic phenotype, but the extent to which this can be achieved is uncertain. To address this question, fluxes through the pathways of central metabolism in a heterotrophic Arabidopsis (Arabidopsis thaliana) cell suspension culture were deduced from the redistribution of label in steady-state 13C-labeling experiments using [1-13C]-, [2-13C]-, and [U-13C6]glucose. Focusing on the pentose phosphate pathway (PPP), multiple data sets were fitted simultaneously to models in which the subcellular compartmentation of the PPP was altered. The observed redistribution of the label could be explained by any one of three models of the subcellular compartmentation of the oxidative PPP, but other biochemical evidence favored the model in which the oxidative steps of the PPP were duplicated in the cytosol and plastids, with flux through these reactions occurring largely in the cytosol. The analysis emphasizes the inherent difficulty of analyzing the PPP without predefining the extent of its compartmentation and the importance of obtaining high-quality data that report directly on specific subcellular processes. The Arabidopsis flux map also shows that the potential ATP yield of respiration in heterotrophic plant cells can greatly exceed the direct metabolic requirements for biosynthesis, highlighting the need for caution when predicting flux through metabolic networks using assumptions based on the energetics of resource utilization. PMID:19939942

Masakapalli, Shyam K.; Le Lay, Pascaline; Huddleston, Joanna E.; Pollock, Naomi L.; Kruger, Nicholas J.; Ratcliffe, R. George

2010-01-01

279

Pb and Sr isotope measurements by inductively coupled plasma mass spectrometer: efficient time management for precision improvement  

NASA Astrophysics Data System (ADS)

One of the factors limiting the precision of inductively coupled plasma mass spectrometry is the counting statistics, which depend upon acquisition time and ion fluxes. In the present study, the precision of the isotopic measurements of Pb and Sr is examined. The time of measurement is optimally shared for each isotope, using a mathematical simulation, to provide the lowest theoretical analytical error. Different algorithms of mass bias correction are also taken into account and evaluated in term of improvement of overall precision. Several experiments allow a comparison of real conditions with theory. The present method significantly improves the precision, regardless of the instrument used. However, this benefit is more important for equipment which originally yields a precision close to that predicted by counting statistics. Additionally, the procedure is flexible enough to be easily adapted to other problems, such as isotopic dilution.

Monna, F.; Loizeau, J.-L.; Thomas, B. A.; Guéguen, C.; Favarger, P.-Y.

1998-08-01

280

ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.  

SciTech Connect

The use of isotope labels has had a fundamentally important role in the determination of mechanisms of homogeneously catalyzed reactions. Mechanistic data is valuable since it can assist in the design and rational improvement of homogeneous catalysts. There are several ways to use isotopes in mechanistic chemistry. Isotopes can be introduced into controlled experiments and followed where they go or don't go; in this way, Libby, Calvin, Taube and others used isotopes to elucidate mechanistic pathways for very different, yet important chemistries. Another important isotope method is the study of kinetic isotope effects (KIEs) and equilibrium isotope effect (EIEs). Here the mere observation of where a label winds up is no longer enough - what matters is how much slower (or faster) a labeled molecule reacts than the unlabeled material. The most careti studies essentially involve the measurement of isotope fractionation between a reference ground state and the transition state. Thus kinetic isotope effects provide unique data unavailable from other methods, since information about the transition state of a reaction is obtained. Because getting an experimental glimpse of transition states is really tantamount to understanding catalysis, kinetic isotope effects are very powerful.

BULLOCK,R.M.; BENDER,B.R.

2000-12-01

281

Hydrogen isotope fractionation during H2\\/CO2 acetogenesis: hydrogen utilization efficiency and the origin of lipid-bound hydrogen  

Microsoft Academic Search

Hydrogen metabolism was studied in the anaerobic bacterium, Sporomusa sp. strain DMG 58, by measuring natural abundance levels of deuterium in H 2 , H 2 O, and individual fatty acids during acetogenic growth on H 2 \\/ CO 2 . Four cultures were grown, each in medium with a distinct hydrogen-isotopic composition ( ? D-H 2 O). The ?

D. L. VALENTINE; A. L. SESSIONS; S. C. TYLER; A. CHIDTHAISONG

2004-01-01

282

Biosynthesis of the iron-guanylylpyridinol cofactor of [Fe]-hydrogenase in methanogenic archaea as elucidated by stable-isotope labeling.  

PubMed

[Fe]-hydrogenase catalyzes the reversible hydride transfer from H(2) to methenyltetrahydromethanoptherin, which is an intermediate in methane formation from H(2) and CO(2) in methanogenic archaea. The enzyme harbors a unique active site iron-guanylylpyridinol (FeGP) cofactor, in which a low-spin Fe(II) is coordinated by a pyridinol-N, an acyl group, two carbon monoxide, and the sulfur of the enzyme's cysteine. Here, we studied the biosynthesis of the FeGP cofactor by following the incorporation of (13)C and (2)H from labeled precursors into the cofactor in growing methanogenic archaea and by subsequent NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FT-ICR-MS) and IR analysis of the isolated cofactor and reference compounds. The pyridinol moiety of the cofactor was found to be synthesized from three C-1 of acetate, two C-2 of acetate, two C-1 of pyruvate, one carbon from the methyl group of l-methionine, and one carbon directly from CO(2). The metabolic origin of the two CO-ligands was CO(2) rather than C-1 or C-2 of acetate or pyruvate excluding that the two CO are derived from dehydroglycine as has previously been shown for the CO-ligands in [FeFe]-hydrogenases. A formation of CO from CO(2) via direct reduction catalyzed by a nickel-dependent CO dehydrogenase or from formate could also be excluded. When the cells were grown in the presence of (13)CO, the two CO-ligands and the acyl group became (13)C-labeled, indicating either that free CO is an intermediate in their synthesis or that free CO can exchange with these iron-bound ligands. Based on these findings, we propose pathways for how the FeGP cofactor might be synthesized. PMID:22260087

Schick, Michael; Xie, Xiulan; Ataka, Kenichi; Kahnt, Jörg; Linne, Uwe; Shima, Seigo

2012-02-15

283

International Isotope Society  

NSDL National Science Digital Library

The international isotope society (IIS) "aims to encourage the synthesis and applications of isotopes and isotopically labeled compounds to benefit of all." Visitors can find information about upcoming international conferences as well as summaries of past symposiums. The website provides copies of the presentation speeches discussing the activities of award winning scientists. Researchers can find out about the society's low level radioactive waste committee's activities to create a positive public image of the use of radioisotopes in research. An online technical report educates students and teachers about photomultipliers and their applications.

284

Determination of depleted uranium in urine via isotope ratio measurements using large-bore direct injection high efficiency nebulizer-inductively coupled plasma mass spectrometry.  

PubMed

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. PMID:15479520

Westphal, Craig S; McLean, John A; Hakspiel, Shelly J; Jackson, William E; McClain, David E; Montaser, Akbar

2004-09-01

285

Assessment of effects of the rising atmospheric nitrogen deposition on nitrogen uptake and long-term water-use efficiency of plants using nitrogen and carbon stable isotopes.  

PubMed

This study assesses the effects of the atmospheric nitrogen (N) deposition on the N uptake and the long-term water-use efficiency of two C(3) plants (Agropyron cristatum and Leymus chinensis) and two C(4) plants (Amaranthus retroflexus and Setaria viridis) using N and C stable isotopes. In addition, this study explores the potential correlation between leaf N isotope (?(15)N) values and leaf C isotope (?(13)C) values. This experiment shows that the atmospheric N deposition has significant effects on the N uptake, ?(15)N and leaf N content (N(m)) of C(3) plants. As the atmospheric N deposition rises, the proportion and the amount of N absorbed from the simulated atmospheric deposition become higher, and the ?(15)N and N(m) of the two C(3) plants both also increase, suggesting that the rising atmospheric N deposition is beneficial for C(3) plants. However, C(4) plants display different patterns in their N uptake and in their variations of ?(15)N and N(m) from those of C(3) plants. C(4) plants absorb less N from the atmospheric deposition, and the leaf N(m) does not change with the elevated atmospheric N deposition. Photosynthetic pathways may account for the differences between C(3) and C(4) plants. This study also shows that atmospheric N deposition does not play a role in determining the ?(13)C and in the long-term water-use efficiency of C(3) and C(4) plants, suggesting that the long-term water-use pattern of the plants does not change with the atmospheric N input. In addition, this study does not observe any relationship between leaf ?(15)N and leaf ?(13)C in both C(3) and C(4) plants. PMID:21638358

Yao, F Y; Wang, G A; Liu, X J; Song, L

2011-07-15

286

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

NASA Astrophysics Data System (ADS)

Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of cross-linked and non-cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone.

Merkley, Eric D.; Baker, Erin S.; Crowell, Kevin L.; Orton, Daniel J.; Taverner, Thomas; Ansong, Charles; Ibrahim, Yehia M.; Burnet, Meagan C.; Cort, John R.; Anderson, Gordon A.; Smith, Richard D.; Adkins, Joshua N.

2013-03-01

287

A comparison of MS/MS-based, stable-isotope-labeled, quantitation performance on ESI-quadrupole TOF and MALDI-TOF/TOF mass spectrometers.  

PubMed

The peptide-based quantitation accuracy and precision of LC-ESI (QSTAR Elite) and LC-MALDI (4800 MALDI TOF/TOF) were compared by analyzing identical Escherichia coli tryptic digests containing iTRAQ-labeled peptides of defined abundances (1:1, 2.5:1, 5:1, and 10:1). Only 51.4% of QSTAR spectra were used for quantitation by ProteinPilot Software versus 66.7% of LC-MALDI spectra. The average protein sequence coverages for LC-ESI and LC-MALDI were 24.0 and 18.2% (14.9 and 8.4 peptides per protein), respectively. The iTRAQ-based expression ratios determined by ProteinPilot from the 57 467 ESI-MS/MS and 26 085 MALDI-MS/MS spectra were analyzed for measurement accuracy and reproducibility. When the relative abundances of peptides within a sample were increased from 1:1 to 10:1, the mean ratios calculated on both instruments differed by only 0.7-6.7% between platforms. In the 10:1 experiment, up to 64.7% of iTRAQ ratios from LC-ESI MS/MS spectra failed S/N thresholds and were excluded from quantitation, while only 0.1% of the equivalent LC-MALDI iTRAQ ratios were rejected. Re-analysis of an archived LC-MALDI sample set stored for 5 months generated 3715 MS/MS spectra for quantitation, compared with 3845 acquired originally, and the average ratios differed by only 3.1%. Overall, MS/MS-based peptide quantitation performance of offline LC-MALDI was comparable with on-line LC-ESI, which required threefold less time. However, offline LC-MALDI allows the re-analysis of archived HPLC-separated samples. PMID:19504495

Kuzyk, Michael A; Ohlund, Leanne B; Elliott, Monica H; Smith, Derek; Qian, Hong; Delaney, Allen; Hunter, Christie L; Borchers, Christoph H

2009-06-01

288

Nutrition Labeling  

NASA Astrophysics Data System (ADS)

Nutrition labeling regulations differ in countries around the world. The focus of this chapter is on nutrition labeling regulations in the USA, as specified by the Food and Drug Administration (FDA) and the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). A major reason for analyzing the chemical components of foods in the USA is nutrition labeling regulations. Nutrition label information is not only legally required in many countries, but also is of increasing importance to consumers as they focus more on health and wellness.

Metzger, Lloyd E.

289

Efficient radiosynthesis of carbon-11 labelled uncharged Thioflavin T derivatives using [ 11C]methyl triflate for ? -amyloid imaging in Alzheimer's Disease with PET  

Microsoft Academic Search

The synthesis of carbon-11 amino function labelled uncharged Thioflavin T derivatives is known to be performed by reaction of the demethyl-precursors with [11C]methyl iodide but the labelling yields are only mediocre. The use of [11C]methyl triflate improved the radiochemical yield of three potential ?-amyloid imaging PET-radiotracers significantly. Performance of the labelling reaction by reacting the corresponding precursor molecules with [11C]methyl

C. Solbach; M. Uebele; G. Reischl; H.-J. Machulla

2005-01-01

290

Nutrition label  

NSDL National Science Digital Library

This label shows that there are some nutrients that should be limited in the diet of humans and there are others that humans need to intake on a daily basis to stay healthy. These labels show the percent daily value that the food provides for each nutrient.

N/A N/A (FDA; )

2007-09-27

291

Labelling Operators (Programming tricks with MAMBA)  

E-print Network

in C (the watershed transform, the geodesic reconstruction, a very efficient particle labelling hints to design sufficiently performing algorithms. Operators involving connected components or region general and can be used with other measures. Other labelling techniques will also be described

Beucher, Serge

292

Elevated CO2 increases tree-level intrinsic water use efficiency: insights from carbon and oxygen isotope analyses in tree rings across three forest FACE sites  

SciTech Connect

Elevated CO2 increases intrinsic water use efficiency (WUEi) of forests, but the magnitude of this effect and its interaction with climate is still poorly understood. We combined tree ring analysis with isotope measurements at three Free Air CO2 Enrichment (FACE, POP-EUROFACE, in Italy; Duke FACE in North Carolina and ORNL in Tennessee, USA) sites, to cover the entire life of the trees. We used 13C to assess carbon isotope discrimination ( 13C ci/ca) and changes in WUEi, while direct CO2 effects on stomatal conductance were explored using 18O as a proxy. Across all the sites, elevated CO2 increased 13C-derived WUEi on average by 73% for Liquidambar styraciflua, 77% for Pinus taeda and 75% for Populus sp., but through different ecophysiological mechanisms. Our findings provide a robust means of predicting WUEi responses from a variety of tree species exposed to variable environmental conditions over time, and species-specific relationships that can help modeling elevated CO2 and climate impacts on forest productivity, carbon and water balances.

Battipaglia, Giovanna [Second University of Naples; Saurer, Matthias [Paul Scherrer Institut, Villigen, Switzerland; Cherubini, Paulo [WSL Swiss Federal Institute for Forest, Snow and Landscape Research; Califapietra, Carlo [University of Tuscia; McCarthy, Heather R [Duke University; Norby, Richard J [ORNL; Cotrufo, M. Francesca [Colorado State University, Fort Collins

2013-01-01

293

Radioactively labelled porphyrin derivatives  

NASA Astrophysics Data System (ADS)

Radioactive labelling of guanidine-bearing tetraphenylporphyrin and Dy—texaphyrin with selected radionuclides (166Ho and 90Y) is described. A basic characterisation of studied porphyrin and texaphyrin, including their behaviour in a wide range of pH values and data on holmium and yttrium complexation with these compounds was probed using UV-VIS absorption spectrometry. The labelling yield of these macrocyclic molecules depends on the pH of the reaction mixture, metal: ligand ratio and time of incubation. Optimal reaction conditions for formation of porphyrin and texaphyrin radioactive complexes were determined by thin layer chromatography with the detection of ?- activity. The ability of porphyrin derivatives to bind anions was examined as well. Our experiments were focused on perrhenate ion (ReO4 -) because radiopharmaceuticals labelled with isotopes 186Re and 188Re play an important role in therapy of numerous tumour diseases. The possibility of applying ReO4 - anion directly for labelling purposes, without the necessity of its reduction to lower oxidation state, was not proved.

Koní?ová, R.; Ernestová, M.; Jedináková-K?ížová, V.; Král, V.

2003-01-01

294

Quantitative Proteomics Using Stable Isotope Labeling with Amino Acids in Cell Culture Reveals Changes in the Cytoplasmic, Nuclear, and Nucleolar Proteomes in Vero Cells Infected with the Coronavirus Infectious Bronchitis Virus*  

PubMed Central

Virus-host interactions involve complex interplay between viral and host factors, rendering them an ideal target for proteomic analysis. Here we detail a high throughput quantitative proteomics analysis of Vero cells infected with the coronavirus infectious bronchitis virus (IBV), a positive strand RNA virus that replicates in the cytoplasm. Stable isotope labeling with amino acids in cell culture (SILAC) was used in conjunction with LC-MS/MS to identify and quantify 1830 cellular and two viral proteins from IBV-infected cells. Fractionation of cells into cytoplasmic, nuclear, and nucleolar extracts was used to reduce sample complexity and provide information on the trafficking of proteins between the different compartments. Each fraction showed a proportion of proteins exhibiting ?2-fold changes in abundance. Ingenuity Pathway Analysis revealed that proteins that changed in response to infection could be grouped into different functional categories. These included proteins regulated by NF-?B- and AP-1-dependent pathways and proteins involved in the cytoskeleton and molecular motors. A luciferase-based reporter gene assay was used to validate the up-regulation of AP-1- and NF-?B-dependent transcription in IBV-infected cells and confirmed using immunofluorescence. Immunofluorescence was used to validate changes in the subcellular localization of vimentin and myosin VI in IBV-infected cells. The proteomics analysis also confirmed the presence of the viral nucleocapsid protein as localizing in the cytoplasm, nucleus, and nucleolus and the viral membrane protein in the cytoplasmic fraction. This research is the first application of SILAC to study total host cell proteome changes in response to positive sense RNA virus infection and illustrates the versatility of this technique as applied to infectious disease research. PMID:20467043

Emmott, Edward; Rodgers, Mark A.; Macdonald, Andrew; McCrory, Sarah; Ajuh, Paul; Hiscox, Julian A.

2010-01-01

295

18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry  

SciTech Connect

Selected reaction monitoring-mass spectrometry (SRM-MS) is an emerging technology for high throughput targeted protein quantification and verification in biological and biomarker discovery studies; however, the cost associated with the use of stable isotope labeled synthetic peptides as internal standards is prohibitive for quantitatively screening large numbers of candidate proteins as often required in the pre-verification phase of biomarker discovery. Herein we present the proof-of-concept experiments of using an 18O-labeled 'universal' reference as comprehensive internal standards for quantitative SRM-MS analysis. With an 18O-labeled whole proteome sample as reference, every peptide of interest will have its own corresponding heavy isotope labeled internal standard, thus providing an ideal approach for quantitative screening of a large number of candidates using SRM-MS. Our results showed that the 18O incorporation efficiency using a recently improved protocol was >99.5% for most peptides investigated, a level comparable to 13C/15N labeled synthetic peptides in terms of heavy isotope incorporation. The accuracy, reproducibility, and linear dynamic range of quantification were further assessed based on known ratios of standard proteins spiked into mouse plasma with an 18O-labeled mouse plasma reference. A dynamic range of four orders of magnitude in relative concentration was obtained with high reproducibility (i.e., coefficient of variance <10%) based on the 16O/18O peak area ratios. Absolute and relative quantification of C-reactive protein and prostate-specific antigen were demonstrated by coupling an 18O-labeled reference with standard additions of protein standards. Collectively, our results demonstrated that the use of 18O-labeled reference provides a convenient and effective strategy for quantitative SRM screening of large number of candidate proteins.

Kim, Jong Seo; Fillmore, Thomas L.; Liu, Tao; Robinson, Errol W.; Hossain, Mahmud; Champion, Boyd L.; Moore, Ronald J.; Camp, David G.; Smith, Richard D.; Qian, Weijun

2011-10-11

296

Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases.  

PubMed

Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions. PMID:24285530

Poraj-Kobielska, Marzena; Atzrodt, Jens; Holla, Wolfgang; Sandvoss, Martin; Gröbe, Glenn; Scheibner, Katrin; Hofrichter, Martin

2013-01-01

297

High-efficiency astatination of antibodies using N-iodosuccinimide as the oxidising agent in labelling of N-succinimidyl 3-(trimethylstannyl)benzoate  

Microsoft Academic Search

Monoclonal antibodies C215, reactive with colorectal carcinomas, and MOv18, reactive with most of the ovarian carcinomas, were radiohalogenated with [211At]astatine. The radiohalogen was conjugate coupled to antibodies via the intermediate labelling reagent N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE) in a two-step, single-pot reaction. Optimisation of the labelling of the reagent was achieved using N-iodosuccinimide, NIS, as the oxidising agent. The yields ranged from 69–95%

S. Lindegren; H. Andersson; T. Bäck; L. Jacobsson; B. Karlsson; G. Skarnemark

2001-01-01

298

Effects of different chelates and labeling media on platelet labeling with In111  

Microsoft Academic Search

Recently, many variations of In-111 platelet labeling have been introduced with no consistent method currently being used. In an effort to determine optimal In-111 labeling conditions. The authors have studied the effects of different chelates and labeling media on In-111 platelet labeling efficiency (LE). Labeling of human platelets in plasma with In-111-oxine, resulted in a mean LE of 27% at

R. L. Hill-Zobel; S. Gannon; B. McCandless; M. F. Tsan

1985-01-01

299

A Simple Procedure for Effective Quenching of Trypsin Activity and Prevention of 18O-Labeling Back-Exchange  

PubMed Central

Trypsin-catalyzed stable isotope 16O/18O-labeling of the C-terminal carboxyl groups of peptides is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by residual trypsin that can catalyze the back-exchange of 18O with 16O after labeling. Here, we demonstrate through a detailed evaluation that boiling the peptide sample for 10 minutes provides a simple means for completely quenching residual trypsin activity and preventing oxygen back-exchange in 18O-labeled samples. We also observed that the presence of organic solvents such as acetonitrile made quenching trypsin activity less efficient. Finally, current 18O-labeling methods that typically employ immobilized trypsin result in significant sample losses due to non-specific binding of peptides on the resin, making their application toward smaller biological samples increasingly impractical. We present here an improved 18O-labeling protocol that is more applicable to microscale biological samples by using solution-phase trypsin instead of immobilized trypsin to overcome the non-specific sample loss issue encountered with the use of immobilized trypsin. The ability to generate stably 18O-labeled samples without back-exchange should enable more effective applications of 18O-labeled toward large-scale biomarker discovery and validations where an 18O-labeled sample can be used as a common reference for quantitation. PMID:19222237

Petritis, Brianne O.; Qian, Wei-Jun; Camp, David G.; Smith, Richard D.

2009-01-01

300

Solution structure of the two RNA recognition motifs of hnRNP A1 using segmental isotope labeling: how the relative orientation between RRMs influences the nucleic acid binding topology.  

PubMed

Human hnRNP A1 is a multi-functional protein involved in many aspects of nucleic-acid processing such as alternative splicing, micro-RNA biogenesis, nucleo-cytoplasmic mRNA transport and telomere biogenesis and maintenance. The N-terminal region of hnRNP A1, also named unwinding protein 1 (UP1), is composed of two closely related RNA recognition motifs (RRM), and is followed by a C-terminal glycine rich region. Although crystal structures of UP1 revealed inter-domain interactions between RRM1 and RRM2 in both the free and bound form of UP1, these interactions have never been established in solution. Moreover, the relative orientation of hnRNP A1 RRMs is different in the free and bound crystal structures of UP1, raising the question of the biological significance of this domain movement. In the present study, we have used NMR spectroscopy in combination with segmental isotope labeling techniques to carefully analyze the inter-RRM contacts present in solution and subsequently determine the structure of UP1 in solution. Our data unambiguously demonstrate that hnRNP A1 RRMs interact in solution, and surprisingly, the relative orientation of the two RRMs observed in solution is different from the one found in the crystal structure of free UP1 and rather resembles the one observed in the nucleic-acid bound form of the protein. This strongly supports the idea that the two RRMs of hnRNP A1 have a single defined relative orientation which is the conformation previously observed in the bound form and now observed in solution using NMR. It is likely that the conformation in the crystal structure of the free form is a less stable form induced by crystal contacts. Importantly, the relative orientation of the RRMs in proteins containing multiple-RRMs strongly influences the RNA binding topologies that are practically accessible to these proteins. Indeed, RRM domains are asymmetric binding platforms contacting single-stranded nucleic acids in a single defined orientation. Therefore, the path of the nucleic acid molecule on the multiple RRM domains is strongly dependent on whether the RRMs are interacting with each other. The different nucleic acid recognition modes by multiple-RRM domains are briefly reviewed and analyzed on the basis of the current structural information. PMID:23247503

Barraud, Pierre; Allain, Frédéric H-T

2013-01-01

301

High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.  

PubMed

The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows. PMID:23033477

Thiede, Bernd; Koehler, Christian J; Strozynski, Margarita; Treumann, Achim; Stein, Robert; Zimny-Arndt, Ursula; Schmid, Monika; Jungblut, Peter R

2013-02-01

302

Rare-isotope and kinetic studies of Pt/SnO2 catalysts  

NASA Technical Reports Server (NTRS)

Closed-cycle pulsed CO2 laser operation requires the use of an efficient CO-O2 recombination catalyst for these dissociation products which otherwise would degrade the laser operation. The catalyst must not only operate at low temperatures but also must operate efficiently for long periods. In the case of the Laser Atmospheric Wind Sounder (LAWS) laser, an operational lifetime of 3 years is required. Additionally, in order to minimize atmospheric absorption and enhance aerosol scatter of laser radiation, the LAWS system will operate at 9.1 micrometers with an oxygen-18 isotope CO2 lasing medium. Consequently, the catalyst must not only operate at low temperatures but must also preserve the isotopic integrity of the rare-isotope composition in the recombination mode. Several years ago an investigation of commercially available and newly synthesized recombination catalysts for use in closed-cycle pulsed common and rare-isotope CO2 lasers was implemented at the NASA Langley Research Center. Since that time, mechanistic efforts utilizing both common and rare oxygen isotopes have been implemented and continue. Rare-isotope studies utilizing commercially available platinum-tin oxide catalyst have demonstrated that the catalyst contributes oxygen-16 to the product carbon dioxide thus rendering it unusable for rare-isotope applications. A technique has been developed for modification of the surface of the common-isotope catalyst to render it usable. Results of kinetic and isotope label studies using plug flow, recycle plug flow, and closed internal recycle plug flow reactor configuration modes are discussed.

Upchurch, Billy T.; Wood, George M.; Schryer, David R.; Hess, Robert V.; Miller, Irvin M.; Kielin, Erik J.

1990-01-01

303

Proteomics meets genetics: SILAC labeling of Drosophila melanogaster larvae and cells for in vivo functional studies.  

PubMed

Stable isotope labeling by amino acids in cell culture (SILAC) is an established and potent method for quantitative proteomics. When combined with high-resolution mass spectrometry (MS) and efficient algorithms for the analysis of quantitative MS data, SILAC has proven to be the strategy of choice for the in-depth characterization of functional states at the protein level. The fruit fly Drosophila melanogaster is one of the most widely used model systems for studies of genetics and developmental biology. Despite this, a global proteomic approach in Drosophila is rarely considered. Here, we describe an adaptation of SILAC for functional investigation of fruit flies by proteomics: We illustrate how to perform efficient SILAC labeling of cells in culture and whole fly larvae. The combination of SILAC, a highly accurate global protein quantification method, and of the fruit fly, the prime genetics and developmental model, represents a unique opportunity for quantitative proteomic studies in vivo. PMID:25059620

Cuomo, Alessandro; Sanfilippo, Roberta; Vaccari, Thomas; Bonaldi, Tiziana

2014-01-01

304

Multiple tag labeling method for DNA sequencing  

DOEpatents

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.

Mathies, Richard A. (Contra Costa County, CA); Huang, Xiaohua C. (Mt. View, CA); Quesada, Mark A. (San Francisco, CA)

1995-01-01

305

Correlated optical and isotopic nanoscopy  

NASA Astrophysics Data System (ADS)

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.

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

2014-04-01

306

Convenient and Efficient Synthesis of a Lanthanide3+-Coordinated, Diethylene Triamine Pentaacetic Acid Labeled Biopolymer as an Assay for the Cholecystokinin B Receptor  

PubMed Central

To develop an assay for the cholecystokinin B receptor with an Eu3+-labeled cholecystokinin peptide via a diethylene triamine pentaacetic acid chelating linker, a commercial dianhydride diethylene triamine pentaacetic acid precursor was directly attached to the N-terminus of cholecystokinin peptides by a solid-phase synthesis method with a satisfactory yield and purity after reverse-phase high-performance liquid chromatography separation. Lanthanide was then coordinated to the peptide via a diethylene triamine pentaacetic acid bifunctional agent. This method is a useful approach to the large-scale synthesis of lanthanide3+-coordinated, diethylene triamine pentaacetic acid labeled biopolymers. This research provides not only a simple and convenient method for the preparation of lanthanide-based peptide ligand libraries but also possible lanthanide-based high-throughput screening of peptide receptors with a timeresolved fluorescence assay system. Five biopolymers were synthesized and characterized with high-resolution electrospray ionization in this study. PMID:19562042

Gao, F.; Handl, H.; Vagner, J.; Hruby, V.; Gillies, R.

2009-01-01

307

Adipose derived stem cells: efficiency, toxicity, stability of BrdU labeling and effects on self-renewal and adipose differentiation  

Microsoft Academic Search

5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling\\u000a of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine\\u000a how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular\\u000a stability, and

Charlotte Lequeux; Georgette Oni; Ali Mojallal; Odile Damour; Spencer A. Brown

2011-01-01

308

Incorporating concentration dependence in stable isotope mixing models  

Microsoft Academic Search

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 linear mixing model assumes that the proportional contribution of a source to a mixture

Donald L. Phillips; Paul L. Koch

2002-01-01

309

Physicochemical isotope anomalies  

SciTech Connect

Isotopic composition of refractory elements can be modified, by physical processes such as distillation and sputtering, in unexpected patterns. Distillation enriches the heavy isotopes in the residue and the light isotopes in the vapor. However, current models appear to be inadequate to describe the detailed mass dependence, in particular for large fractionations. Coarse- and fine-grained inclusions from the Allende meteorite exhibit correlated isotope effects in Mg both as mass-dependent fractionation and residual anomalies. This isotope pattern can be duplicated by high temperature distillation in the laboratory. A ubiquitous property of meteoritic inclusions for Mg as well as for most of the other elements, where measurements exist, is mass-dependent fractionation. In contrast, terrestrial materials such as microtektites, tektite buttons as well as lunar orange and green glass spheres have normal Mg isotopic composition. A subset of interplanetary dust particles labelled as chondritic aggregates exhibit excesses in {sup 26}Mg and deuterium anomalies. Sputtering is expected to be a dominant mechanism in the destruction of grains within interstellar dust clouds. An active proto-sun as well as the present solar-wind and solar-flare flux are of sufficient intensity to sputter significant amounts of material. Laboratory experiments in Mg show widespread isotope effects including residual {sup 26}Mg excesses and mass dependent fractionation. It is possible that the {sup 26}Mg excesses in interplanetary dust is related to sputtering by energetic solar-wind particles. The implication if the laboratory distillation and sputtering effects are discussed and contrasted with the anomalies in meteoritic inclusions the other extraterrestrial materials the authors have access to.

Esat, T.M. (Australian National Univ., Canberra)

1988-06-01

310

Trends in carbon isotope fractionation in atmospheric carbon dioxide constrain water use efficiency of northern ecosystems from the 1980s to 2010  

NASA Astrophysics Data System (ADS)

Atmospheric CO2 concentrations have increased by approximately 120 ppm since preindustrial times and have reached levels higher than any other time during the last three to five million years ago with uncertain consequences for the modern terrestrial biosphere. When plants take up CO2 for photosynthesis from the atmosphere through stomata openings in their leaves, water escapes due to the gradient in water vapor pressure from the leaf interior to the atmospheric boundary layer. The amount of carbon assimilated by photosynthesis per water lost determines the water use efficiency (WUE) of the plant. The extra CO2 in the atmosphere has been shown to increase WUE in growth chamber studies, allowing plants to take up the same or more CO2 with reduced stomatal conductance, thereby reducing water loss. Carbon isotope fractionation by plants is related to the CO2 concentration gradient from inside the leaf (Ci) to that in the atmosphere (Ca) (e.g. Farquhar model). Therefore intrinsic water use efficiency (iWUE) of the biosphere, defined as the amount of net photosynthesis divided by the stomatal conductance, leaves an imprint on the record of ?13C in atmospheric CO2. We will present estimates of the biological carbon isotope fractionation of atmospheric CO2 from the Scripps Institution of Oceanography flask network from the 1980s to 2010 and discuss the constraints it provides on trends in iWUE over this period. Using the seasonal co-variation of 13C and CO2, we calculate effective fractionation. This data can be used to test hypothetical trends in iWUE and Ci. The conventional wisdom in the field has been that the ratio of Ci/Ca would remain approximately constant as CO2 rises, which would result in no change in fractionation but a modest increase in iWUE. Keenan et al. (2013) recently published an analysis of FluxNet eddy covariance measurements suggesting that Ci has stayed nearly constant since the mid-1990s, translating to a large ~3% yr-1 increase in iWUE. Atmospheric records from Barrow, Alaska, and Alert, Canada stations, suggest an increase in fractionation of northern ecosystems over the last few decades. These atmospheric results seem generally consistent with a constant Ca-Ci scenario, which increases fractionation, but corresponds to a constant iWUE. At the same time that fractionation has been increasing, the amplitude of the seasonal cycle of CO2 has increased dramatically (Graven et al., 2013). One possible explanation for both trends is that the deciduous fraction in the boreal forests has increased as these forest species have shorter, more intense periods of CO2 uptake and have been shown to have greater 13C fractionation and lower iWUE than evergreen species.

Welp, L. R.; Piper, S. C.; Graven, H. D.; Bollenbacher, A.; Meijer, H. A.; Keeling, R. F.

2013-12-01

311

Genetic Control of Water Use Efficiency and Leaf Carbon Isotope Discrimination in Sunflower (Helianthus annuus L.) Subjected to Two Drought Scenarios  

PubMed Central

High water use efficiency (WUE) can be achieved by coordination of biomass accumulation and water consumption. WUE is physiologically and genetically linked to carbon isotope discrimination (CID) in leaves of plants. A population of 148 recombinant inbred lines (RILs) of sunflower derived from a cross between XRQ and PSC8 lines was studied to identify quantitative trait loci (QTL) controlling WUE and CID, and to compare QTL associated with these traits in different drought scenarios. We conducted greenhouse experiments in 2011 and 2012 by using 100 balances which provided a daily measurement of water transpired, and we determined WUE, CID, biomass and cumulative water transpired by plants. Wide phenotypic variability, significant genotypic effects, and significant negative correlations between WUE and CID were observed in both experiments. A total of nine QTL controlling WUE and eight controlling CID were identified across the two experiments. A QTL for phenotypic response controlling WUE and CID was also significantly identified. The QTL for WUE were specific to the drought scenarios, whereas the QTL for CID were independent of the drought scenarios and could be found in all the experiments. Our results showed that the stable genomic regions controlling CID were located on the linkage groups 06 and 13 (LG06 and LG13). Three QTL for CID were co-localized with the QTL for WUE, biomass and cumulative water transpired. We found that CID and WUE are highly correlated and have common genetic control. Interestingly, the genetic control of these traits showed an interaction with the environment (between the two drought scenarios and control conditions). Our results open a way for breeding higher WUE by using CID and marker-assisted approaches and therefore help to maintain the stability of sunflower crop production. PMID:24992022

Adiredjo, Afifuddin Latif; Navaud, Olivier; Muños, Stephane; Langlade, Nicolas B.; Lamaze, Thierry; Grieu, Philippe

2014-01-01

312

Genetic control of water use efficiency and leaf carbon isotope discrimination in sunflower (Helianthus annuus L.) subjected to two drought scenarios.  

PubMed

High water use efficiency (WUE) can be achieved by coordination of biomass accumulation and water consumption. WUE is physiologically and genetically linked to carbon isotope discrimination (CID) in leaves of plants. A population of 148 recombinant inbred lines (RILs) of sunflower derived from a cross between XRQ and PSC8 lines was studied to identify quantitative trait loci (QTL) controlling WUE and CID, and to compare QTL associated with these traits in different drought scenarios. We conducted greenhouse experiments in 2011 and 2012 by using 100 balances which provided a daily measurement of water transpired, and we determined WUE, CID, biomass and cumulative water transpired by plants. Wide phenotypic variability, significant genotypic effects, and significant negative correlations between WUE and CID were observed in both experiments. A total of nine QTL controlling WUE and eight controlling CID were identified across the two experiments. A QTL for phenotypic response controlling WUE and CID was also significantly identified. The QTL for WUE were specific to the drought scenarios, whereas the QTL for CID were independent of the drought scenarios and could be found in all the experiments. Our results showed that the stable genomic regions controlling CID were located on the linkage groups 06 and 13 (LG06 and LG13). Three QTL for CID were co-localized with the QTL for WUE, biomass and cumulative water transpired. We found that CID and WUE are highly correlated and have common genetic control. Interestingly, the genetic control of these traits showed an interaction with the environment (between the two drought scenarios and control conditions). Our results open a way for breeding higher WUE by using CID and marker-assisted approaches and therefore help to maintain the stability of sunflower crop production. PMID:24992022

Adiredjo, Afifuddin Latif; Navaud, Olivier; Muños, Stephane; Langlade, Nicolas B; Lamaze, Thierry; Grieu, Philippe

2014-01-01

313

1. Isotope Definitions and terms a) Isotopes and isotope ratios.  

E-print Network

3/24/2011 1 Outline 1. Isotope Definitions and terms a) Isotopes and isotope ratios. Isotopes fractionation c) Simple illustration with the water cycle 2. CO2 isotopes in photosynthesis a) Photosynthetic discrimination in C3 plants b) C3 vs C4 photosynthesis and the distinction in isotopes c) Measuring isotopic

Saleska, Scott

314

Bcl-2-functionalized ultrasmall superparamagnetic iron oxide nanoparticles coated with amphiphilic polymer enhance the labeling efficiency of islets for detection by magnetic resonance imaging  

PubMed Central

Based on their versatile, biocompatible properties, superparamagnetic iron oxide (SPIO) or ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are utilized for detecting and tracing cells or tumors in vivo. Here, we developed an innoxious and concise synthesis approach for a novel B-cell lymphoma (Bcl)-2 monoclonal antibody-functionalized USPIO nanoparticle coated with an amphiphilic polymer (carboxylated polyethylene glycol monooleyl ether [OE-PEG-COOH]). These nanoparticles can be effectively internalized by beta cells and label primary islet cells, at relatively low iron concentration. The biocompatibility and cytotoxicity of these products were investigated by comparison with the commercial USPIO product, FeraSpin™ S. We also assessed the safe dosage range of the product. Although some cases showed a hypointensity change at the site of transplant, a strong magnetic resonance imaging (MRI) was detectable by a clinical MRI scanner, at field strength of 3.0 Tesla, in vivo, and the iron deposition/attached in islets was confirmed by Prussian blue and immunohistochemistry staining. It is noteworthy that based on our synthesis approach, in future, we could exchange the Bcl-2 with other probes that would be more specific for the targeted cells and that would have better labeling specificity in vivo. The combined results point to the promising potential of the novel Bcl-2-functionalized PEG-USPIO as a molecular imaging agent for in vivo monitoring of islet cells or other cells. PMID:24204136

Yang, Bin; Cai, Haolei; Qin, Wenjie; Zhang, Bo; Zhai, Chuanxin; Jiang, Biao; Wu, Yulian

2013-01-01

315

Separation of nitrogen and oxygen isotopes by liquid chromatography  

Microsoft Academic Search

ISOTOPIC enrichment of organic compounds in the stable minor isotopes 15N, 17O and 18O, which are often used as tracers, is commonly achieved by syntheses that involve isotopically enriched simple precursors such as NH3, H2O and CO2. But such labelled compounds are not always commercially available or readily synthesized. Here we report the direct enrichment of nitrogen and oxygen isotopes

Nobuo Tanaka; Ken Hosoya; Kazuhiro Nomura; Tadanori Yoshimura; Takehiro Ohki; Ryohei Yamaoka; Kazuhiro Kimata; Mikio Araki

1989-01-01

316

Failure to label baboon milk intrinsically with iron  

SciTech Connect

The widely held belief that 50% of the iron in human milk is absorbed is based on studies that have used an extrinsic radioactive iron tag. To determine the validity of an extrinsic tag, it is necessary to label the milk intrinsically with one isotope and to compare absorption of this isotope with absorption of another isotope added as the extrinsic tag. We chose the baboon as a model and infused 59Fe intravenously. In each of three attempts we failed to label the milk intrinsically.

Figueroa-Colon, R.; Elwell, J.H.; Jackson, E.; Osborne, J.W.; Fomon, S.J. (Univ. of Iowa, Iowa City (USA))

1989-11-01

317

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

NASA Astrophysics Data System (ADS)

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.

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

318

Simultaneous tracing of {sup 76}Se-selenite and {sup 77}Se-selenomethionine by absolute labeling and speciation  

SciTech Connect

Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ({sup 82}Se-selenite) and then administered {sup 76}Se-selenite and {sup 77}Se-selenomethionine ({sup 77}Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled {sup 76}Se and {sup 77}Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys.

Suzuki, Kazuo T. [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan)]. E-mail: ktsuzuki@p.chiba-u.ac.jp; Somekawa, Layla [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan); Kurasaki, Kazuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan); Suzuki, Noriyuki [Graduate School of Pharmaceutical Sciences, Chiba University, Chuo, Chiba 260-8675 (Japan)

2006-11-15

319

Simultaneous tracing of 76Se-selenite and 77Se-selenomethionine by absolute labeling and speciation.  

PubMed

Nutritional selenocompounds are transformed into the assumed common intermediate selenide, which is utilized for the synthesis of selenoenzymes or transformed into methylated metabolites for excretion. Hence, selenocompound metabolites can be traced only with labeled selenium. Here we applied a new tracer method for the metallomics of biometals using simultaneous speciation of each metallome labeled with different homo-elemental isotopes to metabolism and availability of selenium. Rats were depleted of endogenous natural abundance selenium by feeding a single selenium stable isotope ((82)Se-selenite) and then administered (76)Se-selenite and (77)Se-selenomethionine ((77)Se-SeMet)simultaneously. Biological samples were subjected to quantification and speciation analysis by HPLC-ICPMS. Metabolites of the labeled (76)Se and (77)Se and interaction with endogenous selenium were traced and examined without interference from the corresponding endogenous natural abundance isotopes. Differences in the distribution and metabolism among organs and between the two nutritional selenocompounds were compared under exactly identical biological and analytical conditions: (1) selenite was distributed more efficiently than SeMet in organs and body fluids except the pancreas. (2) SeMet was taken up by organs in its intact form. (3) Selenium of SeMet origin was distributed selectively in the pancreas and mostly bound to a protein together with intact SeMet. (4) Selenosugars A and B but not trimethylselenonium (TMSe) were detected in the liver. (5) Selenosugar B and TMSe were detected in the kidneys. PMID:16956638

Suzuki, Kazuo T; Somekawa, Layla; Kurasaki, Kazuki; Suzuki, Noriyuki

2006-11-15

320

Perdeuteration and methyl-selective (1)H, (13)C-labeling by using a Kluyveromyces lactis expression system.  

PubMed

The production of stable isotope-labeled proteins is critical in structural analyses of large molecular weight proteins using NMR. Although prokaryotic expression systems using Escherichia coli have been widely used for this purpose, yeast strains have also been useful for the expression of functional eukaryotic proteins. Recently, we reported a cost-effective stable isotope-labeled protein expression using the hemiascomycete yeast Kluyveromyces lactis (K. lactis), which allow us to express exogenous proteins at costs comparable to prokaryotic expression systems. Here, we report the successful production of highly deuterated (>90 %) protein in the K. lactis system. We also examined the methyl-selective (1)H, (13)C-labeling of Ile, Leu, and Val residues using commonly used amino acid precursors. The efficiency of (1)H-(13)C-incorporation varied significantly based on the amino acid. Although a high level of (1)H-(13)C-incorporation was observed for the Ile ?1 position, (1)H, (13)C-labeling rates of Val and Leu methyl groups were limited due to the mitochondrial localization of enzymes involved in amino acid biosynthesis and the lack of transporters for ?-ketoisovalerate in the mitochondrial membrane. In line with this notion, the co-expression with branched-chain-amino-acid aminotransferase in the cytosol significantly improved the incorporation rates of amino acid precursors. Although it would be less cost-effective, addition of (13)C-labeled valine can circumvent problems associated with precursors and achieve high level (1)H, (13)C-labeling of Val and Leu. Taken together, the K. lactis system would be a good alternative for expressing large eukaryotic proteins that need deuteration and/or the methyl-selective (1)H, (13)C-labeling for the sensitive detection of NMR resonances. PMID:24146206

Miyazawa-Onami, Mayumi; Takeuchi, Koh; Takano, Toshiaki; Sugiki, Toshihiko; Shimada, Ichio; Takahashi, Hideo

2013-11-01

321

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)

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. Entrainment of air into the plume is seen as evaporation of condensate. In the plume between about -7 and -12C, the ice condensate fraction increases with height, and the isotope ratios are used to discern ice formation from deposition from ice formed from in situ freezing of cloud liquid. The observed profiles demonstrate a new capacity for cloud process studies and provide new insight into the water budget of clouds.

Noone, D. C.; Raudzens Bailey, A.; Toohey, D. W.; Twohy, C. H.; Heymsfield, A.; Rella, C.; Van Pelt, A. D.

2011-12-01

322

Development of an efficient signal amplification strategy for label-free enzyme immunoassay using two site-specific biotinylated recombinant proteins.  

PubMed

Constructing a recombinant protein between a reporter enzyme and a detector protein to produce a homogeneous immunological reagent is advantageous over random chemical conjugation. However, the approach hardly recombines multiple enzymes in a difunctional fusion protein, which results in insufficient amplification of the enzymatic signal, thereby limiting its application in further enhancement of analytical signal. In this study, two site-specific biotinylated recombinant proteins, namely, divalent biotinylated alkaline phosphatase (AP) and monovalent biotinylated ZZ domain, were produced by employing the Avitag-BirA system. Through the high streptavidin (SA)-biotin interaction, the divalent biotinylated APs were clustered in the SA-biotin complex and then incorporated with the biotinylated ZZ. This incorporation results in the formation of a functional macromolecule that involves numerous APs, thereby enhancing the enzymatic signal, and in the production of several ZZ molecules for the interaction with immunoglobulin G (IgG) antibody. The advantage of this signal amplification strategy is demonstrated through ELISA, in which the analytical signal was substantially enhanced, with a 32-fold increase in the detection sensitivity compared with the ZZ-AP fusion protein approach. The proposed immunoassay without chemical modification can be an alternative strategy to enhance the analytical signals in various applications involving immunosensors and diagnostic chips, given that the label-free IgG antibody is suitable for the ZZ protein. PMID:25622607

Tang, Jin-Bao; Tang, Ying; Yang, Hong-Ming

2015-02-15

323

Effects of Nitrogen-Labeled Gel-Based Controlled-Release Fertilizer on Dry-Matter Accumulation and the Nutrient-Uptake Efficiency of Corn  

Microsoft Academic Search

Effects of a new gel-based controlled-release fertilizer (G-CRF) on the growth and nutrient uptake efficiency of corn were investigated based on a pot experiment. The fate of fertilizer nitrogen (N) was also studied by N tracer method. The results showed that, compared with conventional fertilizer, the G-CRF had significant effects on increasing dry matter, N, phosphorus (P), and potassium (K)

Hong Ding; Yushu Zhang; Shengjin Qin; Weihua Li; Shiqing Li

2011-01-01

324

The anatomy of a laser label  

Technology Transfer Automated Retrieval System (TEKTRAN)

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...

325

10 CFR 431.31 - Labeling requirements.  

Code of Federal Regulations, 2012 CFR

...CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN...INDUSTRIAL EQUIPMENT Electric Motors Labeling...nameplate of an electric motor for which...nominal full load efficiency (as of the date...Disclosure of efficiency information in...must appear on an electric motor's...

2012-01-01

326

10 CFR 431.31 - Labeling requirements.  

Code of Federal Regulations, 2014 CFR

...CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN...INDUSTRIAL EQUIPMENT Electric Motors Labeling...nameplate of an electric motor for which...nominal full load efficiency (as of the date...Disclosure of efficiency information in...must appear on an electric motor's...

2014-01-01

327

10 CFR 431.31 - Labeling requirements.  

Code of Federal Regulations, 2013 CFR

...CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN...INDUSTRIAL EQUIPMENT Electric Motors Labeling...nameplate of an electric motor for which...nominal full load efficiency (as of the date...Disclosure of efficiency information in...must appear on an electric motor's...

2013-01-01

328

10 CFR 431.31 - Labeling requirements.  

Code of Federal Regulations, 2011 CFR

...CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN...INDUSTRIAL EQUIPMENT Electric Motors Labeling...nameplate of an electric motor for which...nominal full load efficiency (as of the date...Disclosure of efficiency information in...must appear on an electric motor's...

2011-01-01

329

Synthesis Of Labeled Metabolites  

DOEpatents

The present invention is directed to labeled compounds, for example, isotopically enriched mustard gas metabolites including: [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1-[[2-(methylsulfinyl)ethyl]sulfonyl]-2-(methylthio); [1,1',2,2'-.sup.13 C.sub.4 ]ethane, 1,1'-sulfonylbis[2-(methylsulfinyl)]; and, 2,2'-sulfinylbis([1,2-.sup.13 C.sub.2 ]ethanol of the general formula ##STR1## where Q.sup.1 is selected from the group consisting of sulfide (--S--), sulfone (--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), at least one C* is .sup.13 C, X is selected from the group consisting of hydrogen and deuterium, and Z is selected from the group consisting of hydroxide (--OH), and --Q.sup.2 --R where Q.sup.2 is selected from the group consisting of sulfide (--S--), sulfone(--S(O)--), sulfoxide (--S(O.sub.2)--) and oxide (--O--), and R is selected from the group consisting of hydrogen, a C.sub.1 to C.sub.4 lower alkyl, and amino acid moieties, with the proviso that when Z is a hydroxide and Q.sup.1 is a sulfide, then at least one X is deuterium.

Martinez, Rodolfo A. (Santa Fe, NM); Silks, III, Louis A. (Los Alamos, NM); Unkefer, Clifford J. (Los Alamos, NM); Atcher, Robert (White Rock, NM)

2004-03-23

330

Read the Label First  

MedlinePLUS

... EPA This Area You are here: EPA Home Pesticides Fact Sheets Health & Safety Read the Label First Read the Label First Need help with product labels? Click and explore the generic label. It will help you understand the ... By their nature, many pesticides may pose some risk to humans, animals, or ...

331

Relationships of stable carbon isotopes, plant water potential and growth: an approach to asses water use efficiency and growth strategies of dry land agroforestry species  

Microsoft Academic Search

The relationships between annual wood stable carbon isotope composition (?13C), dry season midday plant water potential, and annual growth rate were investigated to asses the ability of agroforestry\\u000a species to adapt to climate changes. 6–8 stem disks from four co-occurring species (Acacia senegal, A. seyal, A. tortilis and Balanites aegyptiaca) were collected for radial growth measurements using tree-ring analysis spanning

Aster Gebrekirstos; Meine van Noordwijk; Henry Neufeldt; Ralph Mitlöhner

2011-01-01

332

Preparation and properties of clickable amino analogues of the duocarmycins: factors that affect the efficiency of their fluorescent labelling of DNA.  

PubMed

Herein we report the synthesis of three DNA-alkylating amino analogues of the duocarmycins that carry an alkyne functional group suitable for copper-catalysed click chemistry. The alkyne-containing substituents are connected via a side chain position which projects away from the minor groove, and have only a small effect on DNA alkylation and cytotoxicity. The efficiency of click reactions with fluorophore azides was studied using alkylated ctDNA by analysing the adenine adducts produced after thermal depurination. Click reactions "on DNA" were sensitive to steric effects (tether length to the alkyne) and, surprisingly, to the nature of the fluorophore azide. With the best combination of click partners and reagents, adducts could be detected in the nuclei of treated cells by microscopy or flow cytometry, provided that an appropriate detergent (Triton X-100 and not Tween?20) was used for permeabilisation. The method is sensitive enough to detect adducts at physiologically relevant concentrations, and could have application in the development of nitro analogues of the duocarmycins as hypoxia-activated anticancer prodrugs. PMID:25044224

Tercel, Moana; McManaway, Sarah P; Liyanage, H D Sarath; Pruijn, Frederik B

2014-09-01

333

Atom trap trace analysis of krypton isotopes  

SciTech Connect

A new method of ultrasensitive isotope trace analysis has been developed. This method, based on the technique of laser manipulation of neutral atoms, has been used to count individual {sup 85}Kr and {sup 81}Kr atoms present in a natural krypton gas sample with isotopic abundances in the range of 10{sup {minus}11} and 10{sup {minus}13}, respectively. This method is free of contamination from other isotopes and elements and can be applied to several different isotope tracers for a wide range of applications. The demonstrated detection efficiency is 1 x 10{sup {minus}7}. System improvements could increase the efficiency by many orders of magnitude.

Bailey, K.; Chen, C. Y.; Du, X.; Li, Y. M.; Lu, Z.-T.; O'Connor, T. P.; Young, L.

1999-11-17

334

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

ERIC Educational Resources Information Center

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…

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

2010-01-01

335

Determination of Multiple ?-Torsion Angles in Proteins by Selective and Extensive 13C Labeling and Two-Dimensional Solid-State NMR  

NASA Astrophysics Data System (ADS)

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 C ? 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 ? 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.

Hong, Mei

1999-08-01

336

A method combining SPITC and ¹?O labeling for simultaneous protein identification and relative quantification.  

PubMed

The relative quantification and identification of proteins by matrix-assisted laser desorption ionization time-of-flight MS is very important in /MS is very important in protein research and is usually conducted separately. Chemical N-terminal derivatization with 4-sulphophenyl isothiocyanate facilitates de novo sequencing analysis and accurate protein identification, while (18)O labeling is simple, specific and widely applicable among the isotopic labeling methods used for relative quantification. In the present study, a method combining 4-sulphophenyl isothiocyanate derivatization with (18)O isotopic labeling was established to identify and quantify proteins simultaneously in one experiment. Reaction conditions were first optimized using a standard peptide (fibrin peptide) and tryptic peptides from the model protein (bovine serum albumin). Under the optimized conditions, these two independent labeling steps show good compatibility, and the linear relativity of quantification within the ten times dynamic range was stable as revealed by correlation coefficient analysis (R(2) value?=?0.998); moreover, precursor peaks in MS/MS spectrum could provide accurate quantitative information, which is usually acquired from MS spectrum, enabling protein identification and quantification in a single MS/MS spectrum. Next, this method was applied to native peptides isolated from spider venoms. As expected, the de novo sequencing results of each peptide matched with the known sequence precisely, and the measured quantitative ratio of each peptide corresponded well with the theoretical ratio. Finally, complex protein mixtures of spider venoms from male and female species with unknown genome information were analyzed. Differentially expressed proteins were successfully identified, and their quantitative information was also accessed. Taken together, this protein identification and quantification method is simple, reliable and efficient, which has a good potential in the exploration of peptides/proteins from species with unknown genome. PMID:24809901

Zhang, Wenlong; Long, Jia; Zhang, Cheng; Cai, Naixuan; Liu, Zhonghua; Wang, Ying; Wang, Xianchun; Chen, Ping; Liang, Songping

2014-05-01

337

Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry  

PubMed Central

Multi-isotope imaging mass spectrometry (MIMS) is the quantitative imaging of stable isotope labels in cells with a new type of secondary ion mass spectrometer (NanoSIMS). The power of the methodology is attributable to (i) the immense advantage of using non-toxic stable isotope labels, (ii) high resolution imaging that approaches the resolution of usual transmission electron microscopy and (iii) the precise quantification of label down to 1 part-per-million and spanning several orders of magnitude. Here we review the basic elements of MIMS and describe new applications of MIMS to the quantitative study of metabolic processes including protein and nucleic acid synthesis in model organisms ranging from microbes to humans. PMID:23660233

Steinhauser, Matthew L.; Lechene, Claude P.

2014-01-01

338

Imaging of inflammation with indium-111 tropolonate labeled leukocytes  

Microsoft Academic Search

Indium-111 tropolonate has recently been introduced as a new cell-labeling agent. It has the interesting property of labeling cells in plasma with high efficiency, and may therefore promote an improvement in viability of labeled cells. This paper describes researchers initial experience with ¹¹¹In tropolonate as a leukocyte label for abscess imaging. Pure populations of separated granulocytes, as well as crude

A. Michael Peters; Sethna H. Saverymuttu; Helen J. Reavy; Heather J. Danpure; Safiye Osman; J. Peter Lavender

1983-01-01

339

Methodological Considerations for the Use of Stable Isotope Probing in Microbial Ecology  

Microsoft Academic Search

Stable isotope probing (SIP) is a method used for labeling uncultivated microorganisms in environmental samples or directly\\u000a in field studies using substrate enriched with stable isotope (e.g., 13C). After consumption of the substrate, the cells of microorganisms that consumed the substrate become enriched in the isotope.\\u000a Labeled biomarkers, such as phospholipid-derived fatty acid (PLFA), ribosomal RNA, and DNA can be

Josh D. Neufeld; Marc G. Dumont; Jyotsna Vohra; J. Colin Murrell

2007-01-01

340

Insights from Stable Isotopes on the Role of Terrestrial Ecosystems in the Global Carbon Cycle  

Microsoft Academic Search

The use of isotopic tracers in organic matter, water, and atmospheric gases has become an important component of the study of ecology and global change. Physiological and physical processes discriminate against heavy isotopes in predictable ways, so that measurements of isotopes at natural abundance, i.e., naturally occurring concentrations as opposed to artificial labeling experiments, can provide useful information about biological

Diane E. Pataki; Chun-Ta Lai; Charles D. Keeling; James R. Ehleringer

341

Assessment of vitamin A status in rats by isotope dilution: A simplified model  

SciTech Connect

Isotope-dilution analysis of vitamin A status requires giving a known quantity of labeled vitamin A to the subject and measuring the ratio of labeled to unlabeled retinol in the blood after a period for equilibration. To calculate total body stores from the isotopic ratio of plasma retinol, several assumptions must be made. In considering new ways of better calculating liver vitamin A stores from isotope-dilution data, the authors used the data of Green et al. to estimate loss of vitamin A tracer as a function of time and of vitamin A status. This correction markedly improves the correlation between calculated and analyzed liver vitamin A stores and also quantitively explains the hyperbolic relationship between fraction of tracer dose recovered in liver and mass of liver vitamin A stores. Agreement of this model with experimental data suggests that efficiency of absorption and storage of vitamin A is not affected by vitamin A status. This model can be used to estimate both the amount of tracer needed for a given lower limit of detection and an optimum sampling time.

Furr, H.C.; Cooper, D.A.; Olson, J.A. (Iowa State Univ., Ames (United States))

1990-02-26

342

LabelRank: A Stabilized Label Propagation Algorithm for Community Detection in Networks  

E-print Network

An important challenge in big data analysis nowadays is detection of cohesive groups in large-scale networks, including social networks, genetic networks, communication networks and so. In this paper, we propose LabelRank, an efficient algorithm detecting communities through label propagation. A set of operators is introduced to control and stabilize the propagation dynamics. These operations resolve the randomness issue in traditional label propagation algorithms (LPA), stabilizing the discovered communities in all runs of the same network. Tests on real-world networks demonstrate that LabelRank significantly improves the quality of detected communities compared to LPA, as well as other popular algorithms.

Xie, Jierui

2013-01-01

343

Semiotic labelled deductive systems  

SciTech Connect

We review the class of Semiotic Models put forward by Pospelov, as well as the Labelled Deductive Systems developed by Gabbay, and construct an embedding of Semiotic Models into Labelled Deductive Systems.

Nossum, R.T. [Imperial College of Science, Technology and Medicine, London (United Kingdom)

1996-12-31

344

Isotope Science and Production  

E-print Network

Isotope Science and Production 35 years of experience in isotope production, processing. Contact: Kevin John LANL Isotope Program Manager kjohn@lanl.gov 505-667-3602 Sponsored by the Department of Energy National Isotope Program http://www.nuclear.energy.gov/isotopes/nelsotopes2a.html Isotopes

345

Rapid Verification of Candidate Serological Biomarkers Using Gel-based, Label-free Multiple Reaction Monitoring  

PubMed Central

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 ?l 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

Tang, Hsin-Yao; Beer, Lynn A.; Barnhart, Kurt T.; Speicher, David W.

2011-01-01

346

3DSpectra: A 3-dimensional quantification algorithm for LC-MS labeled profile data.  

PubMed

Mass spectrometry-based proteomics can generate highly informative datasets, as profile three-dimensional (3D) LC-MS data: LC-MS separates peptides in two dimensions (time, m/z) minimizing their overlap, and profile acquisition enhances quantification. To exploit both data features, we developed 3DSpectra, a 3D approach embedding a statistical method for peptide border recognition. 3DSpectra efficiently accesses profile data by means of mzRTree, and makes use of a priori metadata, provided by search engines, to quantify the identified peptides. An isotopic distribution model, shaped by a bivariate Gaussian Mixture Model (GMM), which includes a noise component, is fitted to the peptide peaks using the expectation-maximization (EM) approach. The EM starting parameters, i.e., the centers and shapes of the Gaussians, are retrieved from the metadata. The borders of the peaks are delimited by the GMM iso-density curves, and noisy or outlying data are discarded from subsequent analysis. The 3DSpectra program was compared to ASAPRatio for a controlled mixture of Isotope-Coded Protein Labels (ICPL) labeled proteins, which were mixed at predefined ratios and acquired in enhanced profile mode, in triplicate. The 3DSpectra software showed significantly higher linearity, quantification accuracy, and precision than did ASAPRatio in this real use case simulation where the true ratios are known, and it also achieved wider peptide coverage and dynamic range. PMID:25218586

Nasso, S; Hartler, J; Trajanoski, Z; Di Camillo, B; Mechtler, K; Toffolo, G M

2014-09-16

347

Use of recombinantly produced 15N3-labelled nicotianamine for fast and sensitive stable isotope dilution ultra-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry.  

PubMed

Nicotianamine (NA) is an important metal chelator, implicated in the intra- and intercellular trafficking of several transition metal ions in plants. To decipher its roles in physiological processes such as micronutrient acquisition, distribution or storage, fast and sensitive analytical techniques for quantification of this non-proteinogenic amino acid will be required. The use of a recombinant Schizosaccharomyces pombe strain expressing a nicotianamine synthase (NAS) gene allowed for the production of [(15)N(3)]-NA, which was enriched from cell extracts through cation exchange and used for stable isotope dilution analysis of NA. Such an approach should be widely applicable to important bioanalytes that are difficult to synthesize. The analytical procedure comprises mild aqueous extraction and rapid Fmoc derivatization, followed by fast separation using ultra-performance liquid chromatography (UPLC) and sensitive detection by positive ion electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) with a chromatographic cycle time of only 8 min. Derivatization was optimized with respect to incubation time and species suitable for quantification. The limit of detection was 0.14 to 0.23 pmol in biological matrices with the response being linear up to 42 pmol. Recovery rates were between 83% and 104% in various biological matrices including fission yeast cells, fungal mycelium, plant leaves and roots. PMID:21110009

Schmidt, Holger; Böttcher, Christoph; Trampczynska, Aleksandra; Clemens, Stephan

2011-01-01

348

A whole-forest 14C pulse-label study of microbial dynamics and root turnover (EBIS*)  

E-print Network

A whole-forest 14C pulse-label study of microbial dynamics and root turnover (EBIS*) Margaret S, Irvine, USA; 3 Belowground Forest Research, USA, 4 Lawrence Livermore National Laboratory, USA 5 Oak a pulse label of 14C in plant biomass of the local forests. This whole-ecosystem isotopic label presents

349

Heat-shock response in Arabidopsis thaliana explored by multiplexed quantitative proteomics using differential metabolic labeling.  

PubMed

We have developed a general method for multiplexed quantitative proteomics using differential metabolic stable isotope labeling and mass spectrometry. The method was successfully used to study the dynamics of heat-shock response in Arabidopsis thaliana. A number of known heat-shock proteins were confirmed, and some proteins not previously associated with heat shock were discovered. The method is applicable in stable isotope labeling and allows for high degrees of multiplexing. PMID:18189342

Palmblad, Magnus; Mills, Davinia J; Bindschedler, Laurence V

2008-02-01

350

Encoding and transmission of orthogonally optical label switching using DQPSK payload and MD-RZ label  

NASA Astrophysics Data System (ADS)

Optical label switching (OLS) has been regarded as an efficient technique to route and forward IP packets transparently in the optical layer. In label switching system, orthogonal modulation has shown some advantages, such as its high spectral efficiency. One example is the combination of amplitude-shift-keying (ASK) label and differential phase-shift-keying (DPSK) payload. However, differential quadrature phase-shift-keying (DQPSK) payload has not been used in optical label switching system yet. In this letter, we propose and demonstrate a novel optical label switching scheme by the combination modified return zero (MD-RZ) and DQPSK for the first time. The transmitter is set up by cascading two phase modulators (PM) and one dual-arm LiNbO3 modulator (LN-MOD). The signal is first phase modulated by two PMs. The MD-RZ label is then impressed by the subsequent LN-MOD. Duty cycle and extinction ratio of the MD-RZ labels are tunable. The DQPSK payload can be used in a 20Gbit/s optical label switching system, with 2-bit per symbol modulation/demodulation. Within the network, the label information is read, processed and updated, a process known as label switching, until the burst reaches the desired egress edge node. The use of balanced detection is expected to improve performance for the payload. The transmission performance of 20Gbit/s DQPSK payload with 2.5Gb/s MD-RZ-ASK label is also analysed. MD-RZ labeling of a DQPSK payload has been shown to be a promising candidate for the implementation of OLS system.

Shao, Yufeng; Wen, Shuangchun; Chen, Lin; Xiao, Yaoqiang

2008-03-01

351

Towards a "perfect" Penning trap mass spectrometer for unstable isotopes  

NASA Astrophysics Data System (ADS)

A Penning trap mass spectrometer has been set up at the on-line isotope separator ISOLDE/CERN for the mass determination of unstable heavy isotopes. The spectrometer should fulfil the following requirements: capture of external ions in high efficiency, high resolving power and accuracy, general applicability to all elements and isotopes available at the on-line facility.

Bollen, G.; Hartmann, H.; Kluge, H.-J.; König, M.; Otto, T.; Savard, G.; Stolzenberg, H.; ISOLDE Collaboration

1992-12-01

352

Refining the interpretation of hydroclimate proxies via the integration of a new, efficient isotope-enabled AGM and proxy system modeling: a comparative study  

NASA Astrophysics Data System (ADS)

High-resolution proxy data constitute highly valuable archives of regional hydrological cycle dynamics. In particular, hydroclimate proxy archives, such as records of ?18O or ?D in living and fossil corals, tree-ring cellulose, or speleothem calcite, lend insight into past atmospheric and oceanic changes and form the basis of climate reconstructions on decadal to millennial timescales. However, proxies generally contain a multivariate and non-linear response to regional climate forcing, limiting their usefulness. Disentangling the multivariate influences on biological proxies like corals and trees can be complex due to spatiotemporal climate variability, non-stationarity, and threshold dependence. Models which integrate both climate and the processes by which proxy systems respond to climate are needed to distinguish between climate signal and noise recorded in paleodata. We simulate the former using a fast, realistic, water-isotope-enabled atmospheric GCM ';SPEEDY' (modified from Molteni 2003) and the latter using proxy system models for observations made from tree and speleothem archives. We evaluate the skill of the coupled climate-proxy system model skill using forced SSTs from the NOAA ERSSTv2 (Smith and Reynolds, 2004) for the 20th century, and using CCSM4 SST output for the last millennium as inputs, and comparing the output to actual paleodata observations. We identify inter-model parametric sensitivities and structural differences between simulations and observations to constrain mechanistic understanding at the climate and proxy system levels, and to highlight uncertainties in our conceptual framework.

Dee, S. G.; Emile-Geay, J.; Noone, D. C.; Buenning, N. H.; Evans, M. N.

2013-12-01

353

Labeling earthworms uniformly with 13C and 15N: implications for monitoring nutrient fluxes  

Microsoft Academic Search

Stable isotopes hold promise for improving our ability to quantify energy and N released from earthworm populations through metabolic processes and mortality. However, the isotopic labels 13C and 15N must be incorporated uniformly into the structural and labile tissues of earthworms to trace C and N fluxes accurately. We examined the distribution of 13C and 15N in the tissue and

Joann K Whalen; H. Henry Janzen

2002-01-01

354

Mass Spectrometry-Based Label-Free Quantitative Proteomics  

PubMed Central

In order to study the differential protein expression in complex biological samples, strategies for rapid, highly reproducible and accurate quantification are necessary. Isotope labeling and fluorescent labeling techniques have been widely used in quantitative proteomics research. However, researchers are increasingly turning to label-free shotgun proteomics techniques for faster, cleaner, and simpler results. Mass spectrometry-based label-free quantitative proteomics falls into two general categories. In the first are the measurements of changes in chromatographic ion intensity such as peptide peak areas or peak heights. The second is based on the spectral counting of identified proteins. In this paper, we will discuss the technologies of these label-free quantitative methods, statistics, available computational software, and their applications in complex proteomics studies. PMID:19911078

Zhu, Wenhong; Smith, Jeffrey W.; Huang, Chun-Ming

2010-01-01

355

Isotope separation apparatus and method  

DOEpatents

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.

Feldman, Barry J. (Los Alamos, NM)

1985-01-01

356

BIOAVAILABILITY OF LUTEIN IN HUMANS FROM INTRINSICALLY LABELED VEGETABLES DETERMINED BY LC-APCI-MS  

Technology Transfer Automated Retrieval System (TEKTRAN)

The aim of the investigation was to assess a stable isotope method for determining the relative bioavailability of food-derived lutein in humans. Subjects were administered a single dose of deuterium-labeled carotenoids from intrinsically labeled spinach or collard green; 10 mL blood samples were d...

357

Bar Code Labels  

NASA Technical Reports Server (NTRS)

American Bar Codes, Inc. developed special bar code labels for inventory control of space shuttle parts and other space system components. ABC labels are made in a company-developed anodizing aluminum process and consecutively marketed with bar code symbology and human readable numbers. They offer extreme abrasion resistance and indefinite resistance to ultraviolet radiation, capable of withstanding 700 degree temperatures without deterioration and up to 1400 degrees with special designs. They offer high resistance to salt spray, cleaning fluids and mild acids. ABC is now producing these bar code labels commercially or industrial customers who also need labels to resist harsh environments.

1988-01-01

358

Assimilation efficiency for sediment-sorbed benzo(a)pyrene by Diporeia spp.  

USGS Publications Warehouse

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 ??m). The first approach, the direct measurement method, uses total organic carbon as a tracer and yielded AE values ranging from 45.9-60.4%. 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.

Lydy, M.J.; Landrum, P.F.

1993-01-01

359

Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short-strong hydrogen bonded cluster  

E-print Network

Isotope dependent, temperature regulated, energy repartitioning in a low-barrier, short/deuterium isotope effects, in a fundamental organic hydrogen bonded system using multiple experimental infrared the isotopically labeled systems arises from an analysis of the simulated cluster spectroscopy and leads

Iyengar, Srinivasan S.

360

Isotope effect of the phonons mean free path in graphene by micro-Raman measurement  

NASA Astrophysics Data System (ADS)

The isotope labeled graphene was synthesized in the concentration of 13C carbon atom in 1%, 25%, 50%, 75% and 99%. The isotope effect on the phonon behavior in graphene was investigated based on the micro-Raman analysis of 13C isotope labeled graphene samples. We found that the phonon scattering is affected by the isotopic carbon atom as a point defect. Based on the experiment results, the Klemens-Callaway model and uncertainty principle were used to obtain the mean free path of the G and D phonons. The results agree with the thermal conductivity measurement by non-contact optical method and with other theoretical calculations.

Zhang, CanKun; Li, QiongYu; Tian, Bo; Huang, ZhiYi; Lin, WeiYi; Li, HongYang; He, DaHai; Zhou, YingHui; Cai, WeiWei

2014-10-01

361

Tritium labeling of detonation nanodiamonds.  

PubMed

For the first time, the radioactive labeling of detonation nanodiamonds was efficiently achieved using a tritium microwave plasma. According to our measurements, the total radioactivity reaches 9120 ± 120 ?Ci mg(-1), with 93% of (3)H atoms tightly bonded to the surface and up to 7% embedded into the diamond core. Such (3)H doping will ensure highly stable radiolabeled nanodiamonds, on which surface functionalization is still allowed. This breakthrough opens the way to biodistribution and pharmacokinetics studies of nanodiamonds, while this approach can be scalable to easily treat bulk quantities of nanodiamonds at low cost. PMID:24492594

Girard, Hugues A; El-Kharbachi, Abdelouahab; Garcia-Argote, Sébastien; Petit, Tristan; Bergonzo, Philippe; Rousseau, Bernard; Arnault, Jean-Charles

2014-03-18

362

Dynamic Visualization of Graphs with Extended Labels  

SciTech Connect

The paper describes a novel technique to visualize graphs with extended node and link labels. The lengths of these labels range from a short phrase to a full sentence to an entire paragraph and beyond. Our solution is different from all the existing approaches that almost always rely on intensive computational effort to optimize the label placement problem. Instead, we share the visualization resources with the graph and present the label information in static, interactive, and dynamic modes without the requirement for tackling the intractability issues. This allows us to reallocate the computational resources for dynamic presentation of real-time information. The paper includes a user study to evaluate the effectiveness and efficiency of the visualization technique.

Wong, Pak C.; Mackey, Patrick S.; Perrine, Kenneth A.; Eagan, James R.; Foote, Harlan P.; Thomas, Jim

2005-10-23

363

Ideal regularization for learning kernels from labels.  

PubMed

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. PMID:24824969

Pan, Binbin; Lai, Jianhuang; Shen, Lixin

2014-08-01

364

Nutrition Labels and Obesity  

Microsoft Academic Search

The Nutrition Labeling and Education Act (NLEA) imposed significant changes in the information about calories and nutrients that manufacturers of packaged foods must provide to consumers. This paper tests whether the release of this information impacted body weight and obesity among American adults. We estimate the effect of the new label using a difference-in-differences method. We compare the change before

Jayachandran N. Variyam; John Cawley

2006-01-01

365

Monitoring electron donor metabolism under variable electron acceptor conditions using 13C-labeled lactate  

NASA Astrophysics Data System (ADS)

Three sets of flow-through columns constructed with aquifer sediment from Hanford (WA) were used to study reduction of Cr(VI) to poorly soluble Cr(III) under denitrifying, sulfate-reducing/fermentative, and iron-reducing conditions with lactate as the electron donor. In order to understand the relationship between electron donors and biomarkers, and to determine the differences in carbon isotope fractionation resulting from different microbial metabolic processes, we monitored the variation in carbon isotopes in dissolved inorganic carbon (DIC), in total organic carbon (TOC), and in lactate, acetate and propionate. The greatest enrichment in 13C in columns was observed under denitrifying conditions. The ?13C of DIC increased by ~1750 to ~2000‰ fifteen days after supplementation of natural abundance lactate with a 13C-labeled lactate tracer (for an influent ?13C of ~2250‰ for the lactate) indicating almost complete oxidation of the electron donor. The denitrifying columns were among the most active columns and had the highest cell counts and the denitrification rate was highly correlated with Cr(VI) reduction rate. ?13C values of DIC ranged from ~540 to ~1170‰ for iron-reducing conditions. The lower enrichment in iron columns was related to the lower biological activity observed with lower yields of RNA and cell numbers in the column effluents. The carbon isotope shift in the sulfate-reducing ~198 to ~1960‰ for sulfate-reducing conditions reflecting the lower levels of the lactate in these columns. Additionally, in two of the sulfate columns, almost complete fermentation of the lactate occurred, producing acetate and propionate with the labeled carbon signature, but relatively smaller amounts of inorganic carbon. For all electron-accepting conditions, TOC yielded similar ?13C values as lactate stock solutions. Differences in C use efficiency, metabolic rate or metabolic pathway contributed to the differing TOC ?13C to DIC ?13C ratios between treatments. Carbon isotope signatures of DIC can be useful for monitoring the efficiency of 13C-enriched electron donor consumption associated with bioactivity under reducing conditions.

Bill, M.; Conrad, M. E.; Yang, L.; Beller, H. R.; Brodie, E. L.

2010-12-01

366

42 CFR 84.1103 - Approval labels and markings; approval of contents; use.  

Code of Federal Regulations, 2011 CFR

...Efficiency Respirators and Combination Gas Masks § 84.1103 Approval labels and markings...Respirator type Label type Location Gas mask with a particulate filter, including pesticide gas mask Entire Mask and container. Dust,...

2011-10-01

367

42 CFR 84.1103 - Approval labels and markings; approval of contents; use.  

Code of Federal Regulations, 2010 CFR

...Efficiency Respirators and Combination Gas Masks § 84.1103 Approval labels and markings...Respirator type Label type Location Gas mask with a particulate filter, including pesticide gas mask Entire Mask and container. Dust,...

2010-10-01

368

Analytical techniques in biomedical stable isotope applications: (isotope ratio) mass spectrometry or infrared spectrometry?  

PubMed

An overview is presented of biomedical applications of stable isotopes in general, but mainly focused on the activities of the Center for Liver, Digestive and Metabolic Diseases of the University Medical Center Groningen. The aims of metabolic studies in the areas of glucose, fat, cholesterol and protein metabolism are briefly explained, as well as the principle of breath testing and the techniques to study body composition and energy expenditure. Much attention is paid to the analytical considerations based upon metabolite concentrations, sample size restrictions, the availability of stable isotope labelled substrates and dose requirements in relation to compound-specific isotope analysis. The instrumental advantages and limitations of the generally used techniques gas chromatography/reaction/isotope ratio mass spectrometry and gas chromatography/mass spectrometry are described as well as the novelties of the recently commercialised liquid chromatography/combustion/isotope ratio mass spectrometry. The present use and future perspective of infrared (IR) spectrometry for clinical and biomedical stable isotope applications are reviewed. In this respect, the analytical demands on IR spectrometry are discussed to enable replacement of isotope ratio mass spectrometry by IR spectrometry, in particular, for the purpose of compound-specific isotope ratio analysis in biological matrices. PMID:16543190

Stellaard, Frans; Elzinga, Henk

2005-12-01

369

Introduction Balanced Group Labeled Graphs  

E-print Network

Introduction Results Summary Balanced Group Labeled Graphs M. Joglekar N. Shah A.A. Diwan.A.Diwan Balanced Group Labeled Graphs #12;Introduction Results Summary Outline 1 Introduction Group Labeled Graphs Balanced Labellings Characterization 2 Results Counting Number of Balanced labellings Proof Markable Graphs

Diwan, Ajit A

370

Quantification of Peptide m/z Distributions from 13C-Labeled Cultures with High-Resolution Mass Spectrometry  

PubMed Central

Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify 13C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods. PMID:24387081

2014-01-01

371

Selenium as an alternative peptide label - comparison to fluorophore-labelled penetratin.  

PubMed

In the present study, the impact on peptide properties of labelling peptides with the fluorophore TAMRA or the selenium (Se) containing amino acid SeMet was evaluated. Three differently labelled variants of the cell-penetrating peptide (CPP) penetratin (Pen) were synthesized, PenM(Se), TAMRA-PenM(Se) and TAMRA-Pen. The labelled peptides were characterized in terms of hydrodynamic radius, secondary structure during peptide-membrane interaction, effect on membrane leakage induction, uptake efficiency in HeLa cells. Furthermore, stability of peptides and identities of degradation products in cell media and cell lysate were evaluated. TAMRA-labelling increased the hydrodynamic radius of Pen and PenM(Se) significantly. Labelling with Se caused no or minimal changes in size, secondary structure and membrane leakage induction in concentration levels relevant for cellular uptake studies. Similar degradation patterns of all labelled peptides were observed in HBSS media; degradation was mainly due to oxidation. Cellular uptake was significantly higher for the TAMRA labelled peptides as compared to Se-labelled Pen. Extensive degradation was observed in media during cellular uptake studies, however, in all cell lysates, primarily the intact peptide (PenM(Se), TAMRA-PenM(Se) or TAMRA-Pen) was observed. Selenium labelling caused minimal alteration of the physicochemical properties of the peptide and allowed for absolute quantitative determination of cellular uptake by inductively coupled plasma mass spectrometry. Selenium is thus proposed as a promising alternative label for quantification of peptides in general, altering the properties of the peptide to a minor extent as compared to commonly used peptide labels. PMID:25447743

Hyrup Møller, Laura; Bahnsen, Jesper Søborg; Nielsen, Hanne Mørck; Østergaard, Jesper; Stürup, Stefan; Gammelgaard, Bente

2015-01-25

372

International cigarette labelling practices  

PubMed Central

DESIGN—Cross-sectional study.?PARTICIPANTS—Members of GLOBALink (www.globalink.org), an internet listserve for tobacco activists with members in 56 countries, who were asked to provide specific information on cigarette warning requirements in their countries.?MAIN OUTCOME MEASURES—Presence of specific warning labels, overall content score (based on a 0-10 scale with a point for each specific warning mentioned), size of warning label, location of warning label.?RESULTS—Forty-five countries (80%) responded; 40 had mandatory labelling requirements, three had voluntary agreements with the industry and two had no requirements. In general, American companies did no more in foreign countries than required by local law. The average developing country content score was 1.6, compared with 5.0 in developed countries (p = 0.0003). Forty-two per cent of countries either had no warning requirement or had only a very general health warning. The most common warning was for heart disease (49% of countries) and the least common was for addiction (14%). All warnings were more common in developed than developing countries. Warnings in developed country were on average 27% larger than those in developing countries (p = 0.325). Seventy-three per cent of labels in developing countries appeared only on the side of the pack, whereas 78% of labels in developed countries appeared on the front and back (p = 0.003).?CONCLUSIONS—In almost every respect measured, residents in developing countries are receiving inferior information about the hazards of smoking than residents of developed countries. Laws should be promulgated in importing and exporting countries to ensure that, where their labelling laws differ, companies would be required to provide the more comprehensive labelling.???Keywords: health warnings; cigarette pack labelling; legislation PMID:10629241

Aftab, M.; Kolben, D.; Lurie, P.

1999-01-01

373

Capacitive label reader  

DOEpatents

A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label. 5 figs.

Arlowe, H.D.

1985-11-12

374

Capacitive label reader  

DOEpatents

A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

Arlowe, H. Duane (Albuquerque, NM)

1985-01-01

375

Capacitive label reader  

DOEpatents

A capacitive label reader includes an outer ring transmitting portion, an inner ring transmitting portion, and a plurality of insulated receiving portions. A label is the mirror-image of the reader except that identifying portions corresponding to the receiving portions are insulated from only one of two coupling elements. Positive and negative pulses applied, respectively, to the two transmitting rings biased a CMOS shift register positively to either a 1 or 0 condition. The output of the CMOS may be read as an indication of the label.

Arlowe, H.D.

1983-07-15

376

Eco-Labelling under Imperfect Certification: An Economic Analysis  

E-print Network

to pay a higher price for organic food products in U.K. A problem with environmental attributes of such goods include energy efficiency, organically produced food, green electricity, etc. The consumers cannot (1990), U.S. Energy Star (1992), EU Eco-label (1992) and Thai Green Label (1994). There are large

Bandyopadhyay, Antar

377

HAZARDOUS WASTE LABEL DEPAUL UNIVERSITY  

E-print Network

HAZARDOUS WASTE LABEL DEPAUL UNIVERSITY ENVIRONMENTAL HEALTH & SAFETY 5-4170 Corrosive Non- Hazardous Ignitable Reactive Toxic Oxidizer Other ( explain ) Generator Building Dept. HAZARDOUS WASTE LABEL DEPAUL UNIVERSITY ENVIRONMENTAL HEALTH & SAFETY 5-4170 HAZARDOUS WASTE LABEL DEPAUL UNIVERSITY

Schaefer, Marcus

378

How to Read Drug Labels  

MedlinePLUS

... Healthy Aging > Drugs and alternative medicine Healthy Aging How to read drug labels Printer-friendly version How to ... More information on how to read drug labels How to read a prescription drug label View a text ...

379

Comprehensive analysis of metabolic pathways through the combined use of multiple isotopic tracers  

E-print Network

Metabolic Flux Analysis (MFA) has emerged as a tool of great significance for metabolic engineering and the analysis of human metabolic diseases. An important limitation of MFA, as carried out via stable isotope labeling ...

Antoniewicz, Maciek Robert

2006-01-01

380

Stable isotope studies  

SciTech Connect

The research has been in four general areas: (1) correlation of isotope effects with molecular forces and molecular structures, (2) correlation of zero-point energy and its isotope effects with molecular structure and molecular forces, (3) vapor pressure isotope effects, and (4) fractionation of stable isotopes. 73 refs, 38 figs, 29 tabs.

Ishida, T.

1992-01-01

381

Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in proteomics.  

PubMed

Stable-isotope labeling coupled with liquid-phase separation and MS analysis is a powerful technique for comparative proteomics. We developed a dimethyl labeling strategy (Anal. Chem. 2003, 75, 6843-6852 and J. Proteome Res. 2005, 4, 101-108) to label peptide N-terminus and epsilon-amino groups of Lys with water-soluble formaldehyde via reductive methylation, and an isotopic pair of formaldehyde is used for binary labeling on two sets of samples. In this study, this approach is extended to a four sample labeling by combining the binary isotopic reagents of formaldehyde (d0, d2) and the binary isotopic reducing reagents, sodium cyanoborohydride (d0, d3). To ensure sufficient mass difference, this multiplexed labeling is coupled with endoproteinase Lys-C instead of trypsin for digestion, resulting in at least two labeling sites with a mass difference of 4 Da for each pair of peptide digest. Moreover, multiplex dimethyl labeling was proved to have no significant isotopic effect during RP LC elution. This method was further applied for monitoring Lys-C digestion using hemoglobin as a model. Dimethyl labeled digests derived from seven time points (1-30 h) were grouped into two sets of sample mixtures, separated by nano-LC to reduce the complexity, and then analyzed by ESI-MS/MS. The temporal study reveals that Lys-C digestion was completed in 10-15 h for all detected peptides. The multiplex dimethyl method has not only provided a simultaneous detection mean for four sample sets but has also conserved all the advantages associated with the original binary method. PMID:16927424

Hsu, Jue-Liang; Huang, Sheng-Yu; Chen, Shu-Hui

2006-09-01

382

Spectral Label Fusion  

E-print Network

We present a new segmentation approach that combines the strengths of label fusion and spectral clustering. The result is an atlas-based segmentation method guided by contour and texture cues in the test image. This offers ...

Wachinger, Christian

383

CARBON ISOTOPE DISCRIMINATION AND GROWTH RESPONSE TO STAND DENSITY REDUCTIONS IN OLD PINUS PONDEROSA TREES  

EPA Science Inventory

Carbon isotope ratios ( 13C) of tree rings are commonly used for paleoclimatic reconstruction and for inferring canopy water-use efficiency (WUE). However, the responsiveness of carbon isotope discrimination ( ) to site disturbance and resource availability has only rarely been ...

384

Isotope exchange reactions with radiolabeled sulfur compounds in anoxic seawater  

Microsoft Academic Search

The isotope exchange between35S-labeled sulfur compounds of sulfate (SO42-), elemental sulfur (S0), polysulfide (Sn2-), hydrogen sulfide (SHS-: H2S + HS- + S2-), iron sulfide (FeS), and pyrite (FeS2) was studied at pH 7.6 and 20 °C in anoxic, sterile seawater. Isotope exchange was observed between S0, S22- SHS-, and FeS, but not between35S labeled SO42- or FeS2 and the other

Henrik Fossing; Bo Ba Jørgensen

1990-01-01

385

10 CFR 431.30 - Applicability of labeling requirements.  

Code of Federal Regulations, 2010 CFR

...ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability...42 U.S.C. 6315, apply only to electric motors manufactured after October...

2010-01-01

386

10 CFR 431.30 - Applicability of labeling requirements.  

Code of Federal Regulations, 2012 CFR

...ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability...42 U.S.C. 6315, apply only to electric motors manufactured after October...

2012-01-01

387

10 CFR 431.30 - Applicability of labeling requirements.  

Code of Federal Regulations, 2011 CFR

...ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.30 Applicability...42 U.S.C. 6315, apply only to electric motors manufactured after October...

2011-01-01

388

Intermolecular and intramolecular isotope effects in the deamination of putrescine catalyzed by diamine oxidase  

Microsoft Academic Search

Summary The enzymatic deamination of 1,4-diaminobutane (putrescine) catalyzed by hog kidney diamine oxidase was studied with the aid of deuterium labeled substrates and mass spectrometry. An intermolecular deuterium isotope effect for the deamination of putrescine labeled with deuterium in all 4 alpha positions was observed to be 1.26. 1,4-Diaminobutane-1, 1-d2 was synthesized and intramolecular isotope effects determined. The preference of

P. S. Callery; M. S. B. Nayar; E. M. Jakubowski; M. Stogniew

1982-01-01

389

Probes labelled with energy transfer coupled dyes  

DOEpatents

Compositions are provided comprising sets of fluorescent labels carrying pairs of donor and acceptor dye molecules, designed for efficient excitation of the donors at a single wavelength and emission from the acceptor in each of the pairs at different wavelengths. The different molecules having different donor-acceptor pairs can be modified to have substantially the same mobility under separation conditions, by varying the distance between the donor and acceptor in a given pair. Particularly, the fluorescent compositions find use as labels in sequencing nucleic acids. 7 figs.

Mathies, R.A.; Glazer, A.; Ju, J.

1997-11-18

390