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Sample records for efficient isotopic labeling

  1. Efficient segmental isotope labeling of multi-domain proteins using Sortase A.

    PubMed

    Freiburger, Lee; Sonntag, Miriam; Hennig, Janosch; Li, Jian; Zou, Peijian; Sattler, Michael

    2015-09-01

    NMR studies of multi-domain protein complexes provide unique insight into their molecular interactions and dynamics in solution. For large proteins domain-selective isotope labeling is desired to reduce signal overlap, but available methods require extensive optimization and often give poor ligation yields. We present an optimized strategy for segmental labeling of multi-domain proteins using the S. aureus transpeptidase Sortase A. Critical improvements compared to existing protocols are (1) the efficient removal of cleaved peptide fragments by centrifugal filtration and (2) a strategic design of cleavable and non-cleavable affinity tags for purification. Our approach enables routine production of milligram amounts of purified segmentally labeled protein for NMR and other biophysical studies. PMID:26319988

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

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

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

    PubMed Central

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

    2012-01-01

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

  4. Glutathione specifically labeled with isotopes

    SciTech Connect

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

    1985-10-01

    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.

  5. SAIL--stereo-array isotope labeling.

    PubMed

    Kainosho, Masatsune; Gntert, Peter

    2009-11-01

    Optimal stereospecific and regiospecific labeling of proteins with stable isotopes enhances the nuclear magnetic resonance (NMR) method for the determination of the three-dimensional protein structures in solution. Stereo-array isotope labeling (SAIL) offers sharpened lines, spectral simplification without loss of information and the ability to rapidly collect and automatically evaluate the structural restraints required to solve a high-quality solution structure for proteins up to twice as large as before. This review gives an overview of stable isotope labeling methods for NMR spectroscopy with proteins and provides an in-depth treatment of the SAIL technology. PMID:20370954

  6. Optimal design of isotope labeling experiments.

    PubMed

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

    2014-01-01

    Stable isotope labeling experiments (ILE) constitute a powerful methodology for estimating metabolic fluxes. An optimal label design for such an experiment is necessary to maximize the precision with which fluxes can be determined. But often, precision gained in the determination of one flux comes at the expense of the precision of other fluxes, and an appropriate label design therefore foremost depends on the question the investigator wants to address. One could liken ILE to shadows that metabolism casts on products. Optimal label design is the placement of the lamp; creating clear shadows for some parts of metabolism and obscuring others.An optimal isotope label design is influenced by: (1) the network structure; (2) the true flux values; (3) the available label measurements; and, (4) commercially available substrates. The first two aspects are dictated by nature and constrain any optimal design. The second two aspects are suitable design parameters. To create an optimal label design, an explicit optimization criterion needs to be formulated. This usually is a property of the flux covariance matrix, which can be augmented by weighting label substrate cost. An optimal design is found by using such a criterion as an objective function for an optimizer. This chapter uses a simple elementary metabolite units (EMU) representation of the TCA cycle to illustrate the process of experimental design of isotope labeled substrates. PMID:24218214

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

    DOEpatents

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

    2011-10-04

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

  8. Gluconeogenesis from labeled carbon: estimating isotope dilution

    SciTech Connect

    Kelleher, J.K.

    1986-03-01

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

  9. Isotope labeling methods for relaxation measurements.

    PubMed

    Lundstrm, Patrik; Ahlner, Alexandra; Blissing, Annica Theresia

    2012-01-01

    Nuclear magnetic spin relaxation has emerged as a powerful technique for probing molecular dynamics. Not only is it possible to use it for determination of time constant(s) for molecular reorientation but it can also be used to characterize internal motions on time scales from picoseconds to seconds. Traditionally, uniformly (15)N labeled samples have been used for these experiments but it is clear that this limits the applications. For instance, sensitivity for large systems is dramatically increased if dynamics is probed at methyl groups and structural characterization of low-populated states requires measurements on (13)C?, (13)C? or (13)CO or (1)H?. Unfortunately, homonuclear scalar couplings may lead to artifacts in the latter types of experiments and selective isotopic labeling schemes that only label the desired position are necessary. Both selective and uniform labeling schemes for measurements of relaxation rates for a large number of positions in proteins are discussed in this chapter. PMID:23076579

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

    PubMed

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

    2015-03-19

    Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe?O? nanocrystals (Fe?O?-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe?O? nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe?O?-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests. PMID:25757497

  11. The topology of metabolic isotope labeling networks

    PubMed Central

    Weitzel, Michael; Wiechert, Wolfgang; Nh, Katharina

    2007-01-01

    Background Metabolic Flux Analysis (MFA) based on isotope labeling experiments (ILEs) is a widely established tool for determining fluxes in metabolic pathways. Isotope labeling networks (ILNs) contain all essential information required to describe the flow of labeled material in an ILE. Whereas recent experimental progress paves the way for high-throughput MFA, large network investigations and exact statistical methods, these developments are still limited by the poor performance of computational routines used for the evaluation and design of ILEs. In this context, the global analysis of ILN topology turns out to be a clue for realizing large speedup factors in all required computational procedures. Results With a strong focus on the speedup of algorithms the topology of ILNs is investigated using graph theoretic concepts and algorithms. A rigorous determination of all cyclic and isomorphic subnetworks, accompanied by the global analysis of ILN connectivity is performed. Particularly, it is proven that ILNs always brake up into a large number of small strongly connected components (SCCs) and, moreover, there are natural isomorphisms between many of these SCCs. All presented techniques are universal, i.e. they do not require special assumptions on the network structure, bidirectionality of fluxes, measurement configuration, or label input. The general results are exemplified with a practically relevant metabolic network which describes the central metabolism of E. coli comprising 10390 isotopomer pools. Conclusion Exploiting the topological features of ILNs leads to a significant speedup of all universal algorithms for ILE evaluation. It is proven in theory and exemplified with the E. coli example that a speedup factor of about 1000 compared to standard algorithms is achieved. This widely opens the door for new high performance algorithms suitable for high throughput applications and large ILNs. Moreover, for the first time the global topological analysis of ILNs allows to comprehensively describe and understand the general patterns of label flow in complex networks. This is an invaluable tool for the structural design of new experiments and the interpretation of measured data. PMID:17727715

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

    Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3O4 nanocrystals (Fe3O4-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe3O4 nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe3O4-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests.Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe3O4 nanocrystals (Fe3O4-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe3O4 nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe3O4-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests. Electronic supplementary information (ESI) available: Sequence of phosphopeptides from the digests of α- and β-casein percentages of the 4 methylated products from peptide β1 at different labeling reaction times; sequence of serum phosphopeptides; XPS spectra of Nb 3d and Ti 2p in layered oxides and H+-stacked nanosheets; phosphopeptide enrichment sensitivity of bulk oxides, layered oxides and H+-stacked nanosheets; AFM image of TiNbNS; saturated adsorption isotherm for pNPP adsorbed on bulk oxides, layered oxides and H+-stacked nanosheets; XPS spectra of Fe3O4-TiNbNS nitrogen adsorption-desorption isotherms and pore size distribution curves for the Fe3O4 nanocrystals; phosphopeptide enrichment sensitivity, capacity and selectivity of the Fe3O4-TiNbNS composites; MS/MS spectra of phosphopeptides enriched from serum; linear relationship between the logarithms of peak area ratio and loading volume ratio. See DOI: 10.1039/c4nr07041k

  13. Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels.

    PubMed

    Arlinghaus, H F; Kwoka, M N; Guo, X Q; Jacobson, K B

    1997-04-15

    A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched in isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. This new SBH method, which employs stable isotopic labels to locate target DNAs and TOF-RIMS to detect the labels, will be a very versatile and extensive multiplexing method. PMID:9109351

  14. Multiplexed DNA sequencing and diagnostics by hybridization with enriched stable isotope labels

    SciTech Connect

    Arlinghaus, H.F.; Kwoka, M.N.; Guo, X.Q.; Jacobson, K.B.

    1997-04-15

    A new DNA diagnostic and sequencing system has been developed that uses time-of-flight resonance ionization mass spectrometry (TOF-RIMS) to provide a rapid method of analyzing stable isotope-labeled oligonucleotides in form 1 sequencing by hybridization (SBH). With form 1, the DNA is immobilized on a nylon membrane and enriched isotope-labeled individual oligonucleotide probes are free to seek out complementary DNAs during hybridization. The major advantage of this new approach is that multiple oligonucleotides can be labeled with different enriched isotopes and can all be simultaneously hybridized to the genosensor matrix. The probes can then be simultaneously detected with TOF-RIMS with high selectivity, sensitivity, and efficiency. By using isotopically enriched tin labels, up to 10 labeled oligonucleotides could be examined in a single hybridization to the DNA matrix. Greater numbers of labels are available if rare earth isotopes are employed. In the present study, matrices containing three different DNAs were prepared and simultaneously hybridized with two different probes under a variety of conditions. The results show that DNAs, immobilized on nylon surfaces, can be specifically hybridized to probes labeled with different enriched tin isotopes. Discrimination between complementary and noncomplementary sites of better than 100 was obtained in multiplexed samples. 34 refs., 5 figs.

  15. Stable isotope labeling strategy based on coding theory.

    PubMed

    Kasai, Takuma; Koshiba, Seizo; Yokoyama, Jun; Kigawa, Takanori

    2015-10-01

    We describe a strategy for stable isotope-aided protein nuclear magnetic resonance (NMR) analysis, called stable isotope encoding. The basic idea of this strategy is that amino-acid selective labeling can be considered as "encoding and decoding" processes, in which the information of amino acid type is encoded by the stable isotope labeling ratio of the corresponding residue and it is decoded by analyzing NMR spectra. According to the idea, the strategy can diminish the required number of labelled samples by increasing information content per sample, enabling discrimination of 19 kinds of non-proline amino acids with only three labeled samples. The idea also enables this strategy to combine with information technologies, such as error detection by check digit, to improve the robustness of analyses with low quality data. Stable isotope encoding will facilitate NMR analyses of proteins under non-ideal conditions, such as those in large complex systems, with low-solubility, and in living cells. PMID:26293126

  16. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.

    PubMed

    Boersema, Paul J; Raijmakers, Reinout; Lemeer, Simone; Mohammed, Shabaz; Heck, Albert J R

    2009-01-01

    Accurate quantification of protein expression in biological systems is an increasingly important part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments. Although alternative multiplex quantitative proteomics approaches introduce isotope labels at the organism level ('stable isotope labeling by amino acids in cell culture' (SILAC)) or enable the simultaneous analysis of eight samples (isobaric tagging for relative and absolute quantification (iTRAQ)), stable isotope dimethyl labeling is advantageous in that it uses inexpensive reagents and is applicable to virtually any sample. We describe in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams. The labeling steps take approximately 60-90 min, whereas the full protocol including digestion and (two-dimensional) liquid chromatography-mass spectrometry takes approximately 1.5-3 days to complete. PMID:19300442

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

    PubMed

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

    2015-04-01

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

  18. Improving cell-free protein synthesis for stable-isotope labeling.

    PubMed

    Matsuda, Takayoshi; Koshiba, Seizo; Tochio, Naoya; Seki, Eiko; Iwasaki, Noriyuki; Yabuki, Takashi; Inoue, Makoto; Yokoyama, Shigeyuki; Kigawa, Takanori

    2007-03-01

    Cell-free protein synthesis is suitable for stable-isotope labeling of proteins for NMR analysis. The Escherichia coli cell-free system containing potassium acetate for efficient translation (KOAc system) is usually used for stable-isotope labeling, although it is less productive than other systems. A system containing a high concentration of potassium L-glutamate (L-Glu system), instead of potassium acetate, is highly productive, but cannot be used for stable-isotope labeling of Glu residues. In this study, we have developed a new cell-free system that uses potassium D-glutamate (D-Glu system). The productivity of the D-Glu system is approximately twice that of the KOAc system. The cross peak intensities in the 1H-15N HSQC spectrum of the uniformly stable-isotope labeled Ras protein, prepared with the D-Glu system, were similar to those obtained with the KOAc system, except that the Asp intensities were much higher for the protein produced with the D-Glu system. These results indicate that the D-Glu system is a highly productive cell-free system that is especially useful for stable-isotope labeling of proteins. PMID:17237976

  19. Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

    PubMed

    Rappel, Christina; Schaumlffel, Dirk

    2009-01-01

    The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements. PMID:19117464

  20. Quantifying plant phenotypes with isotopic labeling & metabolic flux analysis.

    PubMed

    Allen, Doug K

    2016-02-01

    Analyses of metabolic flux using stable isotopes in plants have traditionally been restricted to tissues with presumed homogeneous cell populations and long metabolic steady states such as developing seeds, cell suspensions, or cultured roots and root tips. It is now possible to describe these and other metabolically more dynamic tissues such as leaves in greater detail using novel methods in mass spectrometry, isotope labeling strategies, and transient labeling-based flux analyses. Such studies are necessary for a systems level description of plant function that more closely represents biological reality, and provides insights into the genes that will need to be modified as natural resources become ever more limited and environments change. PMID:26613198

  1. Synthesis of stable isotope-labeled epothilone D using a degradation-reconstruction approach.

    PubMed

    Burrell, Richard C; Turley, Wesley A; Bonacorsi, Samuel J

    2015-07-01

    The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope-labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope-labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)-3-acetyl-7,11-dihydroxy-8,8,10,12,16-pentamethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n-butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)-16-acetyl-4,8-dihydroxy-5,5,7,9,13-pentamethyloxacyclohexadec-13-ene-2,6-dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)-16-acetyl-5,5,7,9,13-pentamethyl-4,8-bis(triethylsilyloxy)-oxacyclohexadec-13-ene-2,6-dione. This intermediate was coupled to a stable isotope-labeled thiazole using a Wittig reaction as the key step to provide (13)C5, (15)N-labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of (13)C5, (15)N-labeled epothilone D was prepared in an 8% overall yield from (13)C2, (15)N-labeled thioacetamide and (13)C3-labeled ethyl bromopyruvate. PMID:26158758

  2. Isotopic Labeling of Red Cabbage Anthocyanins with Atmospheric 13-CO2

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Isotopic labeling of plants provides a unique opportunity for understanding metabolic processes. A significant challenge of isotopic labeling during plant growth is that isotopes must be administered without disrupting plant development and at sufficient levels for mass spectral analysis. We describ...

  3. Dimethyl isotope labeling assisted de novo peptide sequencing.

    PubMed

    Hennrich, Marco L; Mohammed, Shabaz; Altelaar, A F Maarten; Heck, Albert J R

    2010-12-01

    Here, we explore a de novo sequencing strategy in which we combine Lys-N protein digestion with differential isotopic dimethyl labeling to facilitate the (de novo) identification of multiply charged peptides in ESI-MS, both under CID and ETD conditions. For a large fraction of the Lys-N generated peptides, all primary amines are present at the N-terminal lysine, enabling specific labeling of the N-terminus. Differential derivatization of only the peptide N-terminus in combination with the simultaneous fragmentation of the corresponding isotopologues allows the straightforward distinction of N-terminal fragments from C-terminal and internal fragments. Furthermore, also singly and multiply charged N-terminal fragments can easily be distinguished due to the mass differences of the isotope labeled fragment pairs. As a proof of concept, we applied this approach to proteins isolated from an avocado fruit, and were able to partially de novo sequence and correctly align, with green plant homologues, a previously uncharacterized avocado ascorbate peroxidase. PMID:20850342

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

    PubMed

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

    2015-10-01

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

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

    PubMed Central

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

    2015-01-01

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

  6. ESTIMATION OF LABELING EFFICIENCY IN PSEUDO-CONTINUOUS ARTERIAL SPIN LABELING

    PubMed Central

    Aslan, Sina; Xu, Feng; Wang, Peiying L.; Uh, Jinsoo; Yezhuvath, Uma S.; van Osch, Matthias; Lu, Hanzhang

    2009-01-01

    Pseudo-Continuous Arterial Spin Labeling (pCASL) MRI is a new ASL technique that has the potential of combining advantages of Continuous-ASL and Pulsed-ASL. However, unlike CASL, the labeling process of pCASL is not strictly an adiabatic inversion and the efficiency of labeling may be subject-specific. Here three experiments were performed to study the labeling efficiency in pCASL MRI. First, the optimal labeling position was determined empirically to be approximately 84 mm below Anterior-Commissure Posterior-Commissure line in order to achieve the highest sensitivity. Second, an experimental method was developed to utilize phase-contrast velocity MRI as a normalization factor and to estimate the labeling efficiency in vivo, which was founded to be 0.860.06 (n=10, meanSD). Third, we compared the labeling efficiency of pCASL MRI under normocapnic and hypercapnic (inhalation of 5% CO2) conditions, and showed that a higher flow velocity in the feeding arteries resulted in a reduction in the labeling efficiency. In summary, our results suggest that labeling efficiency is a critical parameter in pCASL MRI not only in terms of achieving highest sensitivity but also in quantification of absolute CBF in ml/min/100g. We propose that the labeling efficiency should be estimated using phase-contrast velocity MRI on a subject-specific basis. PMID:20187183

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

    PubMed Central

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

    2014-01-01

    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

  8. Stable isotope labeling - Liquid chromatography/mass spectrometry for quantitative analysis of androgenic and progestagenic steroids.

    PubMed

    Guo, Ning; Liu, Ping; Ding, Jun; Zheng, Shu-Jian; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-01-28

    Steroid hormones play important roles in mammal at very low concentrations and are associated with numerous endocrinology and oncology diseases. Therefore, quantitative analysis of steroid hormones can provide crucial information for uncovering underlying mechanisms of steroid hormones related diseases. In the current study, we developed a sensitive method for the detection of steroid hormones (progesterone, dehydroepiandrosterone, testosterone, pregnenolone, 17-hydroxyprogesterone, androstenedione and 17α-hydroxypregnenolone) in body fluids by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. In this respect, a pair of isotopes labeling reagents, Girard reagent P (GP) and d5-Girard reagent P (d5-GP), were synthesized and utilized to label steroid hormones in follicular fluid samples and steroid hormone standards, respectively. The heavy labeled standards were used as internal standards for quantification to minimize quantitation deviation in MS analysis due to the matrix and ion suppression effects. The ionization efficiencies of steroid hormones were greatly improved by 4-504 folds through the introduction of a permanent charged moiety of quaternary ammonium from GP. Using the developed method, we successfully quantified steroid hormones in human follicular fluid. We found that the contents of testosterone and androstenedione exhibited significant increase while the content of pregnenolone had significant decrease in follicular fluid of polycystic ovarian syndrome (PCOS) patients compared with healthy controls, indicating that these steroid hormones with significant change may contribute to the pathogenesis of PCOS. Taken together, the developed stable isotope labeling coupled LC-ESI-MS/MS analysis demonstrated to be a promising method for the sensitive and accurate determination of steroid hormones, which may facilitate the in-depth investigation of steroid hormones related diseases. PMID:26755144

  9. Comparing SILAC- and Stable Isotope Dimethyl-Labeling Approaches for Quantitative Proteomics

    PubMed Central

    2015-01-01

    Stable isotope labeling is widely used to encode and quantify proteins in mass-spectrometry-based proteomics. We compared metabolic labeling with stable isotope labeling by amino acids in cell culture (SILAC) and chemical labeling by stable isotope dimethyl labeling and find that they have comparable accuracy and quantitative dynamic range in unfractionated proteome analyses and affinity pull-down experiments. Analyzing SILAC- and dimethyl-labeled samples together in single liquid chromatography–mass spectrometric analyses minimizes differences under analytical conditions, allowing comparisons of quantitative errors introduced during sample processing. We find that SILAC is more reproducible than dimethyl labeling. Because proteins from metabolically labeled populations can be combined before proteolytic digestion, SILAC is particularly suited to studies with extensive sample processing, such as fractionation and enrichment of peptides with post-translational modifications. We compared both methods in pull-down experiments using a kinase inhibitor, dasatinib, and tagged GRB2-SH2 protein as affinity baits. We describe a StageTip dimethyl-labeling protocol that we applied to in-solution and in-gel protein digests. Comparing the impact of post-digest isotopic labeling on quantitative accuracy, we demonstrate how specific experimental designs can benefit most from metabolic labeling approaches like SILAC and situations where chemical labeling by stable isotope-dimethyl labeling can be a practical alternative. PMID:25077673

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

    PubMed Central

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

    2011-01-01

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

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

    DOE PAGESBeta

    O'Maille, Grace; Go, Eden P.; Hoang, Linh; Want, Elizabeth J.; Smith, Colin; O'Maille, Paul; NordstrÖm, Anders; Morita, Hirotoshi; Qin, Chuan; Uritboonthai, Wilasinee; et al

    2008-01-01

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

  12. Multiple segmental and selective isotope labeling of large RNA for NMR structural studies.

    PubMed

    Nelissen, Frank H T; van Gammeren, Adriaan J; Tessari, Marco; Girard, Frederic C; Heus, Hans A; Wijmenga, Sybren S

    2008-08-01

    Multiple segmental and selective isotope labeling of RNA with three segments has been demonstrated by introducing an RNA segment, selectively labeled with (13)C(9)/(15)N(2)/(2)H((1', 3', 4', 5', 5''))-labeled uridine residues, into the central position of the 20 kDa epsilon-RNA of Duck Hepatitis B Virus. The RNA molecules were produced via two efficient protocols: a two-step protocol, which uses T4 DNA ligase and T4 RNA ligase 1, and a one-pot protocol, which uses T4 RNA ligase 1 alone. With T4 RNA ligase 1 all not-to-be-ligated termini are usually protected to prevent formation of side products. We show that such labor-intensive protection of termini is not required, provided segmentation sites can be chosen such that the segments fold into the target structure or target-like structures and thus are not trapped into stable alternate structures. These sites can be reliably predicted via DINAMelt. The simplified NMR spectrum provided evidence for the presence of a U28 H(3)-imino resonance, previously obscured in the fully labeled sample, and thus of the non-canonical base pair U28:C37. The demonstrated multiple segmental labeling protocols are generally applicable to large RNA molecules and can be extended to more than three segments. PMID:18583361

  13. Optimal isotope labelling for NMR protein structure determinations.

    PubMed

    Kainosho, Masatsune; Torizawa, Takuya; Iwashita, Yuki; Terauchi, Tsutomu; Mei Ono, Akira; Güntert, Peter

    2006-03-01

    Nuclear-magnetic-resonance spectroscopy can determine the three-dimensional structure of proteins in solution. However, its potential has been limited by the difficulty of interpreting NMR spectra in the presence of broadened and overlapping resonance lines and low signal-to-noise ratios. Here we present stereo-array isotope labelling (SAIL), a technique that can overcome many of these problems by applying a complete stereospecific and regiospecific pattern of stable isotopes that is optimal with regard to the quality and information content of the resulting NMR spectra. SAIL uses exclusively chemically and enzymatically synthesized amino acids for cell-free protein expression. We demonstrate for the 17-kDa protein calmodulin and the 41-kDa maltodextrin-binding protein that SAIL offers sharpened lines, spectral simplification without loss of information, and the ability to rapidly collect the structural restraints required to solve a high-quality solution structure for proteins twice as large as commonly solved by NMR. It thus makes a large class of proteins newly accessible to detailed solution structure determination. PMID:16511487

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

    SciTech Connect

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

    2014-12-09

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

  15. Isotope-labeling of the fibril binding compound FSB via a Pd-catalyzed double alkoxycarbonylation.

    PubMed

    Burhardt, Mia N; Taaning, Rolf; Nielsen, Niels Chr; Skrydstrup, Troels

    2012-06-15

    We have synthesized two isotopically labeled variants of the ?-amyloid binding compound FSB possessing (13)C-labels on the two terminal aryl carboxylic acid moieties. One of these was also fully deuterated on the olefinic spacers. The (13)C-isotope labeling was achieved applying a Pd-catalyzed methoxycarbonylation of the corresponding aryl chlorides with externally (ex situ) generated (13)C-labeled CO. Application of the Shirakawa-Hayashi protocol for the Pd-catalyzed reduction of a dialkyne intermediate using D(2)O allowed for the selective deuterium labeling of the two trans-C,C double bonds of FSB. PMID:22612598

  16. Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

    PubMed

    Trouillard, Romain; Hubert-Roux, Marie; Tognetti, Vincent; Guilhaudis, Laure; Plasson, Carole; Menu-Bouaouiche, Laurence; Coquet, Laurent; Guerineau, Franois; Hardouin, Julie; Ele Ekouna, Jean-Pierre; Cosette, Pascal; Lerouge, Patrice; Boitel-Conti, Michle; Afonso, Carlos; Sgalas-Milazzo, Isabelle

    2015-06-16

    Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules. PMID:25973921

  17. Isotopic labeling affects 1,25-dihydroxyvitamin D metabolism

    SciTech Connect

    Halloran, B.P.; Bikle, D.D.; Castro, M.E.; Gee, E.

    1989-02-07

    Isotope substitution can change the biochemical properties of vitamin D. To determine the effect of substituting 3H for 1H on the metabolism of 1,25(OH)2D3, we measured the metabolic clearance rate and renal metabolism of unlabeled and 3H-labeled 1,25(OH)2D3. Substitution of 3H for 1H on carbons 26 and 27 (1,25(OH)2(26,27(n)-3H)D3) or on carbons 23 and 24 (1,25(OH)2(23,24(n)-3H)D3) reduced the in vivo metabolic clearance rate of 1,25(OH)2D3 by 36% and 37%, respectively, and reduced the in vitro renal catabolism of 1,25(OH)2D3 by 11% and 54%, respectively. Substitutions of 3H for 1H on carbons 23 and 24 as opposed to carbons 26 and 27 reduced conversion of (3H)1,25(OH)2D3 to (3H)1,24,25(OH)2D3 by 25% and to putative 24-oxo-1,23,25-dihydroxyvitamin D3 by 1600%. These results indicate that substitution of 3H for 1H on carbons 26 and 27 or on carbons 23 and 24 can reduce the metabolic clearance rate and in vitro metabolism of 1,25(OH)2D3 and quantitatively alter the pattern of metabolic products produced.

  18. Ultrafast infrared spectroscopy of an isotope-labeled photoactivatable flavoprotein.

    PubMed

    Haigney, Allison; Lukacs, Andras; Zhao, Rui-Kun; Stelling, Allison L; Brust, Richard; Kim, Ryu-Ryun; Kondo, Minako; Clark, Ian; Towrie, Michael; Greetham, Gregory M; Illarionov, Boris; Bacher, Adelbert; Rmisch-Margl, Werner; Fischer, Markus; Meech, Stephen R; Tonge, Peter J

    2011-03-01

    The blue light using flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense interest in understanding how the BLUF protein matrix senses and responds to flavin photoexcitation. Light absorption is proposed to result in alterations in the hydrogen-bonding network that surrounds the flavin chromophore on an ultrafast time scale, and the structural changes caused by photoexcitation are being probed by vibrational spectroscopy. Here we report ultrafast time-resolved infrared spectra of the AppA BLUF domain (AppA(BLUF)) reconstituted with isotopically labeled riboflavin (Rf) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics. PMID:21218799

  19. Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry

    PubMed Central

    2015-01-01

    Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. Here, an LC-MS approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of MS-silent pseudouridine modifications. The method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature 15N-labeled rRNA standard, and a number of modifications in both 16S and 23S rRNA were present in substoichiometric amounts in the preribosomal particles. The RNA modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that RNA is modified in a schedule comparable to the association of the ribosomal proteins. Importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of RNA modifications and their impact on intracellular processes in the future. PMID:24422502

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

    NASA Astrophysics Data System (ADS)

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

    Light isotopes separation, such as 3He/4He, H2/D2, H2/T2, etc., is crucial for various advanced technologies including isotope labeling, nuclear weapons, cryogenics and power generation. However, their nearly identical chemical properties made the separation challenging. The low productivity of the present isotopes separation approaches hinders the relevant applications. An efficient membrane with high performance for isotopes separation is quite appealing. Based on first-principles calculations, we theoretically demonstrated that highly efficient light isotopes separation, such as 3He/4He, can be reached in a porous graphene-like carbon nitride material via quantum sieving effect. Under moderate tensile strain, the quantum sieving of the carbon nitride membrane can be effectively tuned in a continuous way, leading to a temperature window with high 3He/4He selectivity and permeance acceptable for efficient isotopes harvest in industrial application. This mechanism also holds for separation of other light isotopes, such as H2/D2, H2/T2. Such tunable quantum sieving opens a promising avenue for light isotopes separation for industrial application. PMID:26813491

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

    PubMed

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

    2016-01-01

    Light isotopes separation, such as (3)He/(4)He, H2/D2, H2/T2, etc., is crucial for various advanced technologies including isotope labeling, nuclear weapons, cryogenics and power generation. However, their nearly identical chemical properties made the separation challenging. The low productivity of the present isotopes separation approaches hinders the relevant applications. An efficient membrane with high performance for isotopes separation is quite appealing. Based on first-principles calculations, we theoretically demonstrated that highly efficient light isotopes separation, such as (3)He/(4)He, can be reached in a porous graphene-like carbon nitride material via quantum sieving effect. Under moderate tensile strain, the quantum sieving of the carbon nitride membrane can be effectively tuned in a continuous way, leading to a temperature window with high (3)He/(4)He selectivity and permeance acceptable for efficient isotopes harvest in industrial application. This mechanism also holds for separation of other light isotopes, such as H2/D2, H2/T2. Such tunable quantum sieving opens a promising avenue for light isotopes separation for industrial application. PMID:26813491

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

    PubMed Central

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

    2014-01-01

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

  4. Convenient synthesis of stable deuterium-labeled alkylpyrazines for use in stable isotope dilution assays.

    PubMed

    Fang, Mingchih; Cadwallader, Keith R

    2013-04-17

    Stable isotope dilution assays (SIDA) provide for accurate and precise quantitation of aroma components, such as alkylpyrazines, which are often present in low concentrations in complex food matrices. The unavailability of labeled standards is the main limitation to the widespread use of SIDA. This study describes the chlorination of several alkylpyrazines to form the corresponding chloroalkylpyrazine compounds, which are efficient starting materials for the synthesis of deuterium-labeled alkylpyrazines, namely [H?]-2-methylpyrazine (d-1), [H?]-2-ethylpyrazine (d-2), [H?]-2,3(or 6)-dimethylpyrazine (d-3A, d-3B), [H?]-2,[H?]-6-dimethylpyrazine (d-3C), [H?]-2,[H?]-6-diethylpyrazine (d-4), [H?]-2-ethyl-3(or 6)-methylpyrazine (d-5A, d-5B), 2,[H?]-3,5-trimethylpyrazine (d-6), [H?]-2-ethyl-3,6-dimethylpyrazine (d-7), [H?]-2-ethyl-3,5-dimethylpyrazine (d-8), and 2,3-diethyl-[H?]-5-methylpyrazine (d-9), which were obtained in good yields (57-100%) and high purities (86-98%). These stable isotopes were used as internal standards in SIDA to accurately and precisely determine selected alkylpyrazines in commercial peanut butter, cocoa powder, and instant coffee. 2,3-Diethyl-5-methylpyrazine (p-9) and 2-ethyl-3,5-dimethylpyrazine (p-8), despite their low abundance, had the highest odor-active values among the 13 pyrazines quantified in all products due to their very low odor thresholds. PMID:23528050

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

    DOEpatents

    Goldblatt, M.; McInteer, B.B.

    1974-01-29

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

  6. Fluorescent isotope-coded affinity tag 2: peptide labeling and affinity capture.

    PubMed

    Rivera-Monroy, Zuly; Bonn, Guenther K; Guttman, Andrs

    2009-04-01

    Fluorescent isotope-coded affinity tag (FCAT) is a novel reagent to label cysteine-containing peptides. The fluorescein group on the tag enables absolute quantification by fluorescence detection, also supporting affinity capture of the labeled peptides. In this paper we report the synthesis of the heavy isotopic form of the FCAT reagent and its use in labeling tryptic peptides. The heavy form of the reagent exhibited the same reactivity and chromatographic behavior that of the light version. Effective labeling of tryptic peptides from alpha-lactalbumin, fetuin, BSA and phosphorylase b was attained by using both the heavy and light FCAT tags. Selective capture of the FCAT-labeled peptides was easily performed by pipette tips containing either anti-FITC antibody or iminodiacetic-acid-coated beads. The differently labeled peptides were separated by RP HPLC and analyzed by MALDI-TOF MS. PMID:19288590

  7. Isotopic labelling studies for a gold-catalysed skeletal rearrangement of alkynyl aziridines

    PubMed Central

    Martin, Nicolas; Spencer, Neil

    2011-01-01

    Summary Isotopic labelling studies were performed to probe a proposed 1,2-aryl shift in the gold-catalysed cycloisomerisation of alkynyl aziridines into 2,4-disubstituted pyrroles. Two isotopomers of the expected skeletal rearrangement product were identified using 13C-labelling and led to a revised mechanism featuring two distinct skeletal rearrangements. The mechanistic proposal has been rationalised against the reaction of a range of 13C- and deuterium-labelled substrates. PMID:21804880

  8. Methyl-specific isotopic labeling: a molecular tool box for solution NMR studies of large proteins.

    PubMed

    Kerfah, Rime; Plevin, Michael J; Sounier, Remy; Gans, Pierre; Boisbouvier, Jerome

    2015-06-01

    Nuclear magnetic resonance (NMR) spectroscopy is a uniquely powerful tool for studying the structure, dynamics and interactions of biomolecules at atomic resolution. In the past 15 years, the development of new isotopic labeling strategies has opened the possibility of exploiting NMR spectroscopy in the study of supra-molecular complexes with molecular weights of up to 1MDa. At the core of these isotopic labeling developments is the specific introduction of [(1)H,(13)C]-labeled methyl probes into perdeuterated proteins. Here, we describe the evolution of these approaches and discuss their impact on structural and biological studies. The relevant protocols are succinctly reviewed for single and combinatorial isotopic-labeling of methyl-containing residues, and examples of applications on challenging biological systems, including high molecular weight and membrane proteins, are presented. PMID:25881211

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

    Tao, Shujuan; Orlando, Ron

    2014-01-01

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

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

    SciTech Connect

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

    2002-08-20

    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.

  12. Isotope labeling strategies for the study of high-molecular-weight proteins by solution NMR spectroscopy.

    PubMed

    Tugarinov, Vitali; Kanelis, Voula; Kay, Lewis E

    2006-01-01

    The development of isotope labeling methodology has had a significant impact on NMR studies of high-molecular-weight proteins and macromolecular complexes. Here we review some of this methodology that has been developed and used in our laboratory. In particular, experimental protocols are described for the production of highly deuterated, uniformly 15N- and 13C-labeled samples of large proteins, with optional incorporation of selective isotope labels into methyl groups of isoleucine, leucine and valine residues. Various types of methyl labeling schemes are assessed, and the utility of different methyl labeling strategies is highlighted for studies ranging from protein structure determination to the investigation of side-chain dynamics. In the case of malate synthase G (MSG), the time frame of the whole preparation, including the protein refolding step, is about 70 h. PMID:17406304

  13. A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's ?-amyloid peptides from E. coli for NMR-based structural analysis.

    PubMed

    Sharma, Sudhir C; Armand, Tara; Ball, K Aurelia; Chen, Anna; Pelton, Jeffrey G; Wemmer, David E; Head-Gordon, Teresa

    2015-12-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting millions of people worldwide. AD is characterized by the presence of extracellular plaques composed of aggregated/oligomerized ?-amyloid peptides with A?42 peptide representing a major isoform in the senile plaques. Given the pathological significance of A?42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of A?42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled A?42 for structural studies by NMR. The protocol involves utilization of an auto induction system with (15)N isotope labeled medium, for high-level expression of A?42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-A?42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled A?42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled A?42 peptide (Garai et al., 2009). We obtain a final yield of ? 6 mg/L culture for (15)N isotope-labeled A?42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric A?42 peptide validate the uniform incorporation of the isotopic label. The method described here is equally applicable for the uniform isotope labeling with (15)N and (13)C in A?42 peptide as well as its other variants including any A?42 peptide mutants. PMID:26231074

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

    PubMed

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

    2015-03-01

    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

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

    PubMed

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

    2015-11-01

    Experimental analysis of kinetic isotope effects represents an extremely powerful approach for gaining information about the transition state structure of complex reactions not available through other methodologies. The implementation of this approach to the study of nucleic acid chemistry requires the synthesis of nucleobases and nucleotides enriched for heavy isotopes at specific positions. In this review, we highlight current approaches to the synthesis of nucleic acids enriched site specifically for heavy oxygen and nitrogen and their application in heavy atom isotope effect studies. This article is part of a special issue titled: Enzyme Transition States from Theory and Experiment. PMID:25828952

  16. Profiling of thiol-containing compounds by stable isotope labeling double precursor ion scan mass spectrometry.

    PubMed

    Liu, Ping; Huang, Yun-Qing; Cai, Wen-Jing; Yuan, Bi-Feng; Feng, Yu-Qi

    2014-10-01

    Here we developed a novel strategy of isotope labeling in combination with high-performance liquid chromatography-double precursor ion scan mass spectrometry (IL-LC-DPIS-MS) analysis for nontargeted profiling of thiol-containing compounds. In this strategy, we synthesized a pair of isotope labeling reagents (?-bromoacetonylquinolinium bromide, BQB; ?-bromoacetonylquinolinium-d7 bromide, BQB-d7) that contain a reactive group, an isotopically labeled moiety, and an ionizable group to selectively label thiol-containing compounds. The BQB and BQB-d7 labeled compounds can generate two characteristic product ions m/z 218 and 225, which contain an isotope tag and therefore were used for double precursor ion scans in mass spectrometry analysis. The peak pairs with characteristic mass differences can be readily extracted from the two precursor ion scan (PIS) spectra and assigned as potential thiol-containing candidates, which facilitates the identification of analytes. BQB and BQB-d7 labeled thiol-containing compounds can be clearly distinguished by generating two individual ion chromatograms. Thus, thiol-containing compounds from two samples labeled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, offering good identification and accurate quantification by eliminating the MS response fluctuation and mutual interference from the two labeled samples. Using the IL-LC-DPIS-MS strategy, we profiled the thiol-containing compounds in beer and human urine, and 21 and 103 thiol candidates were discovered in beer and human urine, respectively. In addition, 9 and 17 thiol candidates in beer and human urine were successfully identified by further comparison with thiol standards or tandem mass spectrometry analysis. Taken together, the IL-LC-DPIS-MS method is demonstrated to be a promising strategy in the profiling of compounds with identical groups in metabolomics study. PMID:25222826

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

    PubMed

    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

    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

  18. Metabolic pathway of inorganic and organic selenocompounds labeled with stable isotope in Japanese quail.

    PubMed

    Anan, Yasumi; Ohbo, Ai; Tani, Yuta; Ogra, Yasumitsu

    2014-12-01

    The distribution and metabolism of an inorganic selenium (Se) compound and a selenoamino acid in quails were evaluated by speciation with inductively coupled plasma mass spectrometry (ICP-MS) and a stable isotope. Quails were orally administered stable isotope [(77)Se]-labeled selenite and selenomethionine (SeMet) at the nutritional dose of 10?g Se/bird. Then, the quails were dissected 3, 9, and 24h after the administration to examine the metabolic pathway and the time-dependent change of Se. The concentrations of exogenous Se in all the organs and tissues of the SeMet-administered group were significantly higher than those of the selenite-administered group 3h after the administration. This suggested that SeMet was more rapidly and/or efficiently incorporated into the quail body than selenite. A Se-containing protein in the serum was detected only in the SeMet-administered quails, but not in the selenite-administered quails. The major urinary Se metabolite, i.e., Se-methylseleno-N-acetyl-galactosamine (selenosugar), was detected in the quail serum after the administration of both selenite and SeMet. The endogenous amount of Se-methylated selenosugar (MeSeSug) in the serum of quails seemed to be larger than that of the rodents. We conclude that the metabolic pathway of Se in quails was the same as that in rodents, but the metabolic capacity for Se seemed to be larger in quails than in rodents. PMID:25326891

  19. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins.

    PubMed

    Edfors, Fredrik; Boström, Tove; Forsström, Björn; Zeiler, Marlis; Johansson, Henrik; Lundberg, Emma; Hober, Sophia; Lehtiö, Janne; Mann, Matthias; Uhlen, Mathias

    2014-06-01

    The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed. PMID:24722731

  20. Quantitation of asparagine deamidation by isotope labeling and liquid chromatography coupled with mass spectrometry analysis.

    PubMed

    Liu, Hongcheng; Wang, Fengqiang; Xu, Wei; May, Kimberly; Richardson, Daisy

    2013-01-01

    Nonenzymatic asparagine (Asn) deamidation is one of the commonly observed posttranslational modifications of proteins. Recent development of several specific analytical methods has allowed for efficient identification and differentiation of the deamidation products (i.e., isoaspartate [isoAsp] and aspartate [Asp]). Isotope labeling of isoAsp and Asp that are generated during sample preparation by 18O has been developed and can differentiate isoAsp and Asp as analytical artifacts from those present in the samples prior to sample preparation for an accurate quantitation. However, the 18O labeling procedure has a limitation due to the additional incorporation of up to two 18O atoms into the peptide C-terminal carboxyl groups. Variability in the incorporation of 18O atoms into the peptide C-terminal carboxyl groups results in complicated mass spectra and hinders data interpretation. This limitation can be overcome by the dissection of the complicated mass spectra using a calculation method presented in the current study. The multiple-step calculation procedure has been successfully employed to determine the levels of isoAsp and Asp that are present in the sample prior to sample treatment. PMID:23017877

  1. Efficient Isotope Editing of Proteins for Site-Directed Vibrational Spectroscopy.

    PubMed

    Peuker, Sebastian; Andersson, Hanna; Gustavsson, Emil; Maiti, Kiran Sankar; Kania, Rafal; Karim, Alavi; Niebling, Stephan; Pedersen, Anders; Erdelyi, Mate; Westenhoff, Sebastian

    2016-02-24

    Vibrational spectra contain unique information on protein structure and dynamics. However, this information is often obscured by spectral congestion, and site-selective information is not available. In principle, sites of interest can be spectrally identified by isotope shifts, but site-specific isotope labeling of proteins is today possible only for favorable amino acids or with prohibitively low yields. Here we present an efficient cell-free expression system for the site-specific incorporation of any isotope-labeled amino acid into proteins. We synthesized 1.6 mg of green fluorescent protein with an isotope-labeled tyrosine from 100 mL of cell-free reaction extract. We unambiguously identified spectral features of the tyrosine in the fingerprint region of the time-resolved infrared absorption spectra. Kinetic analysis confirmed the existence of an intermediate state between photoexcitation and proton transfer that lives for 3 ps. Our method lifts vibrational spectroscopy of proteins to a higher level of structural specificity. PMID:26796542

  2. Turnover of Leaf Waxes in Florida Slash Pine: Results of an Isotopic Labeling Experiment

    NASA Astrophysics Data System (ADS)

    Crumsey, J.; Conte, M. H.; Weber, J. C.; Mortazavi, B.; Smith, M.; Chanton, J.

    2006-12-01

    Isotopic discrimination of terrestrial photosynthesis, atmospheric CO2 concentration, and ?13CO2 are important parameters in global carbon models that are employed to estimate global carbon sources and sinks. Yet, terrestrial isotopic discrimination can be highly variable over space and time, yielding large uncertainties of terrestrial fluxes. The isotopic composition of plant wax aerosols in continental air masses can be used as an indirect measure of the spatial and temporal patterns of photosynthetic discrimination integrated over large (subcontinental) spatial scales. However, the temporal offset between wax biosynthesis and the wax aerosol isotopic signal of photosynthetic discrimination is not well constrained. To further our understanding of this temporal lag, this study sought to determine the turnover time of conifer leaf waxes by performing an isotopic labeling experiment. Four clonal pine saplings were placed in a tent and labeled with enriched 13CO2 for one year, while another four control saplings were grown under ambient CO2. At the end of the year long enrichment, the labeled saplings were removed from the tent and placed in ambient air, such that the wax turnover rate could be determined by analyzing the resultant isotopic and molecular changes. The results of this experiment indicated that after 80 days of sequestering ambient CO2, the wax (and soluble sugar) isotopic composition of the labeled saplings varied minimally. The molecular composition of the waxes, however, did change over time. From these results we concluded that waxes are turning over, but rather than being synthesized de novo from recently fixed carbon precursors they are synthesized using carbon from stored (labeled) carbon pools. Therefore, the ?13C of conifer leaf waxes in aerosols may not reflect recent photosynthetic discrimination, but instead represents photosynthetic discrimination integrated over a longer period of time. The implications of these findings are focused on interpreting the wax aerosol ?13C as an integrative measure of past photosynthetic discrimination in global carbon cycling models, and also provide new insights on internal cycling among plant carbon pools.

  3. X13CMS: global tracking of isotopic labels in untargeted metabolomics.

    PubMed

    Huang, Xiaojing; Chen, Ying-Jr; Cho, Kevin; Nikolskiy, Igor; Crawford, Peter A; Patti, Gary J

    2014-02-01

    Studies of isotopically labeled compounds have been fundamental to understanding metabolic pathways and fluxes. They have traditionally, however, been used in conjunction with targeted analyses that identify and quantify a limited number of labeled downstream metabolites. Here we describe an alternative workflow that leverages recent advances in untargeted metabolomic technologies to track the fates of isotopically labeled metabolites in a global, unbiased manner. This untargeted approach can be applied to discover novel biochemical pathways and characterize changes in the fates of labeled metabolites as a function of altered biological conditions such as disease. To facilitate the data analysis, we introduce X(13)CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X(13)CMS uses the XCMS platform to detect metabolite peaks and perform retention-time alignment in liquid chromatography/mass spectrometry (LC/MS) data. With the use of the XCMS output, the program then identifies isotopologue groups that correspond to isotopically labeled compounds. The retrieval of these groups is done without any a priori knowledge besides the following input parameters: (i) the mass difference between the unlabeled and labeled isotopes, (ii) the mass accuracy of the instrument used in the analysis, and (iii) the estimated retention-time reproducibility of the chromatographic method. Despite its name, X(13)CMS can be used to track any isotopic label. Additionally, it detects differential labeling patterns in biological samples collected from parallel control and experimental conditions. We validated the ability of X(13)CMS to accurately retrieve labeled metabolites from complex biological matrices both with targeted LC/MS/MS analysis of a subset of the hits identified by the program and with labeled standards spiked into cell extracts. We demonstrate the full functionality of X(13)CMS with an analysis of cultured rat astrocytes treated with uniformly labeled (U-)(13)C-glucose during lipopolysaccharide (LPS) challenge. Our results show that out of 223 isotopologue groups enriched from U-(13)C-glucose, 95 have statistically significant differential labeling patterns in astrocytes challenged with LPS compared to unchallenged control cells. Only two of these groups overlap with the 32 differentially regulated peaks identified by XCMS, indicating that X(13)CMS uncovers different and complementary information from untargeted metabolomic studies. Like XCMS, X(13)CMS is implemented in R. It is available from our laboratory website at http://pattilab.wustl.edu/x13cms.php . PMID:24397582

  4. Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy

    SciTech Connect

    Unkefer, C.; Hernandez, G.; Springer, P.; Trewhella, J.; Blumenthal, D.; Lidstrom, M.

    1996-04-01

    The project sought to employ stable isotope labeling and NMR spectroscopy to study protein structures and provide insight into important biochemical problems. A methylotrophic bacterial expression system has been developed for uniform deuterium and carbon-13 labeling of proteins for structural studies. These organisms grow using methanol as the sole source of carbon and energy. Because isotopically labeled methanol is relatively inexpensive, the methylotrophs are ideal for expressing proteins labeled uniformly with deuterium and/or carbon-13. This expression system has been employed to prepare deuterated troponin C. NMR spectroscopy measurements have been made on the inhibitory peptide from troponin I (residues 96--115), both as the free peptide and the peptide complexed with deuterated troponin C. Proton-NMR spectroscopy resonance-signal assignments have been made for the free peptide.

  5. Brain quantitative proteomics combining GeLC-MS and isotope-coded protein labeling (ICPL).

    PubMed

    Maccarrone, Giuseppina; Lebar, Maria; Martins-de-Souza, Daniel

    2014-01-01

    Proteomics has been revolutionized by the rapid advance of mass spectrometric instrumentations and techniques. Parallel methodologies for the quantification of proteomes also evolved, including in vitro stable isotope labeling. Here, we present a protocol for employing isotope-coded protein labeling (ICPL) as part of a shotgun proteomics workflow denoting its advantages and disadvantages. This protocol is suitable to studying any proteome of interest, only requiring a specific sample preparation and protein identification. Given our expertise, descriptions here are centered on the study of brain disorders. PMID:24791988

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

    NASA Astrophysics Data System (ADS)

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

    2006-12-01

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

  7. Stable isotope labeling of oligosaccharide cell surface antigens

    SciTech Connect

    Unkefer, C.J.; Silks, L.A. III; Martinez, R.A.

    1998-12-31

    The overall goal of this Laboratory Directed Research and Development (LDRD) project was to develop new methods for synthesis of {sup 13}C-labeled oligosaccharides that are required for nuclear magnetic resonance (NMR) studies of their solution conformation. Oligosaccharides are components of the cell`s outer surface and are involved in important processes such as cell-cell recognition and adhesion. Recently, Danishefsky and coworkers at Slone-Kettering Cancer Center developed a method for the solid-phase chemical synthesis of oligosaccharides. The specific goal of this LDRD project was to prepare uniform {sup 13}C-labeled aldohexose precursors required for the solid-phase synthesis of the Lewis blood-group antigenic determinants. We report the synthesis of {sup 13}C-labeled D-glucal, D-galactal and Fucosyl precursors. We have been collaborating with the Danishefsky group on the synthesis of the Lewis oligosaccharides and the NMR analysis of their solution conformation.

  8. Global Potential of Energy Efficiency Standards and Labeling Programs

    SciTech Connect

    McNeil, Michael A; McNeil, Michael A.; Letschert, Virginie; de la Rue du Can, Stephane

    2008-06-15

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

  9. 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...

  10. Chasing the Elusive Benzofuran Impurity of the THR Antagonist NH-3: Synthesis, Isotope Labeling, and Biological Activity.

    PubMed

    Singh, Latika; Pressly, Brandon; Mengeling, Brenda J; Fettinger, James C; Furlow, J David; Lein, Pamela J; Wulff, Heike; Singh, Vikrant

    2016-03-01

    We have synthesized and established the structure of a long-suspected, but hitherto unknown, benzofuran side product (EBI) formed during the synthesis of NH-3. Understanding the mechanism of its formation has enabled isotope (D) labeling. We further developed a highly efficient method for separating EBI from NH-3. Interestingly, EBI was found to be a very potent thyroid hormone receptor (THR) agonist, while NH-3 is an antagonist. In this process, we have also achieved a significantly improved synthesis of NH-3. PMID:26849160

  11. Differential Isotope Labeling of Glycopeptides for Accurate Determination of Differences in Site-Specific Glycosylation.

    PubMed

    Pabst, Martin; Beneov, Iva; Fagerer, Stephan R; Jacobsen, Mathias; Eyer, Klaus; Schmidt, Gregor; Steinhoff, Robert; Krismer, Jasmin; Wahl, Fabian; Preisler, Jan; Zenobi, Renato

    2016-01-01

    We introduce a stable isotope labeling approach for glycopeptides that allows a specific glycosylation site in a protein to be quantitatively evaluated using mass spectrometry. Succinic anhydride is used to specifically label primary amino groups of the peptide portion of the glycopeptides. The heavy form (D4(13)C4) provides an 8 Da mass increment over the light natural form (H4(12)C4), allowing simultaneous analysis and direct comparison of two glycopeptide profiles in a single MS scan. We have optimized a protocol for an in-solution trypsin digestion, a one-pot labeling procedure, and a post-labeling solid-phase extraction to obtain purified and labeled glycopeptides. We provide the first demonstration of this approach by comparing IgG1 Fc glycopeptides from polyclonal IgG samples with respect to their galactosylation and sialylation patterns using MALDI MS and LC-ESI-MS. PMID:26573365

  12. Expeditious syntheses of stable and radioactive isotope-labeled anticonvulsant agent, JNJ-26990990, and its metabolites.

    PubMed

    Lin, Ronghui; Weaner, Larry E; Hoerr, David C; Salter, Rhys; Gong, Yong

    2013-01-01

    Syntheses of stable and radioactive isotope-labeled anticonvulsant agent, JNJ-26990990, that is, N-(benzo[b]thien-3-ylmethyl)-sulfamide and its metabolites are described. [(13)C(15)N]Benzo[b]thiophene-3-carbonitrile was first prepared by coupling of 3-bromo-benzo[b]thiophene with [(13)C(15)N]-copper cyanide. The resultant [(13)C(15)N]benzo[b]thiophene-3-carbonitrile was reduced with lithium aluminum deuteride to give [(13)CD2(15)N]benzo[b]thiophen-3-yl-methylamine; which was then coupled with sulfamide to afford [(13)CD2(15)N]-N-(benzo[b]thien-3-ylmethyl)-sulfamide, the stable isotope-labeled compound with four stable isotope atoms. Direct oxidation of [(13)CD2(15)N]-N-(benzo[b]thien-3-ylmethyl)-sulfamide with hydrogen peroxide and peracetic acid gave the stable isotope-labeled sulfoxide and sulfone metabolites. On the other hand, radioactive (14)C-labeled N-(benzo[b]thien-3-ylmethyl)-sulfamide was prepared conveniently by sequential coupling of 3-bromo-benzo[b]thiophene with [(14)C]-copper cyanide, reduction of the carbonitrile to carboxaldehyde, and reductive amination with sulfamide. PMID:24285137

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

    NASA Astrophysics Data System (ADS)

    Drożdżewski, Piotr; Kordon, Ewa

    2000-11-01

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  15. Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis, an isotopic-labeling technique for accurate biochemical profiling

    PubMed Central

    de Jong, Felice A; Beecher, Chris

    2013-01-01

    Metabolomics or biochemical profiling is a fast emerging science; however, there are still many associated bottlenecks to overcome before measurements will be considered robust. Advances in MS resolution and sensitivity, ultra pressure LC MS, ESI, and isotopic approaches such as flux analysis and stable-isotope dilution, have made it easier to quantitate biochemicals. The digitization of mass spectrometers has simplified informatic aspects. However, issues of analytical variability, ion suppression and metabolite identification still plague metabolomics investigators. These hurdles need to be overcome for accurate metabolite quantitation not only for in vitro systems, but for complex matrices such as biofluids and tissues, before it is possible to routinely identify biomarkers that are associated with the early prediction and diagnosis of diseases. In this report, we describe a novel isotopic-labeling method that uses the creation of distinct biochemical signatures to eliminate current bottlenecks and enable accurate metabolic profiling. PMID:23046270

  16. Addressing the current bottlenecks of metabolomics: Isotopic Ratio Outlier Analysis™, an isotopic-labeling technique for accurate biochemical profiling.

    PubMed

    de Jong, Felice A; Beecher, Chris

    2012-09-01

    Metabolomics or biochemical profiling is a fast emerging science; however, there are still many associated bottlenecks to overcome before measurements will be considered robust. Advances in MS resolution and sensitivity, ultra pressure LC-MS, ESI, and isotopic approaches such as flux analysis and stable-isotope dilution, have made it easier to quantitate biochemicals. The digitization of mass spectrometers has simplified informatic aspects. However, issues of analytical variability, ion suppression and metabolite identification still plague metabolomics investigators. These hurdles need to be overcome for accurate metabolite quantitation not only for in vitro systems, but for complex matrices such as biofluids and tissues, before it is possible to routinely identify biomarkers that are associated with the early prediction and diagnosis of diseases. In this report, we describe a novel isotopic-labeling method that uses the creation of distinct biochemical signatures to eliminate current bottlenecks and enable accurate metabolic profiling. PMID:23046270

  17. Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes.

    PubMed

    Woo, Christina M; Bertozzi, Carolyn R

    2016-01-01

    Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post-translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this protocol, we describe a novel mass-independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). To use IsoTaG, cell culture samples are metabolically labeled with an azido- or alkynyl-sugar. Metabolically labeled glycoproteins are then tagged using Click chemistry and enriched with an isotopic recoding biotin probe. Intact glycopeptides are recovered by cleavage of the probe, analyzed with directed MS, and assigned by targeted mass-independent data analysis. The outlined procedure is well defined in cell culture and has been executed with over 15 cell lines. © 2016 by John Wiley & Sons, Inc. PMID:26995354

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

    PubMed

    Jung, Joon-Young; Oh, Min-Kyu

    2015-01-01

    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

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

    PubMed

    Schubeis, Tobias; Lhrs, Thorsten; Ritter, Christiane

    2015-01-01

    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

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

    PubMed Central

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

    2010-01-01

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

  1. Determination of complex isotopomer patterns in isotopically labeled compounds by mass spectrometry.

    PubMed

    Jennings, Mark E; Matthews, Dwight E

    2005-10-01

    A classic problem in analytical chemistry has been determination of individual components in a mixture without availability of the pure individual components. Measurement of the distribution of isotopomers in a labeled compound or mixture of labeled compounds is an example of this problem that is commonly encountered when stable isotopically labeled metabolites are used to determine in vivo kinetics and metabolism. We present a method that uses the measured mass spectral data of the unlabeled material to represent any and all combinations of isotopomer variations of that material and to determine abundances of these isotopomers. Although examples of the method are presented for gas chromatography/mass spectrometry, the method is applicable to any type of mass spectrometry data. The method also accounts for errors induced by mass spectrometer ionization and resolution effects. To demonstrate this method, we determined the isotopomer distributions of samples of 13C-labeled leucine and glucose for both highly enriched isotopomers and labeled isotopomers present in low abundance against a natural isotopic abundance background. The method accurately and precisely determined isotopomer identity and abundance in the labeled materials without adding noise or error that was not inherent in the original mass spectral data. In examples shown here, isotopomer uncertainties were calculated with relative standard errors of <1% from good quality mass spectral data. PMID:16194110

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

    PubMed

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

    2010-01-01

    The extensive collection of NOE constraint data involving the aromatic ring signals is essential for accurate protein structure determination, although it is often hampered in practice by the pervasive signal overlapping and tight spin couplings for aromatic rings. We have prepared various types of stereo-array isotope labeled phenylalanines (epsilon- and zeta-SAIL Phe) and tyrosine (epsilon-SAIL Tyr) to overcome these problems (Torizawa et al. 2005), and proven that these SAIL amino acids provide dramatic spectral simplification and sensitivity enhancement for the aromatic ring NMR signals. In addition to these SAIL aromatic amino acids, we recently synthesized delta-SAIL Phe and delta-SAIL Tyr, which allow us to observe and assign delta-(13)C/(1)H signals very efficiently. Each of the various types of SAIL Phe and SAIL Tyr yields well-resolved resonances for the delta-, epsilon- or zeta-(13)C/(1)H signals, respectively, which can readily be assigned by simple and robust pulse sequences. Since the delta-, epsilon-, and zeta-proton signals of Phe/Tyr residues give rise to complementary NOE constraints, the concomitant use of various types of SAIL-Phe and SAIL-Tyr would generate more accurate protein structures, as compared to those obtained by using conventional uniformly (13)C, (15)N-double labeled proteins. We illustrated this with the case of an 18.2 kDa protein, Escherichia coli peptidyl-prolyl cis-trans isomerase b (EPPIb), and concluded that the combined use of zeta-SAIL Phe and epsilon-SAIL Tyr would be practically the best choice for protein structural determinations. PMID:19662334

  3. Comparative pharmacokinetics of unlabeled and deuterium-labeled terbutaline: demonstration of a small isotope effect

    SciTech Connect

    Borgstroem, L.L.; Lindberg, C.; Joensson, S.S.; Svensson, K.

    1988-11-01

    An equimolar mixture of terbutaline and (/sup 2/H6)terbutaline was given as an oral solution to six healthy volunteers (three men and three women). Frequent blood samples were collected during a 24-h period and the plasma concentrations of unlabeled and deuterium-labeled terbutaline were measured by GC-MS. The overall geometric mean plasma concentration ratio of terbutaline to (/sup 2/H6)terbutaline (isotope ratio) was 1.04 and differed significantly from unity. The difference can be explained by a difference in lipophilicity between the analogues, affecting their absorption. No trend in isotope ratio over the experimental time was observed. For unknown reasons, the isotope ratio was higher for women (1.07) than for men (1.00). Deuterium-labeled terbutaline can be used, intravenously or orally, as an absolute reference in bioavailability studies on terbutaline. If deuterium-labeled terbutaline is given orally in a single-day relative bioavailability study, a correlation should be made for the observed isotope effect.

  4. Testing isotopic labeling with [C?]glucose as a method of advanced glycation sites identification.

    PubMed

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

    2012-12-01

    The Maillard reaction occurring between reducing sugars and reactive amino groups of biomolecules leads to the formation of a heterogeneous mixture of compounds: early, intermediate, and advanced glycation end products (AGEs). These compounds could be markers of certain diseases and of the premature aging process. Detection of Amadori products can be performed by various methods, including MS/MS techniques and affinity chromatography on immobilized boronic acid. However, the diversity of the structures of AGEs makes detection of these compounds more difficult. The aim of this study was to test a new method of AGE identification based on isotope (13)C labeling. The model protein (hen egg lysozyme) was modified with an equimolar mixture of [(12)C(6)]glucose and [(13)C(6)]glucose and then subjected to reduction of the disulfide bridges followed by tryptic hydrolysis. The digest obtained was analyzed by LC-MS. The glycation products were identified on the basis of characteristic isotopic patterns resulting from the use of isotopically labeled glucose. This method allowed identification of 38 early Maillard reaction products and five different structures of the end glycation products. This isotopic labeling technique combined with LC-MS is a sensitive method for identification of advanced glycation end products even if their chemical structure is unknown. PMID:22964226

  5. Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

    SciTech Connect

    DeMaeyer-Guignard, J.; Cachard, A.; DeMaeyer, E.

    1982-07-30

    Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either (/sup 35/S)methionine or a /sup 14/C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all (/sup 35/S)methionine-labeled proteins and 2.6% of all /sup 14/C-amino acid-labeled proteins released into the medium.

  6. Stable isotope labeling by amino acids applied to bacterial cell culture.

    PubMed

    Soufi, Boumediene; Macek, Boris

    2014-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a widely used approach in quantitative proteomics; however, due to limitations such as required auxotrophy for the amino acids employed for labeling, it was thus far rarely employed in bacteria. Although limitations of SILAC in microbiological applications are significant and restrict its use exclusively to cells cultured in minimal media, we and others have successfully used it to fully label proteomes of model bacteria and measure their relative expression dynamics under different experimental conditions. Here we provide a brief overview of applications of SILAC in bacteria and describe a detailed protocol for SILAC labeling of Escherichia coli and Bacillus subtilis cells in culture, which in many cases can be applied to other members of both gram-positive and gram-negative bacterial species. PMID:25059601

  7. Quantifying proteomes and their post-translational modifications by stable isotope label-based mass spectrometry

    PubMed Central

    Merrill, Anna E.; Coon, Joshua J.

    2013-01-01

    Stable isotope labeling coupled with mass spectrometry has revolutionized the scope and impact of protein expression studies. Label incorporation can occur metabolically or chemically, and each method bears specific strengths and weaknesses. Quantitative proteomics confidently identifies specific interactions between proteins and other biological species, such as nucleic acids and metabolites. Extending label-based methods to phosphorylation-modified forms of proteins enables the construction of signaling networks and their temporal responses to stimuli. The integration of multiple data types offers systems-level insight on coordinated biological processes. Finally, the development of methods applicable to tissue quantification suggests the emerging role of label-based, quantitative mass spectrometry in translational science. PMID:23835517

  8. Radiogenic Nd isotope labeling of the northern NE Atlantic during MIS 2

    NASA Astrophysics Data System (ADS)

    Roberts, Natalie L.; Piotrowski, Alexander M.

    2015-08-01

    Paleoceanographic reconstructions rely on chemical proxies which are controlled by physical, chemical, and biological marine parameters. The accurate interpretation of proxy records relies on the integrity of proxy-environmental relationships through time, and under changing conditions. In this study we closely examine paleo controls on authigenic Nd isotope records from five cores in the northern NE Atlantic, approximating a depth-transect, allowing spatial and temporal relationships to be reconstructed. We compare our Nd isotope records with other paleocirculation proxies, and consider the sedimentalogical controls on Nd isotope signals, by comparing ice-rafted detritus lithology and counts, detrital sediment chemistry and redox sensitive element concentrations measured on foraminifera authigenic coatings. With this suite of geochemical and sedimentalogical data we show that Nd isotope records in the northern NE Atlantic were labeled by radiogenic sediments, however this modification did not occur in the pore-waters of each core, but instead likely reflects changes in the Nd isotopic composition of deep-waters caused by the input of ice-rafted sediment during Heinrich events and the last glacial maximum. This study has implications for understanding how localized changes in the Nd isotope signal can set a watermass end-member composition, decoupling chemical proxy-circulation relationships locally, but providing a signal which can be potentially traced along the deep-water flowpath. Such scenarios must be considered in future interpretations of glacial Nd isotope records taken from within the ice-rafted detritus belt and downstream along watermass flowpaths.

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

    NASA Astrophysics Data System (ADS)

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

    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.

  10. Use of oxygen-18 isotopic labeling to assay photorespiration in terrestrial plants and algae

    SciTech Connect

    de Veau, E.J.

    1988-01-01

    A new method was devised to quantify photorespiration. The assay utilized {sup 18}O{sub 2} to isotopically label intermediates of the glycolate pathway, specifically glycolate, glycine, and serine, for various time periods. The pathway intermediates were isolated and analyzed on a mass spectrometer to determine molecular percent {sup 18}O-enrichment. Rates of glycolate synthesis were determined from: {sup 18}O-labeling kinetics of the intermediates, derived rate equations, and non-linear regression techniques. The method was adapted to measure photorespiratory rates in both terrestrial plants and algae. Test plants are Triticum aestivum, Zea mays L., Pavlova lutheri and Chlorella pyrenoidosa.

  11. Synthesis of an Isotopically Labeled Naphthalene Derivative That Supports a Long-Lived Nuclear Singlet State

    PubMed Central

    2015-01-01

    The synthesis of an octa-alkoxy substituted isotopically labeled naphthalene derivative, shown to have excellent properties in singlet NMR experiments, is described. This highly substituted naphthalene system, which incorporates an adjacent 13C spin pair, is readily accessed from a commercially available 13C2-labeled building block via sequential thermal alkynyl- and arylcyclobutenone rearrangements. The synthetic route incorporates a simple desymmetrization approach leading to a small difference in the chemical shifts of the 13C spin pair, a design constraint crucial for accessing nuclear singlet order. PMID:25898076

  12. Novel approaches in selective tryptophan isotope labeling by using Escherichia coli overexpression media.

    PubMed

    Schrghuber, Julia; Sra, Tom; Bisaccia, Marilena; Schmid, Walther; Konrat, Robert; Lichtenecker, Roman J

    2015-03-23

    NMR-based investigations of large protein complexes require optimized isotopic labeling schemes. We report new methods to introduce stable isotopes into tryptophan residues; these are fine-tuned to the requirements of the particular protein NMR experiment. Selective backbone labeling was performed by using a new ?-ketoacid precursor as an additive in cell-based overexpression media. Additionally, we developed synthetic routes to certain isotopologues of indole with (13)C-(1)H spin systems surrounded by (12)C and (2)H. The corresponding proteins, overexpressed in the presence of these precursor compounds, can be effectively analyzed for conformational changes in tryptophan residues in response to external stimuli, such as interaction with other proteins or small molecules. PMID:25703586

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

    SciTech Connect

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

    2011-03-04

    We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

  14. Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

    PubMed

    Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

    2012-01-01

    Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills. PMID:22665301

  15. Atlas encoding by randomized forests for efficient label propagation.

    PubMed

    Zikic, Darko; Glocker, Ben; Criminisi, Antonio

    2013-01-01

    We propose a method for multi-atlas label propagation based on encoding the individual atlases by randomized classification forests. Most current approaches perform a non-linear registration between all atlases and the target image, followed by a sophisticated fusion scheme. While these approaches can achieve high accuracy, in general they do so at high computational cost. This negatively affects the scalability to large databases and experimentation. To tackle this issue, we propose to use a small and deep classification forest to encode each atlas individually in reference to an aligned probabilistic atlas, resulting in an Atlas Forest (AF). At test time, each AF yields a probabilistic label estimate, and fusion is done by averaging. Our scheme performs only one registration per target image, achieves good results with a simple fusion scheme, and allows for efficient experimentation. In contrast to standard forest schemes, incorporation of new scans is possible without retraining, and target-specific selection of atlases remains possible. The evaluation on three different databases shows accuracy at the level of the state of the art, at a significantly lower runtime. PMID:24505745

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

    PubMed

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

    2015-01-20

    Human fecal samples contain endogenous human metabolites, gut microbiota metabolites, and other compounds. Profiling the fecal metabolome can produce metabolic information that may be used not only for disease biomarker discovery, but also for providing an insight about the relationship of the gut microbiome and human health. In this work, we report a chemical isotope labeling liquid chromatography-mass spectrometry (LC-MS) method for comprehensive and quantitative analysis of the amine- and phenol-containing metabolites in fecal samples. Differential (13)C2/(12)C2-dansyl labeling of the amines and phenols was used to improve LC separation efficiency and MS detection sensitivity. Water, methanol, and acetonitrile were examined as an extraction solvent, and a sequential water-acetonitrile extraction method was found to be optimal. A step-gradient LC-UV setup and a fast LC-MS method were evaluated for measuring the total concentration of dansyl labeled metabolites that could be used for normalizing the sample amounts of individual samples for quantitative metabolomics. Knowing the total concentration was also useful for optimizing the sample injection amount into LC-MS to maximize the number of metabolites detectable while avoiding sample overloading. For the first time, dansylation isotope labeling LC-MS was performed in a simple time-of-flight mass spectrometer, instead of high-end equipment, demonstrating the feasibility of using a low-cost instrument for chemical isotope labeling metabolomics. The developed method was applied for profiling the amine/phenol submetabolome of fecal samples collected from three families. An average of 1785 peak pairs or putative metabolites were found from a 30 min LC-MS run. From 243 LC-MS runs of all the fecal samples, a total of 6200 peak pairs were detected. Among them, 67 could be positively identified based on the mass and retention time match to a dansyl standard library, while 581 and 3197 peak pairs could be putatively identified based on mass match using MyCompoundID against a Human Metabolome Database and an Evidence-based Metabolome Library, respectively. This represents the most comprehensive profile of the amine/phenol submetabolome ever detected in human fecal samples. The quantitative metabolome profiles of individual samples were shown to be useful to separate different groups of samples, illustrating the possibility of using this method for fecal metabolomics studies. PMID:25486321

  17. 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...

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

    PubMed

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

    2006-07-01

    Recently we have developed the stereo-array isotope labeling (SAIL) technique to overcome the conventional molecular size limitation in NMR protein structure determination by employing complete stereo- and regiospecific patterns of stable isotopes. SAIL sharpens signals and simplifies spectra without the loss of requisite structural information, thus making large classes of proteins newly accessible to detailed solution structure determination. The automated structure calculation program CYANA can efficiently analyze SAIL-NOESY spectra and calculate structures without manual analysis. Nevertheless, the original SAIL method might not be capable of determining the structures of proteins larger than 50 kDa or membrane proteins, for which the spectra are characterized by many broadened and overlapped peaks. Here we have carried out simulations of new SAIL patterns optimized for minimal relaxation and overlap, to evaluate the combined use of SAIL and CYANA for solving the structures of larger proteins and membrane proteins. The modified approach reduces the number of peaks to nearly half of that observed with uniform labeling, while still yielding well-defined structures and is expected to enable NMR structure determinations of these challenging systems. PMID:16602075

  19. Enhanced sample multiplexing for nitrotyrosine-modified proteins using combined precursor isotopic labeling and isobaric tagging.

    PubMed

    Robinson, Ren A S; Evans, Adam R

    2012-06-01

    Current strategies for identification and quantification of 3-nitrotyrosine (3NT) post-translationally modified proteins (PTM) generally rely on biotin/avidin enrichment. Quantitative approaches have been demonstrated which employ isotopic labeling or isobaric tagging in order to quantify differences in the relative abundances of 3NT-modified proteins in two or potentially eight samples, respectively. Here, we present a novel strategy which uses combined precursor isotopic labeling and isobaric tagging (cPILOT) to increase the multiplexing capability of quantifying 3NT-modified proteins to 12 or 16 samples using commercially available tandem mass tags (TMT) or isobaric tags for relative and absolute quantification (iTRAQ), respectively. This strategy employs "light" and "heavy" labeled acetyl groups to block both N-termini and lysine residues of tryptic peptides. Next, 3NT is reduced to 3-aminotyrosine (3AT) using sodium dithionite followed by derivatization of light and heavy labeled 3AT-peptides with either TMT or iTRAQ multiplex reagents. We demonstrate the proof-of-principle utility of cPILOT with in vitro nitrated bovine serum albumin (BSA) and mouse splenic proteins using TMT(0), TMT(6), and iTRAQ(8) reagents and discuss limitations of the strategy. PMID:22509719

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

    PubMed

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

    2014-11-01

    This study reports the convenient, low cost, one-step synthesis of labelled analogues of six volatile phenols, guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-ethylphenol, eugenol and vanillin, using microwave-assisted deuterium exchange, for use as internal standards for stable isotope dilution analysis. The current method improves on previous strategies in that it enables incorporation of deuterium atoms on the aromatic ring, thereby ensuring retention of the isotope label during mass spectrometry fragmentation. When used as standards for SIDA, these labelled volatile phenols will improve the accuracy and reproducibility of quantitative food and beverage analysis. PMID:24874385

  1. Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides.

    PubMed

    Lelyveld, Victor S; Bjrkbom, Anders; Ransey, Elizabeth M; Sliz, Piotr; Szostak, Jack W

    2015-12-16

    High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex. PMID:26583201

  2. Pinpointing RNA-Protein Cross-Links with Site-Specific Stable Isotope-Labeled Oligonucleotides

    PubMed Central

    2015-01-01

    High affinity RNA-protein interactions are critical to cellular function, but directly identifying the determinants of binding within these complexes is often difficult. Here, we introduce a stable isotope mass labeling technique to assign specific interacting nucleotides in an oligonucleotide-protein complex by photo-cross-linking. The method relies on generating site-specific oxygen-18-labeled phosphodiester linkages in oligonucleotides, such that covalent peptide-oligonucleotide cross-link sites arising from ultraviolet irradiation can be assigned to specific sequence positions in both RNA and protein simultaneously by mass spectrometry. Using Lin28A and a let-7 pre-element RNA, we demonstrate that mass labeling permits unambiguous identification of the cross-linked sequence positions in the RNA-protein complex. PMID:26583201

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

    USGS Publications Warehouse

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

    2007-01-01

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

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

    PubMed

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

    2014-01-01

    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

  5. Molecular and mass spectroscopic analysis of isotopically labeled organic residues

    NASA Technical Reports Server (NTRS)

    Mendoza-Gomez, Celia X.; Greenberg, J. Mayo; Mccain, P.; Ferris, J. P.; Briggs, R.; Degroot, M. S.; Schutte, Willem A.

    1989-01-01

    Experimental studies aimed at understanding the evolution of complex organic molecules on interstellar grains were performed. The photolysis of frozen gas mixtures of various compositions containing H2O, CO, NH3, and CH4 was studied. These species were chosen because of their astrophysical importance as deducted from observational as well as theoretical studies of ice mantles on interstellar grains. These ultraviolet photolyzed ices were warmed up in order to produce refractory organic molecules like the ones formed in molecular clouds when the icy mantles are being irradiated and warmed up either by a nearby stellar source or impulsive heating. The laboratory studies give estimates of the efficiency of production of such organic material under interstellar conditions. It is shown that the gradual carbonization of organic mantles in the diffuse cloud phase leads to higher and higher visual absorptivity - yellow residues become brown in the laboratory. The obtained results can be applied to explaining the organic components of comets and their relevance to the origin of life.

  6. METHOD TO TEST ISOTOPIC SEPARATION EFFICIENCY OF PALLADIUM PACKED COLUMNS

    SciTech Connect

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

    2007-06-27

    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.

  7. Tests of isotopic separation efficiency of palladium packed columns

    SciTech Connect

    Heung, L. K.; Staack, G. C.; Klein, J. E.; Jacobs, W. D.

    2008-07-15

    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 presence were tested using an isotope displacement method. A simple computer model was also developed to calculate the number of theoretical separation stages based on the test results. The effects of column diameter, metal foam presence and gas flow rate were identified. (authors)

  8. Isotope-Labeled Amyloids via Synthesis, Expression, and Chemical Ligation for Use in FTIR, 2D IR, and NMR Studies.

    PubMed

    Zhang, Tianqi O; Grechko, Maksim; Moran, Sean D; Zanni, Martin T

    2016-01-01

    This chapter provides protocols for isotope-labeling the human islet amyloid polypeptide (hIAPP or amylin) involved in type II diabetes and γD-crystallin involved in cataract formation. Because isotope labeling improves the structural resolution, these protocols are useful for experiments using Fourier transform infrared (FTIR), two-dimensional infrared (2D IR), and NMR spectroscopies. Our research group specializes in using 2D IR spectroscopy and isotope labeling. 2D IR spectroscopy provides structural information by measuring solvation from 2D diagonal lineshapes and vibrational couplings from cross peaks. Infrared spectroscopy can be used to study kinetics, membrane proteins, and aggregated proteins. Isotope labeling provides greater certainty in the spectral assignment, which enables new structural insights that are difficult to obtain with other methods. For amylin, we provide a protocol for (13)C/(18)O labeling backbone carbonyls at one or more desired amino acids in order to obtain residue-specific structural resolution. We also provide a protocol for expressing and purifying amylin from E. coli, which enables uniform (13)C or (13)C/(15)N labeling. Uniform labeling is useful for measuring the monomer infrared spectrum in an amyloid oligomer or fiber as well as amyloid protein bound to another polypeptide or protein, such as a chaperone or an inhibitor. In addition, our expression protocol results in 2-2.5 mg of amylin peptide per 1 L cell culture, which is a high enough yield to straightforwardly obtain the 2-10 mg needed for high resolution and solid-state NMR experiments. Finally, we provide a protocol to isotope-label either of the two domains of γD-crystallin using expressed protein ligation. Domain labeling makes it possible to resolve the structures of the two halves of the protein in FTIR and 2D IR spectra. With modifications, these strategies and protocols for isotope labeling can be applied to other amyloid polypeptides and proteins. PMID:26453203

  9. Comparison of Acetate Turnover in Methanogenic and Sulfate-Reducing Sediments by Radiolabeling and Stable Isotope Labeling and by Use of Specific Inhibitors: Evidence for Isotopic Exchange

    PubMed Central

    de Graaf, W.; Wellsbury, P.; Parkes, R. J.; Cappenberg, T. E.

    1996-01-01

    Acetate turnover in the methanogenic freshwater anoxic sediments of Lake Vechten, The Netherlands, and in anoxic sediments from the Tamar Estuary, United Kingdom, and the Grosser Jasmunder Bodden, Germany, the latter two dominated by sulfate reduction, was determined. Stable isotopes and radioisotopes, inhibitors (chloroform and fluoroacetate), and methane flux were used to provide independent estimates of acetate turnover. Pore water acetate pool sizes were determined by gas chromatography with a flame ionization detector, and stable isotope-labeled acetate was determined by gas chromatography-mass spectrometry. The appearance of acetates with a different isotope labeling pattern from that initially added demonstrated that isotopic exchange occurred during methanogenic acetate metabolism. The predominant exchange processes were (i) D-H exchange in the methyl group and (ii) (sup13)C-(sup12)C exchange at the carboxyl carbon. These exchanges are most probably caused by the activity of the enzyme complex carbon monoxide dehydrogenase and subsequent methyl group dehydrogenation by tetrahydromethanopterine or a related enzyme. The methyl carbon was not subject to exchange during transformation to methane, and hence acetate with the methyl carbon labeled will provide the most reliable estimate of acetate turnover to methane. Acetate turnover rate estimates with these labels were consistent with independent estimates of acetate turnover (acetate accumulation after inhibition and methane flux). Turnover rates from either radioisotope- or stable isotope-labeled methyl carbon isotopes are, however, dependent on accurate determination of the acetate pool size. The additions of large amounts of stable isotope-labeled acetate elevate the acetate pool size, stimulating acetate consumption and causing deviation from steady-state kinetics. This can, however, be overcome by the application of a non-steady-state model. Isotopic exchange in sediments dominated by sulfate reduction was minimal. PMID:16535268

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

    PubMed

    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

    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

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

    PubMed Central

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

    2013-01-01

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

  12. Segmental isotopic labeling of the Hsp70 molecular chaperone DnaK using expressed protein ligation

    PubMed Central

    Clerico, Eugenia M.; Zhuravleva, Anastasia; Smock, Robert G.; Gierasch, Lila M.

    2010-01-01

    Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including choice of site for ligation with retention of function, optimization of ligation yield, and purification from unreacted domains. Ligated semi-labeled DnaK was successfully produced with a Cys residue at position 383, and the ligated product harboring the Cys mutation was confirmed to be functional and identical to an expressed Cys-containing two-domain construct. The NMR spectrum of the segmentally labeled protein was considerably simplified, enabling unequivocal assignment and enhanced analysis of dynamics, as a prelude to exploring the energy landscape for allostery in the Hsp70 family. PMID:20564022

  13. Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR.

    PubMed

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

    2013-10-01

    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-(13)C] 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-(13)C] 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

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

    SciTech Connect

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

    2006-04-10

    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.

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

    PubMed

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

    2015-09-10

    Two potent glucocorticoid receptor agonists were prepared labeled with carbon-14 and with stable isotopes to perform drug metabolism, pharmacokinetics, and bioanalytical studies. Carbon-14 labeled (1) was obtained from an enantiopure alkyne (5) via a Sonogashira coupling to a previously reported 5-amino-4-iodo-[2-(14) C]pyrimidine [(14) C]-(6), followed by a base-mediated cyclization (1) in 72% overall radiochemical yield. Carbon-14 labeled (2) was prepared in five steps employing a key benzoic acid intermediate [(14) C]-(13), which was synthesized in one pot from enolization of trifluoromethylketone (12), followed by bromine-magnesium exchange and then electrophile trapping reaction with [(14) C]-carbon dioxide. A chiral auxiliary (S)-1-(4-methoxyphenyl)ethylamine was then coupled to this acid to give [(14) C]-(15). Propargylation and separation of diastereoisomers by crystallizations gave the desired diastereomer [(14) C]-(17) in 34% yield. Sonogashira coupling to iodopyridine (10) followed by cyclization to the azaindole [(14) C]-(18) and finally removal of the chiral auxiliary gave [(14) C]-(2) in 7% overall yield. For stable isotope syntheses, [(13) C6 ]-(1) was obtained in three steps using [(13) C4 ]-(6) and trimethylsilylacetylene-[(13) C2 ] in 26% yield, while [(2) H5 ]-(2) was obtained by first preparing the iodopyridine [(2) H5 ]-(10) in five steps. Then, Sonogashira coupling to chiral alkyne (24) and cyclization gave [(2) H5 ]-(2) in 42% overall yield. PMID:26391408

  16. PCR and non-isotopic labeling techniques for plant virus detection.

    PubMed

    Fenby, N S; Scott, N W; Slater, A; Elliott, M C

    1995-07-01

    PCR technology permits the detection of viruses at levels several orders of magnitude lower than is possible by other methods. This high sensitivity facilitates detection of virus sequences during the early stages of infection of plants and in soil and vector samples. Early detection of beet necrotic yellow vein virus (BNYVV) in Beta vulgaris is an important part of the strategy for prevention of the spread of rhizomania, a commercially significant disease of sugar beet. A diagnostic test for BNYVV has been developed. This test involves amplification of the viral genome by PCR coupled with non-isotopic labeling and detection of specific sequences. The PCR amplification of BNYVV sequences has been optimized with respect to primer design, sample preparation and reaction conditions. Several non-isotopic labeling strategies for signal amplification have been compared. Hybridization with digoxigenin-labelled cDNA permits the most sensitive detection of PCR products and is the most appropriate method for routine diagnosis. These observations are discussed in the context of the application of PCR for detecting a wide range of viruses. PMID:7580844

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  18. Pharmacokinetic and metabolism studies using uniformly stable isotope labeled proteins with HPLC/CRIMS detection.

    PubMed

    Osborn, B L; Abramson, F P

    1998-10-01

    We present a novel method for performing pharmacokinetic and metabolism studies on macromolecules that offers advantages over the existing techniques of radiolabeling, immunoassay or bioassays. Our strategy uses macromolecules with stable isotopes uniformly distributed throughout the structure. The stable isotope enrichment is detected using high performance liquid chromatography combined with chemical reaction interface mass spectrometry (HPLC/CRIMS). HPLC/CRIMS is a technique where analytes are first eluted from an HPLC column and then dissociated in a microwave reaction chamber. The dissociated analytes are oxidized using SO2 and the resulting small molecules are detected by the mass spectrometer. The stable-isotope labeled analyte is distinguished from the matrix carbon by monitoring the enrichment of 13CO2. In order to demonstrate the feasibility of performing pharmacokinetic and metabolism studies using this technique, uniformly 13C, 15N-labeled rat growth hormone was administered intravenously to rats and blood samples were collected. Raw plasma samples were analysed by HPLC/CRIMS. Growth hormone was detectable for 1 h following administration. The absolute amounts detected ranged from a high of 66 pmol to a low of 825 fmol in a 20 microL plasma sample. The data were modeled using PCNONLIN and were consistent with a one compartment model. The calculated half-life was 7.7 +/- 0.7 min, with a clearance of 4.5 +/- 0.3 mL min(-1), values consistent with literature reports for growth hormone in rats. No circulating growth hormone metabolites were detected in the plasma. This paper demonstrates a novel technique for performing pharmacokinetic studies of proteins. The uniform labeling strategy also presents a viable comprehensive method for obtaining metabolism data on macromolecules. PMID:9818710

  19. Apolipoprotein B metabolism in humans: studies with stable isotope-labeled amino acid precursors.

    PubMed

    Marsh, Julian B; Welty, Francine K; Lichtenstein, Alice H; Lamon-Fava, Stefania; Schaefer, Ernst J

    2002-06-01

    This article reviews the literature from 1986 to early 2001 relating to apoB100 and apoB48 kinetics in humans using amino acid precursors labeled with stable isotopes. The following subjects are reviewed: (1) methodology; (2) normal individuals and the effects of aging; (3) diet; (4) hereditary dyslipidemias: familial hypercholesterolemia, familial combined hyperlipidemia, cholesteryl ester storage disease, cholesteryl ester transfer protein deficiency, lipoprotein lipase deficiency, familial hypobetalipoproteinemia, and truncated forms of apoB; (5) hormonal perturbations: estrogen, insulin, diabetes, obesity, and growth hormone; (6) the nephrotic syndrome; and (7) the effects of the statin class of drugs. Because of the advances which have been made in mass spectrometry techniques, the advantages of using non-radioactive tracers in humans have made stable isotope kinetic studies the present day standard in this area of research. PMID:11996942

  20. Isotopically labeled CO{sub 2} from stratosphere: A tracer of carbon biogeochemistry

    SciTech Connect

    Yung, Yuk L.; Thiemens, M.H.

    1993-11-01

    It has been recently discovered that it the stratosphere is a source of isotopically enriched CO{sub 2}: CO{sup 18}O and CO{sub 17}O. The cause of this isotopic enrichment is exchange between heavy O{sub 3} and CO{sub 2} via the excited radical O({sup 1D}). The research effort consists of a coordinated laboratory and model surfaces of isotopomers of CO{sub 2}. The laboratory study yields data on the chemical kinetics of oxygen exchange between CO{sub 2} and O{sub 3}. The modeling study uses the laboratory results as well as atmospheric measurements to model the source and sinks of CO{sub 2} isotopomers in the stratosphere and troposphere. It is expected that this combined study will bring new insights on the exchange of CO{sub 2} between the atmosphere and the biosphere. The goals of this study are to study the kinetic pathways for isotopic exchange between O{sub 2} and CO{sub 2} and to study O{sub 3}: the exchange rate of isotopically labelled CO{sub 2} between the stratosphere and the troposphere.

  1. Quantitation of protein phosphorylation in pregnant rat uteri using stable isotope dimethyl labeling coupled with IMAC.

    PubMed

    Huang, Sheng-Yu; Tsai, Mei-Ling; Wu, Chin-Jen; Hsu, Jue-Liang; Ho, Shih-Hsin; Chen, Shu-Hui

    2006-03-01

    Quantitative analysis of protein phosphorylation provides important insights into molecular signaling mechanisms and a better understanding of many cellular processes. In this study, we coupled stable isotope dimethyl labeling with immobilized metal affinity chromatography (IMAC) enrichment to quantify protein phosphorylation at MS-determined phosphorylation sites. The proposed method was first characterized using alpha- and beta-casein as two model phosphoproteins, and further applied to the analysis of pregnant rat uteri with and without treatment with 8-bromo-cGMP. Dimethyl labeling has several significant advantages: global, fast (within 5 min) and complete (near 100%). Our results indicate that the labeling has no adverse effect on the IMAC enrichment for tryptic peptides having single and multiple phosphorylation sites. Moreover, the enhanced a1 signal and the complete reaction by dimethyl labeling provide unequivocal identification of both the N-terminal amino acid and the number of the labeling site. Using these two criteria in data validation, which is particularly important for identifying phosphoproteins, we found that the confidence in interpreting dimethyl-labeled peptides had greatly increased. In the analysis of late gestation rat uteri, the abundance ratio between treated and un-treated phosphopeptide signals ranged from 0.51 to 1.69 with an average of around 1.01 +/- 0.25. The obtained ratio of the phosphorylation levels at Ser 15 of HSP27 was further confirmed by the consistent results obtained from Western blot analyses. Based on the analysis of the results, it is interesting to note that the activated cGMP dependent protein kinase G (PKG) seems to affect the phosphorylation of proteins associated with the inhibition of cell migration and proliferation, redistribution of actin-associated proteins, and the increase of protein synthesis in late-gestation uteri. These observations provide important evidence suggesting that activated PKG may play a critical role in the shift of pregnant uteri from proliferative to hypertrophic states. PMID:16470654

  2. Dansylation isotope labeling liquid chromatography mass spectrometry for parallel profiling of human urinary and fecal submetabolomes.

    PubMed

    Su, Xiaoling; Wang, Nan; Chen, Deying; Li, Yunong; Lu, Yingfeng; Huan, Tao; Xu, Wei; Li, Liang; Li, Lanjuan

    2016-01-15

    Human urine and feces can be non-invasively collected for metabolomics-based disease biomarker discovery research. Because urinary and fecal metabolomes are thought to be different, analysis of both biospecimens may generate a more comprehensive metabolomic profile that can be better related to the health state of an individual. Herein we describe a method of using differential chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS) for parallel metabolomic profiling of urine and feces. Dansylation labeling was used to quantify the amine/phenol submetabolome changes among different samples based on (12)C-labeling of individual samples and (13)C-labeling of a pooled urine or pooled feces and subsequent analysis of the (13)C-/(12)C-labeled mixture by LC-MS. The pooled urine and pooled feces are further differentially labeled, mixed and then analyzed by LC-MS in order to relate the metabolite concentrations of the common metabolites found in both biospecimens. This method offers a means of direct comparison of urinary and fecal submetabolomes. We evaluated the analytical performance and demonstrated the utility of this method in the analysis of urine and feces collected daily from three healthy individuals for 7 days. On average, 2534113 (n=126) peak pairs or metabolites could be detected from a urine sample, while 250777 (n=63) peak pairs were detected from a fecal sample. In total, 5372 unique peak pairs were detected from all the samples combined; 3089 and 3012 pairs were found in urine and feces, respectively. These results reveal that the urine and fecal metabolomes are very different, thereby justifying the consideration of using both biospecimens to increase the probability of finding specific biomarkers of diseases. Furthermore, the CIL LC-MS method described can be used to perform parallel quantitative analysis of urine and feces, resulting in more complete coverage of the human metabolome. PMID:26709303

  3. A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling.

    PubMed

    Guan, Shenheng; Price, John C; Prusiner, Stanley B; Ghaemmaghami, Sina; Burlingame, Alma L

    2011-12-01

    In a recent study, in vivo metabolic labeling using (15)N traced the rate of label incorporation among more than 1700 proteins simultaneously and enabled the determination of individual protein turnover rate constants over a dynamic range of three orders of magnitude (Price, J. C., Guan, S., Burlingame, A., Prusiner, S. B., and Ghaemmaghami, S. (2010) Analysis of proteome dynamics in the mouse brain. Proc. Natl. Acad. Sci. U. S. A. 107, 14508-14513). These studies of protein dynamics provide a deeper understanding of healthy development and well-being of complex organisms, as well as the possible causes and progression of disease. In addition to a fully labeled food source and appropriate mass spectrometry platform, an essential and enabling component of such large scale investigations is a robust data processing and analysis pipeline, which is capable of the reduction of large sets of liquid chromatography tandem MS raw data files into the desired protein turnover rate constants. The data processing pipeline described in this contribution is comprised of a suite of software modules required for the workflow that fulfills such requirements. This software platform includes established software tools such as a mass spectrometry database search engine together with several additional, novel data processing modules specifically developed for (15)N metabolic labeling. These fulfill the following functions: (1) cross-extraction of (15)N-containing ion intensities from raw data files at varying biosynthetic incorporation times, (2) computation of peptide (15)N isotopic incorporation distributions, and (3) aggregation of relative isotope abundance curves for multiple peptides into single protein curves. In addition, processing parameter optimization and noise reduction procedures were found to be necessary in the processing modules in order to reduce propagation of errors in the long chain of the processing steps of the entire workflow. PMID:21937731

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

    PubMed

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

    2015-06-01

    Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an ?-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies. PMID:25740604

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

    PubMed Central

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

    2014-01-01

    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

  6. Proteome Scale-Protein Turnover Analysis Using High Resolution Mass Spectrometric Data from Stable-Isotope Labeled Plants.

    PubMed

    Fan, Kai-Ting; Rendahl, Aaron K; Chen, Wen-Ping; Freund, Dana M; Gray, William M; Cohen, Jerry D; Hegeman, Adrian D

    2016-03-01

    Protein turnover is an important aspect of the regulation of cellular processes for organisms when responding to developmental or environmental cues. The measurement of protein turnover in plants, in contrast to that of rapidly growing unicellular organismal cultures, is made more complicated by the high degree of amino acid recycling, resulting in significant transient isotope incorporation distributions that must be dealt with computationally for high throughput analysis to be practical. An algorithm in R, ProteinTurnover, was developed to calculate protein turnover with transient stable isotope incorporation distributions in a high throughput automated manner using high resolution MS and MS/MS proteomic analysis of stable isotopically labeled plant material. ProteinTurnover extracts isotopic distribution information from raw MS data for peptides identified by MS/MS from data sets of either isotopic label dilution or incorporation experiments. Variable isotopic incorporation distributions were modeled using binomial and beta-binomial distributions to deconvolute the natural abundance, newly synthesized/partial-labeled, and fully labeled peptide distributions. Maximum likelihood estimation was performed to calculate the distribution abundance proportion of old and newly synthesized peptides. The half-life or turnover rate of each peptide was calculated from changes in the distribution abundance proportions using nonlinear regression. We applied ProteinTurnover to obtain half-lives of proteins from enriched soluble and membrane fractions from Arabidopsis roots. PMID:26824330

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

    NASA Astrophysics Data System (ADS)

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

    2010-12-01

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

  8. Evaluation of the accuracy of protein quantification using isotope TMPP-labeled peptides.

    PubMed

    Shen, Hongyan; An, Mingrui; Zou, Xiao; Zhao, Xuyang; Wang, Qingsong; Xing, Guowen; Ji, Jianguo

    2015-09-01

    N-succinimidyloxycarbonylmethyl tris(2,4,6-trimethoxyphenyl) phosphonium bromide (TMPP-Ac-OSu) reacts rapidly, mildly, and specifically with the N-terminals of proteins and peptides. Thus, it can be developed as an ideal isotope-coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP-based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on-tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP-labeled peptides were combined at different ratios and subsequently analyzed by LC-MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope-coded TMPP method achieved accurate quantification and compatibility with IEF-based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis. PMID:25930663

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

    PubMed

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

    2011-10-01

    Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and non-cleaved proteins by peptide isotope quantification and bioinformatics search criteria. PMID:21959240

  10. Determination of protein conformation by isotopically labelled cross-linking and dedicated software

    NASA Astrophysics Data System (ADS)

    Nielsen, Tina; Thaysen-Andersen, Morten; Larsen, Nanna; Jørgensen, Flemming S.; Houen, Gunnar; Højrup, Peter

    2007-12-01

    Chemical cross-linking in conjunction with mass spectrometry (MS) can be used for sensitive and rapid investigation of the three-dimensional structure of proteins at low resolution. However, the resulting data are very complex, and on the bioinformatic side, there still exists an urgent need for improving computer software for (semi-) automated cross-linking data analysis. In this study, we have developed dedicated software for rapid and confident identification and validation of cross-linked species using an isotopic labelled cross-linker approach in combination with MS. Deuterated (+4 Da) and non-deuterated (+0 Da) bis(sulfosuccinimidyl)suberate, BS3, was used as homobifunctional cross-linker to tag the cross-linked regions. Peptides generated from proteolysis were separated using high performance liquid chromatography, and peptide mass fingerprinting was obtained for the individual fractions using matrix-assisted laser-desorption ionisation time-of-flight (MALDI TOF) MS. The resulting peptide mass lists were combined and transferred to the program, ProteinXXX, which generated the theoretical mass values of all combinations of cross-linked peptides and dead-end cross-links and compared this to the obtained mass lists. In addition, screening for 4 Da-separated signals aided the identification of potential cross-linked species. Sequence information of these candidates was then obtained using MALDI TOF TOF. The cross-linked peptides could then be validated based on the match of the fragmentation pattern and the theoretical values produced by ProteinXXX. This semi-automated interpretation provided a high analysis speed of cross-linking data, with efficient and confident identification of cross-linked species. Four experiments using different conditions showed a high degree of reproducibility as only 1 and 2 cross-links out of 36 identified was not observed in two experiments. The method was tested using human placenta calreticulin (CRT). Based on the identified cross-links, a few corrections to a model of calreticulin obtained by homology modelling using calnexin as template can be suggested. Furthermore, the cross-links show that the C-terminal of the protein continues along the core region opposite the P-domain for at least 11 residues beyond the known structure. In addition, it was observed that the conformation of CRT does not change significantly in the presence or absence of the divalent ions, Ca2+ and Zn2+.

  11. Isotope labeling studies on the electron impact mass spectral fragmentation patterns of chloropropanol acetates.

    PubMed

    Rahn, Anja K K; Yaylayan, Varoujan A

    2013-09-18

    Chloropropanol (CP) esters are part of an emerging group of process-induced toxicants that are considered as potential health hazards particularly in palm oil. Mass spectrometry-based methodologies for identification of CP esters in food are critical in overcoming the challenges associated with direct detection methods. In the present study, a convenient strategy was employed to generate all possible CP acetates through reacting acetic anhydride with either glycerol in the presence of a chloride source or the corresponding CPs, such as 3-chloro-, 1,3-dichloro-, 2-chloro-, and 1,2-dichloropropanols, allowing for the identification of the individual CP acetates and assignment of their mass spectral fragmentations. Mass spectral fragmentations were confirmed through the use of the isotopic signature of chlorine in addition to the isotope labeling experiments performed using isotopically labeled precursors, such as [C-U?] glycerol, [C-U?] acetic anhydride, [C-2,2'] acetic anhydride, and [d?] 3-monochloropropane-1,2-diol (3-MCPD) as reactants. Such studies have indicated that all CP esters can undergo two general fragmentations under electron impact (EI) conditions, one generating the acylium ion at m/z 45 and the other generating a chlorinated cyclic acyloxonium ion at m/z 135.6. Considering the fact that such ions can also be generated from any fatty acid containing CP esters after undergoing McLafferty rearrangement, the ion at m/z 135.6 can therefore be considered as a universal marker for the presence of CP esters undergoing EI fragmentation. Furthermore, these studies have also indicated the formation of ions characteristic of CP diesters, monoesters, and dichloro esters. PMID:23964824

  12. Identification of Biologically-Produced Organic Matter in an Aquifer System using Stable Isotope Labeling

    NASA Astrophysics Data System (ADS)

    Kujawinski, E. B.; Mailloux, B. J.; Morrison, L.

    2004-12-01

    The carbon cycle in aquifer systems is poorly understood. In particular, the role of prokaryotic and eukaryotic microbes in the cycling of organic matter (OM) has not been well documented. The goal of this work was to utilize stable isotopes in combination with geochemical and microbial methods to study microbially-mediated OM transformations in aquifers and aquifer sediments. A laboratory flow-through column experiment was conducted with sediment collected from a pristine, shallow, coastal plain aquifer. The groundwater medium was amended with low levels of an isotopically labeled nutrient, 13C-acetate. At pre-determined time points, organic matter (OM) was isolated from the sediment and groundwater. OM components were analyzed by isotope-ratio GC-MS and electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). The incorporation of 13C was examined for both the aqueous phase and sediment bound OM. Analyzing both dissolved and adsorbed fractions enabled us to determine the relative importance of each on microbe-mediated OM transformations. The incorporation of 13C allowed us to estimate residence times of different organic matter fractions and to answer fundamental questions about organic matter lability in the subsurface environment.

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

    NASA Astrophysics Data System (ADS)

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

    2011-08-01

    Isoindoline nitroxides are potentially useful probes for viable biological systems, exhibiting low cytotoxicity, moderate rates of biological reduction and favorable Electron Paramagnetic Resonance (EPR) characteristics. We have evaluated the anionic (5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl; CTMIO), cationic (5-( N, N, N-trimethylammonio)-1,1,3,3-tetramethylisoindolin-2-yloxyl iodide, QATMIO) and neutral (1,1,3,3-tetramethylisoindolin-2-yloxyl; TMIO) nitroxides and their isotopically labeled analogs ( 2H 12- and/or 2H 12- 15N-labeled) as potential EPR oximetry probes. An active ester analogue of CTMIO, designed to localize intracellularly, and the azaphenalene nitroxide 1,1,3,3-tetramethyl-2,3-dihydro-2-azaphenalen-2-yloxyl (TMAO) were also studied. While the EPR spectra of the unlabeled nitroxides exhibit high sensitivity to O 2 concentration, deuteration resulted in a loss of superhyperfine features and a subsequent reduction in O 2 sensitivity. Labeling the nitroxides with 15N increased the signal intensity and this may be useful in decreasing the detection limits for in vivo measurements. The active ester nitroxide showed approximately 6% intracellular localization and low cytotoxicity. The EPR spectra of TMAO nitroxide indicated an increased rigidity in the nitroxide ring, due to dibenzo-annulation.

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

    PubMed

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

    2006-01-01

    Most proteomics approaches for relative quantification of protein expression use a combination of stable-isotope labeling and mass spectrometry. Traditionally, researchers have used difference gel electrophoresis (DIGE) from stained 1D and 2D gels for relative quantification. While differences in protein staining intensity can often be visualized, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. A method is presented for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using In-gel Stable-Isotope Labeling (ISIL). Proteins extracted from any source (tissue, cell line, immunoprecipitate, etc.), treated under two experimental conditions, are resolved in separate lanes by gel electrophoresis. The regions of interest (visualized by staining) are reacted separately with light versus heavy isotope-labeled reagents, and the gel slices are then mixed and digested with proteases. The resulting peptides are then analyzed by LC-MS to determine relative abundance of light/heavy isotope pairs and analyzed by LC-MS/MS for identification of sequence and modifications. The strategy compares well with other relative quantification strategies, and in silico calculations reveal its effectiveness as a global relative quantification strategy. An advantage of ISIL is that visualization of gel differences can be used as a first quantification step followed by accurate and sensitive protein level stable-isotope labeling and mass spectrometry-based relative quantification. PMID:16396506

  15. Respiratory carbon metabolism following illumination in intact French bean leaves using (13)C/(12)C isotope labeling.

    PubMed

    Nogués, Salvador; Tcherkez, Guillaume; Cornic, Gabriel; Ghashghaie, Jaleh

    2004-10-01

    The origin of the carbon atoms in the CO(2) respired by French bean (Phaseolus vulgaris) leaves in the dark has been studied using (13)C/(12)C isotopes as tracers. The stable isotope labeling was achieved through a technical device that uses an open gas-exchange system coupled online to an elemental analyzer and linked to an isotope ratio mass spectrometer. The isotopic analysis of the CO(2) respired in the dark after a light period revealed that the CO(2) was labeled, but the labeling level decreased progressively as the dark period increased. The pattern of disappearance depended on the amount of carbon fixed during the labeling and indicated that there were several pools of respiratory metabolites with distinct turnover rates. We demonstrate that the carbon recently assimilated during photosynthesis accounts for less than 50% of the carbon in the CO(2) lost by dark respiration and that the proportion is not influenced by leaf starvation in darkness before the labeling. Therefore, most of the carbon released by dark respiration after illumination does not come from new photosynthates. PMID:15377781

  16. Determination of Polychlorinated Biphenyls in Solid Samples by Isotope Dilution Mass Spectrometry Using ?Cl-Labeled Analogues.

    PubMed

    Somoano-Blanco, Lourdes; Rodriguez-Gonzalez, Pablo; Garca Fonseca, Sergio; Alonso, J Ignacio Garcia

    2015-08-01

    This work describes the first application of (37)Cl-labeled compounds to isotope dilution mass spectrometry (IDMS). The synthesis of 12 (37)Cl-labeled polychlorinated biphenyls (PCBs) was carried out by the chlorination of biphenyl with isotopically enriched chlorine gas, generated by the direct oxidation of Na(37)Cl with potassium peroxymonosulfate. After an exhaustive purification due to the presence of other congeners, the concentration and the isotopic enrichment of all (37)Cl-labeled PCBs in the mixture was determined. The proposed procedure allows the simultaneous quantification of every isotope diluted PCB congener in a single gas chromatography-tandem mass spectrometry (GC-MS/MS) injection without resorting to a methodological calibration graph. The results obtained here demonstrate that the use of (37)Cl-labeled analogues provides results in agreement with the certified values of three different Certified Reference Materials (marine sediment SRM 1944, fish tissue 1947, and loamy soil CRM 962-50) and analytical figures of merit comparable to those obtained using regular IDMS procedures based on the use of commercially available (13)C-labeled analogues. PMID:26165349

  17. Identification and comparative quantitation of glycation by stable isotope labeling and LC-MS.

    PubMed

    Liu, Hongcheng; Ponniah, Gomathinayagam; Neill, Alyssa; Patel, Rekha; Andrien, Bruce

    2014-05-01

    Glycation is a common modification of proteins both in vitro and in vivo. To aid identification and comparative quantitation, a method of stable isotope labeling followed by LC-MS analysis was proposed. The samples were reduced using sodium borohydride or sodium borodeuteride. Reduction of the Schiff base between the amine group and the reducing sugars resulted in a molecular weight increase of 2Da using sodium borohydride or a molecular weight increase of 3Da using sodium borodeuteride. The molecular weight difference of 1Da between peptides containing glycated lysine residue reduced using sodium borohydride or sodium borodeuteride was used to identify glycated peptides and to calculate the glycation difference between samples. The method was used to investigate glycation of a recombinant human IgG1 antibody under native and denaturing conditions. The result demonstrated a good correlation between glycation propensity of lysine residues and their solvent exposure levels. PMID:24705536

  18. Glycoproteomics of Trypanosoma cruzi trypomastigotes using subcellular fractionation, lectin affinity, and stable isotope labeling.

    PubMed

    Atwood, James A; Minning, Todd; Ludolf, Fernanda; Nuccio, Arthur; Weatherly, Daniel B; Alvarez-Manilla, Gerardo; Tarleton, Rick; Orlando, Ron

    2006-12-01

    Herein we detail the first glycoproteomic analysis of a human pathogen. We describe an approach that enables the identification of organelle and cell surface N-linked glycoproteins from Trypanosoma cruzi, the causative agent of Chagas' disease. This approach is based on a subcellular fractionation protocol to produce fractions enriched in either organelle or plasma membrane/cytoplasmic proteins. Through lectin affinity capture of the glycopeptides from each subcellular fraction and stable isotope labeling of the glycan attachment sites with H(2)18O, we unambiguously identified 36 glycosylation sites on 35 glycopeptides which mapped to 29 glycoproteins. We also present the first expression evidence for 11 T. cruzi specific glycoproteins and provide experimental data indicating that the mucin associated surface protein family (MASP) and dispersed gene family (DGF-1) are post-translationally modified by N-linked glycans. PMID:17137339

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

    NASA Astrophysics Data System (ADS)

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

    2009-07-01

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

  20. Stable Isotope Labeling Strategy for Protein-Ligand Binding Analysis in Multi-Component Protein Mixtures

    NASA Astrophysics Data System (ADS)

    DeArmond, Patrick D.; West, Graham M.; Huang, Hai-Tsang; Fitzgerald, Michael C.

    2011-03-01

    Described here is a stable isotope labeling protocol that can be used with a chemical modification- and mass spectrometry-based protein-ligand binding assay for detecting and quantifying both the direct and indirect binding events that result from protein-ligand binding interactions. The protocol utilizes an H{2/16}O2 and H{2/18}O2 labeling strategy to evaluate the chemical denaturant dependence of methionine oxidation in proteins both in the presence and absence of a target ligand. The differential denaturant dependence to the oxidation reactions performed in the presence and absence of ligand provides a measure of the protein stability changes that occur as a result of direct interactions of proteins with the target ligand and/or as a result of indirect interactions involving other protein-ligand interactions that are either induced or disrupted by the ligand. The described protocol utilizes the 18O/16O ratio in the oxidized protein samples to quantify the ligand-induced protein stability changes. The ratio is determined using the isotopic distributions observed for the methionine-containing peptides used for protein identification in the LC-MS-based proteomics readout. The strategy is applied to a multi-component protein mixture in this proof-of-principle experiment, which was designed to evaluate the technique's ability to detect and quantify the direct binding interaction between cyclosporin A and cyclophilin A and to detect the indirect binding interaction between cyclosporin A and calcineurin (i.e., the protein-protein interaction between cyclophilin A and calcineurin that is induced by cyclosporin A binding to cyclophilin A).

  1. Labeled trees and the efficient computation of derivations

    NASA Technical Reports Server (NTRS)

    Grossman, Robert; Larson, Richard G.

    1989-01-01

    The effective parallel symbolic computation of operators under composition is discussed. Examples include differential operators under composition and vector fields under the Lie bracket. Data structures consisting of formal linear combinations of rooted labeled trees are discussed. A multiplication on rooted labeled trees is defined, thereby making the set of these data structures into an associative algebra. An algebra homomorphism is defined from the original algebra of operators into this algebra of trees. An algebra homomorphism from the algebra of trees into the algebra of differential operators is then described. The cancellation which occurs when noncommuting operators are expressed in terms of commuting ones occurs naturally when the operators are represented using this data structure. This leads to an algorithm which, for operators which are derivations, speeds up the computation exponentially in the degree of the operator. It is shown that the algebra of trees leads naturally to a parallel version of the algorithm.

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

    NASA Astrophysics Data System (ADS)

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

    2006-12-01

    We used 14C and 13C labeling to assess the in situ respiration of alanine-, starch-, and lignocellulose-derived carbon from the sporocarps of particular fungal species fruiting in a boreal forest in Alaska. By measuring isotopically-labeled respiration of sporocarps, which can be identified to species, we were able to attribute turnover of carbon compounds to specific fungal groups. Moreover, collection of sporocarp respiration is non-destructive, so we could return to the same sporocarps to collect a time series of measurements that spanned hours to days. We tested the hypotheses that alanine and starch turn over more quickly than lignocellulose, and that saprotrophic fungi would use starch-C and lignocellulose-C but ectomycorrhizal fungi would not. Small amounts of 14C-labeled alanine (about 100,000 permil) were dispensed into the soil within three meters of sporocarps of the ectomycorrhizal fungus Lactarius alnicola. Δ14CO2 values of sporocarp respiration climbed from 75.8 +/- 6.3 permil to 7855 +/- 3940 permil within one hour of additions, indicating that the fungus quickly acquired, transported, and transformed the alanine-C. In a separate approach, a mixture of 13C-labeled starch (about 15,000 permil) and 14C-labeled lignocellulose (about 36,000 permil) was applied in 9 m2 plots containing sporocarps of the ectomycorrhizal genera Phellodon and Sarcodon and the saprotrophic genera Lycoperdon and Polyporus. An unlabeled control plot was also established. We observed no detectable increase in 14CO2 or 13CO2 over a 144 hour period, suggesting that neither ectomycorrhizal nor saprotrophic fungi significantly broke down starch or lignocellulose during this time. The alanine experiment is one of the first to indicate that ectomycorrhizal fungi can influence the spatial distribution and storage of soil carbon over short time scales. This influence may be restricted to carbon of organic compounds like amino acids. In contrast, starch was not transformed quickly even by saprotrophic fungi, which may be due to an absence or lack of activity of starch-degrading fungal species during the study period. Potential activity of the starch-metabolizing enzyme alpha-glucosidase was only 0.59 +/- 0.17 ìmol h-1 g dry soil-1, which was 7 times less than activity of beta-glucosidase, which breaks down cellulose. The slow turnover of lignocellulose-C was consistent with slow decomposition rates of plant litter in this biome.

  3. CTA coronary labeling through efficient geodesics between trees using anatomy priors.

    PubMed

    Glsn, Mehmet A; Funka-Lea, Gareth; Zheng, Yefeng; Eckert, Matthias

    2014-01-01

    We present an efficient realization of recent work on unique geodesic paths between tree shapes for the application of matching coronary arteries to a standard model of coronary anatomy in order to label the coronary arteries. Automatically labeled coronary arteries would speed reporting for physicians. The efficiency of the approach and the quality of the results are enhanced using the relative position of detected cardiac structures. We explain how to efficiently compute the geodesic paths between tree shapes using Dijkstra's algorithm and we present a methodology to account for missing side branches during matching. For nearly all labels our approach shows promise compared with recent work and we results for 8 additional labels. PMID:25485419

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  5. 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

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

    2014-01-01

    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 cells natural metabolic pathways to label proteins with isotopically heavy amino acids. Incorporation of these heavy amino acids effectively labels a cells 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 proteinprotein 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

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

    PubMed Central

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

    2013-01-01

    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 organismsincluding humansare 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

  7. Stable isotope labeling, in vivo, of D- and L-tryptophan pools in lemna gibba and the low incorporation of label into indole-3-acetic acid

    SciTech Connect

    Baldi, B.G. ); Maher, B.R. ); Slovin, J.P.; Cohen, J.D. Univ. of Maryland, College Park )

    1991-04-01

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

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

    PubMed Central

    2016-01-01

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

  9. Efficient catalytic synthesis of dendritic polymers having internal fluorescence labels for bioconjugation.

    PubMed

    Chen, Guanghui; Felgner, Philip L; Guan, Zhibin

    2008-07-01

    Here we present an efficient synthesis of functional dendritic polymers carrying internal fluorescence labels for bioconjugation. Specifically, dendritic polymers having pyrene as fluorescence label in the core and N-hydroxysuccinimide (NHS) functional groups at the periphery were synthesized by coupling heterobifunctional PEG to hydroxyl functionalized dendritic polyethylene core. The dendritic polyethylene cores containing one pyrene label per polymer molecule were prepared through a one-step transition-metal-catalyzed polymerization using a pyrene-labeled Pd(II)-alpha-diimine chain walking catalyst. A series of pyrene-labeled dendritic scaffolds were obtained with different molecular weights and sizes. NHS active end groups were introduced to the periphery of the dendritic scaffolds through end-group functionalization. Those NHS-functionalized dendritic scaffolds were successfully used to conjugate a model protein, ovalbumin, to yield protein-polymer conjugates carrying multiple copies of protein attached to each scaffold. PMID:18517245

  10. Characterization of Reduced Magmatic C-O-H-N Volatiles By Isotopic Labeling

    NASA Astrophysics Data System (ADS)

    Falksen, E.; Armstrong, L. S.; Hirschmann, M. M.

    2014-12-01

    Characterization of COHN species in silicate melts [1-10] is required to understand the role of reduced volatiles in planetary and early Earth processes, including partitioning between planetary cores, mantles, and atmospheres during early differentiation. Vibrational spectroscopy has been used to examine volatile speciation, but for a number of absorptions there is uncertainty as to whether they relate to species containing N, C, or both [1,3]. In particular, an IR band at 3370 cm-1 is commonly attributed to N-H stretching [1,4,5,7], but associated Raman bands near 3280 cm-1 have also been attributed to alkyne (C-H) bonds [8-10]. The 3370 cm-1 IR band appears even in nominally N-free experiments owing to trapped air and is accompanied by a feature at 1615 cm-1 which could be caused by C=O or N-H bonds [1,3,8]. We sought to determine whether N and C were responsible for various IR bands by dissolving different isotopes of N and C in basaltic melts at high pressure and temperature and observing the shift in position of the resulting absorptions. Experiments were conducted at 1.2 GPa and 1400 oC and volatiles were added to a basaltic oxide mix in the form of unlabeled, 13C labeled, and 15N labeled urea [(NH2)2CO]. The resulting glasses were analyzed using FTIR and the theoretical band shifts were predicted based on a classical approximation of a diatomic molecule. Relative to isotopically normal glasses, bands at both 3370 cm-1 and 1615 cm-1 decrease by 4-8 wavenumbers for 15N and not at all for 13C, consistent with origination by N-H bonds in amines or metal-ammine complexes. [1] Stanley et al. (2014) GCA 129, 54-76. [2] Wetzel et al. (2013) PNAS 110, 8010-8013. [3] Armstrong et al. (in prep). [4] Kadik et al. (2011) Geochem. Int. 49, 429-438. [5] Kadik et al. (2013) PEPI 214, 14-24. [6]Mysen (2013) Chem. Geo. 346, 113-124. [7] Mysen et al. (2008) Am. Min. 93, 1760-1770. [8] Mysen et al. (2009) GCA 73, 1696-1710. [9] Dasgupta et al. (2013) GCA 102, 191-212. [10] Chi et al. (2014) 139, 447-471. [11] Socrates (2001).

  11. Isotope label-aided mass spectrometry reveals the influence of environmental factors on metabolism in single eggs of fruit fly.

    PubMed

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

    2012-01-01

    In order to investigate the influence of light/dark cycle on the biosynthesis of metabolites during oogenesis, here we demonstrate a simple experimental protocol which combines in-vivo isotopic labeling of primary metabolites with mass spectrometric analysis of single eggs of fruit fly (Drosophila melanogaster). First, fruit flies were adapted to light/dark cycle using artificial white light. Second, female flies were incubated with an isotopically labeled sugar ((13)C(6)-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

  12. Biocompatible Supramolecular Catalytic One-Dimensional Nanofibers for Efficient Labeling of Live Cells.

    PubMed

    Khalily, Mohammad Aref; Gulseren, Gulcihan; Tekinay, Ayse B; Guler, Mustafa O

    2015-12-16

    Understanding complex cellular functions requires study and tracking of biomolecules such as proteins, glycans, and lipids in their natural environment. Herein, we report the first supramolecular nanocatalyst for bioorthogonal click reaction to label live cells. This biocompatible and biodegradable nanocatalyst was formed by self-assembled peptide nanofibers complexed with copper ions. The supramolecular nanocatalyst enhanced azide-alkyne cycloaddition reaction rate under physiological conditions and was shown to be useful for efficient bioorthogonal labeling of live cells. PMID:26457765

  13. Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement.

    PubMed

    Thurlow, Sophie E; Kilgour, David P; Campopiano, Dominic J; Mackay, C Logan; Langridge-Smith, Pat R R; Clarke, David J; Campbell, Colin J

    2016-03-01

    Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential (E(?)) for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods, but top-down mass spectrometry recently has been shown to enable the quantification of E(?) for thiol oxidations. In this paper, we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E(?) for the thiols in both E. coli Thioredoxin 1 and human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using liquid chromatography-mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E(?) measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists. PMID:26881737

  14. Investigation of bn-44 peptide fragments using high resolution mass spectrometry and isotope labeling.

    PubMed

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

    2014-12-01

    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. PMID:25280401

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

    N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

  16. Shape-Controlled Synthesis of Isotopic Yttrium-90-Labeled Rare Earth Fluoride Nanocrystals for Multimodal Imaging.

    PubMed

    Paik, Taejong; Chacko, Ann-Marie; Mikitsh, John L; Friedberg, Joseph S; Pryma, Daniel A; Murray, Christopher B

    2015-09-22

    Isotopically labeled nanomaterials have recently attracted much attention in biomedical research, environmental health studies, and clinical medicine because radioactive probes allow the elucidation of in vitro and in vivo cellular transport mechanisms, as well as the unambiguous distribution and localization of nanomaterials in vivo. In addition, nanocrystal-based inorganic materials have a unique capability of customizing size, shape, and composition; with the potential to be designed as multimodal imaging probes. Size and shape of nanocrystals can directly influence interactions with biological systems, hence it is important to develop synthetic methods to design radiolabeled nanocrystals with precise control of size and shape. Here, we report size- and shape-controlled synthesis of rare earth fluoride nanocrystals doped with the β-emitting radioisotope yttrium-90 ((90)Y). Size and shape of nanocrystals are tailored via tight control of reaction parameters and the type of rare earth hosts (e.g., Gd or Y) employed. Radiolabeled nanocrystals are synthesized in high radiochemical yield and purity as well as excellent radiolabel stability in the face of surface modification with different polymeric ligands. We demonstrate the Cerenkov radioluminescence imaging and magnetic resonance imaging capabilities of (90)Y-doped GdF3 nanoplates, which offer unique opportunities as a promising platform for multimodal imaging and targeted therapy. PMID:26257288

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

    NASA Technical Reports Server (NTRS)

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

    2004-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Loy, Alexander

    2010-05-01

    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.

  19. ANIBAL, stable isotope-based quantitative proteomics by aniline and benzoic acid labeling of amino and carboxylic groups.

    PubMed

    Panchaud, Alexandre; Hansson, Jenny; Affolter, Michael; Bel Rhlid, Rachid; Piu, Stéphane; Moreillon, Philippe; Kussmann, Martin

    2008-04-01

    Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states. PMID:18083701

  20. Stable isotope labeling tandem mass spectrometry (SILT): Integration with peptide identification and extension to data-dependent scans

    PubMed Central

    Elbert, Donald L.; Mawuenyega, Kwasi G.; Scott, Evan A.; Wildsmith, Kristin R.; Bateman, Randall J.

    2009-01-01

    Quantitation of relative or absolute amounts of proteins by mass spectrometry can be prone to large errors. The use of MS/MS ion intensities and stable isotope labeling, which we term stable isotope labeling tandem mass spectrometry (SILT), decreases the effects of contamination from unrelated compounds. We present a software package (SILTmass) that automates protein identification and quantification by the SILT method. SILTmass has the ability to analyze the kinetics of protein turnover, in addition to relative and absolute protein quantitation. Instead of extracting chromatograms to find elution peaks, SILTmass uses only scans in which a peptide is identified and that meet an ion intensity threshold. Using only scans with identified peptides, the accuracy and precision of SILT is shown to be superior to precursor ion intensities, particularly at high or low dilutions of the isotope labeled compounds or with low amounts of protein. Using example scans, we demonstrate likely reasons for the improvements in quantitation by SILT. The appropriate use of variable modifications in peptide identification is described for measurement of protein turnover kinetics. The combination of identification with SILT facilitates quantitation without peak detection and helps to ensure the appropriate use of variable modifications for kinetics experiments. PMID:18774841

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

    PubMed Central

    2013-01-01

    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

  2. Intracellular Isotope Localization in Ammonia sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments.

    PubMed

    Nomaki, Hidetaka; Bernhard, Joan M; Ishida, Akizumi; Tsuchiya, Masashi; Uematsu, Katsuyuki; Tame, Akihiro; Kitahashi, Tomo; Takahata, Naoto; Sano, Yuji; Toyofuku, Takashi

    2016-01-01

    Some benthic foraminiferal species are reportedly capable of nitrate storage and denitrification, however, little is known about nitrate incorporation and subsequent utilization of nitrate within their cell. In this study, we investigated where and how much (15)N or (34)S were assimilated into foraminiferal cells or possible endobionts after incubation with isotopically labeled nitrate and sulfate in dysoxic or anoxic conditions. After 2 weeks of incubation, foraminiferal specimens were fixed and prepared for Transmission Electron Microscopy (TEM) and correlative nanometer-scale secondary ion mass spectrometry (NanoSIMS) analyses. TEM observations revealed that there were characteristic ultrastructural features typically near the cell periphery in the youngest two or three chambers of the foraminifera exposed to anoxic conditions. These structures, which are electron dense and ~200-500 nm in diameter and co-occurred with possible endobionts, were labeled with (15)N originated from (15)N-labeled nitrate under anoxia and were labeled with both (15)N and (34)S under dysoxia. The labeling with (15)N was more apparent in specimens from the dysoxic incubation, suggesting higher foraminiferal activity or increased availability of the label during exposure to oxygen depletion than to anoxia. Our results suggest that the electron dense bodies in Ammonia sp. play a significant role in nitrate incorporation and/or subsequent nitrogen assimilation during exposure to dysoxic to anoxic conditions. PMID:26925038

  3. Intracellular Isotope Localization in Ammonia sp. (Foraminifera) of Oxygen-Depleted Environments: Results of Nitrate and Sulfate Labeling Experiments

    PubMed Central

    Nomaki, Hidetaka; Bernhard, Joan M.; Ishida, Akizumi; Tsuchiya, Masashi; Uematsu, Katsuyuki; Tame, Akihiro; Kitahashi, Tomo; Takahata, Naoto; Sano, Yuji; Toyofuku, Takashi

    2016-01-01

    Some benthic foraminiferal species are reportedly capable of nitrate storage and denitrification, however, little is known about nitrate incorporation and subsequent utilization of nitrate within their cell. In this study, we investigated where and how much 15N or 34S were assimilated into foraminiferal cells or possible endobionts after incubation with isotopically labeled nitrate and sulfate in dysoxic or anoxic conditions. After 2 weeks of incubation, foraminiferal specimens were fixed and prepared for Transmission Electron Microscopy (TEM) and correlative nanometer-scale secondary ion mass spectrometry (NanoSIMS) analyses. TEM observations revealed that there were characteristic ultrastructural features typically near the cell periphery in the youngest two or three chambers of the foraminifera exposed to anoxic conditions. These structures, which are electron dense and ~200–500 nm in diameter and co-occurred with possible endobionts, were labeled with 15N originated from 15N-labeled nitrate under anoxia and were labeled with both 15N and 34S under dysoxia. The labeling with 15N was more apparent in specimens from the dysoxic incubation, suggesting higher foraminiferal activity or increased availability of the label during exposure to oxygen depletion than to anoxia. Our results suggest that the electron dense bodies in Ammonia sp. play a significant role in nitrate incorporation and/or subsequent nitrogen assimilation during exposure to dysoxic to anoxic conditions. PMID:26925038

  4. Monitoring protein conformational changes and dynamics using stable-isotope labeling and mass spectrometry (CDSiL-MS)

    PubMed Central

    Kahsai, Alem W.; Rajagopal, Sudarshan; Sun, Jinpeng; Xiao, Kunhong

    2015-01-01

    Understanding the mechanism accompanying functional conformational changes associated with protein activation has important implications for drug design. Here, we describe a powerful method, CDSiL-MS (conformational changes and dynamics using stable-isotope labeling and mass-spectrometry), which involves chemical-labeling by isotope-coded forms of N-ethylmaleimide or succinic anhydride to site-specifically label the side-chains of cysteines or lysines, respectively, in native proteins. Subsequent MS-analysis allows the quantitative monitoring of reactivity of residues as a function of time, providing a measurement of the labeling kinetics, thereby enabling elucidation of conformational changes of proteins. We demonstrate the utility of this method using a model G-protein coupled receptor, the ?2-adrenergic receptor including experiments that characterize the functional conformational-changes associated with activation of distinct signaling pathways induced by different ?-adrenoceptor ligands. The procedure requires five days and can easily be adapted to systems where soluble and detergent-solubilized membrane protein targets, which undergo function-dependent conformational-changes, can be interrogated structurally to allow drug screening. PMID:24810039

  5. Efficient isotope separation by single-photon atomic sorting

    SciTech Connect

    Jerkins, M.; Chavez, I.; Raizen, M. G.; Even, U.

    2010-09-15

    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.

  6. Energy Efficiency Standards and Labels in North America: Opportunities for Harmonization

    SciTech Connect

    Vanwiemcgrory, Laura; Wiel, Stephen; Van Wie McGrory, Laura; Harrington, Lloyd

    2002-05-16

    To support the North American Energy Working Group's Expert Group on Energy Efficiency (NAEWG-EE), USDOE commissioned 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 reached the following conclusions: Out of 24 energy-using products for which at least one of the three countries has energy efficiency regulations, three products -- refrigerators/freezers, split system central air conditioners, and room air conditioners -- have similar or identical minimum energy performance standards (MEPS) in the three countries. These same three products, as well as three-phase motors, have similar or identical test procedures throughout the region. There are 10 products with different MEPS and test procedures, but which have the short-term potential to develop common test procedures, MEPS, and/or labels. Three other noteworthy areas where possible energy efficiency initiatives have potential for harmonization are standby losses, uniform endorsement labels, and a new standard or label on windows. This paper explains these conclusions and presents the underlying comparative data.

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

    PubMed

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

    2015-04-01

    Protein immuno-detection encompasses a broad range of analytical methodologies, including western blotting, flow cytometry, and microscope-based applications. These assays which detect, quantify, and/or localize expression for one or more proteins in complex biological samples, are reliant upon fluorescent or enzyme-tagged target-specific antibodies. While small molecule labeling kits are available with a range of detection moieties, the workflow is hampered by a requirement for multiple dialysis-based buffer exchange steps that are both time-consuming and subject to sample loss. In a previous study, we briefly described an alternative method for small-scale protein labeling with small molecule dyes whereby all phases of the conjugation workflow could be performed in a single centrifugal diafiltration device. Here, we expand on this foundational work addressing functionality of the device at each step in the workflow (sample cleanup, labeling, unbound dye removal, and buffer exchange/concentration) and the implications for optimizing labeling efficiency. When compared to other common buffer exchange methodologies, centrifugal diafiltration offered superior performance as measured by four key parameters (process time, desalting capacity, protein recovery, retain functional integrity). Originally designed for resin-based affinity purification, the device also provides a platform for up-front antibody purification or albumin carrier removal. Most significantly, by exploiting the rapid kinetics of NHS-based labeling reactions, the process of continuous diafiltration minimizes reaction time and long exposure to excess dye, guaranteeing maximal target labeling while limiting the risks associated with over-labeling. Overall, the device offers a simplified workflow with reduced processing time and hands-on requirements, without sacrificing labeling efficiency, final yield, or conjugate performance. PMID:25813016

  8. Leukocyte labeling in plasma made simple and efficient: Preparation and evaluation

    SciTech Connect

    Thakur, M.L.; McKenney, S.; Seifert, C.; Madsen, M.; Suntherlingam, M.; Desai, A.; Patel, J.; Park, C.H.

    1984-01-01

    Over the past few years the use of In-111 leukocytes (In-111-L) for abscess localization has become increasingly popular. Using less toxic Mercaptopyridine-N-oxide or its Na-salt (Merc, LD/sub 50/ 340 mg/kg b.w.) rather than oxine (LD/sub 50/ 88.8 mg/kg b.w.), In-111-L could be prepared either in salt balance media or preferably by a kit procedure in plasma. In the plasma preparation L concentrated in 0.5 ml plasma are added to 25 ..mu..g dry Merc and subsequently incubated at an ambient temperature for 20 minutes with a weak chelate of In-111 either dry or in solution. Greater than 90/sup 0/ labeling efficiency is achieved which compared favorably to 10 ..mu..g and 50 ..mu..g each of oxine and tropolone. Phagocytic ability of 10 million L exposed to up to 200 ..mu..g Merc remained unaffected. In-111-L and Ga-67 citrate were given to abscess bearing dogs and abscess/blood (24 hr.) ratios of 75 and 12 respectively were achieved. Those were similar to those obtained with In-111-oxine labeled L (75.2 vs. 75.8). Abscess/liver and abscess/spleen ratios obtained with In-111-merc labeled L (14.6 and 4.2 respectively) were more favorable than those obtained with In-111-oxine labeled L (4.2d and 1.8 respectively). These indicated that less quantity of L labeled with In-111-Merc was deposited in these organs. Furthermore, L labeled with In-111-Merc cleared more rapidly from human lungs than L labeled with In-111-oxine. Data indicate that use of Merc permits efficient L labeling in plasma, provides a kit method, and preserves L physiologic function.

  9. Absolute quantification of human tear lactoferrin using multiple reaction monitoring technique with stable-isotopic labeling.

    PubMed

    You, Jingjing; Willcox, Mark; Fitzgerald, Anna; Schiller, Belinda; Cozzi, Paul J; Russell, Pamela J; Walsh, Bradley J; Wasinger, Valerie C; Graham, Peter H; Li, Yong

    2016-03-01

    The mass spectrometry technique of multiple reaction monitoring (MRM) was used to quantify and compare the expression level of lactoferrin in tear films among control, prostate cancer (CaP), and benign prostate hyperplasia (BPH) groups. Tear samples from 14 men with CaP, 15 men with BPH, and 14 controls were analyzed in the study. Collected tears (2 μl) of each sample were digested with trypsin overnight at 37 °C without any pretreatment, and tear lactoferrin was quantified using a lactoferrin-specific peptide, VPSHAVVAR, both using natural/light and isotopic-labeled/heavy peptides with MRM. The average tear lactoferrin concentration was 1.01 ± 0.07 μg/μl in control samples, 0.96 ± 0.07 μg/μl in the BPH group, and 0.98 ± 0.07 μg/μl in the CaP group. Our study is the first to quantify tear proteins using a total of 43 individual (non-pooled) tear samples and showed that direct digestion of tear samples is suitable for MRM studies. The calculated average lactoferrin concentration in the control group matched that in the published range of human tear lactoferrin concentration measured by enzyme-linked immunosorbent assay (ELISA). Moreover, the lactoferrin was stably expressed across all of the samples, with no significant differences being observed among the control, BPH, and CaP groups. PMID:26717899

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

    SciTech Connect

    McMahon, James E.; Wiel, Stephen

    2001-02-16

    Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and the United Nations Foundation (UNF) recognize the need to support policy makers in their efforts to implement energy-efficiency standards and labeling programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This guidebook was prepared over the course of the past year with significant contribution from the authors and reviewers mentioned previously. Their diligent participation has made this the international guidance tool it was intended to be. The lead authors would also like to thank the following individuals for their support in the development, production, and distribution of the guidebook: Marcy Beck, Elisa Derby, Diana Dhunke, Ted Gartner, and Julie Osborn of Lawrence Berkeley National Laboratory as well as Anthony Ma of Bevilacqua-Knight, Inc. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards-setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design, development, implementation, maintenance, and evaluation of the programs and on the design of the labels and standards themselves. In addition, it directs the reader to references and other resources likely to be useful in conducting the activities described and includes a chapter on energy policies and programs that complement appliance efficiency labels and standards. This guidebook attempts to reflect the essential framework of labeling and standards programs. It is the intent of the authors and sponsors to distribute copies of this book worldwide at no charge for the general public benefit. The guidebook is also available on the web at www.CLASPonline.org and can be downloaded to be used intact or piecemeal for whatever beneficial purposes readers may conceive.

  11. Mechanistic investigations aided by isotopic labeling. 10. Investigations of novel furan-2,3-dione rearrangements by oxygen-17 labeling

    SciTech Connect

    Kollenz, G.; Sterk, H.; Hutter, G. )

    1991-01-04

    The oxa 1,3-diene moiety in 4-benzoyl-5-phenylfuran-2,3-dione (1) adds aryl isocyanides or heterocumulenes via formal (4 + 1) or (4 + 2) cycloaddition processes. The unstable primary adducts undergo novel furandione rearrangements to intermediates in which the two oxygen atoms of the lactone moiety in (1) are equivalent. This equivalence was confirmed by {sup 17}O-labeling experiments using {sup 17}O NMR spectroscopic and mass spectroscopic measurements. Comparison of the {sup 17}O chemical shifts in (1), labeled either at the benzoyl and ring oxygens (1a-{sup 17}O) or at both exocyclic ring-carbonyl oxygens (1b-{sup 17}O), with those in the products (2-4) confirmed the proposed pathways of these rearrangements. Reactions involving carbodiimides, isocyanates, and ketene imines were investigated.

  12. Probing Protein 3D Structures and Conformational Changes Using Electrochemistry-Assisted Isotope Labeling Cross-Linking Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zheng, Qiuling; Zhang, Hao; Wu, Shiyong; Chen, Hao

    2016-02-01

    This study presents a new chemical cross-linking mass spectrometry (MS) method in combination with electrochemistry and isotope labeling strategy for probing both protein three-dimensional (3D) structures and conformational changes. For the former purpose, the target protein/protein complex is cross-linked with equal mole of premixed light and heavy isotope labeled cross-linkers carrying electrochemically reducible disulfide bonds (i.e., DSP-d0 and DSP-d8 in this study, DSP = dithiobis[succinimidyl propionate]), digested and then electrochemically reduced followed with online MS analysis. Cross-links can be quickly identified because of their reduced intensities upon electrolysis and the presence of doublet isotopic peak characteristics. In addition, electroreduction converts cross-links into linear peptides, facilitating MS/MS analysis to gain increased information about their sequences and modification sites. For the latter purpose of probing protein conformational changes, an altered procedure is adopted, in which the protein in two different conformations is cross-linked using DSP-d0 and DSP-d8 separately, and then the two protein samples are mixed in 1:1 molar ratio. The merged sample is subjected to digestion and electrochemical mass spectrometric analysis. In such a comparative cross-linking experiment, cross-links could still be rapidly recognized based on their responses to electrolysis. More importantly, the ion intensity ratios of light and heavy isotope labeled cross-links reveal the conformational changes of the protein, as exemplified by examining the effect of Ca2+ on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology.

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

    PubMed Central

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

    2013-01-01

    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 δ13C (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 δ13C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics. PMID:24124493

  14. An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids.

    PubMed

    Nikonowicz, E P; Pardi, A

    1993-08-20

    An efficient method is presented for the assignment of the proton, carbon, and nitrogen resonances in the NMR spectra of isotopically labeled nucleic acids. The assignment strategy starts by identifying all protons and carbons belonging to the same sugar ring through application of a set of 2D or 3D heteronuclear HCCH NMR experiments. Next the individual sugar rings are connected to their corresponding bases through intra-residue 1H-1H nuclear Overhauser effects (NOEs) observed in a 3D (1H, 13C, 1H) NOESY-HMQC experiment. Sequential NOE connectivities observed in this experiment are then used to assign each residue in the nucleotide sequence. The imino protons and nitrogens, and the cytidine amino protons and nitrogens, are assigned by 2D (15N, 1H) HMQC and 3D (1H, 15N, 1H) NOESY-HMQC experiments in H2O. This assignment procedure is illustrated on the 99% 13C/15N labeled RNA duplex r(GGCGCUUGCGUC)2. The application of these multi-dimensional heteronuclear magnetic resonance experiments enormously simplifies the resonance assignment of nucleic acids and allows assignment of many more protons, carbons and nitrogens than was possible using standard techniques on unlabeled molecules. Since a larger percentage of the protons can now be assigned by these experiments, much more NMR structural information can be obtained which will significantly extend the size limit for solution structure determinations of RNAs. PMID:8396648

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

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

    SciTech Connect

    Zhou, Nan

    2008-03-01

    China first adopted minimum energy performance standards (MEPS) in 1989. Today, there are standards for a wide range of domestic, commercial and selected industrial equipment. In 1999, China launched a voluntary endorsement label, which has grown to cover over 40 products including water-saving products (See Figure 1). Further, in 2005, China started a mandatory energy information label (also referred to as the 'Energy Label'). Today, the Energy Label is applied to four products including: air conditioners; household refrigerators; clothes washers; and unitary air conditioners (See Figure 2). MEPS and the voluntary endorsement labeling specifications have been updated and revised 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.

  17. Nic1 inactivation enables stable isotope labeling with 13C615N4-arginine in Schizosaccharomyces pombe.

    PubMed

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

    2015-01-01

    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, (13)C(6) (15)N(4)-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 (13)C(6) (15)N(4)-arginine is catabolized by arginase and urease activity to (15)N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni(2+)-dependent urease activity, through deletion of the sole Ni(2+) transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable (13)C(6) (15)N(4)-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

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

    PubMed Central

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

    2015-01-01

    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

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

    SciTech Connect

    Mark E. Fuller; Tullis C. Onstott

    2003-12-17

    This report summarizes the results of a research project conducted to develop new methods to label bacterial cells so that they could be tracked and enumerated as they move in the subsurface after they are introduced into the groundwater (i.e., during bioaugmentation). Labeling methods based on stable isotopes of carbon (13C) and vital fluorescent stains were developed. Both approaches proved successful with regards to the ability to effectively label bacterial cells. Several methods for enumeration of fluorescently-labeled cells were developed and validated, including near-real time microplate spectrofluorometry that could be performed in the field. However, the development of a 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).

  20. CK-LPA: Efficient community detection algorithm based on label propagation with community kernel

    NASA Astrophysics Data System (ADS)

    Lin, Zhen; Zheng, Xiaolin; Xin, Nan; Chen, Deren

    2014-12-01

    With the rapid development of Web 2.0 and the rise of online social networks, finding community structures from user data has become a hot topic in network analysis. Although research achievements are numerous at present, most of these achievements cannot be adopted in large-scale social networks because of heavy computation. Previous studies have shown that label propagation is an efficient means to detect communities in social networks and is easy to implement; however, some drawbacks, such as low accuracy, high randomness, and the formation of a monster community, have been found. In this study, we propose an efficient community detection method based on the label propagation algorithm (LPA) with community kernel (CK-LPA). We assign a corresponding weight to each node according to node importance in the whole network and update node labels in sequence based on weight. Then, we discuss the composition of weights, the label updating strategy, the label propagation strategy, and the convergence conditions. Compared with the primitive LPA, existing drawbacks are solved by CK-LPA. Experiments and benchmarks reveal that our proposed method sustains nearly linear time complexity and exhibits significant improvements in the quality aspect of static community detection. Hence, the algorithm can be applied in large-scale social networks.

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

    PubMed

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

    2014-02-15

    The contamination of Pb in agricultural soils is one of the most important ecological problems, which potentially results in serious health risk on human health through food chain. Hence, the fate of exogenous Pb contaminated in agricultural soils is needed to be deeply explored. By spiking soils with the stable enriched isotopes of (206)Pb, the contamination of exogenous Pb(2+) ions in three agricultural soils sampled from the estuary areas of Jiulong River, China was simulated in the present study, and the distribution, mobility and bioavailability of exogenous Pb in the soils were investigated using the isotopic labeling method coupled with a four-stage BCR (European Community Bureau of Reference) sequential extraction procedure. Results showed that about 60-85% of exogenous Pb was found to distribute in reducible fractions, while the exogenous Pb in acid-extractable fractions was less than 1.0%. After planting, the amounts of exogenous Pb presenting in acid-extractable, reducible and oxidizable fractions in rhizospheric soils decreased by 60-66%, in which partial exogenous Pb was assimilated by plants while most of the metal might transfer downward due to daily watering and applying fertilizer. The results show that the isotopic labeling technique coupled with sequential extraction procedures enables us to explore the distribution, mobility and bioavailability of exogenous Pb contaminated in soils, which may be useful for the further soil remediation. PMID:24412626

  2. Isotopic labeling as a tool to establish intramolecular vibrational coupling: The reaction of 2-propanol on Mo(110)

    NASA Astrophysics Data System (ADS)

    Uvdal, P.; Wiegand, Benjamin C.; Serafin, J. G.; Friend, C. M.

    1992-12-01

    The reactions of 2-propanol on Mo(110) were investigated using temperature programmed reaction, high resolution electron energy loss, and x-ray photoelectron spectroscopies. 2-Propanol forms 2-propoxide upon adsorption at 120 K on Mo(110). The 2-propoxide intermediate deoxygenates via selective ? C-H bond scission to eliminate propene as well as C-O bond hydrogenolysis to form trace amounts of propane. The C-O bond of 2-propoxide is estimated to be nearly perpendicular to the surface. Selective isotopic labeling was used to establish the coupling between the C-O stretch and modes associated with the hydrocarbon framework. The degree of coupling was strongly affected by bonding to the surface, primarily due to weakening of the C-O bond when 2-propoxide is bound to Mo(110). Selective isotopic labeling was, therefore, essential in making vibrational assignments and in identifying key reaction steps. Only a small kinetic isotope effect was observed during reaction of (CD3)(CH3)CHOH, consistent with a substantial component of C-O bond breaking in the transition state for propene elimination. Coupling of the C-O stretch to motion of the methyl group is also suggested to be important in the transition state for propene elimination.

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

    NASA Astrophysics Data System (ADS)

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

    2013-09-01

    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.

  4. Subcellular imaging of isotopically labeled carbon compounds in a biological sample by ion microprobe (NanoSIMS).

    PubMed

    Clode, Peta L; Stern, Richard A; Marshall, Alan T

    2007-03-01

    Here we demonstrate the technique of nanoscale secondary ion mass spectrometry, utilizing the Cameca NanoSIMS50 ion microprobe, to detect and image the metabolism of an isotopically labeled compound (NaH(13)CO(3)) in a biological sample. In particular, we have designed and verified protocols for imaging the subcellular distribution and determining the relative abundance of labeled (13)C, within the coral Galaxea fascicularis. Analyses were conducted on 1-mum thick sections of resin-embedded material, using both scanned (mapping) and static (spot analysis) Cs(+) primary ion beam of approximately 100 nm diameter. Using these samples we establish that NanoSIMS has adequate mass resolution to reliably distinguish (13)C from potential isobaric interference by (12)C(1)H and that data extracted from ion maps are comparable to those acquired by spot analyses. Independent of the method of acquisition, ratioing of (13)C to the naturally abundant (12)C is essential if meaningful data, which can be statistically compared to standard and control samples, are to be obtained. These results highlight the potential of NanoSIMS for intracellular tracking of a variety of organic and inorganic compounds labeled with stable isotopes of C, N, O, S, P, and halogens. PMID:17279515

  5. Simultaneous determination of ?-, ?- and ?-hexabromocyclododecane diastereoisomers in water samples by isotope dilution mass spectrometry using (81)Br-labeled analogs.

    PubMed

    Somoano-Blanco, Lourdes; Rodriguez-Gonzalez, Pablo; Centineo, Giusepe; Fonseca, Sergio Garca; Garcia Alonso, J Ignacio

    2016-01-15

    This work describes the synthesis, characterization and application of three (81)Br-labeled diastereosiomers of hexabromocyclododecane (HBCD) for the accurate and precise determination of ?-, ?- and ?-HBCD in water samples by isotope dilution mass spectrometry. The synthesis of the labeled analogs was carried out by bromination of cis, trans, trans-1,5,9-cyclododecatriene with (81)Br-enriched bromine. After isolation and purification by semipreparative HPLC, each diastereoisomer was characterized in terms of concentration and isotopic enrichment. Then, they were added to the samples to simultaneously quantify the three HBCD diastereoisomers in a single LC-MS/MS injection without resorting to a methodological calibration graph. The results obtained here demonstrate that the use of (81)Br-labeled analogs provides accurate and precise determinations of ?-, ?- and ?-HBCD in real water samples. The limits of quantification obtained in real samples for ?-, ?- and ?-HBCD were 0.022, 0.073 and 0.015ngL(-1), respectively, significantly lower than those required by the European Directive 2013/39/EC. PMID:26739916

  6. Combining UHPLC-High Resolution MS and Feeding of Stable Isotope Labeled Polyketide Intermediates for Linking Precursors to End Products.

    PubMed

    Klitgaard, Andreas; Frandsen, Rasmus J N; Holm, Dorte K; Knudsen, Peter B; Frisvad, Jens C; Nielsen, Kristian F

    2015-07-24

    We present the results from stable isotope labeled precursor feeding studies combined with ultrahigh performance liquid chromatography-high resolution mass spectrometry for the identification of labeled polyketide (PK) end-products. Feeding experiments were performed with (13)C8-6-methylsalicylic acid (6-MSA) and (13)C14-YWA1, both produced in-house, as well as commercial (13)C7-benzoic acid and (2)H7-cinnamic acid, in species of Fusarium, Byssochlamys, Aspergillus, and Penicillium. Incorporation of 6-MSA into terreic acid or patulin was not observed in any of six evaluated species covering three genera, because the 6-MSA was shunted into (2Z,4E)-2-methyl-2,4-hexadienedioic acid. This indicates that patulin and terreic acid may be produced in a closed compartment of the cell and that (2Z,4E)-2-methyl-2,4-hexadienedioic acid is a detoxification product toward terreic acid and patulin. In Fusarium spp., YWA1 was shown to be incorporated into aurofusarin, rubrofusarin, and antibiotic Y. In A. niger, benzoic acid was shown to be incorporated into asperrubrol. Incorporation levels of 0.7-20% into the end-products were detected in wild-type strains. Thus, stable isotope labeling is a promising technique for investigation of polyketide biosynthesis and possible compartmentalization of toxic metabolites. PMID:26132344

  7. Cost-efficient THz-resonators for label-free detection of DNA hybridization

    NASA Astrophysics Data System (ADS)

    Nagel, M.; Kurz, H.

    2005-08-01

    THz-technology provides new ground for label-free detection of biomolecules by taking advantage of existing hybridization techniques. Recent developments for label-free analysis of genetic material with femtomol-sensitivity using integrated functionalized THz sensors are presented. In comparison to former planar strip line approaches, the modular concept based on parallel-plate waveguides presented here provides a cost-efficient solution given the exchangeability, disposability or reusability of components. Three different fabrication technologies for disposable THz resonator chips are investigated.

  8. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry for Applications in Stable Isotope Probing

    PubMed Central

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L.

    2014-01-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating 13C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography–tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% 13C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation. PMID:25217022

  9. Sensitive, Efficient Quantitation of 13C-Enriched Nucleic Acids via Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry for Applications in Stable Isotope Probing.

    PubMed

    Wilhelm, Roland; Szeitz, András; Klassen, Tara L; Mohn, William W

    2014-12-01

    Stable isotope probing (SIP) of nucleic acids is a powerful tool for studying the functional traits of microbial populations within complex communities, but SIP involves a number of technical challenges. Many of the difficulties in DNA-SIP and RNA-SIP experiments can be effectively overcome with an efficient, sensitive method for quantitating the isotopic enrichment of nucleic acids. Here, we present a sensitive method for quantitating (13)C enrichment of nucleic acids, requiring a few nanograms of sample, and we demonstrate its utility in typical DNA-SIP and RNA-SIP experiments. All five nucleobases (adenine, guanine, cytosine, thymine, and uracil) were separated and detected by using ultrahigh-performance liquid chromatography-tandem mass spectrometry. We detected all isotopic species in samples with as low as 1.5 atom% (13)C above natural abundance, using 1-ng loadings. Quantitation was used to characterize the isotopic enrichment kinetics of cellulose- and lignin-based microcosm experiments and to optimize the recovery of enriched nucleic acids. Application of our method will minimize the quantity of expensive isotopically labeled substrates required and reduce the risk of failed experiments due to insufficient recovery of labeled nucleic acids for sequencing library preparation. PMID:25217022

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

    PubMed

    Becker, Harry; Walker, Richard J

    2003-09-11

    The abundances of elements and their isotopes in our Galaxy show wide variations, reflecting different nucleosynthetic processes in stars and the effects of Galactic evolution. These variations contrast with the uniformity of stable isotope abundances for many elements in the Solar System, which implies that processes efficiently homogenized dust and gas from different stellar sources within the young solar nebula. However, isotopic heterogeneity has been recognized on the subcentimetre scale in primitive meteorites, indicating that these preserve a compositional memory of their stellar sources. Small differences in the abundance of stable molybdenum isotopes in bulk rocks of some primitive and differentiated meteorites, relative to terrestrial Mo, suggest large-scale Mo isotopic heterogeneity between some inner Solar System bodies, which implies physical conditions that did not permit efficient mixing of gas and dust. Here we report Mo isotopic data for bulk samples of primitive and differentiated meteorites that show no resolvable deviations from terrestrial Mo. This suggests efficient mixing of gas and dust in the solar nebula at least to 3 au from the Sun, possibly induced by magnetohydrodynamic instabilities. These mixing processes must have occurred before isotopic fractionation of gas-phase elements and volatility-controlled chemical fractionations were established. PMID:12968172

  11. Studies of strontium biokinetics in humans. Part 1: optimisation of intrinsic labelling of foodstuffs with stable isotopes of strontium.

    PubMed

    Hllriegl, Vera; Werner, Eckhard; Roth, Paul; Schramel, Peter; Wendler, Iris

    2002-09-01

    The radioactive isotopes of strontium, mainly (90)Sr, which are common fission products, may significantly contribute to the internal exposure of the population in case of their accidental release into the environment and transfer to the food chain. For (90)Sr, the internal radiation dose is significantly dependent on the fractional absorption of the ingested activity (f(1)-value). Human data on the absorption of dietary strontium and of soluble forms of the element give values ranging from about 0.15 to 0.45 (up to 1.0) for adults. The International Commission on Radiological Protection (ICRP) has adopted f(1)-values of 0.6 for children of less than 1 year of age, 0.4 for children between 1 and 15 years and 0.3 for adolescents above 15 years of age. This study was aimed at investigating how far these values correspond to the actual uptake of strontium from contaminated foodstuffs. A methodology is presented that has been developed for preparing foodstuffs intrinsically labelled with stable isotopes and that will be used in tracer kinetic investigations. The results show that cress and salad can be adequately labelled, i.e. a strontium concentration of 1.36+/-0.47 g per kg of cress (wet weight) and of 0.29+/-0.04 g per kg of salad (wet weight) may be obtained within 15 days and 24 days, respectively. For the biokinetic investigations on humans, applying stable isotopes of Sr as tracers, about 0.1-1 mg strontium is required per volunteer, i.e. a few grams of the edible parts of the labelled material are sufficient. PMID:12373326

  12. Stable isotope-labeled vitamin D, metabolites and chemical analogs: Synthesis and use in mass spectrometric studies

    SciTech Connect

    Coldwell, R.D.; Trafford, D.J.; Varley, M.J.; Kirk, D.N.; Makin, H.L. )

    1990-10-01

    Methods for the measurement of vitamin D and its metabolites using stable isotope-labeled internal standards and mass spectrometry are reviewed. The synthesis of both labeled and unlabeled standards is illustrated, and details of the synthesis of (26,26,27,27,27(-2)H5)-25,26-dihydroxyvitamin D3 and (28,28,28(-2)H3)-24,25-dihydroxyvitamin D2 are given. The use of in vitro biologic systems for the production of further metabolites of deuterated 25-hydroxyvitamin D3 is discussed. Use of deuterated 25-hydroxydihydrotachysterol3 as a substrate in the isolated perfused rat kidney has provided valuable data for the assignment of structure to a number of metabolites of 25-hydroxydihydrotachysterol3 formed in this system. 51 refs.

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

    PubMed Central

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

    2015-01-01

    Fluorophore labeling of proteins while preserving native functions is essential for bulk Frster resonance energy transfer (FRET) interaction and single molecule imaging analysis. Here we describe a versatile, efficient, specific, irreversible, gentle and low-cost method for labeling proteins with fluorophores that appears substantially more robust than a similar but chemically distinct procedure. The method employs the controlled enzymatic conversion of a central Cys to a reactive formylglycine (fGly) aldehyde within a six amino acid Formylglycine Generating Enzyme (FGE) recognition sequence in vitro. The fluorophore is then irreversibly linked to the fGly residue using a Hydrazinyl-Iso-Pictet-Spengler (HIPS) ligation reaction. We demonstrate the robust large-scale fluorophore labeling and purification of E.coli (Ec) mismatch repair (MMR) components. Fluorophore labeling did not alter the native functions of these MMR proteins in vitro or in singulo. Because the FGE recognition sequence is easily portable, FGE-HIPS fluorophore-labeling may be easily extended to other proteins. PMID:26582263

  14. Spatially tracking (13) C-labelled substrate (bicarbonate) accumulation in microbial communities using laser ablation isotope ratio mass spectrometry.

    PubMed

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

    2014-12-01

    Microbial mats are characterized by extensive metabolic interactions, rapidly changing internal geochemical gradients, and prevalent microenvironments within tightly constrained physical structures. We present laser ablation isotope ratio mass spectrometry (LA-IRMS) as a culture-independent, spatially specific technology for tracking the accumulation of (13) C-labelled substrate into heterogeneous microbial mat communities. This study demonstrates the novel LA-IRMS approach by tracking labeled bicarbonate incorporation into a cyanobacteria-dominated microbial mat system. The spatial resolution of 50??m was sufficient for distinguishing different mat strata and the approach effectively identified regions of greatest label incorporation. Sample preparation for LA-IRMS is straightforward and the spatial selectivity of LA-IRMS minimizes the volume of mat consumed, leaving material for complimentary analyses. We present analysis of DNA extracted from a sample post-ablation and suggest pigments, lipids or other biomarkers could similarly be extracted following ablation. LA-IRMS is well positioned to spatially resolve the accumulation of any (13) C-labelled substrate provided to a mat, making this a versatile tool for studying carbon transfer and interspecies exchanges within the limited spatial confines of such systems. PMID:25155264

  15. 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 ...

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

    SciTech Connect

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

    2011-09-26

    As part of its commitment to promoting and improving the local enforcement of appliance energy efficiency standards and labeling, the China National Institute of Standardization (CNIS) launched the National and Local Enforcement of Energy Efficiency Standards and Labeling project on August 14, 2009. The project’s short-term goal is to expand the effort to improve enforcement of standards and labeling requirements to the entire country within three years, with a long-term goal of perfecting overall enforcement. For this project, Jiangsu, Shandong, Sichuan and Shanghai were selected as pilot locations. This report provides information on the local enforcement project’s recent background, 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.

  17. Deletion of Genes Encoding Arginase Improves Use of “Heavy” Isotope-Labeled Arginine for Mass Spectrometry in Fission Yeast

    PubMed Central

    Borek, Weronika E.; Zou, Juan; Rappsilber, Juri; Sawin, Kenneth E.

    2015-01-01

    The use of “heavy” isotope-labeled arginine for stable isotope labeling by amino acids in cell culture (SILAC) mass spectrometry in the fission yeast Schizosaccharomyces pombe is hindered by the fact that under normal conditions, arginine is extensively catabolized in vivo, resulting in the appearance of “heavy”-isotope label in several other amino acids, most notably proline, but also glutamate, glutamine and lysine. This “arginine conversion problem” significantly impairs quantification of mass spectra. Previously, we developed a method to prevent arginine conversion in fission yeast SILAC, based on deletion of genes involved in arginine catabolism. Here we show that although this method is indeed successful when 13C6-arginine (Arg-6) is used for labeling, it is less successful when 13C615N4-arginine (Arg-10), a theoretically preferable label, is used. In particular, we find that with this method, “heavy”-isotope label derived from Arg-10 is observed in amino acids other than arginine, indicating metabolic conversion of Arg-10. Arg-10 conversion, which severely complicates both MS and MS/MS analysis, is further confirmed by the presence of 13C515N2-arginine (Arg-7) in arginine-containing peptides from Arg-10-labeled cells. We describe how all of the problems associated with the use of Arg-10 can be overcome by a simple modification of our original method. We show that simultaneous deletion of the fission yeast arginase genes car1+ and aru1+ prevents virtually all of the arginine conversion that would otherwise result from the use of Arg-10. This solution should enable a wider use of heavy isotope-labeled amino acids in fission yeast SILAC. PMID:26075619

  18. Untargeted Profiling of Tracer-Derived Metabolites Using Stable Isotopic Labeling and Fast Polarity-Switching LCESI-HRMS

    PubMed Central

    2014-01-01

    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 LCHRMS-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

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

    PubMed Central

    Araujo, Paulo T.; Frank, Otakar; Mafra, Daniela L.; Fang, Wenjing; Kong, Jing; Dresselhaus, Mildred S.; Kalbac, Martin

    2013-01-01

    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 approaches, isotopic labeling and electronic doping, is powerful tool and allows to tailor, independently and distinctly, the thermal-related and transport-related phenomena in materials, since one can impose different symmetries for electrons and phonons in these systems. Variations in the system's phonon self-energy renormalizations due to the charge distribution and doping changes could be analyzed separately for each individual layer. Symmetry arguments together with first-order Raman spectra show that the single layer graphene (1LG), which is directly contacted to the electrode, has a higher concentration of charge carriers than the second graphene layer, which is not contacted by the electrode. These different charge distributions are reflected and demonstrated by different phonon self-energy renormalizations of the G modes for AB-2LG and for t-2LG. PMID:23792906

  20. Extrinsic Labeling of Staple Food Crops with Isotopic Iron Does Not Consistently Result in Full Equilibration: Revisiting the Methodology.

    PubMed

    Glahn, Raymond P; Cheng, Zhiqiang; Giri, Shree

    2015-11-01

    Extrinsic isotopic labeling of food Fe has been used for over 50 years to measure Fe absorption. This method assumes that complete equilibration occurs between the extrinsic and the intrinsic Fe prior to intestinal absorption. The present study tested this assumption via in vitro digestion of varieties of maize, white beans, black beans, red beans, and lentils. Prior to digestion, foods were extrinsically labeled with (58)Fe at concentrations of 1, 10, 50, and 100% of the intrinsic (56)Fe. Following an established in vitro digestion protocol, the digest was centrifuged and the Fe solubilities of the extrinsic (58)Fe and the intrinsic (56)Fe were compared as a measure of extrinsic/intrinsic equilibration. In the beans, significantly more of the extrinsic Fe (up to 2-3 times, p < 0.001) partitioned into the supernatant. The effect varied depending upon the seed coat color, the harvest, and the concentration of the extrinsic Fe. For lentils and maize the extrinsic Fe tended to partition into the insoluble fraction and also varied depending on variety and harvest. There was no crop that consistently demonstrated full equilibration of the extrinsic Fe with the intrinsic Fe. These observations challenge the accuracy of Fe absorption studies in which isotopic extrinsic Fe was used to evaluate Fe absorption and bioavailability. PMID:26456842

  1. Hydrogen release kinetics during reactive magnetron sputter depostion of a-Si:H: An isotope labeling study

    NASA Astrophysics Data System (ADS)

    Abelson, J. R.; Mandrell, L.; Doyle, J. R.

    1994-08-01

    The release of moleculear hydrogen from the growing surface of hydrogenated amorphous silicon films is determined using an isotope labeling technique. The results demonstrate that surface-bonded H atoms are readily abstracted by atomic hydrogen arriving from the gas phase. The films are deposited by dc reactive magnetron sputtering of a silicon target in an argon-hydrogen atmosphere. To achieve isotope labeling, we first deposit a deuterated amorphous silicon film, then commence growth of hydrogenated amorphous silicon and measure the transient release of HD and D2 from the growing surface using mass spectrometry. Release occurs when the supply of reactive hydrogen in the growth flux exceeds the incorporation rate into the film, and is observed under all experimental conditions. The net rate of H incorporation is known from ex situ measurments of film growth and hydrogen content. We combine the H release and incorporation data in a mass balance argument to determine the H-surface kinetics. Under conditions which produce electronically useful films, (1) 0.5-1.0 hydrogen atoms react with the growing surface per incorporated silicon atom, (2) the near surface of the growing film contains 1-3 x 10(exp 15)/sq cm pf excess hydrogen, the dominant hydrogen release mechanism is by direct abstraction to form H2 molecules, and the kinetics of H release and incorportation can be described by constant rate coefficients. These data are supported by studies of H interactions with single-crystal silicon and amorphous carbon surfaces.

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

    PubMed

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

    2016-03-01

    Recently, γ-glutamyl dipeptides (γ-GPs) were found to be responsible for the attractive kokumi flavor of Parmesan cheese (PC). Quantitation of γ-GPs and their parent amino acids in 13-, 24-, and 30-month ripened PC samples by LC-MS/MS and stable isotope dilution analysis (SIDA), in-cheese (13)C-labeling studies, followed by analysis of the γ-glutamyl transferase (GGT) activity revealed γ-GPs to be generated most efficiently after 24 months of ripening by a GGT-catalyzed transfer of the γ-glutamyl moiety of l-glutamine onto various acceptor amino acids released upon casein proteolysis. Following the identification of milk as a potential GGT source in PC, the functionality of the milk's GGT to generate the target γ-GPs was validated by stable isotope double-labeling (SIDL) experiments. Therefore, raw and heat-treated milk samples were incubated with l-glutamine-[U-(13)C] and acceptor amino acids (X) and the hetero- (γ-Glu-[(13)C5]-X) and homotranspeptidation products (γ-Glu-Gln-[(13)C10]) were quantitated by LC-MS/MS-SIDA using γ-Glu-Ala-[(13)C3] as the internal standard. High GGT activity to generate the γ-GPs and preference for l-phenylalanine and l-methionine as acceptor amino acids were found in raw milk and milk samples heat-treated for 10 min up to a maximum of 65 °C. In comparison, GGT activity and SIDL studies performed with inoculated Lactobacillus strains, including Lactobacillus harbinensis and Lactobacillus casei identified in PC by means of 16S rRNA gene sequencing, did not show any significant GGT activity and unequivocally demonstrated unpasteurized cow's milk, rather than microorganisms, as a key factor in γ-glutamyl dipeptide generation in Parmesan cheese. PMID:26866784

  3. Innovative method for carbon dioxide determination in human postmortem cardiac gas samples using headspace-gas chromatography-mass spectrometry and stable labeled isotope as internal standard.

    PubMed

    Varlet, V; Smith, F; de Froidmont, S; Dominguez, A; Rinaldi, A; Augsburger, M; Mangin, P; Grabherr, S

    2013-06-19

    A novel approach to measure carbon dioxide (CO2) in gaseous samples, based on a precise and accurate quantification by (13)CO2 internal standard generated in situ is presented. The main goal of this study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable in the routine determination of CO2. The main drawback of the GC methods discussed in the literature for CO2 measurement is the lack of a specific internal standard necessary to perform quantification. CO2 measurement is still quantified by external calibration without taking into account analytical problems which can often occur considering gaseous samples. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in situ an internal labeled standard gas ((13)CO2) on the basis of the stoichiometric formation of CO2 by the reaction of hydrochloric acid (HCl) with sodium hydrogen carbonate (NaH(13)CO3). This method allows a precise measurement of CO2 concentration and was validated on various human postmortem gas samples in order to study its efficiency. PMID:23746406

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

    DOEpatents

    Martinez, Rodolfo A.; Unkefer, Clifford J.; Alvarez, Marc A.

    2008-01-22

    The present invention is directed to asymmetric chiral labeled glycerols including at least one chiral atom, from one to two .sup.13C atoms and from zero to four deuterium atoms bonded directly to a carbon atom, e.g., (2S) [1,2-.sup.13C.sub.2]glycerol and (2R) [1,2-.sup.13C.sub.2]glycerol, and to the use of such chiral glycerols in the preparation of labeled amino acids.

  5. Differential Isotope Labeling of 38 Dietary Polyphenols and Their Quantification in Urine by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry.

    PubMed

    Achaintre, David; Buleté, Audrey; Cren-Olivé, Cécile; Li, Liang; Rinaldi, Sabina; Scalbert, Augustin

    2016-03-01

    A large number of polyphenols are consumed with the diet and may contribute to the prevention of chronic diseases such as cardiovascular diseases, diabetes, cancers, and neurodegenerative diseases. More comprehensive methods are needed to measure exposure to this complex family of bioactive plant compounds in epidemiological studies. We report here a novel method enabling the simultaneous measurement in urine of 38 polyphenols representative of the main classes and subclasses found in the diet. This method is based on differential (12)C-/(13)C-isotope labeling of polyphenols through derivatization with isotopic dansyl chloride reagents and on the analysis of the labeled polyphenols by tandem mass spectrometry. This derivatization approach overcomes the need for costly labeled standards. Different conditions for enzyme hydrolysis of polyphenol glucuronides and sulfate esters, extraction, and dansylation of unconjugated aglycones were tested and optimized. Limits of quantification varied from 0.01 to 1.1 μM depending on polyphenols. Intrabatch coefficients of variation varied between 3.9% and 9.6%. Interbatch variations were lower than 15% for 31 compounds and lower than 29% for 6 additional polyphenols out of the 38 tested. Thirty seven polyphenols were validated and then analyzed in 475, 24 h urine samples from the European Prospective Investigation on Cancer and Nutrition (EPIC) study. Thirty four polyphenols could be detected and successfully estimated and showed large interindividual variations of concentrations (2-3 orders of magnitude depending on the compound), with median concentrations spanning from 0.01 to over 1000 μM for all 34 compounds. PMID:26814424

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

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

    Understanding the soil organic matter (SOM) dynamics is one of the most important challenges in soil science. Transformation of low molecular weight organic substances (LMWOS) is a key step in biogeochemical cycles because 1) all high molecular substances pass this stage during their decomposition and 2) only LMWOS will be taken up by microorganisms. Previous studies on LMWOS were focused on determining net fluxes through the LMWOS pool, but they rarely identified transformations. As LMWOS are the preferred C and energy source for microorganisms, the transformations of LMWOS are dominated by biochemical pathways of the soil microorganisms. Thus, understanding fluxes and transformations in soils requires a detailed knowledge on the biochemical pathways and its controlling factors. Tracing C fate in soil by isotopes became on of the most applied and promising biogeochemistry tools. Up to now, studies on LMWOS were nearly exclusively based on uniformly labeled organic substances i.e. all C atoms in the molecules were labeled with 13C or 14C. However, this classical approach did not allow the differentiation between use of intact initial substances in any process, or whether they were transformed to metabolites. The novel tool of position-specific labeling enables to trace molecule atoms separately and thus to determine the cleavage of molecules - a prerequisite for metabolic tracing. Position-specific labeling of LMWOS and quantification of 13CO2 and 13C in bulk soil enabled following the basic metabolic pathways of soil microorganisms. However, only the combination of position-specific 13C labeling with compound-specific isotope analysis of microbial biomarkers and metabolites allowed 1) tracing specific anabolic pathways in diverse microbial communities in soils and 2) identification of specific pathways of individual functional microbial groups. So, these are the prerequisites for soil fluxomics. Our studies combining position-specific labeled glucose with amino sugar 13C analysis showed that oxidizing catabolic pathways and anabolic pathways, i.e. building-up new cellular compounds, occurred in soils simultaneously. This involved an intensive C recycling within the microorganisms that was observed not only for cytosolic compounds but also for cell wall polymers. Fungal metabolism and fluxes were slower than bacterial intracellular C recycling and turnover. Furthermore, position-specific labeling of glutamate and subsequent 13C analysis of microbial phospholipid fatty acids (PLFA) revealed starvation pathways, which were only active in specific microbial groups in soils. These studies revealed that position-specific labeling enables the reconstruction of metabolic pathways of LMWOS within diverse microbial communities in complex media such as soil. Processes occurring simultaneously in soil i.e. 1) within individual, reversible metabolic pathways and 2) in various microbial groups could be traced by position-specific labeling in soils in situ. Tracing these pathways and understanding their regulating factors are crucial for soil C fluxomics, the extremely complex network of transformations towards mineralization versus the formation of microbial biomass compounds. Quantitative models to assess microbial group specific metabolic networks can be generated and parameterized by this approach. The submolecular knowledge of transformation steps and biochemical pathways in soils and their regulating factors is essential for understanding C cycling and long-term C storage in soils.

  7. Probing the Metabolic Network in Bloodstream-Form Trypanosoma brucei Using Untargeted Metabolomics with Stable Isotope Labelled Glucose

    PubMed Central

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

    2015-01-01

    Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate. PMID:25775470

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

    PubMed

    Paj?k, Ma?gorzata; Pa?ka, Katarzyna; Winnicka, El?bieta; Ka?ska, Marianna

    2016-01-01

    Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L-tryptophan, i.e. 5'-bromo-[2-(3) H]-, 5'-bromo-[2-(2) H/(3) H]-, 5'-fluoro-[2-(3) H]-5'-fluoro-[2-(2) H/(3) H]-, 6'-fluoro-[2-(3) H]-, 6'-fluoro-[2-(2) H/(3) H]-L-tryptophan, as well as, L-tyrosine, i.e. 3'-fluoro-[2-(3) H]-, 3'-fluoro-[2-(2) H/(3) H]-, 3'-chloro-[2-(3) H]-, and 3'-chloro-[2-(2) H/(3) H]-L-tyrosine, and also L-phenylalanine, i.e. 2'-fluoro-[(3S)-(3) H]-, 2'-fluoro-[(3S)-(2) H/(3) H]-, 2'-chloro-[(3S)-(3) H]-, 2'-chloro-[(3S)-(2) H/(3) H]-, 4'-chloro-[(3S)-(3) H]-, and 4'-chloro-[(3S)-(2) H/(3) H]-L-phenylalanine were synthesized using enzymatic methods. Isotopomers of L-tryptophan were synthesized by coupling of halogenated indoles with S-methyl-L-cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L-tyrosine were obtained by the enzymatically supported exchange between halogenated L-tyrosine and isotopic water. Labeled halogenated isotopologues of L-Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D2 O) with addition of HTO were used. PMID:26586485

  9. Sulfur-34S Stable Isotope Labeling of Amino Acids for Quantification (SULAQ34) of Proteomic Changes in Pseudomonas fluorescens during Naphthalene Degradation*

    PubMed Central

    Herbst, Florian-Alexander; Taubert, Martin; Jehmlich, Nico; Behr, Tobias; Schmidt, Frank; von Bergen, Martin; Seifert, Jana

    2013-01-01

    The relative quantification of proteins is one of the major techniques used to elucidate physiological reactions. Because it allows one to avoid artifacts due to chemical labeling, the metabolic introduction of heavy isotopes into proteins and peptides is the preferred method for relative quantification. For eukaryotic cells, stable isotope labeling by amino acids in cell culture (SILAC) has become the gold standard and can be readily applied in a vast number of scenarios. In the microbial realm, with its highly versatile metabolic capabilities, SILAC is often not feasible, and the use of other 13C or 15N labeled substrates might not be practical. Here, the incorporation of heavy sulfur isotopes is shown to be a useful alternative. We introduce 34S stable isotope labeling of amino acids for quantification and the corresponding tools required for spectra extraction and disintegration of the isotopic overlaps caused by the small mass shift. As proof of principle, we investigated the proteomic changes related to naphthalene degradation in P. fluorescens ATCC 17483 and uncovered a specific oxidative-stress-like response. PMID:23603340

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

    Metabolic rates of microbial communities constitute a key physiological parameter for understanding the in situ growth constraints for life in any environment. Isotope labeling techniques provide a powerful approach for measuring such biological activity, due to the use of isotopically enriched substrate tracers whose incorporation into biological materials can be detected with high sensitivity by isotope-ratio mass spectrometry. Nano-meter scale secondary ion mass spectrometry (NanoSIMS) combined with stable isotope labeling provides a unique tool for studying the spatiometabolic activity of microbial populations at the single cell level in order to assess both community structure and population diversity. However, assessing the distribution and range of microbial activity in complex environmental systems with slow-growing organisms, diverse carbon and nitrogen sources, or heterotrophic subpopulations poses a tremendous technical challenge because the introduction of isotopically labeled substrates frequently changes the nutrient availability and can inflate or bias measures of activity. Here, we present the use of hydrogen isotope labeling with deuterated water as an important new addition to the isotopic toolkit and apply it for the determination of single cell microbial activities by NanoSIMS imaging. This tool provides a labeling technique that minimally alters any aquatic chemical environment, can be administered with strong labels even in minimal addition (natural background is very low), is an equally universal substrate for all forms of life even in complex, carbon and nitrogen saturated systems, and can be combined with other isotopic tracers. The combination of heavy water labeling with the most commonly used NanoSIMS tracer, 15N, is technically challenging but opens up a powerful new set of multi-tracer experiments for the study of microbial activity in complex communities. We present the first truly simultaneous single cell triple isotope system measurements of 2H/1H, 13C/12C and 15N/14N and apply it to study of microbial metabolic heterogeneity and nitrogen metabolism in a continuous culture case study. Our data provide insight into both the diversity of microbial activity rates, as well as patterns of ammonium utilization at the single cell level.

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

    PubMed Central

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-08-01

    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.

  13. Efficient Analysis of Mass Spectrometry Data Using the Isotope Wavelet

    NASA Astrophysics Data System (ADS)

    Hussong, Rene; Tholey, Andreas; Hildebrandt, Andreas

    2007-09-01

    Mass spectrometry (MS) has become today's de-facto standard for high-throughput analysis in proteomics research. Its applications range from toxicity analysis to MS-based diagnostics. Often, the time spent on the MS experiment itself is significantly less than the time necessary to interpret the measured signals, since the amount of data can easily exceed several gigabytes. In addition, automated analysis is hampered by baseline artifacts, chemical as well as electrical noise, and an irregular spacing of data points. Thus, filtering techniques originating from signal and image analysis are commonly employed to address these problems. Unfortunately, smoothing, base-line reduction, and in particular a resampling of data points can affect important characteristics of the experimental signal. To overcome these problems, we propose a new family of wavelet functions based on the isotope wavelet, which is hand-tailored for the analysis of mass spectrometry data. The resulting technique is theoretically well-founded and compares very well with standard peak picking tools, since it is highly robust against noise spoiling the data, but at the same time sufficiently sensitive to detect even low-abundant peptides.

  14. Efficient Analysis of Mass Spectrometry Data Using the Isotope Wavelet

    SciTech Connect

    Hussong, Rene; Hildebrandt, Andreas; Tholey, Andreas

    2007-09-18

    Mass spectrometry (MS) has become today's de-facto standard for high-throughput analysis in proteomics research. Its applications range from toxicity analysis to MS-based diagnostics. Often, the time spent on the MS experiment itself is significantly less than the time necessary to interpret the measured signals, since the amount of data can easily exceed several gigabytes. In addition, automated analysis is hampered by baseline artifacts, chemical as well as electrical noise, and an irregular spacing of data points. Thus, filtering techniques originating from signal and image analysis are commonly employed to address these problems. Unfortunately, smoothing, base-line reduction, and in particular a resampling of data points can affect important characteristics of the experimental signal. To overcome these problems, we propose a new family of wavelet functions based on the isotope wavelet, which is hand-tailored for the analysis of mass spectrometry data. The resulting technique is theoretically well-founded and compares very well with standard peak picking tools, since it is highly robust against noise spoiling the data, but at the same time sufficiently sensitive to detect even low-abundant peptides.

  15. Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

    PubMed Central

    Alexandrino, Maria; Knief, Claudia; Lipski, Andr

    2001-01-01

    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

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

    SciTech Connect

    Zhou, Nan; Zheng, Nina; Fridley, David

    2012-02-28

    Appliance energy efficiency standards and labeling (S&L) programs have been important policy tools for regulating the efficiency of energy-using products for over 40 years and continue to expand in terms of geographic and product coverage. The most common S&L programs include mandatory minimum energy performance standards (MEPS) that seek to push the market for efficient products, and energy information and endorsement labels that seek to pull the market. This study seeks to review and compare some of the earliest and most well-developed S&L programs in three countries and one region: the U.S. MEPS and ENERGY STAR, Australia MEPS and Energy Label, European Union MEPS and Ecodesign requirements and Energy Label and Japanese Top Runner programs. For each program, key elements of S&L programs are evaluated and comparative analyses across the programs undertaken to identify best practice examples of individual elements as well as cross-cutting factors for success and lessons learned in international S&L program development and implementation. The international review and comparative analysis identified several overarching themes and highlighted some common factors behind successful program elements. First, standard-setting and programmatic implementation can benefit significantly from a legal framework that stipulates a specific timeline or schedule for standard-setting and revision, product coverage and legal sanctions for non-compliance. Second, the different MEPS programs revealed similarities in targeting efficiency gains that are technically feasible and economically justified as the principle for choosing a standard level, in many cases at a level that no product on the current market could reach. Third, detailed survey data such as the U.S. Residential Energy Consumption Survey (RECS) and rigorous analyses provide a strong foundation for standard-setting while incorporating the participation of different groups of stakeholders further strengthen the process. Fourth, sufficient program resources for program implementation and evaluation are critical to the effectiveness of standards and labeling programs and cost-sharing between national and local governments can help ensure adequate resources and uniform implementation. Lastly, check-testing and punitive measures are important forms of enforcement while the cancellation of registration or product sales-based fines have also proven effective in reducing non-compliance. The international comparative analysis also revealed the differing degree to which the level of government decentralization has influenced S&L programs and while no single country has best practices in all elements of standards and labeling development and implementation, national examples of best practices for specific elements do exist. For example, the U.S. has exemplified the use of rigorous analyses for standard-setting and robust data source with the RECS database while Japan�s Top Runner standard-setting principle has motivated manufacturers to exceed targets. In terms of standards implementation and enforcement, Australia has demonstrated success with enforcement given its long history of check-testing and enforcement initiatives while mandatory information-sharing between EU jurisdictions on compliance results is another important enforcement mechanism. These examples show that it is important to evaluate not only the drivers of different paths of standards and labeling development, but also the country-specific context for best practice examples in order to understand how and why certain elements of specific S&L programs have been effective.

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

    ERIC Educational Resources Information Center

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

    2004-01-01

    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.

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

    NASA Astrophysics Data System (ADS)

    Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo

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

  19. Encoding atlases by randomized classification forests for efficient multi-atlas label propagation.

    PubMed

    Zikic, D; Glocker, B; Criminisi, A

    2014-12-01

    We propose a method for multi-atlas label propagation (MALP) based on encoding the individual atlases by randomized classification forests. Most current approaches perform a non-linear registration between all atlases and the target image, followed by a sophisticated fusion scheme. While these approaches can achieve high accuracy, in general they do so at high computational cost. This might negatively affect the scalability to large databases and experimentation. To tackle this issue, we propose to use a small and deep classification forest to encode each atlas individually in reference to an aligned probabilistic atlas, resulting in an Atlas Forest (AF). Our classifier-based encoding differs from current MALP approaches, which represent each point in the atlas either directly as a single image/label value pair, or by a set of corresponding patches. At test time, each AF produces one probabilistic label estimate, and their fusion is done by averaging. Our scheme performs only one registration per target image, achieves good results with a simple fusion scheme, and allows for efficient experimentation. In contrast to standard forest schemes, in which each tree would be trained on all atlases, our approach retains the advantages of the standard MALP framework. The target-specific selection of atlases remains possible, and incorporation of new scans is straightforward without retraining. The evaluation on four different databases shows accuracy within the range of the state of the art at a significantly lower running time. PMID:25042602

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

    PubMed

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

    2011-01-01

    A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-(2)H(4);1,2,3,4,5,6-(13)C(6);2,6-(15)N(2)]lysine, was synthesized by the "head-to-tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-(2)H(2);1,2,3,4,5-(13)C(5);2-(15)N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins. PMID:21121622

  1. Assessing water-use-efficiency from stable isotopes and eddy covariance data

    NASA Astrophysics Data System (ADS)

    Knohl, A.; Barthel, M.; Kutsch, W. L.; Salmon, Y.; Sturm, P.

    2009-04-01

    The water and carbon cycles in terrestrial ecosystems play an essential role in the earth system by modifying the atmospheric carbon dioxide and water vapor composition and by partitioning energy at the land surface. Carbon uptake during photosynthesis and water release during transpiration are closely linked through stomatal conductance. The efficiency of carbon uptake versus water loss is called water-use-efficiency and is a key factor for agricultural irrigation practices and species competition in water limited ecosystems. Here we review various concepts to assess water-use-efficiency at plant and ecosystem scale and present results from different biomes. We will show that inherent water-use-efficiency (estimated from eddy covariance measurements as the ratio of gross primary productivity over evapotranspiration times vapor pressure deficit) is a fairly conservative ecosystem property and only changes under sever drought conditions. Another approach to assess water-use-efficiency is the stable carbon isotope composition (^13C) of plant material. Typically, in a first approximation a simply linear relationship between leaf ^13C and intrinsic water-use-efficiency (the ratio of assimilation to stomata conductance) is assumed. More recent studies, however, show that various factors influence leaf ^13C independently of the exchange through the stomata leading to incorrect water-use-efficiency estimates. Based on laboratory and field experiments using stable carbon isotopes in various plant components as well as recently developed laser spectroscopy for continuous stable isotope measurements we discuss possibilities and limitations of using stable carbon isotopes to approximate plant water-use-efficiency.

  2. Insight into the Formation of Molecular Species in Laser-Induced Plasma of Isotopically Labeled Organic Samples.

    PubMed

    Glaus, Reto; Riedel, Jens; Gornushkin, Igor

    2015-10-01

    Recently, the detection of molecular species in laser-induced breakdown spectroscopy (LIBS) has gained increasing interest, particularly for isotopic analysis. In LIBS of organic materials, it is predominantly CN and C2 species that are formed, and multiple mechanisms may contribute to their formation. To gain deeper insight into the formation of these species, laser-induced plasma of (13)C and (15)N labeled organic materials was investigated in a temporally and spatially resolved manner. LIBS on fumaric acid with a (13)C labeled double bond allowed the formation mechanism of C2 to be investigated by analyzing relative signal intensities of (12)C2, (12)C(13)C, and (13)C2 molecules. In the early plasma (<5 ?s), the majority of C2 originates from association of completely atomized target molecules, whereas in the late plasma, the increased concentration of (13)C2 is due to incomplete dissociation of the carbon double bond. The degree of this fragmentation was found to be up to 80% and to depend on the type of the atmospheric gas. Spatial distributions of C2 revealed distinct differences for plasma generated in nitrogen and argon. A study of the interaction of ablated organics with ambient nitrogen showed that the ambient nitrogen contributed mainly to CN formation. The pronounced anisotropy of the C(15)N to C(14)N ratio across the diameter of the plasma was observed in the early plasma, indicating poor initial mixing of the plasma with the ambient gas. Overall, for accurate isotope analysis of organics, LIBS in argon with relatively short integration times (<10 ?s) provides the most robust results. On the other hand, if information about the original molecular structure is of interest, then experiments in nitrogen (or air) with long integration times appear to be the most promising. PMID:26402464

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

    PubMed

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

    2014-12-17

    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/mLh for Pro-Hyp-Gly to 163 1 nmol/mLh 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

  4. Effect of reversible reactions on isotope label redistribution--analysis of the pentose phosphate pathway.

    PubMed

    Follstad, B D; Stephanopoulos, G

    1998-03-15

    The pentose phosphate pathway plays several key roles in metabolism including supply of biosynthetic carbon skeletons and reducing power. Previous research has focused on determining the fluxes through the reactions of this pathway using carbon-labeled substrates and models that make certain assumptions about the reversibility of the transketolase and transaldolase reactions in the nonoxidative pathway. These assumptions, however, have resulted in inconsistencies between the predicted carbon label distributions using these models and those determined experimentally. A general metabolic reaction network model developed in this paper and applied to the pentose phosphate pathway not only incorporates reaction reversibility but also accounts for the effect of individually varying extents of reaction reversibility on labeled carbon fractional enrichment values for intermediate metabolites. In addition, an algorithm is presented that can be used to calculate the three individual transaldolase and transketolase extents of reversibility. The results of this method show that varying extents of reaction reversibility have an observable effect on the metabolite carbon label distributions which can in turn affect flux calculation for other parts of the metabolic network such as the tricarboxylic acid cycle. In addition, the observability of reversibility extent and accuracy of flux calculations depend on the particular choice of metabolite carbon enrichments measured. In particular, [6-13C]hexose 6-phosphate and [4-13C]erythrose 4-phosphate carbon enrichment values resulting from [1-13C]glucose feeding contained more information as compared to those from ribose 5-phosphate. This analysis was applied to literature data of metabolite carbon labeling that resulted from supplying either 13C- or 14C-enriched substrates to several cell types growing under various conditions. The specific activities of metabolite carbon atoms taken from rat epididymal adipose tissue, goosefish islet cells, Corynebacterium glutamicum, and Escherichia coli supplied with either [2-14C]glucose or [1-13C]glucose demonstrate how reversibility is present in the pentose phosphate pathway and the extents of reversibility can be estimated from labeled carbon data sets. PMID:9546650

  5. Correction: NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro.

    PubMed

    Lee, Ronald F S; Escrig, Stphane; Croisier, Marie; Clerc-Rosset, Stphanie; Knott, Graham W; Meibom, Anders; Davey, Curt A; Johnsson, Kai; Dyson, Paul J

    2015-11-28

    Correction for 'NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro' by Ronald F. S. Lee et al., Chem. Commun., 2015, DOI: 10.1039/c5cc06983a. PMID:26507472

  6. Efficient Total Chemical Synthesis of (13) C=(18) O Isotopomers of Human Insulin for Isotope-Edited FTIR.

    PubMed

    Dhayalan, Balamurugan; Fitzpatrick, Ann; Mandal, Kalyaneswar; Whittaker, Jonathan; Weiss, Michael A; Tokmakoff, Andrei; Kent, Stephen B H

    2016-03-01

    Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2 D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable (13) C=(18) O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis-via a key ester insulin intermediate-of 97 % enriched [(1-(13) C=(18) O)Phe(B24) ] human insulin: stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-(13) C=(18) O)Phe(B24) ] human insulin showed that it had full insulin receptor binding activity. Linear and 2 D IR spectra revealed a distinct red-shifted amide I carbonyl band peak at 1595 cm(-1) resulting from the (1-(13) C=(18) O)Phe(B24) backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function. PMID:26715336

  7. Dual isotope study of iodine-125 and indium-111-labeled antibody in athymic mice

    SciTech Connect

    Carney, P.L.; Rogers, P.E.; Johnson, D.K. )

    1989-03-01

    Monoclonal antibody B72.3 was coupled to a benzylisothiocyanate derivative of diethylenetriaminepentaacetic acid (DTPA). The maximum substitution achievable without loss of immunoreactivity was three DTPA groups per immunoglobulin molecule. The resulting conjugate was labeled with {sup 111}In by brief incubation with {sup 111}InCl{sub 3}, giving a mean radiochemical yield of {sup 111}In-labeled antibody of 96%. The ({sup 111}In)B72.3 preparation was mixed with an ({sup 125}I) B72.3 preparation, obtained by the chloramine-T method, and the mixture administered to athymic mice bearing subcutaneous LS174T colon carcinoma xenografts. There were no significant differences (p greater than 0.1) in the biodistributions of the two labels at 1, 2, 5, and 7 days postinjection. These results are contrasted with prior studies showing elevated levels of {sup 111}In in liver, spleen, and kidneys using B72.3-DTPA conjugates prepared via the bicyclic anhydride. It is concluded that protein cross-linking and/or the formation of unstable chelate sites in anhydride coupled conjugates underlie these disparities.

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

    PubMed

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

    2016-01-01

    Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1-11.3 folds. In addition, the concentration of homocysteine, ?-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines. PMID:26888486

  9. Fatty Acid Labeling from Glutamine in Hypoxia Can Be Explained by Isotope Exchange without Net Reductive Isocitrate Dehydrogenase (IDH) Flux

    PubMed Central

    Fan, Jing; Kamphorst, Jurre J.; Rabinowitz, Joshua D.; Shlomi, Tomer

    2013-01-01

    Acetyl-CoA is an important anabolic precursor for lipid biosynthesis. In the conventional view of mammalian metabolism, acetyl-CoA is primarily derived by the oxidation of glucose-derived pyruvate in mitochondria. Recent studies have employed isotope tracers to show that in cancer cells grown in hypoxia or with defective mitochondria, a major fraction of acetyl-CoA is produced via another route, reductive carboxylation of glutamine-derived α-ketoglutarate (catalyzed by reverse flux through isocitrate dehydrogenase, IDH). Here, we employ a quantitative flux model to show that in hypoxia and in cells with defective mitochondria, oxidative IDH flux persists and may exceed the reductive flux. Therefore, IDH flux may not be a net contributor to acetyl-CoA production, although we cannot rule out net reductive IDH flux in some compartments. Instead of producing large amounts of net acetyl-CoA reductively, the cells adapt by reducing their demand for acetyl-CoA by importing rather than synthesizing fatty acids. Thus, fatty acid labeling from glutamine in hypoxia can be explained by spreading of label without net reductive IDH flux. PMID:24030823

  10. Conformational changes of recombinant monoclonal antibodies by limited proteolytic digestion, stable isotope labeling, and liquid chromatography-mass spectrometry.

    PubMed

    Ponniah, Gomathinayagam; Nowak, Christine; Kita, Adriana; Cheng, Guilong; Kori, Yekaterina; Liu, Hongcheng

    2016-03-15

    Limited proteolytic digestion is a method with a long history that has been used to study protein domain structures and conformational changes. A method of combining limited proteolytic digestion, stable isotope labeling, and mass spectrometry was established in the current study to investigate protein conformational changes. Recombinant monoclonal antibodies with or without the conserved oligosaccharides, and with or without oxidation of the conserved methionine residues, were used to test the newly proposed method. All of the samples were digested in ammonium bicarbonate buffer prepared in normal water. The oxidized deglycosylated sample was also digested in ammonium bicarbonate buffer prepared in (18)O-labeled water. The sample from the digestion in (18)O-water was spiked into each sample digested in normal water. Each mixed sample was subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). The molecular weight differences between the peptides digested in normal water versus (18)O-water were used to differentiate peaks from the samples. The relative peak intensities of peptides with or without the C-terminal incorporation of (18)O atoms were used to determine susceptibility of different samples to trypsin and chymotrypsin. The results demonstrated that the method was capable of detecting local conformational changes of the recombinant monoclonal antibodies caused by deglycosylation and oxidation. PMID:26747642

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

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

    PubMed Central

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

    2009-01-01

    Prochlorococcus and Synechococcus are the two most abundant marine cyanobacteria. They represent a significant fraction of the total primary production of the world oceans and comprise a major fraction of the prey biomass available to phagotrophic protists. Despite relatively rapid growth rates, picocyanobacterial cell densities in open-ocean surface waters remain fairly constant, implying steady mortality due to viral infection and consumption by predators. There have been several studies on grazing by specific protists on Prochlorococcus and Synechococcus in culture, and of cell loss rates due to overall grazing in the field. However, the specific sources of mortality of these primary producers in the wild remain unknown. Here, we use a modification of the RNA stable isotope probing technique (RNA-SIP), which involves adding labelled cells to natural seawater, to identify active predators that are specifically consuming Prochlorococcus and Synechococcus in the surface waters of the Pacific Ocean. Four major groups were identified as having their 18S rRNA highly labelled: Prymnesiophyceae (Haptophyta), Dictyochophyceae (Stramenopiles), Bolidomonas (Stramenopiles) and Dinoflagellata (Alveolata). For the first three of these, the closest relative of the sequences identified was a photosynthetic organism, indicating the presence of mixotrophs among picocyanobacterial predators. We conclude that the use of RNA-SIP is a useful method to identity specific predators for picocyanobacteria in situ, and that the method could possibly be used to identify other bacterial predators important in the microbial food-web. PMID:19196281

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

    PubMed Central

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

    2016-01-01

    Precursor ion scan and multiple reaction monitoring scan (MRM) are two typical scan modes in mass spectrometry analysis. Here, we developed a strategy by combining stable isotope labeling (IL) with liquid chromatography-mass spectrometry (LC-MS) under double precursor ion scan (DPI) and MRM for analysis of thiols in 5 types of human cancer urine. Firstly, the IL-LC-DPI-MS method was applied for non-targeted profiling of thiols from cancer samples. Compared to traditional full scan mode, the DPI method significantly improved identification selectivity and accuracy. 103 thiol candidates were discovered in all cancers and 6 thiols were identified by their standards. It is worth noting that pantetheine, for the first time, was identified in human urine. Secondly, the IL-LC-MRM-MS method was developed for relative quantification of thiols in cancers compared to healthy controls. All the MRM transitions of light and heavy labeled thiols were acquired from urines by using DPI method. Compared to DPI method, the sensitivity of MRM improved by 2.1–11.3 folds. In addition, the concentration of homocysteine, γ-glutamylcysteine and pantetheine enhanced more than two folds in cancer patients compared to healthy controls. Taken together, the method demonstrated to be a promising strategy for identification and comprehensive quantification of thiols in human urines. PMID:26888486

  14. geoRge: A Computational Tool To Detect the Presence of Stable Isotope Labeling in LC/MS-Based Untargeted Metabolomics.

    PubMed

    Capellades, Jordi; Navarro, Miriam; Samino, Sara; Garcia-Ramirez, Marta; Hernandez, Cristina; Simo, Rafael; Vinaixa, Maria; Yanes, Oscar

    2016-01-01

    Studying the flow of chemical moieties through the complex set of metabolic reactions that happen in the cell is essential to understanding the alterations in homeostasis that occur in disease. Recently, LC/MS-based untargeted metabolomics and isotopically labeled metabolites have been used to facilitate the unbiased mapping of labeled moieties through metabolic pathways. However, due to the complexity of the resulting experimental data sets few computational tools are available for data analysis. Here we introduce geoRge, a novel computational approach capable of analyzing untargeted LC/MS data from stable isotope-labeling experiments. geoRge is written in the open language R and runs on the output structure of the XCMS package, which is in widespread use. As opposed to the few existing tools, which use labeled samples to track stable isotopes by iterating over all MS signals using the theoretical mass difference between the light and heavy isotopes, geoRge uses unlabeled and labeled biologically equivalent samples to compare isotopic distributions in the mass spectra. Isotopically enriched compounds change their isotopic distribution as compared to unlabeled compounds. This is directly reflected in a number of new m/z peaks and higher intensity peaks in the mass spectra of labeled samples relative to the unlabeled equivalents. The automated untargeted isotope annotation and relative quantification capabilities of geoRge are demonstrated by the analysis of LC/MS data from a human retinal pigment epithelium cell line (ARPE-19) grown on normal and high glucose concentrations mimicking diabetic retinopathy conditions in vitro. In addition, we compared the results of geoRge with the outcome of X(13)CMS, since both approaches rely entirely on XCMS parameters for feature selection, namely m/z and retention time values. To ensure data traceability and reproducibility, and enabling for comparison with other existing and future approaches, raw LC/MS files have been deposited in MetaboLights (MTBLS213) and geoRge is available as an R script at https://github.com/jcapelladesto/geoRge . PMID:26639619

  15. Stable carbon isotope labeling reveals different carry-over effects between functional types of tropical trees in an Ethiopian mountain forest.

    PubMed

    Krepkowski, Julia; Gebrekirstos, Aster; Shibistova, Olga; Bruning, Achim

    2013-07-01

    We present an intra-annual stable carbon isotope (?(13)C) study based on a labeling experiment to illustrate differences in temporal patterns of recent carbon allocation to wood structures of two functional types of trees, Podocarpus falcatus (a late-successional evergreen conifer) and Croton macrostachyus (a deciduous broadleaved pioneer tree), in a tropical mountain forest in Ethiopia. Dendrometer data, wood anatomical thin sections, and intra-annual ?(13)C analyses were applied. Isotope data revealed a clear annual growth pattern in both studied species. For P. falcatus, it was possible to synchronize annual ?(13) C peaks, wood anatomical structures and monthly precipitation patterns. The labeling signature was evident for three consecutive years. For C. macrostachyus, isotope data illustrate a rapid decline of the labeling signal within half a year. Our ?(13)C labeling study indicates a distinct difference in carryover effects between trees of different functional types. A proportion of the labeled ?(13)C is stored in reserves of wood parenchyma for up to 3 yr in P. falcatus. By contrast, C. macrostachyus shows a high turnover of assimilates and a carbon carryover effect is only detectable in the subsequent year. PMID:23586968

  16. Multi-isotope labelling (13C, 18O, 2H) of fresh assimilates to trace organic matter dynamics in the plant-soil system

    NASA Astrophysics Data System (ADS)

    Studer, M. S.; Siegwolf, R. T. W.; Leuenberger, M.; Abiven, S.

    2014-11-01

    Isotope labelling is a powerful tool to study elemental cycling within terrestrial ecosystems. Here we describe a new multi-isotope technique to label organic matter (OM). We exposed poplars (Populus deltoides x nigra) for 14 days to an atmosphere enriched in 13CO2 and depleted in 2H218O. After one week, the water-soluble leaf OM (?13C = 1346 162) and the leaf water were strongly labelled (?18O = -63 8, ?2H = -156 15). The leaf water isotopic composition was between the atmospheric and stem water, indicating a considerable diffusion of vapour into the leaves (58-69%). The atomic ratios of the labels recovered (18O/13C, 2H/13C) were 2-4 times higher in leaves than in the stems and roots. This either indicates the synthesis of more condensed compounds (lignin vs. cellulose) in roots and stems, or be the result of O and H exchange and fractionation processes during transport and biosynthesis. We demonstrate that the three major OM elements (C, O, H) can be labelled and traced simultaneously within the plant. This approach could be of interdisciplinary interest for the fields of plant physiology, paleoclimatic reconstruction or soil science.

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  18. An Efficient Method to Calculate the Aggregated Isotopic Distribution and Exact Center-Masses

    NASA Astrophysics Data System (ADS)

    Claesen, Jrgen; Dittwald, Piotr; Burzykowski, Tomasz; Valkenborg, Dirk

    2012-04-01

    In this article, we present a computation- and memory-efficient method to calculate the probabilities of occurrence and exact center-masses of the aggregated isotopic distribution of a molecule. The method uses fundamental mathematical properties of polynomials given by the Newton-Girard theorem and Viete's formulae. The calculation is based on the atomic composition of the molecule and the natural abundances of the elemental isotopes in normal terrestrial matter. To evaluate the performance of the proposed method, which we named BRAIN, we compare it with the results obtained from five existing software packages ( IsoPro, Mercury, Emass, NeutronCluster, and IsoDalton) for 10 biomolecules. Additionally, we compare the computed mass centers with the results obtained by calculating, and subsequently aggregating, the fine isotopic distribution for two of the exemplary biomolecules. The algorithm will be made available as a Bioconductor package in R, and is also available upon request.

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

    SciTech Connect

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

    1987-10-01

    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.

  20. Design and operation of a continuous 13C and 15N labeling chamber for uniform or differential, metabolic and structural, plant tissue isotope labeling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tracing heavy stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O o...

  1. NMR characterization of a 72 kDa transcription factor using differential isotopic labeling.

    PubMed

    Mukherjee, Sulakshana P; Borin, Brendan; Quintas, Pedro O; Dyson, H Jane

    2016-03-01

    NF-κB is a major transcription factor that mediates a number of cellular signaling pathways. Crystal structure analysis gives an incomplete picture of the behavior of the protein, particularly in the free state; free monomers or dimers of NF-κB have never been crystallized. NMR analysis gives insights into the structure and dynamics of the protein in solution, but a necessary first step is the assignment of resonances. The size of the heterodimer of the Rel homology regions of the NF-κB monomers p65 and p50 (72 kDa) prohibits the straightforward use of triple-resonance spectroscopy to obtain the assignments. However, the dynamic nature of the free heterodimer, in particular the independence of the DNA-binding and dimerization domains of each monomer, allows the assignments made on differentially labeled smaller domains to be mapped successfully onto the spectrum of the larger full-length RHR. Problematic areas such as the p65 nuclear localization sequence, which is disordered in the free protein, can be approached by residue-specific labeling and comparison with previously-published spectra of a short peptide with the same sequence. Overall, this NMR analysis of NF-κB has given valuable insights into the highly dynamic nature of the free state, which is likely to play an important role in the functional cycle of NF-κB in the cell. PMID:26647230

  2. Isolation of aminoacyl-tRNA and its labeling with stable-isotope tracers: Use in studies of human tissue protein synthesis

    SciTech Connect

    Watt, P.W.; Lindsay, Y.; Scrimgeour, C.M.; Chien, P.A.F.; Taylor, D.J.; Rennie, M.J. ); Gibson, J.N.A. Univ. of Edinbergh )

    1991-07-01

    The authors isolated aminoacyl-tRNA from human and rat tissues and measured, by GC/MS, its labeling in vivo by ({sup 15}N)-and ({sup 13}C)leucine. Tracer dilution artifacts seemed unlikely since, after infusion of L-(1-{sup 13}C, {sup 15}N)leucine into rats, (1) muscle leucyl-tRNA labeling exceeded tissue free leucine labeling, (2) values were largely unaffected by storing over 5 min at 22C, and (3) L-(2,4,5-methyl-{sup 13}C)leucine was not incorporated into leucyl-tRNA during homogenization. Leucyl-tRNA labeling in liver and muscle suggested charging from extra- and intracellular pools: e.g., after infusing L-(1-{sup 13}C, {sup 15}N)leucine, rat muscle tissue free leucine {sup 13}C labeling exceeded that by {sup 15}N and both were significantly lower than venous plasma indicating tracer dilution by transamination and by proteolysis; however, leucyl-tRNA labeling by either isotope was significantly above mixed tissue free leucing. Human placental leucyl-tRNA labeling (after predelivery tracer infusion) was 37% lower than maternal uterine vein labeling but not significantly different from placental free leucine or umbilical arterial leucine.

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

    PubMed

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

    2014-11-01

    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

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

    PubMed

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

    2016-01-20

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

  5. A coding method for efficient subgraph querying on vertex- and edge-labeled graphs.

    PubMed

    Zhu, Lei; Song, Qinbao; Guo, Yuchen; Du, Lei; Zhu, Xiaoyan; Wang, Guangtao

    2014-01-01

    Labeled graphs are widely used to model complex data in many domains, so subgraph querying has been attracting more and more attention from researchers around the world. Unfortunately, subgraph querying is very time consuming since it involves subgraph isomorphism testing that is known to be an NP-complete problem. In this paper, we propose a novel coding method for subgraph querying that is based on Laplacian spectrum and the number of walks. Our method follows the filtering-and-verification framework and works well on graph databases with frequent updates. We also propose novel two-step filtering conditions that can filter out most false positives and prove that the two-step filtering conditions satisfy the no-false-negative requirement (no dismissal in answers). Extensive experiments on both real and synthetic graphs show that, compared with six existing counterpart methods, our method can effectively improve the efficiency of subgraph querying. PMID:24853266

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

    Much research effort is focused on identifying global CH4 sources and sinks to estimate their current and potential strength in response to land-use change and global warming. Aerobic CH4 oxidation is regarded as the key process reducing the strength of CH4 emissions in wetlands, but is hitherto difficult to quantify. Recent studies quantify the efficiency of CH4 oxidation based on CH4 stable isotope signatures. The approach utilizes the fact that a significant isotope fractionation occurs when CH4 is oxidized. Moreover, it also considers isotope fractionation by diffusion. For field applications the 'open-system equation' is applied to determine the CH4 oxidation efficiency: fox = (δE - δP)/ (αox - αtrans) where fox is the fraction of CH4 oxidized; δE is δ13C of emitted CH4; δP is δ13C of produced CH4; αox is the isotopic fractionation factor of oxidation; αtrans is the isotopic fractionation factor of transport. We quantified CH4 oxidation in polygonal tundra soils of Russia's Lena River Delta analyzing depth profiles of CH4 concentrations and stable isotope signatures. Therefore, both fractionation factors αox and αtrans were determined for three polygon centers with differing water table positions and a polygon rim. While most previous studies on landfill cover soils have assumed a gas transport dominated by advection (αtrans = 1), other CH4 transport mechanisms as diffusion have to be considered in peatlands and αtrans exceeds a value of 1. At our study we determined αtrans = 1.013 ± 0.003 for CH4 when diffusion is the predominant transport mechanism. Furthermore, results showed that αox differs widely between sites and horizons (αox = 1.013 ± 0.012) and has to be determined for each case. The impact of both fractionation factors on the quantification of CH4 oxidation was estimated by considering both the potential diffusion rate at different water contents and potential oxidation rates. Calculations for a water saturated tundra soil indicated a CH4 oxidation efficiency of 88% in the upper horizon. Using carbon isotope fractionation improves the in situ quantification of CH4 oxidation in wetlands and thus the assessment of current and potential CH4 sources and sinks in these ecosystems.

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

    PubMed

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

    2009-07-01

    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

  8. Synthesis of a stable isotopically labeled universal surrogate peptide for use as an internal standard in LC-MS/MS bioanalysis of human IgG and Fc-fusion protein drug candidates.

    PubMed

    Voronin, Kimberly; Allentoff, Alban J; Bonacorsi, Samuel J; Mapelli, Claudio; Gong, Sharon X; Lee, Ving; Riexinger, Douglas; Sanghvi, Nishith; Jiang, Hao; Zeng, Jianing

    2014-07-01

    The synthesis of a 16-residue, stable isotopically labeled peptide is described for use as a LC-MS/MS (Liquid chromatography-mass spectrometry/mass spectrometry) internal standard in bioanalytical studies. This peptide serves as a single universal surrogate peptide capable of quantifying a wide variety of immunoglobulin G and Fc-fusion protein drug candidates in animal species used in pre-clinical drug development studies. An efficient synthesis approach for this peptide was developed using microwave-assisted solid phase peptide synthesis (SPPS) techniques, which included the use of a pseudoproline dipeptide derivative. The corresponding conventional room temperature SPPS was unsuccessful and gave only mixtures of truncated products. Stable-labeled leucine was incorporated as a single residue via manual coupling of commercially available Fmoc-[(13) C6 , (15) N]-l-leucine onto an 11-unit segment followed by automated microwave-assisted elaboration of the final four residues. Using this approach, the desired labeled peptide was prepared in high purity and in sufficient quantities for long-term supplies as a bioanalytical internal standard. The results strongly demonstrate the importance of utilizing both microwave-assisted peptide synthesis and pseudoproline dipeptide techniques to allow the preparation of labeled peptides with highly lipophilic and sterically hindered side-chains. PMID:25089024

  9. Differential proteomic analysis using isotope-coded protein-labeling strategies: comparison, improvements and application to simulated microgravity effect on Cupriavidus metallidurans CH34.

    PubMed

    Leroy, Baptiste; Rosier, Caroline; Erculisse, Vanessa; Leys, Natalie; Mergeay, Max; Wattiez, Ruddy

    2010-06-01

    Among differential proteomic methods based on stable isotopic labeling, isotope-coded protein labeling (ICPL) is a recent non-isobaric technique devised to label primary amines found in proteins. ICPL overcomes some of the disadvantages found in other chemical-labeling techniques, such as iTRAQ or ICAT. However, previous analyses revealed that more than 30% of the proteins identified in regular ICPL generally remain unquantified. In this study, we describe a modified version of ICPL, named Post-digest ICPL, that makes it possible to label and thus to quantify all the peptides in a sample (bottom-up approach). Optimization and validation of this Post-digest ICPL approach were performed using a standard protein mixture and complex protein samples. Using this strategy, the number of proteins that were identified and quantified was greatly increased in comparison with regular ICPL and cICAT approaches. The pros and cons of this improvement are discussed. This complementary approach to traditional ICPL was applied to the analysis of modification of protein abundances in the model bacterium Cupriavidus metallidurans CH34 after cultivation under simulated microgravity. In this context, two different systems - a 2-D clinorotation and 3-D random positioning device - were used and the results were compared and discussed. PMID:20391527

  10. Limits of the efficiency of isotope fractionation processes in the solar wind derived from the magnesium isotopic composition as observed with the WIND/MASS experiment

    NASA Astrophysics Data System (ADS)

    Boschler, P.; Balsiger, H.; Bodmer, R.; Kern, O.; Zurbuchen, Th.; Gloeckler, G.; Hamilton, D. C.; Collier, M. R.; Hovestadt, D.

    From geochemical evidence it is assumed that the isotopic composition of solar magnesium agrees within fractions of a per mill with terrestrial magnesium. This provides a unique opportunity to derive upper limits on the efficiency of isotope fractionation processes in the solar wind. We analyze results from the first year of operation of the WIND/MASS instrument, and we find that the isotopic composition of magnesium in the solar wind agrees within the experimental uncertainty of a few percent with the terrestrial composition. We observe no significant variations between different solar wind regimes. Our results are discussed in the context of recent theoretical models of solar wind feeding and acceleration.

  11. NanoSIMS analysis of an isotopically labelled organometallic ruthenium(II) drug to probe its distribution and state in vitro.

    PubMed

    Lee, Ronald F S; Escrig, Stphane; Croisier, Marie; Clerc-Rosset, Stphanie; Knott, Graham W; Meibom, Anders; Davey, Curt A; Johnsson, Kai; Dyson, Paul J

    2015-11-28

    The in vitro inter- and intra-cellular distribution of an isotopically labelled ruthenium(II)-arene (RAPTA) anti-metastatic compound in human ovarian cancer cells was imaged using nano-scale secondary ion mass spectrometry (NanoSIMS). Ultra-high resolution isotopic images of (13)C, (15)N, and Ru indicate that the phosphine ligand remains coordinated to the ruthenium(II) ion whereas the arene detaches. The complex localizes mainly on the membrane or at the interface between cells which correlates with its anti-metastatic effects. PMID:26426486

  12. Stable Isotope Labeled Tracers for Metabolic Pathway Elucidation by GC-MS and FT-MS

    PubMed Central

    Higashi, Richard M.; Fan, Teresa W-M.; Lorkiewicz, Pawel K.; Moseley, Hunter N.B.; Lane, Andrew N.

    2015-01-01

    Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics is poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, Stable Isotope Resolved Metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks, in a wide range of experimental systems, including human subjects. MS offers a wide range of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS affordable by many individual laboratories, to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter will focus on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM. PMID:25270929

  13. Simultaneous determination of seven ?2-agonists in human and bovine urine by isotope dilution liquid chromatography-tandem mass spectrometry using compound-specific minimally (13)C-labelled analogues.

    PubMed

    Gonzlez-Antua, Ana; Rodrguez-Gonzlez, Pablo; Centineo, Giuseppe; Garca Alonso, J Ignacio

    2014-10-29

    Seven ?2-agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC-ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally (13)C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg(-1) levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg(-1) ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg(-1) for all compounds, except to salbutamol in bovine urine which was of 0.029ngg(-1). The use of compound-specific isotopically labelled analogues minimally labelled in (13)C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of ?2-agonists in human and bovine urine. PMID:25468499

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

    SciTech Connect

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

    2012-03-26

    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 (CO2)-C-13 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.

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

    SciTech Connect

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

    2012-03-01

    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.

  16. Metabolic Tracing Using Stable Isotope-Labeled Substrates and Mass Spectrometry in the Perfused Mouse Heart.

    PubMed

    Ruiz, Matthieu; Gélinas, Roselle; Vaillant, Fanny; Lauzier, Benjamin; Des Rosiers, Christine

    2015-01-01

    There has been a resurgence of interest for the field of cardiac metabolism catalyzed by evidence demonstrating a role of metabolic dysregulation in the pathogenesis of heart disease as well as the increased need for new therapeutic targets for patients with these diseases. In this regard, measuring substrate fluxes is critical in providing insight into the dynamics of cellular metabolism and in delineating the regulation of metabolite production and utilization. This chapter provides a comprehensive description of concepts, guidelines, and tips to assess metabolic fluxes relevant to energy substrate metabolism using (13)C-labeled substrates and (13)C-isotopomer analysis by gas chromatography-mass spectrometry (GC-MS), and the ex vivo working heart as study model. The focus will be on the mouse and on flux parameters, which are commonly assessed in the field, namely, those relevant to substrate selection for energy metabolism, specifically the relative contribution of carbohydrate (glucose, lactate, and pyruvate) and fatty acid oxidation to acetyl-CoA formation for citrate synthesis, glycolysis, as well as anaplerosis. We provide detailed procedures for the heart isolation and perfusion in the working mode as well as for sample processing for metabolite extraction and analysis by GC-MS and subsequent data processing for calculation of metabolic flux parameters. Finally, we address practical considerations and discuss additional applications and future challenges. PMID:26358903

  17. Deuterium Nuclear Spin-Lattice Relaxation Times and Quadrupolar Coupling Constants in Isotopically Labeled Saccharides

    NASA Astrophysics Data System (ADS)

    Bose-Basu, Bidisha; Zajicek, Jaroslav; Bondo, Gail; Zhao, Shikai; Kubsch, Meredith; Carmichael, Ian; Serianni, Anthony S.

    2000-06-01

    13C and 2H spin-lattice relaxation times have been determined by inversion recovery in a range of site-specific 13C- and 2H-labeled saccharides under identical solution conditions, and the data were used to calculate deuterium nuclear quadrupolar coupling constants (2H NQCC) at specific sites within cyclic and acyclic forms in solution. 13C T1 values ranged from ?0.6 to 8.2 s, and 2H T1 values ranged from ?79 to 450 ms, depending on molecular structure (0.4 M sugar in 5 mM EDTA (disodium salt) in 2H2O-depleted H2O, pH 4.8, 30C). In addition to providing new information on 13C and 2H relaxation behavior of saccharides in solution, the resulting 2H1 NQCC values reveal a dependency on anomeric configuration within aldopyranose rings, whereas 2H NQCC values at other ring sites appear less sensitive to configuration at C1. In contrast, 2H NQCC values at both anomeric and nonanomeric sites within aldofuranose rings appear to be influenced by anomeric configuration. These experimental observations were confirmed by density functional theory (DFT) calculations of 2H NQCC values in model aldopyranosyl and aldofuranosyl rings.

  18. Preventing arginine-to-proline conversion in a cell-line-independent manner during cell cultivation under stable isotope labeling by amino acids in cell culture (SILAC) conditions.

    PubMed

    Lössner, Christopher; Warnken, Uwe; Pscherer, Armin; Schnölzer, Martina

    2011-05-01

    Quantitative proteomics has increasingly gained impact in life science research as a tool to describe changes in protein expression between different cellular states. Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful technique for relative quantification of proteins. However, the accuracy of quantification is impaired by the metabolic conversion of arginine to proline resulting in additional heavy labeled proline peptide satellites. Here we reinvestigated the addition of unlabeled proline during cell cultivation under SILAC conditions considering several thousand peptides and demonstrated that the arginine-to-proline conversion is prevented independent of the cell line used. PMID:21241653

  19. Limited Proteolysis Combined with Stable Isotope Labeling Reveals Conformational Changes in Protein (Pseudo)kinases upon Binding Small Molecules.

    PubMed

    Di Michele, Michela; Stes, Elisabeth; Vandermarliere, Elien; Arora, Rohit; Astorga-Wells, Juan; Vandenbussche, Jonathan; van Heerde, Erika; Zubarev, Roman; Bonnet, Pascal; Linders, Joannes T M; Jacoby, Edgar; Brehmer, Dirk; Martens, Lennart; Gevaert, Kris

    2015-10-01

    Likely due to conformational rearrangements, small molecule inhibitors may stabilize the active conformation of protein kinases and paradoxically promote tumorigenesis. We combined limited proteolysis with stable isotope labeling MS to monitor protein conformational changes upon binding of small molecules. Applying this method to the human serine/threonine kinase B-Raf, frequently mutated in cancer, we found that binding of ATP or its nonhydrolyzable analogue AMP-PNP, but not ADP, stabilized the structure of both B-Raf(WT) and B-Raf(V600E). The ATP-competitive type I B-Raf inhibitor vemurafenib and the type II inhibitor sorafenib stabilized the kinase domain (KD) but had distinct effects on the Ras-binding domain. Stabilization of the B-Raf(WT) KD was confirmed by hydrogen/deuterium exchange MS and molecular dynamics simulations. Our results are further supported by cellular assays in which we assessed cell viability and phosphorylation profiles in cells expressing B-Raf(WT) or B-Raf(V600E) in response to vemurafenib or sorafenib. Our data indicate that an overall stabilization of the B-Raf structure by specific inhibitors activates MAPK signaling and increases cell survival, helping to explain clinical treatment failure. We also applied our method to monitor conformational changes upon nucleotide binding of the pseudokinase KSR1, which holds high potential for inhibition in human diseases. PMID:26293246

  20. Stable isotope dimethyl labeling combined with LTQ mass spectrometric detection, a quantitative proteomics technology used in liver cancer research

    PubMed Central

    TANG, BO; LI, YANG; ZHAO, LIANG; YUAN, SHENGGUANG; WANG, ZHENRAN; LI, BO; CHEN, QIAN

    2013-01-01

    Liver cancer is a common malignant disease, with high incidence and mortality rates. The study on the proteomics of liver cancer has attracted particular attention. The quantitative study method of proteomics depends predominantly on two-dimensional (2D) gel electrophoresis. In the present study we reported a rapid and accurate proteomics quantitative study method of high repeatability that includes the use of stable isotope labeling for the extraction of proteins and peptides via enzymolysis to achieve new type 2D capillary liquid chromatography-mass spectrometry separation using the separation mode of cation-exchange chromatography in conjunction with reversed-phase chromatography. LTQ OrbiTrap mass spectrometry detection was also performed. A total of 188 differential proteins were analyzed, including 122 upregulating [deuterium/hydrogen ratio (D/H) >1.5)] and 66 downregulating proteins (D/H<0.67). These proteins may play an important role in the occurrence, drug resistance, metastasis and recurrence of cancer or other pathological processes. Such a proteomics technology may provide biological data as well as a new methodological basis for liver cancer research. PMID:24648984

  1. Stable isotope labelling reveals that NaCl stress decreases the production of Ensifer (Sinorhizobium) arboris lipochitooligosaccharide signalling molecules.

    PubMed

    Penttinen, Petri; Rsnen, Leena A; Lortet, Gilles; Lindstrm, Kristina

    2013-12-01

    Ensifer (Sinorhizobium) arboris is a symbiont of salt-tolerant leguminous trees in the genera Acacia and Prosopis that are utilized in the prevention of soil erosion and desertification and in phytoremediation of salinized soil. Signalling between the plant and the rhizobia is essential for the formation of effective symbiosis that increases the success of reclaiming saline sites. We assessed the effect of salt stress on the growth and the production of lipochitooligosaccharide signalling molecules (LCOs) of S. arboris HAMBI 2361, an LCO-overproducing derivative of the S. arboris type strain HAMBI 1552. The strain tolerated NaCl up to 750 mM. To obtain both qualitative and quantitative information on the LCO production under salt stress, we devised a method where LCOs were differentially labelled by stable isotopes of nitrogen, (14)N and (15)N, and analysed by mass spectrometry. Under control conditions, the strain produced altogether 27 structural LCO variants. In 380 mM NaCl, 13 LCO variants were produced in detectable amounts, and six of these were reliably quantified, ranging from one-tenth to one-third of the non-stressed one. PMID:24256411

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

    PubMed Central

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

    2015-01-01

    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. PMID:25699032

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

    PubMed

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

    2015-01-01

    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 (13)C-labeled substrates (bicarbonate, formate, acetate, and glucose) to determine the relative uptake of these different carbon sources. Highest (13)C 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. (13)C-glucose showed a similar, but a 10-30 times lower uptake across most fatty acids. (13)C-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 (13)C-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. (13)C-uptake into archaeal lipids occurred predominantly with (13)C-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. PMID:25699032

  4. Crystallization of Enantiomerically Pure Proteins from Quasi-Racemic Mixtures: Structure Determination by X-Ray Diffraction of Isotope-Labeled Ester Insulin and Human Insulin.

    PubMed

    Mandal, Kalyaneswar; Dhayalan, Balamurugan; Avital-Shmilovici, Michal; Tokmakoff, Andrei; Kent, Stephen B H

    2016-03-01

    As a part of a program aimed towards the study of the dynamics of human insulin-protein dimer formation using two-dimensional infrared spectroscopy, we used total chemical synthesis to prepare stable isotope labeled [(1-(13) C=(18) O)Phe(B24) )] human insulin, via [(1-(13) C=(18) O)Phe(B24) )] ester insulin as a key intermediate product that facilitates folding of the synthetic protein molecule (see preceding article). Here, we describe the crystal structure of the synthetic isotope-labeled ester insulin intermediate and the product synthetic human insulin. Additionally, we present our observations on hexamer formation with these two proteins in the absence of phenol derivatives and/or Zn metal ions. We also describe and discuss the fractional crystallization of quasi-racemic protein mixtures containing each of these two synthetic proteins. PMID:26707939

  5. In-depth qualitative and quantitative profiling of tyrosine phosphorylation using a combination of phosphopeptide immunoaffinity purification and stable isotope dimethyl labeling.

    PubMed

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

    2010-01-01

    Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated. PMID:19770167

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

    PubMed Central

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

    2010-01-01

    Several mass spectrometry-based assays have emerged for the quantitative profiling of cellular tyrosine phosphorylation. Ideally, these methods should reveal the exact sites of tyrosine phosphorylation, be quantitative, and not be cost-prohibitive. The latter is often an issue as typically several milligrams of (stable isotope-labeled) starting protein material are required to enable the detection of low abundance phosphotyrosine peptides. Here, we adopted and refined a peptidecentric immunoaffinity purification approach for the quantitative analysis of tyrosine phosphorylation by combining it with a cost-effective stable isotope dimethyl labeling method. We were able to identify by mass spectrometry, using just two LC-MS/MS runs, more than 1100 unique non-redundant phosphopeptides in HeLa cells from about 4 mg of starting material without requiring any further affinity enrichment as close to 80% of the identified peptides were tyrosine phosphorylated peptides. Stable isotope dimethyl labeling could be incorporated prior to the immunoaffinity purification, even for the large quantities (mg) of peptide material used, enabling the quantification of differences in tyrosine phosphorylation upon pervanadate treatment or epidermal growth factor stimulation. Analysis of the epidermal growth factor-stimulated HeLa cells, a frequently used model system for tyrosine phosphorylation, resulted in the quantification of 73 regulated unique phosphotyrosine peptides. The quantitative data were found to be exceptionally consistent with the literature, evidencing that such a targeted quantitative phosphoproteomics approach can provide reproducible results. In general, the combination of immunoaffinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling provides a cost-effective approach that can alleviate variation in sample preparation and analysis as samples can be combined early on. Using this approach, a rather complete qualitative and quantitative picture of tyrosine phosphorylation signaling events can be generated. PMID:19770167

  7. 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

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

    1985-12-01

    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).

  8. Diet-animal fractionation of nitrogen stable isotopes reflects the efficiency of nitrogen assimilation in ruminants.

    PubMed

    Cantalapiedra-Hijar, G; Ortigues-Marty, I; Sepchat, B; Agabriel, J; Huneau, J F; Fouillet, H

    2015-04-14

    The natural abundance of ?N in animal proteins (??Nanimal) is greater than that in the diet consumed by the animals (??Ndiet), with a discrimination factor (??N = ??Nanimal - ??Ndiet) that is known to vary according to nutritional conditions. The objectives of the present study were to test the hypothesis that ??N variations depend on the efficiency of nitrogen utilisation (ENU) in growing beef cattle, and to identify some of the physiological mechanisms responsible for this N isotopic fractionation in ruminants. Thus, we performed the regression of the ??N of plasma proteins obtained from thirty-five finishing beef cattle fed standard and non-conventional diets against different feed efficiency indices, including ENU. We also performed the regression of the ??N of different ruminant N pools (plasma and milk proteins, urine and faeces) against different splanchnic N fluxes obtained from multi-catheterised lactating dairy cows. The ??N of plasma proteins was negatively correlated with feed efficiency indices in beef cattle, especially ENU (body protein gain/N intake) and efficiency of metabolisable protein (MP) utilisation (body protein gain/MP intake). Although ??N obtained from different N pools in dairy cows were all negatively correlated with ENU, the highest correlation was found when ??N was calculated from plasma proteins. ??N showed no correlation with urea-N recycling or rumen NH? absorption, but exhibited a strong correlation with liver urea synthesis and splanchnic amino acid metabolism, which points to a dominant role of splanchnic tissues in the present N isotopic fractionation study. PMID:25716533

  9. Overcoming matrix effects in electrospray: quantitation of ?-agonists in complex matrices by isotope dilution liquid chromatography-mass spectrometry using singly (13)C-labeled analogues.

    PubMed

    Gonzlez-Antua, Ana; Domnguez-Romero, Juan C; Garca-Reyes, Juan F; Rodrguez-Gonzlez, Pablo; Centineo, Giuseppe; Garca Alonso, J Ignacio; Molina-Daz, Antonio

    2013-05-01

    In this work, the implementation of isotope dilution mass spectrometry (IDMS) using minimal labeling and isotope pattern deconvolution (IPD) is evaluated as a strategy for the minimization of matrix effects during trace determination of ?2-agonists in complex matrices by liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). First, the parameters affecting the measurement of isotopic composition of organic compounds by liquid chromatography electrospray ionization high resolution mass spectrometry with a time-of-flight analyzer were evaluated using as a case of study three different ?2-agonists: clenbuterol, clenproperol and brombuterol. Then, a calibration graph-free IDMS methodology was evaluated in order to overcome matrix effects in LC-ESI-MS in complex samples. In this procedure singly (13)C-labeled analogues of clenbuterol, clenproperol and brombuterol were employed in combination with IPD. Using this approach accurate and precise results were obtained in the simultaneous quantification of ?2-agonists in human urine and bovine liver, even at the sub ngg(-1) and particularly in spite of the previously reported matrix effects. Recovery rates in the range of 97-114% in fortified human urine and from 95% to 111% in fortified bovine liver were obtained with RSD (%) of independent recovery experiments always lower than 6%. These results demonstrate that the proposed methodology based on the use of (13)C1-labeled standards and IPD is a reliable approach for accurate LC-MS quantitation of small molecules and compatible with full-scan high-resolution mass spectrometry. PMID:23523066

  10. Non-homogeneity of isotopic labelling in 15N gas flux studies: theory, some observations and possible lessons

    NASA Astrophysics Data System (ADS)

    Well, Reinhard; Buchen, Caroline; Deppe, Marianna; Eschenbach, Wolfram; Gattinger, Andreas; Giesemann, Anette; Krause, Hans-Martin; Lewicka-Szczebak, Dominika

    2015-04-01

    Quantifying dinitrogen (N2) and nitrous oxide (N2O) fluxes from different soil N pools and processes can be accomplished using the 15N tracer technique but this is subject to four different sources of bias (i. - iv.). This approach includes 15N labelling of selected N pools in soil and subsequent isotope analysis of all relevant N pools as well as of gas samples from enclosures, i.e. mixtures of soil-derived and atmospheric N2 and N2O. Depending on the processes of interest, there may be 15N labelling of one or several N pools, were several labelling treatment are needed in the latter case (e.g. Müller et al., 2004). Measuring pool-derived N2 or N2O has been shown to include two calculation problems, (i.) arising from multiple pools (e.g. Arah, 1992) and (ii.) dealing with the non-random distribution of N2 and N2O mole masses (Hauck et al., 1958). Non-randomness can be solved if m/z 28, 29 and 30 are correctly analysed and the 15N enrichment of one (to distinguish two pools, i.e. soil and atmosphere) or two pools (in case of three pools) is known (Spott & Stange, 2008). Moreover (iii.), NO3- pools generating N2 and N2O via denitrification can be identical or different, e.g. if N2O evolved from higher enriched NO3- in deeper soil was more reduced to N2 compared to N2O evolved from N2O from shallow soil with lower enrichment, or vice versa. Apportioning N2O fluxes to NH4+ (nitrification and/or nitrifier denitrification) and NO3- (denitrification) is often conducted by NO3-labeling, measuring δ15N of emitted N2O and applying mixing equations were the measured 15N enrichment of NH4+and NO3-pool is used. However, this assumes that the average 15N enrichment of NH4+and NO3-in the soil is identical to the enrichment in the active soil domain producing N2 and/or N2O. Violation of this precondition must lead to bias in source apportionment (iv.), but to our knowledge this has not been investigated until now. Here we present conceptual models and model calculations addressing cases iii. and iv.. Furthermore we present some experimental data illustrating this. These include two data sets from denitrification experiments exhibiting substantial deviations in 15N enrichment between the N pools producing N2 and N2O. Moreover, results from a lab incubation study to quantify NH4+-derived N2O with increasing NH4+ amendment under conditions favouring nitrification are shown, were non-labelled NH4+ was added together with 15N labelled NO3-. Here we found large deviations between the 15N enrichment of NO3- in extracted soil water and the 15N enrichment of the labelled N pool as calculated from N2O isotopologues (Bergsma et al., 2001). We think that this reflects type iv. bias, probably because enrichment of NO3- in anoxic micro-sites was less diluted by non-labelled NO3- from nitrification compared to NO3- in oxic zones. Our data analysis provides a means to overcome bias iv. and thus to obtain correct source apportionment. References: Arah, J.R.M. (1992): Soil Sci. Soc. Am. J. 56, 795 - 800, 1992. Bergsma, T. et al. (2001): Env. Sci. & Technol. 35(21): 4307-4312. Hauck, R.D., et al.(1958): Soil Science 86, 287 - 291, 1958. Lewicka-Szczebak, D. et al.(2013): Rapid Comm. Mass Spectrom., 27 1548-1558. Müller, C. et al. (2004): Soil Biol. Biochem. 36(4): 619-632. Mulvaney, R.L.(1984):. Soil Sci. Soc. Am. J. 48:690 - 692. Spott, O, et al.. (2006): Rapid Comm. Mass Spectrom., 20: 3267-3274. Spott, O. and C. F. Stange (2007): Rapid Comm. Mass Spectrom., 21: 2398-2406.

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

    SciTech Connect

    Wiel, Stephen; McMahon, James E.

    2005-04-28

    Energy-performance improvements in consumer products are an essential element in any government's portfolio of energy-efficiency and climate change mitigation programs. Governments need to develop balanced programs, both voluntary and regulatory, that remove cost-ineffective, energy-wasting products from the marketplace and stimulate the development of cost-effective, energy-efficient technology. Energy-efficiency labels and standards for appliances, equipment, and lighting products deserve to be among the first policy tools considered by a country's energy policy makers. The U.S. Agency for International Development (USAID) and several other organizations identified on the cover of this guidebook recognize the need to support policy makers in their efforts to implement energy-efficiency standards and labeling programs and have developed this guidebook, together with the Collaborative Labeling and Appliance Standards Program (CLASP), as a primary reference. This second edition of the guidebook was prepared over the course of the past year, four years after the preparation of the first edition, with a significant contribution from the authors and reviewers mentioned previously. Their diligent participation helps maintain this book as the international guidance tool it has become. The lead authors would like to thank the members of the Communications Office of the Environmental Energy Technologies Division, Lawrence Berkeley National Laboratory for their support in the development, production, and distribution of the guidebook. This guidebook is designed as a manual for government officials and others around the world responsible for developing, implementing, enforcing, monitoring, and maintaining labeling and standards setting programs. It discusses the pros and cons of adopting energy-efficiency labels and standards and describes the data, facilities, and institutional and human resources needed for these programs. It provides guidance on the design, development, implementation, maintenance, and evaluation of the programs and on the design of the labels and standards themselves. In addition, it directs the reader to references and other resources likely to be useful in conducting the activities described and includes a chapter on energy policies and programs that complement appliance efficiency labels and standards. This guidebook attempts to reflect the essential framework of labeling and standards programs. It is the intent of the authors and sponsor to distribute copies of this book worldwide, at no charge, for the general public benefit. The guidebook is also available on the web at www.clasponline.org and may be downloaded to be used intact or piecemeal for whatever beneficial purposes readers may conceive.

  12. Relative quantitation of protein nitration by liquid chromatographymass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation

    PubMed Central

    Guo, Jia; Prokai-Tatrai, Katalin; Prokai, Laszlo

    2012-01-01

    Protein nitration has been recognized as an important biomarker for nitroxidative stress associated with various diseases. While identification of protein targets for nitration is important, its quantitative profiling also is necessary to understand the biological impact of this low-abundance posttranslational modification. We have previously reported an efficient and straightforward enrichment method for nitropeptides to reduce sample complexity and permit unambiguous site-specific identifications by LCMS analyses. This approach relies on two chemical derivatization steps: specifically reductive methylation of aliphatic amines and, then, conversion of nitrotyrosines to the corresponding aminotyrosines before their selective capture by a solid-phase reagent we introduced previously. Hence, the method inherently offers the opportunity for relative quantitation of nitropeptides by using isotopic variants of formaldehyde for reductive methylation. This simple method was tested via LCMS analyses of differently N-methylated nitropeptides and nitroubiquitin as a model nitroprotein enriched from human serum albumin digest and from human plasma, respectively. PMID:22285050

  13. Development of isotope labeling liquid chromatography-mass spectrometry for metabolic profiling of bacterial cells and its application for bacterial differentiation.

    PubMed

    Wu, Yiman; Li, Liang

    2013-06-18

    Quantitative and comprehensive profiling of cellular metabolites is currently a challenging task in bacterial metabolomics. In this work, a simple and robust method for profiling the amine- and phenol-containing metabolome of bacterial cells is described. The overall workflow consists of methanol-based cell lysis and metabolite extraction with ultrasonication, differential isotope dansylation labeling of cellular metabolites, and analysis of the labeled metabolites by liquid chromatography-mass spectrometry (LC-MS). Over a thousand peak pairs or putative metabolites can be detected from bacterial cells in a 25 min LC-MS run and near 2500 putative metabolites can be found in one bacterium from combined results of multiple analyses. After careful examination and optimization of the sample preparation process, this method is shown to be effective for both Gram-positive and Gram-negative bacteria. An idea of applying LC-ultraviolet (UV) detection to quantify the total amount of labeled metabolites is shown to be effective for normalizing the amounts of metabolites present in different samples for metabolome comparison. The use of differential isotopic labeling allows relative quantification of each individual metabolite, which facilitates comparative metabolomics studies and the generation of a metabolic fingerprint of a bacterium. Finally, this method is demonstrated to be useful for the differentiation of three bacterial species in cultured media and spiked human urine samples. PMID:23495969

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

    PubMed

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

    2014-07-01

    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 β-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

  15. Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry

    SciTech Connect

    Shimizu, K.; Yamaga, N.; Kohara, H.

    1988-03-01

    A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with /sup 2/H/sub 2/O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using (/sup 2/H)diethylene glycol and (/sup 2/H)hydrazine hydrate afforded (11,11,12,12,23,23(-2)H)lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-(11,11,12,12(-2)H)pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-(11,11,12,12(-2)H)pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-(11,11,12,12(-2)H) progesterone (XIV), consisting of 0.3% /sup 2/H0-, 1.1% /sup 2/H/sub 1/-, 8.6% /sup 2/H/sub 2/-, 37.1% /sup 2/H/sub 3/-, 52.1% /sup 2/H/sub 4/-, and 0.8% /sup 2/H/sub 5/-species.

  16. High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

    PubMed Central

    Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

    2008-01-01

    Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

  17. Accurate and Efficient Resolution of Overlapping Isotopic Envelopes in Protein Tandem Mass Spectra

    PubMed Central

    Xiao, Kaijie; Yu, Fan; Fang, Houqin; Xue, Bingbing; Liu, Yan; Tian, Zhixin

    2015-01-01

    It has long been an analytical challenge to accurately and efficiently resolve extremely dense overlapping isotopic envelopes (OIEs) in protein tandem mass spectra to confidently identify proteins. Here, we report a computationally efficient method, called OIE_CARE, to resolve OIEs by calculating the relative deviation between the ideal and observed experimental abundance. In the OIE_CARE method, the ideal experimental abundance of a particular overlapping isotopic peak (OIP) is first calculated for all the OIEs sharing this OIP. The relative deviation (RD) of the overall observed experimental abundance of this OIP relative to the summed ideal value is then calculated. The final individual abundance of the OIP for each OIE is the individual ideal experimental abundance multiplied by 1 + RD. Initial studies were performed using higher-energy collisional dissociation tandem mass spectra on myoglobin (with direct infusion) and the intact E. coli proteome (with liquid chromatographic separation). Comprehensive data at the protein and proteome levels, high confidence and good reproducibility were achieved. The resolving method reported here can, in principle, be extended to resolve any envelope-type overlapping data for which the corresponding theoretical reference values are available. PMID:26439836

  18. Analysis of cytochrome P450 metabolites of arachidonic acid by stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry.

    PubMed

    Zhu, Quan-Fei; Hao, Yan-Hong; Liu, Ming-Zhou; Yue, Jiang; Ni, Jian; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-09-01

    Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia. PMID:26253834

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

    PubMed Central

    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.

    2014-01-01

    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 15N-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 (R2 = 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. PMID:25293624

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

    PubMed

    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

    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. PMID:25293624

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

    PubMed

    Michael, Claudia; Rizzi, Andreas M

    2015-02-27

    Glycan reductive isotope labeling (GRIL) using (12)C6-/(13)C6-aniline as labeling reagent is reported with the aim of quantitative N-glycan fingerprinting. Porous graphitized carbon (PGC) as stationary phase in capillary scale HPLC coupled to electrospray mass spectrometry with time of flight analyzer was applied for the determination of labeled N-glycans released from glycoproteins. The main benefit of using stable isotope-coding in the context of comparative glycomics lies in the improved accuracy and precision of the quantitative analysis in combined samples and in the potential of correcting for structure-dependent incomplete enzymatic release of oligosaccharides when comparing identical target proteins. The method was validated with respect to mobile phase parameters, reproducibility, accuracy, linearity and limit of detection/quantification (LOD/LOQ) using test glycoproteins. It is shown that the developed method is capable of determining relative amounts of N-glycans (including isomers) comparing two samples in one single HPLC-MS run. The analytical potential and usefulness of GRIL in combination with PGC-ESI-TOF-MS is demonstrated comparing glycosylation in human monoclonal antibodies produced in Chinese hamster ovary cells (CHO) and hybridoma cell lines. PMID:25638265

  2. Xanthine oxidoreductase activity assay in tissues using stable isotope-labeled substrate and liquid chromatography high-resolution mass spectrometry.

    PubMed

    Murase, Takayo; Nampei, Mai; Oka, Mitsuru; Ashizawa, Naoki; Matsumoto, Koji; Miyachi, Atsushi; Nakamura, Takashi

    2016-01-01

    Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.10.7, 1585, 92825pmol/min/mg of protein, respectively (meanSD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter. PMID:26673227

  3. Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis

    PubMed Central

    Croner, Roland S.; Sevim, Müzeyyen; Metodiev, Metodi V.; Jo, Peter; Ghadimi, Michael; Schellerer, Vera; Brunner, Maximillian; Geppert, Carol; Rau, Tilman; Stürzl, Michael; Naschberger, Elisabeth; Matzel, Klaus E.; Hohenberger, Werner; Lottspeich, Friedrich; Kellermann, Josef

    2016-01-01

    Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%–15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients’ informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort. PMID:26861291

  4. Identification of Predictive Markers for Response to Neoadjuvant Chemoradiation in Rectal Carcinomas by Proteomic Isotope Coded Protein Label (ICPL) Analysis.

    PubMed

    Croner, Roland S; Sevim, Müzeyyen; Metodiev, Metodi V; Jo, Peter; Ghadimi, Michael; Schellerer, Vera; Brunner, Maximillian; Geppert, Carol; Rau, Tilman; Stürzl, Michael; Naschberger, Elisabeth; Matzel, Klaus E; Hohenberger, Werner; Lottspeich, Friedrich; Kellermann, Josef

    2016-01-01

    Neoadjuvant chemoradiation (nCRT) is an established procedure in stage union internationale contre le cancer (UICC) II/III rectal carcinomas. Around 53% of the tumours present with good tumor regression after nCRT, and 8%-15% are complete responders. Reliable selection markers would allow the identification of poor or non-responders prior to therapy. Tumor biopsies were harvested from 20 patients with rectal carcinomas, and stored in liquid nitrogen prior to therapy after obtaining patients' informed consent (Erlangen-No.3784). Patients received standardized nCRT with 5-Fluoruracil (nCRT I) or 5-Fluoruracil ± Oxaliplatin (nCRT II) according to the CAO/ARO/AIO-04 protocol. After surgery, regression grading (Dworak) of the tumors was performed during histopathological examination of the specimens. Tumors were classified as poor (Dworak 1 + 2) or good (Dworak 3 + 4) responders. Laser capture microdissection (LCM) for tumor enrichment was performed on preoperative biopsies. Differences in expressed proteins between poor and good responders to nCRT I and II were identified by proteomic analysis (Isotope Coded Protein Label, ICPL™) and selected markers were validated by immunohistochemistry. Tumors of 10 patients were classified as histopathologically poor (Dworak 1 or 2) and the other 10 tumor samples as histopathologically good (Dworak 3 or 4) responders to nCRT after surgery. Sufficient material in good quality was harvested for ICPL analysis by LCM from all biopsies. We identified 140 differentially regulated proteins regarding the selection criteria and the response to nCRT. Fourteen of these proteins were synchronously up-regulated at least 1.5-fold after nCRT I or nCRT II (e.g., FLNB, TKT, PKM2, SERINB1, IGHG2). Thirty-five proteins showed a complete reciprocal regulation (up or down) after nCRT I or nCRT II and the rest was regulated either according to nCRT I or II. The protein expression of regulated proteins such as PLEC1, TKT, HADHA and TAGLN was validated successfully by immunohistochemistry. ICPL is a valid method to identify differentially expressed proteins in rectal carcinoma tissue between poor vs. good responders to nCRT. The identified protein markers may act as selection criteria for nCRT in the future, but our preliminary findings must be reproduced and validated in a prospective cohort. PMID:26861291

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

    PubMed Central

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

    2014-01-01

    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 15N 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

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

    PubMed

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

    2014-11-01

    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

  7. Isolation, In-111 labeling, and abscess detection efficiency of rabbit polymorphonuclear leukocytes (PMN) from blood and peritoneal fluid

    SciTech Connect

    Bettin, K.M.; Elson, M.K.; Gerding, D.N.; Bamberger, D.M.; Forstrom, L.A.; Shafer, R.B.

    1984-01-01

    In-111 labeled blood and peritoneal exudate PMN were compared for labeling efficiency and ability to migrate to sites of experimental abscesses using both direct sampling and visual imaging techniques. Blood PMN were prepared by combining heparinized blood with 6% Hetastarch for 1 hour and layering the plasma over a double density Ficoll-Hy-paque gradient (S.G. 1.076 over 1.141). The PMN layer (90-99% PMN) at the interface yielded 10/sup 6/-10/sup 7/ PMN from 80-120 ml of blood. Peritoneal PMN were obtained by infusion of 0.1% glycogen, followed by infusion of saline after 4 or 18 hours. The exudate yielded 10/sup 7/-10/sup 8/ PMN (80-99% PMN). PMN suspensions were labeled for 30 minutes by addition of 100 ..mu..Ci of In-111-oxine, then washed twice. Percent cell-associated radioactivity of the labeled blood, 4 hour, and 18 hour peritoneal PMN was 89%, 88%, and 86%. The labeled PMN were injected intravenously into rabbits which had two of three abdominal capsules (table tennis balls drilled with 250 1.5 mm holes) inoculated with Staphylococcus aureus 4 hours earlier. Peak venous recovery of circulating labeled PMN, for blood, 4 hour and 18 hour peritoneal PMN was 60%, 43%, and 19%. Gamma camera images 24 hours after infusion into infected rabbits were superior with 4 hour peritoneal PMN. The peritoneal PMN harvested 4 hours after glycogen stimulation are simple to prepare, are obtainable in greater numbers than blood PMN, and result in better abscess visualization.

  8. Convenient preparation of deuterium-labeled analogs of peptides containing N-substituted glycines for a stable isotope dilution LC-MS quantitative analysis.

    PubMed

    B?chor, Remigiusz; D?bowski, Dawid; ??gowska, Anna; Stefanowicz, Piotr; Rolka, Krzysztof; Szewczuk, Zbigniew

    2015-11-01

    N-substituted glycines constitute mimics of natural amino acids that are of great interest in the peptide-based drug development. Peptoids-oligo(N-substituted glycines) have been recently demonstrated to be highly active peptidomimetics in biological systems, resistant to proteolytic degradation. We developed a method of the deuterium labeling of peptidomimetics containing N-substituted glycine residues via H/D exchange of their ?-carbon hydrogen atoms. The labeling was shown to be easy, inexpensive, and without the use of derivatization reagents or the need for a further purification. The deuterons introduced at the ?-carbon atoms do not undergo a back exchange under acidic conditions during liquid chromatography mass spectrometry (LC-MS) analysis. The LC-MS analysis of a mixture of isotopologues revealed a co-elution of deuterated and nondeuterated forms of the peptidomimetics, which may be useful in the quantitative isotope dilution analysis of peptoids and other derivatives of N-substituted glycines. PMID:26415697

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

    PubMed

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

    2016-02-18

    Benzylic rearrangement stable isotope labeling (BRSIL) was explored to quantify the guanidino and ureido compounds (GCs and UCs). This method employed a common reagent, benzil, to label the guanidino and ureido groups through nucleophilic attacking then benzylic migrating. The use of BRSIL was investigated in the analysis of five GCs (creatine, l-arginine, homoarginine, 4-guanidinobutyric acid, and methylguanidine) and two UCs (urea and citrulline). The labeling was found simple and specific. The introduction of bi-phenyl group and the generation of nitrogen heterocyclic ring in the benzil-d0/d5 labeled GCs and UCs improved the retention behaviors in liquid chromatography (LC) and increased the sensitivity of electrospray ionization mass spectrometry (ESI MS) detection. The fragment ion pairs of m/z 182/187 and m/z 210/215 from the benzil-d0/d5 tags facilitated the discovery of potential GCs and UCs candidates residing in biological matrices. The use of BRSIL combined with LC-ESI MS was applied for simultaneously quantitation of GCs and UCs in thyroid tissues. It was demonstrated that nine GCs and UCs were detected, six of which were further quantified based on corresponding standards. It was concluded that five GCs and UCs (l-arginine, homoarginine, 4-guanidinobutyric acid, methylguanidine, and citrulline) were statistically significantly different (p<0.05) between the para-carcinoma and carcinoma thyroid tissue samples. PMID:26826695

  10. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source.

    PubMed

    Ostroumov, P N; Barcikowski, A; Dickerson, C A; Perry, A; Pikin, A I; Sharamentov, S I; Vondrasek, R C; Zinkann, G P

    2015-08-01

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstrate stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this paper, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz. PMID:26329185

  11. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    NASA Astrophysics Data System (ADS)

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; Perry, A.; Pikin, A. I.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.

    2015-08-01

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstrate stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this paper, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.

  12. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    SciTech Connect

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; Perry, A.; Pikin, A. I.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.

    2015-08-28

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstrate stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this study, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.

  13. Fast and efficient charge breeding of the Californium rare isotope breeder upgrade electron beam ion source

    DOE PAGESBeta

    Ostroumov, P. N.; Barcikowski, A.; Dickerson, C. A.; Perry, A.; Pikin, A. I.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.

    2015-08-28

    The Electron Beam Ion Source (EBIS), developed to breed Californium Rare Isotope Breeder Upgrade (CARIBU) radioactive beams at Argonne Tandem Linac Accelerator System (ATLAS), is being tested off-line. A unique property of the EBIS is a combination of short breeding times, high repetition rates, and a large acceptance. Overall, we have implemented many innovative features during the design and construction of the CARIBU EBIS as compared to the existing EBIS breeders. The off-line charge breeding tests are being performed using a surface ionization source that produces singly charged cesium ions. The main goal of the off-line commissioning is to demonstratemore » stable operation of the EBIS at a 10 Hz repetition rate and a breeding efficiency into single charge state higher than 15%. These goals have been successfully achieved and exceeded. We have measured (20% ± 0.7%) breeding efficiency into the single charge state of 28+ cesium ions with the breeding time of 28 ms. In general, the current CARIBU EBIS operational parameters can provide charge breeding of any ions in the full mass range of periodic table with high efficiency, short breeding times, and sufficiently low charge-to-mass ratio, 1/6.3 for the heaviest masses, for further acceleration in ATLAS. In this study, we discuss the parameters of the EBIS and the charge breeding results in a pulsed injection mode with repetition rates up to 10 Hz.« less

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

    PubMed

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

    2014-10-01

    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 C-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 C-dansylation labeling. The C-labeled individual sample was mixed with an equal volume of the C-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

  15. Fast and efficient (18) F-labeling by [(18) f]fluorophenylazocarboxylic esters.

    PubMed

    Fehler, Stefanie K; Maschauer, Simone; Hfling, Sarah B; Bartuschat, Amelie L; Tschammer, Nuska; Hbner, Harald; Gmeiner, Peter; Prante, Olaf; Heinrich, Markus R

    2014-01-01

    Introduction of [(18) F]fluoride ion into the aromatic core of phenylazocarboxylic esters was achieved in only 30?seconds, with radiochemical yields of up to 95?% (85(10)?%). For labeling purposes, the resulting (18) F-substituted azoester can be further converted in radical-arylation reactions to give biaryls, or in substitutions at its carbonyl unit to produce azocarboxamides. PMID:24339325

  16. Efficient approximate and dynamic matching of patterns using a labeling paradigm

    SciTech Connect

    Cenk Sahinalp, S.; Vishkin, U.

    1996-12-31

    A key approach in string processing algorithmics has been the labeling paradigm [KMR72], which is based on assigning labels to some of the substrings of a given string. If these labels are chosen consistently, they can enable fast comparisons of substrings. Until the first optimal parallel algorithm for suffix tree construction was given in [SV94], the labeling paradigm was considered not to be competitive with other approaches. In this paper we show that, this general method is also useful for several central problems in the area of string processing: (1) Approximate String Matching, (2) Dynamic Dictionary Matching, (3) Dynamic Text Indexing. The approximate string matching problem deals with finding all substrings of a text which match a pattern {open_quotes}approximately{close_quotes}, i.e., with at most m differences. The differences can be in the form of inserted, deleted, or replaced characters. The text indexing problem deals with finding all occurrences of a pattern in a text, after the text is preprocessed. In the dynamic text indexing problem, updates to the text in the form of insertions and deletions of substrings are permitted. The dictionary matching problem deals with finding all occurrences of each pattern out of a set of patterns in a text, after the pattern set is preprocessed. In the dynamic dictionary matching problem, insertions and deletions of patterns to the pattern set are permitted.

  17. Overexpression of biotin synthase and biotin ligase is required for efficient generation of sulfur-35 labeled biotin in E. coli

    PubMed Central

    2010-01-01

    Background Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin. Results In this study, we produced [35S]-biotin from Na35SO4 and desthiobiotin with a specific activity of 30.7 Ci/mmol, two orders of magnitude higher than previously published methods. The biotinylation domain (PfBCCP-79) from the Plasmodium falciparum acetyl-CoA carboxylase (ACC) was expressed in E. coli as a biotinylation substrate. We found that overexpression of the E. coli biotin synthase, BioB, and biotin ligase, BirA, increased PfBCCP-79 biotinylation 160-fold over basal levels. Biotinylated PfBCCP-79 was purified by affinity chromatography, and free biotin was liberated using acid hydrolysis. We verified that we had produced radiolabeled biologically active [D]-biotin that specifically labels biotinylated proteins through reuptake in E. coli. Conclusions The strategy described in our report provides a simple and effective method for the production of [35S]-biotin in E. coli based on affinity chromatography. PMID:20937134

  18. Cobalt-Catalyzed Enantioselective Hydrogenation of Minimally Functionalized Alkenes: Isotopic Labeling Provides Insight into the Origin of Stereoselectivity and Alkene Insertion Preferences.

    PubMed

    Friedfeld, Max R; Shevlin, Michael; Margulieux, Grant W; Campeau, Louis-Charles; Chirik, Paul J

    2016-03-16

    The asymmetric hydrogenation of cyclic alkenes lacking coordinating functionality with a C1-symmetric bis(imino)pyridine cobalt catalyst is described and has been applied to the synthesis of important substructures found in natural products and biologically active compounds. High activities and enantioselectivities were observed with substituted benzo-fused five-, six-, and seven-membered alkenes. The stereochemical outcome was dependent on both the ring size and exo/endo disposition. Deuterium labeling experiments support rapid and reversible 2,1-insertion that is unproductive for generating alkane product but accounts for the unusual isotopic distribution in deuterated alkanes. Analysis of the stereochemical outcome of the hydrogenated products coupled with isotopic labeling, stoichiometric, and kinetic studies established 1,2-alkene insertion as both turnover limiting and enantiodetermining with no evidence for erosion of cobalt alkyl stereochemistry by competing β-hydrogen elimination processes. A stereochemical model accounting for the preferred antipodes of the alkanes is proposed and relies on the subtle influence of the achiral aryl imine substituent on the cobalt catalyst. PMID:26854359

  19. Accurate Determination of Amino Acids in Serum Samples by Liquid Chromatography-Tandem Mass Spectrometry Using a Stable Isotope Labeling Strategy.

    PubMed

    Song, Cuihua; Zhang, Shijuan; Ji, Zhongyin; Li, Yipeng; You, Jinmao

    2015-10-01

    An accurate and sensitive liquid chromatography-tandem mass spectrometry method was developed for the analysis of amino acids (isoleucine, leucine, valine, tyrosine, phenylalanine and tryptophan) in serum samples using a stable isotope labeling strategy. Amino acid samples and standards were, respectively, derivatized by 10-methyl-acridone-2-sulfonyl chloride (d0-MASC) and its deuterated counterpart d3-MASC to form isotopic pairs which co-eluted and were detected by an MS detector at the same time. Accurate internal standard-based quantification was thereby achieved without the use of any internal standard analogy. The labeling reaction of MASC with amino acids is fast, simple and robust. Besides, derivatization increased the molecular weight of amino acids, and therefore they were shifted out of the background noise which was often observed in low mass region. The instrument LODs were in the range of 1.0-2.5 nmol/L. Linearities calculated by comparing theoretical peak area ratios of d0-/d3-MASC derivatives with the experimental peak area ratios were excellent with correlation coefficients of >0.995. The proposed method was successfully applied to the analysis of amino acids in serum samples with high sensitivity and accuracy. PMID:25940231

  20. Measurement of unlabeled and stable isotope-labeled homoarginine, arginine and their metabolites in biological samples by GC-MS and GC-MS/MS.

    PubMed

    Kayacelebi, Arslan Arinc; Knfel, Ann-Kathrin; Beckmann, Bibiana; Hanff, Erik; Warnecke, Gregor; Tsikas, Dimitrios

    2015-09-01

    Circulating and excretory L-homoarginine (hArg) and asymmetric dimethylarginine (ADMA) are cardiovascular risk factors. L-Arginine (Arg) is the common precursor of hArg and ADMA. This protocol describes gas chromatography-mass spectrometry (GC-MS) and gas chromatography-mass spectrometry-mass spectrometry (GC-MS/MS) methods for the quantitative determination of hArg, Arg and ADMA in biological samples, including human plasma, urine and sputum. Aliquots (10 L) of native urine, plasma or serum ultrafiltrate (cutoff, 10 kDa), and acetone-deproteinized sputum samples are evaporated to dryness. Then, amino acids are derivatized to their methyl ester N-pentafluoropropionyl derivatives. In parallel, trideuteromethyl ester N-pentafluoropropionyl derivatives of hArg, Arg and ADMA are de novo synthesized from the unlabelled amino acids and used as internal standards. Alternatively, commercially available stable isotope-labeled analogs of hArg, Arg and ADMA are used as internal standards, and they are added to the native biological samples. Quantification is performed by selected ion monitoring in GC-MS and selected reaction monitoring in GC-MS/MS. By these protocols, unlabelled and stable isotope-labeled hArg, Arg and their metabolites including ADMA and ornithine can be measured equally accurately and precisely by GC-MS and GC-MS/MS in several different biological fluids in experimental and clinical settings. PMID:25957527

  1. 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...

  2. Nitrogen use efficiency evaluation of aerobic rice under field capacity water potential using 15N isotopic tracer technique

    NASA Astrophysics Data System (ADS)

    Wahid, Ahmad Nazrul Abd; Rahim, Sahibin Abd; Rahim, Khairuddin Abdul; Harun, Abdul Rahim

    2015-09-01

    This study was carried out to evaluate the efficiency use of the nitrogen fertilizer on aerobic rice varieties MR219-4 and MR219-9 which were grown aerobically under field capacity water potential at the controlled environment area or shield house. Direct 15N isotope tracer method was used in this study, whereby the 15N isotope was utilized as a tracer for nitrogen nutrient uptake. 15N isotope presence in the samples is determined by using emission spectrometer analysis and percentage of total nitrogen is determined by using Kjeldahl method. 15N atom access value contained in the sample will be used in determining the effectiveness of the use of nitrogen in fertilizers through the specific calculation formulas. In this work, the data several data of nitrogen derived from fertilizer (Ndff), total nitrogen, nitrogen uptake and nitrogen use efficiency was obtained.

  3. 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)

    Leroy, Fanny; Riera, Pascal; Jeanthon, Christian; Edmond, Frdrique; Leroux, Cdric; Comtet, Thierry

    2012-05-01

    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.

  4. 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

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

    2013-01-01

    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 μg/kg and a limit of quantitation (LOQ) of 2.89 μg/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 μg/kg. PMID:23230907

  5. 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

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

    2014-06-01

    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

  6. Phosphorus cycling in the Sargasso Sea: Investigation using the oxygen isotopic composition of phosphate, enzyme-labeled fluorescence, and turnover times

    NASA Astrophysics Data System (ADS)

    McLaughlin, Karen; Sohm, Jill A.; Cutter, Gregory A.; Lomas, Michael W.; Paytan, Adina

    2013-04-01

    Dissolved inorganic phosphorus (DIP) concentrations in surface water of vast areas of the ocean are extremely low (<10 nM) and phosphorus (P) availability could limit primary productivity in these regions. We explore the use of oxygen isotopic signature of dissolved phosphate (δ18OPO4) to investigate biogeochemical cycling of P in the Sargasso Sea, Atlantic Ocean. Additional techniques for studying P dynamics including 33P-based DIP turnover time estimates and percent of cells expressing alkaline phosphatase (AP) activity as measured by enzyme-labeling fluorescence are also used. In surface waters, δ18OPO4 values were lower than equilibrium by 3-6‰, indicative of dissolved organic phosphorous (DOP) remineralization by extracellular enzymes. An isotope mass balance model using a variety of possible combinations of enzymatic pathways and substrates indicates that DOP remineralization in the euphotic zone can account for a large proportion on P utilized by phytoplankton (as much as 82%). Relatively short DIP turnover times (4-8 h) and high expression of AP (38-77% of the cells labeled) are consistent with extensive DOP utilization and low DIP availability in the euphotoc zone. In deep water where DOP utilization rates are lower, δ18OPO4 values approach isotopic equilibrium and DIP turnover times are longer. Our data suggests that in the euphotic zone of the Sargasso Sea, DOP may be appreciably remineralized and utilized by phytoplankton and bacteria to supplement cellular requirements. A substantial fraction of photosynthesis in this region is supported by DOP uptake.

  7. The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

    PubMed Central

    Adamczyk, Justyna; Hesselsoe, Martin; Iversen, Niels; Horn, Matthias; Lehner, Angelika; Nielsen, Per Halkjaer; Schloter, Michael; Roslev, Peter; Wagner, Michael

    2003-01-01

    A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment. PMID:14602652

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

    PubMed

    Shen, Junyi; Saijo, Tatsuyoshi

    2009-08-01

    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

  9. Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.

    PubMed Central

    Ludlam, C F; Arkin, I T; Liu, X M; Rothman, M S; Rath, P; Aimoto, S; Smith, S O; Engelman, D M; Rothschild, K J

    1996-01-01

    Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain. PMID:8785331

  10. Earthworm Uptake Routes and Rates of Ionic Zn and ZnO Nanoparticles at Realistic Concentrations, Traced Using Stable Isotope Labeling.

    PubMed

    Laycock, Adam; Diez-Ortiz, Maria; Larner, Fiona; Dybowska, Agnieszka; Spurgeon, David; Valsami-Jones, Eugenia; Rehkmper, Mark; Svendsen, Claus

    2016-01-01

    The environmental behavior of ZnO nanoparticles (NPs), their availability to, uptake pathways by, and biokinetics in the earthworm Lumbricus rubellus were investigated using stable isotope labeling. Zinc isotopically enriched to 99.5% in (68)Zn ((68)Zn-E) was used to prepare (68)ZnO NPs and a dissolved phase of (68)Zn for comparison. These materials enabled tracing of environmentally relevant (below background) NP additions to soil of only 5 mg (68)Zn-E kg(-1). Uptake routes were isolated by introducing earthworms with sealed and unsealed mouthparts into test soils for up to 72 h. The Zn isotope compositions of the soils, pore waters and earthworms were then determined using multiple collector inductively coupled plasma mass spectrometry. Detection and quantification of (68)Zn-E in earthworm tissue was possible after only 4 h of dermal exposure, when the uptake of (68)Zn-E had increased the total Zn tissue concentration by 0.03. The results demonstrate that at these realistic exposure concentrations there is no distinguishable difference between the uptake of the two forms of Zn by the earthworm L. rubellus, with the dietary pathway accounting for ?95% of total uptake. This stands in contrast to comparable studies where high dosing levels were used and dermal uptake is dominant. PMID:26588002

  11. Time-shared experiments for efficient assignment of triple-selectively labeled proteins

    NASA Astrophysics Data System (ADS)

    Lhr, Frank; Laguerre, Aisha; Bock, Christoph; Reckel, Sina; Connolly, Peter J.; Abdul-Manan, Norzehan; Tumulka, Franz; Abele, Rupert; Moore, Jonathan M.; Dtsch, Volker

    2014-11-01

    Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90 pulses allows to either select or discriminate against 13C? or 13C? spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.

  12. Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells.

    PubMed

    Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T; Henningsen, Jeanette; Kratchmarova, Irina; Kassem, Moustapha; Blagoev, Blagoy

    2009-05-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations. PMID:19151416

  13. Luminescent dye-doped or rare-earth-doped monodisperse silica nanospheres as efficient labels in DNA microarrays

    NASA Astrophysics Data System (ADS)

    Enrichi, F.; Ricc, R.; Meneghello, A.; Pierobon, R.; Marinello, F.; Schiavuta, P.

    2009-08-01

    Luminescent nanoparticles are gaining more and more interest in bio-labeling and bio-imaging applications, like for example DNA microarray. This is a high-throughput technology used for detection and quantification of nucleic acid molecules and other ones of biological interest. The analysis is resulting by specific hybridization between probe sequences deposited in array and a target ss-DNA usually expressed by PCR and functionalized by a fluorescent dye. These organic labels have well known disadvantages like photobleaching and limited sensitivity. Quantum dots may be used as alternatives, but they present troubles like blinking, toxicity and excitation wavelengths out of the usual range of commercial instruments, lowering their efficiency. Therefore in this work we investigate a different strategy, based on the use of inorganic silica nanospheres incorporating standard luminescent dyes or rare earth doped nanocrystals. In the first case it is possible to obtain a high luminescence emission signal, due to the high number of dye molecules that can be accommodated into each nanoparticle, reduced photobleaching and environmental protection of the dye molecules thanks to the encapsulation in the silica matrix. In the second case, rare earths exhibit narrow emission bands (easy identification), large Stokes shifts (efficient discrimination of excitation and emission) and long luminescence lifetimes (possibility to perform time-delayed analysis) which can be efficiently used for the improvement of signal to noise ratio. The synthesis and characterization of good luminescent silica spheres either by organic dye-doping or by rare-earth-doping are investigated and reported. Moreover, their application in the DNA microarray technology in comparison to the use of standard molecular fluorophores or commercial quantum dots is discussed. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalization and bio-compatibility makes them very promising for present and future applications in bio-labeling and bio-imaging.

  14. Impact of isotopic effect on density limit and LHCD efficiency in the FT-2 experiments

    NASA Astrophysics Data System (ADS)

    Lashkul, S. I.; Altukhov, A. B.; Gurchenko, A. D.; Gusakov, E. Z.; Dyachenko, V. V.; Esipov, L. A.; Irzak, M. A.; Kantor, M. Yu.; Kouprienko, D. V.; Perevalov, A. A.; Saveliev, A. N.; Shatalin, S. V.; Stepanov, A. Yu.

    2015-07-01

    Current drive by lower hybrid waves (LHCD) is the most effective method to sustain the plasma current, but it is feasible only at the plasma density not exceeding some density limit nDL. In the present work the main attention is paid to the investigation of this effect on the FT-2 (R = 0.55 m, a = 0.08 m, BT ⩽ 3 T, Ipl = 19-40 kA, f0 = 920 MHz) tokamak. The dependence of LHCD efficiency on isotopic plasma content (hydrogen/deuterium) is studied. Characteristic features of such an experiment are a strong influence of the isotope plasma composition on the LH resonance density nLH. For hydrogen plasma nLH ≈ 3.5 × 1019 m-3, while for deuterium plasma nLH ≈ 2 × 1020 m-3. The suppression of the LHCD and beginning of the interaction of LH waves with ions are determined by the hydrogen/deuterium plasma density rise. In the hot hydrogen plasma (Te(r = 0 cm) ≈ 700 eV) the density limit nDL of LHCD is approximately equal to the resonance value nLH ≈ nLC, where nLC is the point of linear conversion. In the hot deuterium plasma one could expect an increase of nDL because of a much higher value of nLH ⩾ nLC ≈ 1020 m-3. However it appeared that the observed density limit for LHCD generation nDL ≈ (3.5-4) × 1019 m-3 is not determined by nLH. The role of parametric instabilities in CD switch-off is considered in both cases. The cooling of the plasma column and density rise could lead to a reduction of the threshold for the parametric decay of f0 and result in early suppression of LHCD. In both cases the LHCD was inversely proportional to the density, which corresponds to the theoretical predictions. In order to analyse the experimentally observed LHCD efficiency the GRILL3D and FRTC codes have been used.

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

    PubMed Central

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

    2015-01-01

    We present a general approach in 1H-detected 13C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 5257, 2006]. First, we demonstrate that 1H indirect detection improves the sensitivity and resolution of 13C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. 1H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by 1H-detected 2D 1H/13C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over 13C-detected 2D 1H/13C SSNMR and 1D 13C CPMAS, demonstrating that 2D 1H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D 13C detection for the first time. High 1H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A 1H-detected 3D 13C/13C/1H experiment on SAIL-ubiquitin provided nearly complete 1H and 13C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems. PMID:25856081

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

    SciTech Connect

    Fridley, David; Zheng, Nina; Zhou, Nan

    2010-06-01

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

  17. Qualitative Metabolome Analysis of Human Cerebrospinal Fluid by 13C-/12C-Isotope Dansylation Labeling Combined with Liquid Chromatography Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Guo, Kevin; Bamforth, Fiona; Li, Liang

    2011-02-01

    Metabolome analysis of human cerebrospinal fluid (CSF) is challenging because of low abundance of metabolites present in a small volume of sample. We describe and apply a sensitive isotope labeling LC-MS technique for qualitative analysis of the CSF metabolome. After a CSF sample is divided into two aliquots, they are labeled by 13C-dansyl and 12C-dansyl chloride, respectively. The differentially labeled aliquots are then mixed and subjected to LC-MS using Fourier-transform ion cyclotron resonance mass spectrometry (FTICR MS). Dansylation offers significant improvement in the performance of chromatography separation and detection sensitivity. Moreover, peaks detected in the mass spectra can be readily analyzed for ion pair recognition and database search based on accurate mass and/or retention time information. It is shown that about 14,000 features can be detected in a 25-min LC-FTICR MS run of a dansyl-labeled CSF sample, from which about 500 metabolites can be profiled. Results from four CSF samples are compared to gauge the detectability of metabolites by this method. About 261 metabolites are commonly detected in replicate runs of four samples. In total, 1132 unique metabolite ion pairs are detected and 347 pairs (31%) matched with at least one metabolite in the Human Metabolome Database. We also report a dansylation library of 220 standard compounds and, using this library, about 85 metabolites can be positively identified. Among them, 21 metabolites have never been reported to be associated with CSF. These results illustrate that the dansylation LC-FTICR MS method can be used to analyze the CSF metabolome in a more comprehensive manner.

  18. Urinary profiling of cis-diol-containing metabolites in rats with bisphenol A exposure by liquid chromatography-mass spectrometry and isotope labeling.

    PubMed

    Li, Shangfu; Jin, Yibao; Wang, Jue; Tang, Zhi; Xu, Shunqing; Wang, Tiejie; Cai, Zongwei

    2016-02-01

    Exposure to bisphenol A (BPA), an environmental contaminant, has been linked to metabolic disorders. However, there are no reports describing the effects of BPA on the profiling of cis-diol metabolites. It is challenge to detect these metabolites in biological samples because of their low abundance, high polarity and serious matrix interference. In this study, a chemical isotope-labeling method was applied to solve these problems. Acetone and deuterated acetone (acetone-d6) were used as chemical tags to label the rat urine samples, respectively. The light and heavy labeling products were recognized using the ShiftedIonsFinder software. The selected cis-diol metabolite signals were used to build a data set. The data set was applied to evaluate the changes in the urinary profiling of cis-diol-containing metabolites in rats with BPA exposure. The results showed that chromatographic separation and mass spectrometry detection of cis-diol metabolites were improved after acetone labeling. Using this method, the cis-diol metabolites were recognized easily from the urine samples. By comparing different dose administration on rats, the influence of BPA exposure on cis-diol metabolites was investigated. The analytes showing noticeable differences were identified. It was found that high-dose BPA exposure had strong effects on the cis-diol compound metabolism. The influences were mostly related to the metabolism of galactose and nucleoside and its analogues. The disturbance of the galactose metabolism by BPA is reported for the first time, to the best of our knowledge. This may have some implications for exploring the toxic effects of BPA exposure. PMID:26739229

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

    SciTech Connect

    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

    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.

  20. 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 ...

  1. ISOTOPIC LABELING AND LC-APCI-MS QUANTIFICATION FOR INVESTIGATING ABSORPTION OF CAROTENOIDS AND VITAMIN K1 FROM KALE

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability to study bioavailability of nutrients from plant-based foods is an important step in determining the potential health impact of those nutrients. This work describes a new method for studying bioavailability of nutrients from green, leafy vegetables by labeling the nutrients in kale with ...

  2. Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis.

    PubMed

    Savas, Jeffrey N; Park, Sung Kyu; Yates, John R

    2016-01-01

    The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after "pulse" labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein's half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo.Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide. PMID:26867752

  3. 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)

    Fujinuma, R.; Balster, N. J.

    2009-12-01

    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.

  4. Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.

    PubMed

    Onorato, Joelle M; Chu, Ching-Hsuen; Ma, Zhengping; Kopcho, Lisa M; Chao, Hannguang J; Lawrence, R Michael; Cheng, Dong

    2015-03-01

    To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes. PMID:25598079

  5. Mass Spectra and Cross-Contribution of Ion Intensity Between Drug Analytes and Their Isotopically Labelled Analogs - Common Opioids and Their Derivatives.

    PubMed

    Chen, B G; Wu, M Y; Liu, R H; Wang, S M; Lewis, R J; Ritter, R M; Canfield, D V

    2008-07-01

    For the quantitation of most drugs and their metabolites, GC-MS is currently the preferred method and isotopically labeled analogs of the analytes are the internal standards (ISs) of choice. Under this analytical setting, chemical derivatization (CD) plays a critical role in the sample preparation process. In addition to meeting the conventional objectives of CD, products derived from the selected CD method must generate ions suitable for designating the analyte and the IS; these ions cannot have significant cross-contribution (CC), i.e., contribution to the intensity of the ions designating the analyte by the IS, and vice versa. With this in mind, the authors have reviewed literature and information provided by manufacturers, searching for suitable CD reagents, CD methods, and isotopically labeled analogs of the analytes related to the following 11 opioids: heroin, 6-acetylmorphine, morphine, hydromorphone, oxymorphone, 6-acetylcodeine, codeine, hydrocodone, dihydrocodeine, oxycodone, and noroxycodone. These analytes and ISs were derivatized with various derivatization groups, followed by GCMS analysis. The resulting MS data are systematically presented in two forms: (a) full-scan mass spectra; and (b) CC data of ion-pairs with potential for designating the analytes and their respective ISs. Many (if not most) of these full-scan mass spectra are not yet available in the literature and should be of reference value to laboratories engaged in the analysis of these drugs/metabolites. Full-scan MS data were further used to select ion-pairs with potential for designating the analytes and ISs in quantitative analysis protocols. The CC data of these ion-pairs were evaluated using data collected in selected ion monitoring mode and systematically tabulated, readily available for analysts searching for this important analytical parameter. PMID:26247421

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

    PubMed

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

    2009-08-01

    Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13C/15N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional "through-bond" spectrum (and 2D HSQC spectra) in addition to the 13C-edited and 15N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods. PMID:19597942

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

    PubMed Central

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

    2012-01-01

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

  8. Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.

    PubMed

    Daumar, Pierre; Wanger-Baumann, Cindy A; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D; Andreev, Oleg A; Reshetnyak, Yana K; Lewis, Jason S

    2012-08-15

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 C with a radiochemical yield of 27.5 6.6% (decay corrected) with high radiochemical purity ?98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers. PMID:22784215

  9. Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation

    PubMed Central

    Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.

    2012-01-01

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known click methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC click chemistry for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 C with a radiochemical yield of 27.5 6.6% (decay corrected) with high radiochemical purity ? 98%. The subsequent CuI catalyzed click reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 520% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative control [18F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the 18F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [18F]-pHLIP analogues as potential PET tracers. PMID:22784215

  10. Quantitative Serum Proteomics Using Dual Stable Isotope Coding and nano LC-MS/MSMS

    PubMed Central

    Wang, Hong; Wong, Chee-Hong; Chin, Alice; Kennedy, Jacob; Zhang, Qing; Hanash, Samir

    2015-01-01

    Stable isotope coding technique in combination with mass spectrometry has emerged as a powerful tool to accurately identify and differentially quantify proteins within complex protein mixtures. We present a novel methodology to increase the yield of quantified proteins while maintaining a high stable-isotopic labeling efficacy. With this approach, intact proteins in complex biological sample such as sera are labeled with the designated dual stable isotope coding (DSIC) systems. In brief, intact proteins are coded sequentially with acrylamide to label Cysteine residues (Cys) and with succinic anhydride to label Lysine residues (Lys). Protein samples coded with this dual stable isotope are subjected to an online 2D-HPLC fractionation. The resolved protein fractions are individually digested with trypsin and analyzed with nano LC-MS/MSMS. Our results show that the DSIC labeling efficiency is 100% for Cysteine (Cys) labeled with acrylamide and 98% for Lysine (Lys) labeled with succinic anhydride. A comparative analysis of DSIC labeling and single labeling of Cysteine residues was made. Analysis of an entire anion-exchange chromatography subfraction of sera yielded 165 identified proteins (criteria: error rate <5% and unique peptides ?2), 104 of which were quantified using the single labeling method (i.e., Cysteine acrylamide labeling only). In contrast, using same criteria for identification, a total 185 proteins were identified and 174 proteins were quantified using the DSIC labeling technique. PMID:19817497

  11. Proteins with high turnover rate in barley leaves estimated by proteome analysis combined with in planta isotope labeling.

    PubMed

    Nelson, Clark J; Alexova, Ralitza; Jacoby, Richard P; Millar, A Harvey

    2014-09-01

    Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used (15)N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of (15)N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of (14)N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until (15)N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that (15)N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis. PMID:25082890

  12. Combined Gel Probe and Isotope Labeling Technique for Measuring Dissimilatory Nitrate Reduction to Ammonium in Sediments at Millimeter-Level Resolution?

    PubMed Central

    Stief, Peter; Behrendt, Anna; Lavik, Gaute; De Beer, Dirk

    2010-01-01

    Dissimilatory NO3? reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO3? concentrations yields potential rather than actual activities of dissimilatory NO3? reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO3? consumption. The water column of the sediment core is amended with 15NO3? at the in situ 14NO3? concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH4+ dissolved in the gel slices is chemically converted by hypobromite to N2 in reaction vials. The isotopic composition of N2 is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of 15NH4+. The results were compared to the NH4+ microsensor profiles measured in freshwater sediment and quartz sand and to the N2O microsensor profiles measured in acetylene-amended sediments to trace denitrification. PMID:20656865

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

    SciTech Connect

    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

    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.

  14. A Method Revealing Bacterial Cell-wall Architecture by Time-dependent Isotope Labeling and Quantitative Liquid Chromatography/Mass Spectrometry

    PubMed Central

    Patti, Gary J.; Chen, Jiawei; Gross, Michael L.

    2009-01-01

    The molecular details of the biosynthesis and resulting architecture of the bacterial cell wall remain unclear but are essential to understanding the activity of glycopeptide antibiotics, the recognition of pathogens by hosts, and the processes of bacterial growth and division. Here we report a new strategy to elucidate bacterial cell-wall architecture based on time-dependent isotope labeling of bacterial cells quantified by liquid chromatography/accurate mass measurement mass spectrometry. The results allow us to track the fate of cell-wall precursors (which contain the vancomycin-binding site) in Enterococcus faecium, a leading antibiotic-resistant pathogen. By comparing isotopic enrichments of post-insertionally modified cell-wall precursors, we find that tripeptides and species without Asx bridges are specific to mature cell wall. Additionally, we find that the sequence of cell-wall maturation varies throughout a cell cycle. We suggest that actively dividing E. faecium cells have three zones of unique peptidoglycan processing. Our results reveal new organizational characteristics of the bacterial cell wall that are important to understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens. PMID:19281243

  15. Combined gel probe and isotope labeling technique for measuring dissimilatory nitrate reduction to ammonium in sediments at millimeter-level resolution.

    PubMed

    Stief, Peter; Behrendt, Anna; Lavik, Gaute; De Beer, Dirk

    2010-09-01

    Dissimilatory NO(3)(-) reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO(3)(-) concentrations yields potential rather than actual activities of dissimilatory NO(3)(-) reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO(3)(-) consumption. The water column of the sediment core is amended with (15)NO(3)(-) at the in situ (14)NO(3)(-) concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH(4)(+) dissolved in the gel slices is chemically converted by hypobromite to N(2) in reaction vials. The isotopic composition of N(2) is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of (15)NH(4)(+). The results were compared to the NH(4)(+) microsensor profiles measured in freshwater sediment and quartz sand and to the N(2)O microsensor profiles measured in acetylene-amended sediments to trace denitrification. PMID:20656865

  16. Quantitative analysis using gas chromatography/combustion/isotope ratio mass spectrometry and standard addition of intrinsically labelled standards (SAIL)--application to isoflavones in foods.

    PubMed

    Bluck, Leslie J C; Jones, Kerry S; Thomas, Jane; Liggins, Jason; Harding, Marilyn; Bingham, Sheila A; Coward, W Andrew

    2002-01-01

    We have investigated a novel application of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) for the quantitative analysis of the isoflavones in food matrices. Previous methods have been hampered by the lack of analytical standards to introduce early enough in the extraction procedure to allow compensations for losses at all stages of the procedure. In this work we have produced standard materials that can be added at the initial extraction, by intrinsically labelling soya plants by growing them in an atmosphere enriched in the stable isotope of carbon in CO(2). On analysis these plants were shown to contain phytoestrogens at a high (up to 20%) level of enrichment. The dried plant material has been used to estimate the isoflavone concentrations of a set of spiked flours. For daidzein the methodology was shown to produce results comparable to those achieved by GC/MS techniques. The method was less successful for genistein, possibly due to the greater fragility of this compound under the conditions required for the analysis. PMID:12478567

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

    SciTech Connect

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

    1982-01-01

    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.

  18. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  19. Multi-isotope labelling of organic matter by diffusion of 2H/18O-H2O vapour and 13C-CO2 into the leaves and its distribution within the plant

    NASA Astrophysics Data System (ADS)

    Studer, M. S.; Siegwolf, R. T. W.; Leuenberger, M.; Abiven, S.

    2015-03-01

    Isotope labelling is a powerful tool to study elemental cycling within terrestrial ecosystems. Here we describe a new multi-isotope technique to label organic matter (OM). We exposed poplars (Populus deltoides nigra) for 14 days to an atmosphere enriched in 13CO2 and depleted in 2H218O. After 1 week, the water-soluble leaf OM (?13C = 1346 162) and the leaf water were strongly labelled (?18O = -63 8, ?2H = -156 15). The leaf water isotopic composition was between the atmospheric and stem water, indicating a considerable back-diffusion of vapour into the leaves (58-69%) in the opposite direction to the net transpiration flow. The atomic ratios of the labels recovered (18O/13C, 2H/13C) were 2-4 times higher in leaves than in the stems and roots. This could be an indication of the synthesis of more condensed compounds in roots and stems (e.g. lignin vs. cellulose) or might be the result of O and H exchange and fractionation processes during phloem transport and biosynthesis. We demonstrate that the three major OM elements (C, O, H) can be labelled and traced simultaneously within the plant. This approach could be of interdisciplinary interest in the fields of plant physiology, palaeoclimatic reconstruction or soil science.

  20. High levels of isotope elimination improve precision and allow individual-based measurements of metabolic rates in animals using the doubly labeled water method

    PubMed Central

    Shirai, Masaki; Niizuma, Yasuaki; Yamamoto, Maki; Oda, Emiko; Ebine, Naoyuki; Oka, Nariko; Yoda, Ken

    2015-01-01

    Doubly labeled water (DLW) can be used to measure energy expenditure in free-ranging animals, but questions have been raised about its accuracy in different species or contexts. We investigated whether differences in the extent of isotope elimination affects the precision and accuracy of the DLW method, which can vary according to the experimental design or metabolic rate of the species. Estimated total energy expenditure by the DLW method (TEEdlw) was compared with actual total energy expenditure simultaneously measured via respirometry (TEEresp) in streaked shearwaters Calonectris leucomelas, a pelagic seabird. Subjects were divided into three groups with different experimental conditions: at rest on the ground for 24 h (Group A) or for 48 h (Group B), and at rest on the water for 24 h (Group C). TEEdlw in Group A matched TEEresp, whereas there was an overestimation of TEEdlw in both Groups B and C compared with TEEresp. However, compared with Group A, TEEdlw in Groups B and C had reduced the isotopic analytical variability and thus higher precision. The best regression model (TEEdlw = 1.37 TEEresp − 14.12) showed a high correlation (R2 = 0.82) between TEEdlw and TEEresp and allows a correction factor for field metabolic rates in streaked shearwaters. Our results demonstrate that the commonly made assumption that the DLW method is not appropriate for individual-based estimates may be incorrect in certain circumstances. Although a correction factor may be necessary when using the DLW method to estimate metabolic rate, greater levels of isotope eliminations provides DLW estimates with high precision, which can adequately represent relative individual estimates. Nevertheless, the DLW method, should be used with caution when characterizing interspecies difference of energy expenditures. PMID:26611463

  1. The effect of FISH and CARD-FISH on the isotopic composition of (13)C- and (15)N-labeled Pseudomonas putida cells measured by nanoSIMS.

    PubMed

    Musat, Niculina; Stryhanyuk, Hryhoriy; Bombach, Petra; Adrian, Lorenz; Audinot, Jean-Nicolas; Richnow, Hans H

    2014-06-01

    The use of nanoSIMS for the exploration of microbial activities in natural habitats often implies that stable isotope tracer experiments are combined with in situ hybridization techniques (i.e. fluorescence in situ hybridization (FISH) or catalyzed reporter deposition (CARD)-FISH). In this study, Pseudomonas putida grown on (13)C- and (15)N-labeled carbon and nitrogen, collected in exponential growth and stationary phases, was hybridized and analyzed by nanoSIMS. It was shown that (13)C and (15)N fractions decreased after FISH and CARD-FISH in comparison to chemically untreated cells. However, the fractions were influenced differently by various treatments. After paraformaldehyde fixation of exponentially growing cells, a reduction of the (13)C and (15)N fractions was measured from 941.2% and 89.53.8% to 90.20.8% and 644.6%, respectively, indicating that nitrogen isotopic composition was most influenced. A further decrease of the (13)C and (15)N fractions to 80.76.5 and 59.54.1%, respectively, was measured after FISH, while CARD-FISH decreased the fractions to 57.43.0% and 47.14.1%, respectively. The analysis of cells collected in different growth phases revealed that the effect of various treatments seemed to be dependent on the cell's physiological state. In addition, a mathematical model that can be used in further studies was developed in order to calculate the amount of carbon introduced into the cells by chemical treatments. These results can be valuable for environmental FISH-nanoSIMS studies where the isotopic composition of single cells will be used to quantitatively assess the importance of specific populations to certain biochemical processes and determine budget estimations. PMID:24702905

  2. High levels of isotope elimination improve precision and allow individual-based measurements of metabolic rates in animals using the doubly labeled water method.

    PubMed

    Shirai, Masaki; Niizuma, Yasuaki; Yamamoto, Maki; Oda, Emiko; Ebine, Naoyuki; Oka, Nariko; Yoda, Ken

    2015-11-01

    Doubly labeled water (DLW) can be used to measure energy expenditure in free-ranging animals, but questions have been raised about its accuracy in different species or contexts. We investigated whether differences in the extent of isotope elimination affects the precision and accuracy of the DLW method, which can vary according to the experimental design or metabolic rate of the species. Estimated total energy expenditure by the DLW method (TEEdlw) was compared with actual total energy expenditure simultaneously measured via respirometry (TEEresp) in streaked shearwaters Calonectris leucomelas, a pelagic seabird. Subjects were divided into three groups with different experimental conditions: at rest on the ground for 24h (Group A) or for 48h (Group B), and at rest on the water for 24h (Group C). TEEdlw in Group A matched TEEresp, whereas there was an overestimation of TEEdlw in both Groups B and C compared with TEEresp. However, compared with Group A, TEEdlw in Groups B and C had reduced the isotopic analytical variability and thus higher precision. The best regression model (TEEdlw=1.37 TEEresp-14.12) showed a high correlation (R(2)=0.82) between TEEdlw and TEEresp and allows a correction factor for field metabolic rates in streaked shearwaters. Our results demonstrate that the commonly made assumption that the DLW method is not appropriate for individual-based estimates may be incorrect in certain circumstances. Although a correction factor may be necessary when using the DLW method to estimate metabolic rate, greater levels of isotope eliminations provides DLW estimates with high precision, which can adequately represent relative individual estimates. Nevertheless, the DLW method, should be used with caution when characterizing interspecies difference of energy expenditures. PMID:26611463

  3. 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...

  4. Determination of isotopic ratios of L-leucine and L-phenylalanine and their stable isotope labeled analogues in biological samples by gas chromatography/triple-stage quadrupole mass spectrometry.

    PubMed

    Schweer, H; Watzer, B; Seyberth, H W; Steinmetz, A; Schaefer, J R

    1996-07-01

    A gas chromatographic/triple-stage quadrupole mass spectrometric (GC/MS/MS) method for measuring very low levels of enrichment of [5,5,5-2H3]-L-leucine and [ring-13C6]-L-phenylalanine in plasma and lipoprotein hydrolysates is described. The amino acids were derivatized to their N-heptafluorobutyryl isobutyl ester derivatives and the isotope ratio was determined by GC/MS/MS in the negative-ion chemical ionization mode. Parent ions were the [M-HF]- ions and fragment ions used for quantification were [P-2HF-C3H7]- (leucine) and [P-HF-OC4H9]- (phenylalanine), respectively. The limit of quantification was about 10 pg of the labeled compound co-eluting with 20 ng of the endogenous compound. The calibration curves were linear in the investigated range from 0.1% to 100% of the labeled compound. In biological samples, the higher selectivity of GC/MS/MS compared with GC/MS was demonstrated. PMID:8799305

  5. Development of an isotope labeling ultra-high performance liquid chromatography mass spectrometric method for quantification of acylglycines in human urine.

    PubMed

    Stanislaus, Avalyn; Guo, Kevin; Li, Liang

    2012-10-31

    Acylglycines play a crucial regulatory and detoxification role in the accumulation of the corresponding acyl CoA esters and are an important class of metabolites in the diagnoses of inborn errors of metabolism. Sensitive quantification of a large number of acylglycines not only improves diagnosis but also enables the discovery of potential new biomarkers of diseases. We report an ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS) method for quantifying acylglycines in human urine with high sensitivity. This method is based on the use of a newly developed isotope labeling reagent, p-dimethylaminophenacyl (DmPA) bromide, to label acylglycines to improve detection sensitivity. Eighteen acylglycines, namely acetylglycine, propionylglycine, isobutyrylglycine, butyrylglycine, 4-hydroxyphenylacetylglycine, 2-furoylglycine, tiglylglycine, 2-methybutyrylglycine, 3-methylcrotonylglycine, isovalerylglycine, valerylglycine, hexanoylglycine, phenylacetylglycine, phenylpropionylglycine, glutarylglycine, heptanoylglycine, octanoylglycine and suberylglycine, were measured. This method uses calibration standards prepared in surrogate matrix (un-derivatized urine) and stable-isotope labeled analytes as the internal standards. The analysis was carried out in the positive ion detection mode using multiple reaction monitoring (MRM) survey scans. The calibration curves were validated over the range of 1.0-500 nM. The method achieved a lower limit of quantitation (LLOQ) of 1-5 nM for all analytes, as measured by the standard derivations associated with calibration curves and confirmed in surrogate matrix; the signal-to-noise ratio at LLOQ ranged from 12.50 to 156.70. Both accuracy (% RE or relative error) and precision (% CV) were <15%. Matrix effects were minimized using the surrogate matrix. All eighteen analytes were stable in urine for at least 5h at room temperature, autosampler (4 °C) for 24 h, 7 weeks at -20 °C and after three freeze/thaw cycles. This surrogate matrix approach was validated using a standard addition experiment. As an example of applications, the endogenous concentrations of all eighteen analytes in urine samples of 20 healthy individuals collected in three consecutive days (i.e., 60 samples) were determined; there was no significant correlation found between the acylglycine profile and gender or body mass indices. PMID:23062437

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

    PubMed Central

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

    2009-01-01

    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

  7. Relationship between efficiency of nitrogen utilization and isotopic nitrogen fractionation in dairy cows: contribution of digestion v. metabolism?

    PubMed

    Cantalapiedra-Hijar, G; Fouillet, H; Huneau, J F; Fanchone, A; Doreau, M; Nozire, P; Ortigues-Marty, I

    2016-02-01

    Animal tissues are naturally 15N enriched relative to their diet and the extent of this difference (?15Nanimal-diet) has been correlated to the efficiency of N assimilation in different species. The rationale is that transamination and deamination enzymes, involved in amino acid metabolism are likely to preferentially convert amino groups containing 14N over 15N. However, in ruminants the contribution of rumen bacterial metabolism relative to animal tissues metabolism to naturally enrich animal proteins in terms of 15N has been not assessed yet. The objective of this study was to assess the impact of rumen and digestion processes on the relationship between ?15Nanimal-diet and efficiency of N utilization for milk protein yield (milk N efficiency (MNE); milk N yield/N intake) as well as the relationship between the 15N natural abundance of rumen bacteria and the efficiency of N use at the rumen level. Solid- and liquid-associated rumen bacteria, duodenal digesta, feces and plasma proteins were obtained (n=16) from four lactating Holstein cows fed four different diets formulated at two metabolizable protein supplies (80% v. 110% of protein requirements) crossed by two different dietary energy source (diets rich in starch v. fiber). We measured the isotopic N fractionation between animal and diet (?15Nanimal-diet) in these different body pools. The ?15Nanimal-diet was negatively correlated with MNE when measured in solid-associated rumen bacteria, duodenal digesta, feces and plasma proteins, with the strongest correlation found for the latter. However, our results showed a very weak 15N enrichment of duodenal digesta (?15Nduodenal digesta-diet mean value=0.42) compared with that observed in plasma proteins (?15Nplasma protein-diet mean value=2.41). These data support the idea that most of the isotopic N fractionation observed in ruminant proteins (?15Nplasma protein-diet) has a metabolic origin with very little direct impact of the overall digestion process on the existing relationship between ?15Nplasma protein-diet and MNE. The 15N natural abundance of rumen bacteria was not related to either rumen N efficiency (microbial N/available N) or digestive N efficiency (metabolizable protein supply/CP intake), but showing a modest positive correlation with rumen ammonia concentration. When using diets not exceeding recommended protein levels, the contribution of rumen bacteria and digestion to the isotopic N fractionation between animal proteins and diet is low. In our conditions, most of the isotopic N fractionation (?15Nplasma protein-diet) could have a metabolic origin, but more studies are warranted to confirm this point with different diets and approaches. PMID:26776494

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

    PubMed

    de la Escosura-Muiz, Alfredo; Plichta, Zden?k; Hork, Daniel; Merkoi, Arben

    2015-05-15

    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

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

    PubMed

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

    2014-09-01

    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

  10. 4-Phenylaminomethyl-Benzeneboric Acid Modified Tip Extraction for Determination of Brassinosteroids in Plant Tissues by Stable Isotope Labeling-Liquid Chromatography-Mass Spectrometry.

    PubMed

    Yu, Lei; Ding, Jun; Wang, Ya-Lan; Liu, Ping; Feng, Yu-Qi

    2016-01-19

    Monitoring brassinosteroids (BRs) has been of major interest of researchers as these substances play a crucial role in a variety of phytological processes in plants. However, the determination of endogenous BRs in plant tissue is still a challenging task due to their low abundance and the complex matrix of plant tissues. In this study, a single step strategy by combining tip extraction and in situ derivatization was proposed for BR analysis. In the proposed strategy, a mixed mode sorbent (C8-SO3H) in tip was modified with 4-phenylaminomethyl-benzeneboric acid (4-PAMBA) through cation exchange and hydrophobic interactions, and then used as a boronate affinity media to selectively capture and purify BRs from plant extract through the reaction of boric acid groups of 4-PAMBA and cis-diol on BRs. The BRs-4-PAMBA derivatives formed were easily eluted from the C8-SO3H tip by nullifying the ion exchange and hydrophobic interactions using ammonia acetonitrile, followed by LC-MS/MS analysis. BR standards, isotopically labeled with d5-4-phenylaminomethyl-benzeneboric acid (4-PAMBA-d5) were introduced to improve the assay precision of LC-MS/MS. Under the optimized conditions, the overall process could be completed within 1 h, which is greatly improved in speed compared with previously reported protocols. In addition, the detection sensitivities of labeled BRs were improved by over 2000-fold compared with unlabeled BRs, thus the consumption of plant materials was reduced to 50 mg. Finally, the proposed method was applied for the investigation of BRs response in rice toward Cd stress. PMID:26650986

  11. Stable isotope-labeled tracers for metabolic pathway elucidation by GC-MS and FT-MS.

    PubMed

    Higashi, Richard M; Fan, Teresa W-M; Lorkiewicz, Pawel K; Moseley, Hunter N B; Lane, Andrew N

    2014-01-01

    Advances in analytical methodologies, principally nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS), over the last decade have made large-scale analysis of the human metabolome a reality. This is leading to the reawakening of the importance of metabolism in human diseases, particularly widespread metabolic diseases such as cancer, diabetes, and obesity. Emerging NMR and MS atom-tracking technologies and informatics are poised to revolutionize metabolomics-based research because they deliver the high information throughput (HIT) that is needed for deciphering systems biochemistry. In particular, stable isotope-resolved metabolomics (SIRM) enables unambiguous tracking of individual atoms through compartmentalized metabolic networks in a wide range of experimental systems, including human subjects. MS offers a wide range of instrumental capabilities involving different levels of initial capital outlay and operating costs, ranging from gas-chromatography (GC) MS that is affordable by many individual laboratories to the HIT-supporting Fourier-transform (FT) class of MS that rivals NMR in cost and infrastructure support. This chapter focuses on sample preparation, instrument, and data processing procedures for these two extremes of MS instrumentation used in SIRM. PMID:25270929

  12. Intraoperative beta probe: A device for detecting tissue labeled with positron or electron emitting isotopes during surgery

    SciTech Connect

    Daghighian, F. ); Mazziotta, J.C. ); Hoffman, E.J. ); Shenderov, P.; Eshaghian, B. ); Siegel, S.; Phelps, M.E. )

    1994-01-01

    An intraoperative beta probe was designed, built, and tested for detection of radio-labeled malignant tissues that has the advantage of being selectively sensitive to beta while insensitive to gamma radiation. Since beta radiation (electrons or positrons) has a short range in tissue, this probe is ideal for detecting tracers in tumors at the surface of the surgical field. This probe contains a plastic scintillation detector sensitive to beta rays and to a lesser degree some background gamma rays. A second detector counts spurious gamma rays and allows for their subtraction from the activity measured by the first detector. Sensitivity of the dual probe for I-131 and F-18 was measured to be 108 counts/s/kBq (4000 counts/s/[mu]Ci). The dual-detector probe faithfully measured the 10:1 tumor'' to background ratio of radioactivity concentrations in a simulated environment of a tumor in the presence of intense background 511 keV photons. In another phantom experiment, simulating abdominal tumor deposits with various realistic I-131 radioactive concentrations, the probe was able to accurately identify tumors of approximately 50 mg with a tumor/normal radioactivity concentration of 3/1 in 10 s.

  13. Identification of potential genomic biomarkers of hepatotoxicity caused by reactive metabolites of N-methylformamide: Application of stable isotope labeled compounds in toxicogenomic studies.

    PubMed

    Mutlib, Abdul; Jiang, Ping; Atherton, Jim; Obert, Leslie; Kostrubsky, Seva; Madore, Steven; Nelson, Sidney

    2006-10-01

    The inability to predict if a metabolically bioactivated compound will cause toxicity in later stages of drug development or post-marketing is of serious concern. One approach for improving the predictive success of compound toxicity has been to compare the gene expression profile in preclinical models dosed with novel compounds to a gene expression database generated from compounds with known toxicity. While this guilt-by-association approach can be useful, it is often difficult to elucidate gene expression changes that may be related to the generation of reactive metabolites. In an effort to address this issue, we compared the gene expression profiles obtained from animals treated with a soft-electrophile-producing hepatotoxic compound against corresponding deuterium labeled analogues resistant to metabolic processing. Our aim was to identify a subset of potential biomarker genes for hepatotoxicity caused by soft-electrophile-producing compounds. The current study utilized a known hepatotoxic compound N-methylformamide (NMF) and its two analogues labeled with deuterium at different positions to block metabolic oxidation at the formyl (d(1)) and methyl (d(3)) moieties. Groups of mice were dosed with each compound, and their livers were harvested at different time intervals. RNA was prepared and analyzed on Affymetrix GeneChip arrays. RNA transcripts showing statistically significant changes were identified, and selected changes were confirmed using TaqMan RT-PCR. Serum clinical chemistry and histopathologic evaluations were performed on selected samples as well. The data set generated from the different groups of animals enabled us to determine which gene expression changes were attributed to the bioactivating pathway. We were able to selectively modulate the metabolism of NMF by labeling various positions of the molecule with a stable isotope, allowing us to monitor gene changes specifically due to a particular metabolic pathway. Two groups of genes were identified, which were associated with the metabolism of a certain part of the NMF molecule. The metabolic pathway leading to the production of reactive methyl isocyanate resulted in distinct expression patterns that correlated with histopathologic findings. There was a clear correlation between the expression of certain genes involved in the cell cycle/apoptosis and inflammatory pathways and the presence of reactive metabolite. These genes may serve as potential genomic biomarkers of hepatotoxicity induced by soft-electrophile-producing compounds. However, the robustness of these potential genomic biomarkers will need to be validated using other hepatotoxicants (both soft- and hard-electrophile-producing agents) and compounds known to cause idiosyncratic liver toxicity before being adopted into the drug discovery screening process. PMID:17040096

  14. 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

    Svensson, Jan; Karolinska Institutet, Department of Clinical Sciences, Danderyd Hospital, SE-182 88 Stockholm ; Bergman, Ann-Charlotte; Adamson, Ulf; Blombaeck, Margareta; Wallen, Hakan; Joerneskog, Gun

    2012-05-04

    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.

  15. Efficient sampling of early signal arrival for estimation of perfusion and transit time in whole-brain arterial spin labeling.

    PubMed

    Lee, Wayne; Janik, Rafal; Scouten, Amy; Stefanovic, Bojana; Sled, John G

    2012-07-01

    Arterial spin labeling can be used to measure both cerebral perfusion and arterial transit time. However, accurate estimation of these parameters requires adequate temporal sampling of the arterial spin labeling difference signal. In whole-brain multislice acquisitions, two factors reduce the accuracy of the parameter estimates: saturation of labeled blood in transit and inadequate sampling of early difference signal in superior slices. Label saturation arises when slices are acquired inferior-to-superior such that slice selection in proximal slices spoils the label for a distal slice. Inadequate sampling arises when the time spent acquiring inferior slices is too long to allow early sampling of the difference signal in superior slices. A novel approach to multislice imaging is proposed to address these two issues. In round-robin arterial spin labeling, slices are acquired in a different order after every pair of control-label acquisitions. Round-robin arterial spin labeling enables the acquisitions of all slices across the same range of postlabel delays in a descending superior-to-inferior order. This eliminates the temporal sampling problem and greatly reduces label saturation. Arterial transit time estimates obtained for the whole brain with round-robin arterial spin labeling show better agreement with a single-slice acquisition than do conventional multislice acquisitions. PMID:22189961

  16. Biosynthesis of 15N-labeled cylindrospermopsin and its application as internal standard in stable isotope dilution analysis.

    PubMed

    Kittler, Katrin; Hoffmann, Holger; Lindemann, Franziska; Koch, Matthias; Rohn, Sascha; Maul, Ronald

    2014-09-01

    Cylindrospermopsin (CYN) is a cyanobacterial toxin associated with human and animal poisonings. Due to its toxicity in combination with its widespread occurrence, the development of reliable methods for selective, sensitive detection and accurate quantification is mandatory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis using stable isotope dilution analysis (SIDA) represents an ideal tool for this purpose. U-[(15)N5]-CYN was synthesized by culturing Aphanizomenon flos-aquae in Na(15)NO3-containing cyanobacteria growth medium followed by a cleanup using graphitized carbon black columns and mass spectrometric characterization. Subsequently, a SIDA-LC-MS/MS method for the quantification of CYN in freshwater and Brassica matrices was developed showing satisfactory performance data. The recovery ranged between 98 and 103%; the limit of quantification was 15ng/L in freshwater and 50?g/kg dry weight in Brassica samples. The novel SIDA was applied for CYN determination in real freshwater samples as well as in kale and in vegetable mustard exposed to toxin-containing irrigation water. Two of the freshwater samples taken from German lakes were found to be CYN-contaminated above limit of quantification (17.9 and 60.8ng/L). CYN is systemically available to the examined vegetable species after exposure of the rootstock leading to CYN mass fractions in kale and vegetable mustard leaves of 15.0?g/kg fresh weight and 23.9?g/kg fresh weight, respectively. CYN measurements in both matrices are exemplary for the versatile applicability of the developed method in environmental analysis. PMID:25064600

  17. Comprehensive and highly sensitive urinary steroid hormone profiling method based on stable isotope-labeling liquid chromatography-mass spectrometry.

    PubMed

    Dai, Weidong; Huang, Qiang; Yin, Peiyuan; Li, Jia; Zhou, Jia; Kong, Hongwei; Zhao, Chunxia; Lu, Xin; Xu, Guowang

    2012-12-01

    Steroid hormones are crucial substances that mediate a wide range of vital physiological functions of the body. Determination of the levels of steroid hormones plays an important role in understanding the mechanism of the steroid hormone-related diseases. In this study, we present a novel targeted metabolic profiling method based on the introduction of an easily protonated stable isotope tag to a hydroxyl-containing steroid hormone with a synthesized derivatization reagent, deuterium 4-(dimethylamino)-benzoic acid (d(4)-DMBA), and liquid chromatography-mass spectrometry (LC-MS). Different from other reported derivatization reagents that have been used to enhance the sensitivities for estrogens or androgens, our method is comprehensive with the capability of covering hydroxyl-containing androgens, estrogens, corticoids, and progestogens. Furthermore, the nonderivatized steroid hormones (e.g., 17α-hydroxyprogesterone, progesterone, and androstenedione) were not destroyed during the derivatization process, and their levels could still be obtained in one LC-MS run. We were able to detect 24 steroid hormones at subng/mL levels (the lower limit of detection could reach 5 pg/mL for estrone and 16α-hydroxy estrone, which is equivalent to 0.1 pg on column) with maximum sensitivity enhancement factors of more than 10(3)- to 10(4)-fold after derivatization. The method was successfully applied to the measurement of free (unconjugated) steroid hormones in urine samples of males, females, and pregnant women. Because the significant role the steroid hormone pathway plays in humans, a comprehensive, sensitive, specific, and accurate method for profiling the steroid hormone metabolome shall offer new insights into hormone-related diseases. PMID:23110480

  18. Characterization and quantification of histidine degradation in therapeutic protein formulations by size exclusion-hydrophilic interaction two dimensional-liquid chromatography with stable-isotope labeling mass spectrometry.

    PubMed

    Wang, Chunlei; Chen, Sike; Brailsford, John A; Yamniuk, Aaron P; Tymiak, Adrienne A; Zhang, Yingru

    2015-12-24

    Two dimensional liquid chromatography (2D-LC) coupling size exclusion (SEC) and hydrophilic interaction chromatography (HILIC) is demonstrated as a useful tool to study polar excipients, such as histidine and its degradant, in protein formulation samples. The SEC-HILIC setup successfully removed interferences from complex sample matrices and enabled accurate mass measurement of the histidine degradation product, which was then determined to be trans-urocanic acid. Because the SEC effluent is a strong solvent for the second dimension HILIC, experimental parameters needed to be carefully chosen, i.e., small transferring loop, fast gradient at high flow rates for the second dimension gradient, in order to mitigate the solvent mismatch and to ensure good peak shapes for HILIC separations. In addition, the generation of trans-urocanic acid was quantified by single heart-cutting SEC-HILIC 2D-LC combined with stable-isotope labeling mass spectrometry. Compared with existing 2D quantification methods, the proposed approach is fast, insensitive to solvent mismatch between dimensions, and tolerant of small retention time shifts in the first dimension. Finally, the first dimension diode array detector was found to be a potential degradation source for photolabile analytes such as trans-urocanic acid. PMID:26674608

  19. Analysis of the membrane proteome of ciprofloxacin-resistant macrophages by stable isotope labeling with amino acids in cell culture (SILAC).

    PubMed

    Caceres, Nancy E; Aerts, Maarten; Marquez, Batrice; Mingeot-Leclercq, Marie-Paule; Tulkens, Paul M; Devreese, Bart; Van Bambeke, Franoise

    2013-01-01

    Overexpression of multidrug transporters is a well-established mechanism of resistance to chemotherapy, but other changes may be co-selected upon exposure to drugs that contribute to resistance. Using a model of J774 macrophages made resistant to the fluoroquinolone antibiotic ciprofloxacin and comparing it with the wild-type parent cell line, we performed a quantitative proteomic analysis using the stable isotope labeling with amino acids in cell culture technology coupled with liquid chromatography electrospray ionization Fourier transform tandem mass spectrometry (LC-ESI-FT-MS/MS) on 2 samples enriched in membrane proteins (fractions F1 and F2 collected from discontinuous sucrose gradient). Nine hundred proteins were identified with at least 3 unique peptides in these 2 pooled fractions among which 61 (F1) and 69 (F2) showed a significantly modified abundance among the 2 cell lines. The multidrug resistance associated protein Abcc4, known as the ciprofloxacin efflux transporter in these cells, was the most upregulated, together with Dnajc3, a protein encoded by a gene located downstream of Abcc4. The other modulated proteins are involved in transport functions, cell adhesion and cytoskeleton organization, immune response, signal transduction, and metabolism. This indicates that the antibiotic ciprofloxacin is able to trigger a pleiotropic adaptative response in macrophages that includes the overexpression of its efflux transporter. PMID:23505477

  20. Quantitative proteomics using stable isotope labeling with amino acids in cell culture reveals protein and pathway regulation in porcine circovirus type 2 infected PK-15 cells.

    PubMed

    Fan, Huiying; Ye, Yu; Luo, Yongwen; Tong, Tiezhu; Yan, Guangrong; Liao, Ming

    2012-02-01

    The infection of host cells by porcine circovirus type 2 (PCV2) leads to extensive modulation of the gene expression levels of target cells. To uncover the pathogenesis and virus-host interactions of PCV2, a quantitative proteomic study using the stable isotope labeling with amino acids in cell culture (SILAC), coupled with mass spectrometry, was performed on PCV2-infected PK-15 cells. The SILAC-based approach identified 1341 proteins, 163 of which showed significant change in level at 72 h after infection (79 up-regulated and 84 down-regulated). The modulated proteins included a number of proteins involved in substrate transport, cytoskeletal changes, and the stress response. Changes in the expression levels of selected proteins were verified by Western blot analysis. Ingenuity Pathway Analysis was used to reveal protein and interactive pathway regulation in response to PCV2 infection. Functional network and pathway analyses could provide insights into the complexity and dynamics of virus-host cell interactions and may accelerate our understanding of the mechanisms of PCV2 infection. PMID:22148862

  1. Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

    PubMed

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

    2016-08-01

    Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods. PMID:26988485

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

    PubMed

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

    2012-02-15

    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

  3. Identifying natural substrates for dipeptidyl peptidases 8 and 9 using terminal amine isotopic labeling of substrates (TAILS) reveals in vivo roles in cellular homeostasis and energy metabolism.

    PubMed

    Wilson, Claire H; Indarto, Dono; Doucet, Alain; Pogson, Lisa D; Pitman, Melissa R; McNicholas, Kym; Menz, R Ian; Overall, Christopher M; Abbott, Catherine A

    2013-05-17

    Dipeptidyl peptidases (DP) 8 and 9 are homologous, cytoplasmic N-terminal post-proline-cleaving enzymes that are anti-targets for the development of DP4 (DPPIV/CD26) inhibitors for treating type II diabetes. To date, DP8 and DP9 have been implicated in immune responses and cancer biology, but their pathophysiological functions and substrate repertoire remain unknown. This study utilizes terminal amine isotopic labeling of substrates (TAILS), an N-terminal positional proteomic approach, for the discovery of in vivo DP8 and DP9 substrates. In vivo roles for DP8 and DP9 in cellular metabolism and homeostasis were revealed via the identification of more than 29 candidate natural substrates and pathways affected by DP8/DP9 overexpression. Cleavage of 14 substrates was investigated in vitro; 9/14 substrates for both DP8 and DP9 were confirmed by MALDI-TOF MS, including two of high confidence, calreticulin and adenylate kinase 2. Adenylate kinase 2 plays key roles in cellular energy and nucleotide homeostasis. These results demonstrate remarkable in vivo substrate overlap between DP8/DP9, suggesting compensatory roles for these enzymes. This work provides the first global investigation into DP8 and DP9 substrates, providing a number of leads for future investigations into the biological roles and significance of DP8 and DP9 in human health and disease. PMID:23519473

  4. Bio-generation of stable isotope-labeled internal standards for absolute and relative quantitation of phase II drug metabolites in plasma samples using LC-MS/MS.

    PubMed

    Li, Pei; Li, Zi; Beck, Wayne D; Callahan, Patrick M; Terry, Alvin V; Bar-Peled, Maor; Bartlett, Michael G

    2015-05-01

    Quantification of drug metabolites in biological samples has been of great interest in current pharmaceutical research, since metabolite concentrations and pharmacokinetics can contribute to a better understanding of the toxicity of drug candidates. Two major categories of Phase II metabolites, glucuronide conjugates and glutathione conjugates, may cause significant drug toxicity and therefore require close monitoring at early stages of drug development. In order to achieve high precision, accuracy, and robustness, stable isotope-labeled (SIL) internal standards (IS) are widely used in quantitative bioanalytical methods using liquid chromatography and tandem mass spectrometry (LC-MS/MS), due to their capability of compensating for matrix effects, extraction variations and instrument response fluctuations. However, chemical synthesis of SIL analogues of Phase II metabolites can often be very difficult and require extensive exploratory research, leading to higher cost and significant delays in drug research and development. To overcome these challenges, we have developed a generic method which can synthesize SIL analogues of Phase II metabolites from more available SIL parent drugs or SIL conjugation co-factors, using in vitro biotransformation. This methodology was successfully applied to the bio-generation of SIL glucuronide conjugates and glutathione conjugates. The method demonstrated satisfactory performance in both absolute quantitation and assessment of relative exposure coverage across species in safety tests of drug metabolites (MIST). This generic technique can be utilized as an alternative to chemical synthesis and potentially save time and cost for drug research and development. PMID:25804729

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

    PubMed Central

    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

    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

  6. Preliminary quantitative profile of differential protein expression between rat L6 myoblasts and myotubes by stable isotope labeling with amino acids in cell culture.

    PubMed

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

    2009-03-01

    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 muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence 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 extracellular 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 summary, our results provide new clues for understanding the mechanism of myogenesis. PMID:19253283

  7. Beta-lactamase-catalyzed aminolysis of depsipeptides: Proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping

    SciTech Connect

    Pazhanisamy, S.; Pratt, R.F. )

    1989-08-22

    The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-(((phenylacetyl)glycyl)oxy)benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.

  8. Novel tracer method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies.

    PubMed

    Wu, Dianming; Kampf, Christopher J; Pschl, Ulrich; Oswald, Robert; Cui, Junfang; Ermel, Michael; Hu, Chunsheng; Trebs, Ivonne; Srgel, Matthias

    2014-07-15

    Gaseous nitrous acid (HONO), the protonated form of nitrite, contributes up to ?60% to the primary formation of hydroxyl radical (OH), which is a key oxidant in the degradation of most air pollutants. Field measurements and modeling studies indicate a large unknown source of HONO during daytime. Here, we developed a new tracer method based on gas-phase stripping-derivatization coupled to liquid chromatography-mass spectrometry (LC-MS) to measure the 15N relative exceedance, ?(15N), of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye, purified by solid phase extraction (SPE), and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the optimal working range of ?(15N)=0.2-0.5, the relative standard deviation of ?(15N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO15NO emissions from soil in a dynamic chamber with and without spiking 15) labeled urea. The identification of HO15NO from soil with 15N urea addition confirmed biogenic emissions of HONO from soil. The method enables a new approach of studying the formation pathways of HONO and its role for atmospheric chemistry (e.g., ozone formation) and environmental tracer studies on the formation and conversion of gaseous HONO or aqueous NO2- as part of the biogeochemical nitrogen cycle, e.g., in the investigation of fertilization effects on soil HONO emissions and microbiological conversion of NO2- in the hydrosphere. PMID:24954648

  9. Osmium isotopes suggest fast and efficient mixing in the oceanic upper mantle.

    NASA Astrophysics Data System (ADS)

    Bizimis, Michael; Salters, Vincent

    2010-05-01

    The depleted upper mantle (DUM; the source of MORB) is thought to represent the complementary reservoir of continental crust extraction. Previous studies have calculated the "average" DUM composition based on the geochemistry of MORB. However the Nd isotope compositions of abyssal peridotites have been shown to extend to more depleted compositions than associated MORB. While this argues for the presence of both relatively depleted and enriched material within the upper mantle, the extent of compositional variability, length scales of heterogeneity and timescales of mixing in the upper mantle are not well constrained. Model calculations show that 2Ga is a reasonable mean age of depletion for DUM while Hf - Nd isotopes show the persistence of a depleted terrestrial reservoir by the early Archean (3.5-3.8Ga). U/Pb zircon ages of crustal rocks show three distinct peaks at 1.2, 1.9, and 2.7Ga and these are thought to represent the ages of three major crustal growth events. A fundamental question therefore is whether the present day upper mantle retains a memory of multiple ancient depletion events, or has been effectively homogenized. This has important implications for the nature of convection and time scales of survival of heterogeneities in the upper mantle. Here we compare published Os isotope data from abyssal peridotites and ophiolitic Os-Ir alloys with new data from Hawaiian spinel peridotite xenoliths. The Re-Os isotope system has been shown to yield useful depletion age information in peridotites, so we use it here to investigate the distribution of Re-depletion ages (TRD) in these mantle samples as a proxy for the variability of DUM. The probability density functions (PDF) of TRD from osmiridiums, abyssal and Hawaiian peridotites are all remarkably similar and show a distinct peak at 1.2-1.3 Ga (errors for TRD are set at 0.2Ga to suppress statistically spurious age peaks). The Hawaiian peridotites further show a distinct peak at 1.9-2Ga, but no oceanic mantle samples with TRD older than 2Ga have been reported. The TRD age peaks overlap with two major crustal building events recorded in the U/Pb crustal zircon ages. Therefore, peridotites from the convecting upper mantle can retain some memory of ancient depletion events, and these depletions are perhaps linked to major crustal building or large-scale mantle melting events. In the case of the Hawaiian peridotites, an ancient depletion event is further supported by some extremely radiogenic Hf isotope compositions. However, the vast majority of oceanic mantle samples show a narrow rage of Os isotope compositions (187Os/188Os = 0.123-0.126) with TRDs at 300-600 Ma. If the upper mantle has been produced continuously (or episodically) since at least the early Archean, it is then surprising that almost all oceanic mantle samples record such young depletion ages. We suggest that convective mixing in the mantle is rigorous enough that effectively re-homogenizes and resets the Os isotope composition of previously depleted peridotites within short time scales (<500Ma). Similarly recent ages have been derived from modeling the Sr, Nd, Hf, Pb isotopic composition of MORBs. This resetting and homogenization can be due to re-equilibration of depleted mantle with enriched components, e.g. recycled basaltic crust or more fertile mantle. Ancient depletion events are only effectively preserved in the sublithospheric mantle samples (e.g. Kaapval, Slave, Wyoming cratons) because they remain isolated from the convective mantle.

  10. Plasma-treated polystyrene film that enhances binding efficiency for sensitive and label-free protein biosensing

    NASA Astrophysics Data System (ADS)

    Guo, Bihong; Li, Shaopeng; Song, Lusheng; Yang, Mo; Zhou, Wenfei; Tyagi, Deependra; Zhu, Jinsong

    2015-08-01

    A plasma-treated ultrathin polystyrene (PS) film surface was explored as a simple, robust, and low-cost surface chemistry solution for protein biosensing applications. This surface could dramatically improve the binding efficiency of the protein-protein interactions, which is defined as the binding signal per immobilized ligand. The PS-modified protein biosensor was readily fabricated by spin coating and plasma treatment. Various parameters for fabrication, including the concentration of the PS solution, rate of spin coating, and duration of plasma treatment, were systematically optimized based on the improvement of fluorescence signal yielded by the microfluidic network-aided fluorescence immunoassay. The performance of the label-free protein detection on the optimized surfaces was further evaluated by surface plasmon resonance imaging (SPRi). PS surfaces with optimal fabrication parameters exhibited up to an 620% enhancement of the protein binding response and approximately 210% of the protein binding per immobilized protein ligand compared with a self-assembled monolayer (SAM) surface of 11-mercapto undecanoic acid (MUA). The relationship between the fabrication parameters used and changes to the surface chemistry and the morphological properties were characterized with atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). It was revealed that the morphological changes observed in the plasma-treated PS film were the dominant factor for the improvement of the protein bioassay performance, rather than the chemical changes.

  11. Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

    PubMed Central

    Starkey, Jonathan M.; Zhao, Yingxin; Sadygov, Rovshan G.; Haidacher, Sigmund J.; LeJeune, Wanda S.; Dey, Nilay; Luxon, Bruce A.; Kane, Maureen A.; Napoli, Joseph L.; Denner, Larry; Tilton, Ronald G.

    2010-01-01

    Background Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. Methodology/Principal Findings Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, 18O- and 16O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ?1.5 and p?0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPAR?/? mRNA. Conclusions/Significance Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in type 2 diabetic renal disease. Our observations provide novel insights into potential links between altered lipid metabolism and other gene networks controlled by retinoic acid in the diabetic kidney, and demonstrate the utility of using systems biology to gain new insights into diabetic nephropathy. PMID:20559430

  12. A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling.

    PubMed

    Lopez, Juan; Ahuja, Puneet; Gerard, Melanie; Wieruszeski, Jean-Michel; Lippens, Guy

    2013-11-01

    We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional (15)N-(1)H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a H?-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for ?-synuclein. PMID:24018100

  13. Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimers ?-amyloid peptide for NMR-based structural analysis

    PubMed Central

    Long, Fei; Cho, Wonhwa; Ishii, Yoshitaka

    2011-01-01

    Amyloid fibrils of Alzheimers ?-amyloid peptide (A?) are a primary component of amyloid plaques, a hallmark of Alzheimers disease (AD). Enormous attention has been given to the structural features and functions of A? in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue A?(140), the most abundant A? in brains, for structural studies by NMR spectroscopy. Over-expression of A?(140) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR?) in E. Coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-A? fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ~7 mg and ~1.5 mg of unlabeled A?(140) and uniformly 15N- and/or 13C-protein A?(140) from 1 L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled A? and 1H/15N HSQC solution NMR spectrum of the obtained 15N-labeled A? in the monomeric form confirmed the expression of native A?(140). It was also confirmed by electron micrography and solid-state NMR analysis that the purified A?(140) self-assembles into ?-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant A?(140) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are 13C- and 15N-labeled for NMR experiments. PMID:21640828

  14. Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis.

    PubMed

    Long, Fei; Cho, Wonhwa; Ishii, Yoshitaka

    2011-09-01

    Amyloid fibrils of Alzheimer's ?-amyloid peptide (A?) are a primary component of amyloid plaques, a hallmark of Alzheimer's disease (AD). Enormous attention has been given to the structural features and functions of A? in amyloid fibrils and other type of aggregates in associated with development of AD. This report describes an efficient protocol to express and purify high-quality 40-residue A?(1-40), the most abundant A? in brains, for structural studies by NMR spectroscopy. Over-expression of A?(1-40) with glutathione S-transferase (GST) tag connected by a Factor Xa recognition site (IEGR(?)) in Escherichia coli resulted in the formation of insoluble inclusion bodies even with the soluble GST tag. This problem was resolved by efficient recovery of the GST-A? fusion protein from the inclusion bodies using 0.5% (w/v) sodium lauroyl sarcosinate as solubilizing agent and subsequent purification by affinity chromatography using a glutathione agarose column. The removal of the GST tag by Factor Xa enzymatic cleavage and purification by HPLC yielded as much as ?7 mg and ?1.5mg of unlabeled A?(1-40) and uniformly (15)N- and/or (13)C-protein A?(1-40) from 1L of the cell culture, respectively. Mass spectroscopy of unlabeled and labeled A? and (1)H/(15)N HSQC solution NMR spectrum of the obtained (15)N-labeled A? in the monomeric form confirmed the expression of native A?(1-40). It was also confirmed by electron micrography and solid-state NMR analysis that the purified A?(1-40) self-assembles into ?-sheet rich amyloid fibrils. To the best of our knowledge, our protocol offers the highest yields among published protocols for production of recombinant A?(1-40) samples that are amendable for an NMR-based structural analysis. The protocol may be applied to efficient preparation of other amyloid-forming proteins and peptides that are (13)C- and (15)N-labeled for NMR experiments. PMID:21640828

  15. Comparison of Test Procedures and Energy Efficiency Criteria in Selected International Standards & Labeling Programs for Copy Machines, External Power Supplies, LED Displays, Residential Gas Cooktops and Televisions

    SciTech Connect

    Zheng, Nina; Zhou, Nan; Fridley, David

    2012-03-01

    This report presents a technical review of international minimum energy performance standards (MEPS), voluntary and mandatory energy efficiency labels and test procedures for five products being considered for new or revised MEPS in China: copy machines, external power supply, LED displays, residential gas cooktops and flat-screen televisions. For each product, an overview of the scope of existing international standards and labeling programs, energy values and energy performance metrics and description and detailed summary table of criteria and procedures in major test standards are presented.

  16. Mass spectrometric analysis of free fatty acids in infant milk powders by frozen pretreatment coupled with isotope-labeling derivatization.

    PubMed

    Zhou, Tianxiao; Leng, Jiapeng; Peng, Yaoshan; Zhang, Lei; Guo, Yinlong

    2016-03-01

    In combination with frozen pretreatment and carboxyl group derivatization, a novel workflow was developed for the determination of free fatty acids in milk powder. The workflow showed a significantly enhanced performance for comprehensive free fatty acid analysis owing to a highly efficient frozen extraction method. In addition, the advantages of the workflow also involved high sensitivity and great tolerance to a complex matrix. Characteristic fragment ions of derivatization reagents also provide clear evidence for the qualitative analysis of free fatty acids. Fourteen types of free fatty acids in a number of domestic and overseas infant milk powders have been successfully detected. The content of free fatty acids in the different samples was different, which probably indicates the diverse quality of infant milk powder. The workflow is expected to be a pragmatic tool for the analysis of free fatty acids in intricate matrices. PMID:26718016

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

    PubMed Central

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

    2013-01-01

    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- (50100 ?g/mL nanotube concentration) and time- (1248 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 110 ?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 (108109 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

  18. Uptake and distribution of soil applied zinc by citrus trees-addressing fertilizer use efficiency with 68Zn labeling.

    PubMed

    Hippler, Franz Walter Rieger; Boaretto, Rodrigo Marcelli; Quaggio, Jos Antnio; Boaretto, Antonio Enedi; Abreu-Junior, Cassio Hamilton; Mattos, Dirceu

    2015-01-01

    The zinc (Zn) supply increases the fruit yield of Citrus trees that are grown, especially in the highly weathered soils of the tropics due to the inherently low nutrient availability in the soil solution. Leaf sprays containing micronutrients are commonly applied to orchards, even though the nutrient supply via soil could be of practical value. This study aimed to evaluate the effect of Zn fertilizers that are applied to the soil surface on absorption and partitioning of the nutrient by citrus trees. A greenhouse experiment was conducted with one-year-old sweet orange trees. The plants were grown in soils with different textures (18.1 or 64.4% clay) that received 1.8 g Zn per plant, in the form of either ZnO or ZnSO4 enriched with the stable isotope 68Zn. Zinc fertilization increased the availability of the nutrient in the soil and the content in the orange trees. Greater responses were obtained when ZnSO4 was applied to the sandy loam soil due to its lower specific metal adsorption compared to that of the clay soil. The trunk and branches accumulated the most fertilizer-derived Zn (Zndff) and thus represent the major reserve organ for this nutrient in the plant. The trees recovered up to 4% of the applied Zndff. Despite this relative low recovery, the Zn requirement of the trees was met with the selected treatment based on the total leaf nutrient content and increased Cu/Zn-SOD activity in the leaves. We conclude that the efficiency of Zn fertilizers depends on the fertilizer source and the soil texture, which must be taken into account by guidelines for fruit crop fertilization via soil, in substitution or complementation of traditional foliar sprays. PMID:25751056

  19. Uptake and Distribution of Soil Applied Zinc by Citrus Trees—Addressing Fertilizer Use Efficiency with 68Zn Labeling

    PubMed Central

    Hippler, Franz Walter Rieger; Boaretto, Rodrigo Marcelli; Quaggio, José Antônio; Boaretto, Antonio Enedi; Abreu-Junior, Cassio Hamilton; Mattos, Dirceu

    2015-01-01

    The zinc (Zn) supply increases the fruit yield of Citrus trees that are grown, especially in the highly weathered soils of the tropics due to the inherently low nutrient availability in the soil solution. Leaf sprays containing micronutrients are commonly applied to orchards, even though the nutrient supply via soil could be of practical value. This study aimed to evaluate the effect of Zn fertilizers that are applied to the soil surface on absorption and partitioning of the nutrient by citrus trees. A greenhouse experiment was conducted with one-year-old sweet orange trees. The plants were grown in soils with different textures (18.1 or 64.4% clay) that received 1.8 g Zn per plant, in the form of either ZnO or ZnSO4 enriched with the stable isotope 68Zn. Zinc fertilization increased the availability of the nutrient in the soil and the content in the orange trees. Greater responses were obtained when ZnSO4 was applied to the sandy loam soil due to its lower specific metal adsorption compared to that of the clay soil. The trunk and branches accumulated the most fertilizer-derived Zn (Zndff) and thus represent the major reserve organ for this nutrient in the plant. The trees recovered up to 4% of the applied Zndff. Despite this relative low recovery, the Zn requirement of the trees was met with the selected treatment based on the total leaf nutrient content and increased Cu/Zn-SOD activity in the leaves. We conclude that the efficiency of Zn fertilizers depends on the fertilizer source and the soil texture, which must be taken into account by guidelines for fruit crop fertilization via soil, in substitution or complementation of traditional foliar sprays. PMID:25751056

  20. Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)

    PubMed Central

    Konvalinka, Ana; Zhou, Joyce; Dimitromanolakis, Apostolos; Drabovich, Andrei P.; Fang, Fei; Gurley, Susan; Coffman, Thomas; John, Rohan; Zhang, Shao-Ling; Diamandis, Eleftherios P.; Scholey, James W.

    2013-01-01

    Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models. PMID:23846697

  1. Systematic Optimization of C-Terminal Amine-Based Isotope Labeling of Substrates Approach for Deep Screening of C-Terminome.

    PubMed

    Zhang, Yang; He, Quanze; Ye, Juanying; Li, Yanhong; Huang, Lin; Li, Qingqing; Huang, Jingnan; Lu, Jianan; Zhang, Xumin

    2015-10-20

    It is well-known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structures and understanding protein functions. Although many efforts have been made in the field during the latest 2 decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we report an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in an Escherichia coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to the number of lysine in peptides, more reproducible and with higher MASCOT scores. Moreover, the introduction of the Single-Charge Ion Inclusion (SCII) method to alleviate the charge deficiency of small peptides allowed an additional 26% increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. coli in a triplicate experiments using 80 ?g each. Our optimized method would benefit the deep screening of C-terminome and possibly help discover some novel C-terminal modifications. Data are available via ProteomeXchange with identifier PXD002409. PMID:26361894

  2. An isotope-labeled chemical derivatization method for the quantitation of short-chain fatty acids in human feces by liquid chromatography-tandem mass spectrometry.

    PubMed

    Han, Jun; Lin, Karen; Sequeira, Carita; Borchers, Christoph H

    2015-01-01

    Short-chain fatty acids (SCFAs) are produced by anaerobic gut microbiota in the large bowel. Qualitative and quantitative measurements of SCFAs in the intestinal tract and the fecal samples are important to understand the complex interplay between diet, gut microbiota and host metabolism homeostasis. To develop a new LC-MS/MS method for sensitive and reliable analysis of SCFAs in human fecal samples, 3-nitrophenylhydrazine (3NPH) was employed for pre-analytical derivatization to convert ten C2-C6 SCFAs to their 3-nitrophenylhydrazones under a single set of optimized reaction conditions and without the need of reaction quenching. The derivatives showed excellent in-solution chemical stability. They were separated on a reversed-phase C18 column and quantitated by negative-ion electrospray ionization - multiple-reaction monitoring (MRM)/MS. To achieve accurate quantitation, the stable isotope-labeled versions of the derivatives were synthesized in a single reaction vessel from (13)C6-3NPH, and were used as internal standard to compensate for the matrix effects in ESI. Method validation showed on-column limits of detection and quantitation over the range from low to high femtomoles for the ten SCFAs, and the intra-day and inter-day precision for determination of nine of the ten SCFAs in human fecal samples was ≤8.8% (n=6). The quantitation accuracy ranged from 93.1% to 108.4% (CVs≤4.6%, n=6). This method was used to determine the SCFA concentrations and compositions in six human fecal samples. One of the six samples, which was collected from a clinically diagnosed type 2 diabetes patient showed a significantly high molar ratio of branch-chain SCFAs to straight-chain SCFAs than the others. In summary, this work provides a new LC-MS/MS method for precise and accurate quantitation of SCFAs in human feces. PMID:25479871

  3. Atmospheric CO2 level affects plants' carbon use efficiency: insights from a 13C labeling experiment on sunflower stands

    NASA Astrophysics Data System (ADS)

    Gong, Xiaoying; Schäufele, Rudi; Schnyder, Hans

    2015-04-01

    The increase of atmospheric CO2 concentration has been shown to stimulate plant photosynthesis and (to a lesser extent) growth, thereby acting as a possible sink for the additional atmospheric CO2. However, this effect is dependent on the efficiency with which plants convert atmospheric carbon into biomass carbon, since a considerable proportion of assimilated carbon is returned to the atmosphere via plant respiration. As a core parameter for carbon cycling, carbon use efficiency of plants (CUE, the ratio of net primary production to gross primary production) quantifies the proportion of assimilated carbon that is incorporated into plant biomass. CUE has rarely been assessed based on measurements of complete carbon balance, due to methodological difficulties in measuring respiration rate of plants in light. Moreover, foliar respiration is known to be inhibited in light, thus foliar respiration rate is generally lower in light than in dark. However, this phenomenon, termed as inhibition of respiration in light (IRL), has rarely been assessed at the stand-scale and been incorporated into the calculation of CUE. Therefore, how CUE responses to atmospheric CO2 levels is still not clear. We studied CUE of sunflower stands grown at sub-ambient CO2 level (200 μmol mol-1) and elevated CO2 level (1000 μmol mol-1) using mesocosm-scale gas exchange facilities which enabled continuous measurements of 13CO2/12CO2 exchange. Appling steady-state 13C labeling, fluxes of respiration and photosynthesis in light were separated, and tracer kinetic in respiration was analyzed. This study provides the first data on CUE at a mesocosm-level including respiration in light in different CO2 environments. We found that CUE of sunflower was lower at an elevated CO2 level than at a sub-ambient CO2 level; and the ignorance of IRL lead to erroneous estimations of CUE. Variation in CUE at atmospheric CO2 levels was attributed to several mechanisms. In this study, CO2 enrichment i) affected the size of respiratory substrate pools and the relative contribution of temporary storage pools and current assimilation pools; ii) affected the extent of inhibition of stands' respiration in light, which was related to leaf-level re-fixation of respired CO2; and iii) influenced the ratio of leaf mass to total plant mass. Our study highlights the necessity of integrating measurement of respiration in light in assessing carbon cycling. If the decrease of CUE by CO2 enrichment is a general response of terrestrial ecosystems, the buffering effect of plants C acquisition to the rise of atmospheric CO2 is lower than estimated so far.

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

    NASA Astrophysics Data System (ADS)

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

    2009-09-01

    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.

  5. Precipitation efficiency derived from isotope ratios in water vapor distinguishes dynamical and microphysical influences on subtropical atmospheric constituents

    NASA Astrophysics Data System (ADS)

    Bailey, A.; Nusbaumer, J.; Noone, D.

    2015-09-01

    With water vapor and clouds expected to effect significant feedbacks on climate, moisture transport through convective processes has important implications for future temperature change. The precipitation efficiency—the ratio of the rates at which precipitation and condensation form (e = P/C)—is useful for characterizing how much boundary layer moisture recycles through precipitation versus mixes into the free troposphere through cloud detrainment. Yet it is a difficult metric to constrain with traditional observational techniques. This analysis characterizes the precipitation efficiency of convection near the Big Island of Hawaii, USA, using a novel tracer: isotope ratios in water vapor. The synoptic circulation patterns associated with high and low precipitation efficiency are identified, and the importance of large-scale dynamics and local convective processes in regulating vertical distributions of atmospheric constituents important for climate is evaluated. The results suggest that high e days are correlated with plume-like transport originating from the relatively clean tropics, while low e days are associated with westerly transport, generated by a branching of the jet stream. Differences in transport pathway clearly modify background concentrations of water vapor and other trace gases measured at Mauna Loa Observatory; however, local convective processes appear to regulate aerosols there. Indeed, differences between observed and simulated diurnal cycles of particle number concentration indicate that precipitation scavenges aerosols and possibly facilitates new particle formation when e is high. As measurements of isotope ratios in water vapor expand across the subtropics, the techniques presented here can further our understanding of how synoptic weather, precipitation processes, and climate feedbacks interrelate.

  6. The Semiquinone at the Qi Site of the bc1 Complex Explored Using HYSCORE Spectroscopy and Specific Isotopic Labeling of Ubiquinone in Rhodobacter sphaeroides via 13C Methionine and Construction of a Methionine Auxotroph

    PubMed Central

    2015-01-01

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe 13C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group 13C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the 13C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of 13C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  7. The semiquinone at the Qi site of the bc1 complex explored using HYSCORE spectroscopy and specific isotopic labeling of ubiquinone in Rhodobacter sphaeroides via (13)C methionine and construction of a methionine auxotroph.

    PubMed

    Hong, Sangjin; de Almeida, Wagner B; Taguchi, Alexander T; Samoilova, Rimma I; Gennis, Robert B; O'Malley, Patrick J; Dikanov, Sergei A; Crofts, Antony R

    2014-09-30

    Specific isotopic labeling at the residue or substituent level extends the scope of different spectroscopic approaches to the atomistic level. Here we describe (13)C isotopic labeling of the methyl and methoxy ring substituents of ubiquinone, achieved through construction of a methionine auxotroph in Rhodobacter sphaeroides strain BC17 supplemented with l-methionine with the side chain methyl group (13)C-labeled. Two-dimensional electron spin echo envelope modulation (HYSCORE) was applied to study the (13)C methyl and methoxy hyperfine couplings in the semiquinone generated in situ at the Qi site of the bc1 complex in its membrane environment. The data were used to characterize the distribution of unpaired spin density and the conformations of the methoxy substituents based on density functional theory calculations of (13)C hyperfine tensors in the semiquinone of the geometry-optimized X-ray structure of the bc1 complex (Protein Data Bank entry 1PP9 ) with the highest available resolution. Comparison with other proteins indicates individual orientations of the methoxy groups in each particular case are always different from the methoxy conformations in the anion radical prepared in a frozen alcohol solution. The protocol used in the generation of the methionine auxotroph is more generally applicable and, because it introduces a gene deletion using a suicide plasmid, can be applied repeatedly. PMID:25184535

  8. StableIsotope Labeling with Amino Acids in Cell Culture (SILAC)-based strategy for proteome-wide thermodynamic analysis of protein-ligand binding interactions.

    PubMed

    Tran, Duc T; Adhikari, Jagat; Fitzgerald, Michael C

    2014-07-01

    Described here is a quantitative mass spectrometry-based proteomics method for the large-scale thermodynamic analysis of protein-ligand binding interactions. The methodology utilizes a chemical modification strategy termed, Stability of Proteins from Rates of Oxidation (SPROX), in combination with a Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) approach to compare the equilibrium folding/unfolding properties of proteins in the absence and presence of target ligands. The method, which is general with respect to ligand, measures the ligand-induced changes in protein stability associated with protein-ligand binding. The methodology is demonstrated in a proof-of-principle study in which the well-characterized protein-drug interaction between cyclosporine A (CsA) and cyclophilin A was successfully analyzed in the context of a yeast cell lysate. A control experiment was also performed to assess the method's false positive rate of ligand discovery, which was found to be on the order of 0.4 - 3.5%. The new method was utilized to characterize the adenosine triphosphate (ATP)-interactome in Saccharomyces cerevisiae using the nonhydrolyzable ATP analog, adenylyl imidodiphosphate (AMP-PNP), and the proteins in a yeast cell lysate. The new methodology enabled the interrogation of 526 yeast proteins for interactions with ATP using 2035 peptide probes. Ultimately, 325 peptide hits from 139 different proteins were identified. Approximately 70% of the hit proteins identified in this work were not previously annotated as ATP binding proteins. However, nearly two-thirds of the newly discovered ATP interacting proteins have known interactions with other nucleotides and co-factors (e.g. NAD and GTP), DNA, and RNA based on GO-term analyses. The current work is the first proteome-wide profile of the yeast ATP-interactome, and it is the largest proteome-wide profile of any ATP-interactome generated, to date, using an energetics-based method. The data is available via ProteomeXchange with identifiers PXD000858, DOI 10.6019/PXD000858, and PXD000860. PMID:24741112

  9. Quantitation of anacetrapib, stable-isotope labeled-anacetrapib (microdose), and four metabolites in human plasma using liquid chromatography tandem mass spectrometry.

    PubMed

    Chavez-Eng, C M; Lutz, R W; Li, H; Goykhman, D; Bateman, K P; Woolf, E

    2016-02-01

    An ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of (4S,5R)-5-[3,5-bis (trifluoromethyl)phenyl]-3-{[4'-fluoro-5'-isopropyl-2'-methoxy-4-(trifluoromethyl)biphenyl-2-yl] methyl}-4-methyl-1,3-oxazolidin-2-one (anacetrapib, I) and [(13)C5(15)N]-anacetrapib, II in human plasma has been developed to support a clinical study to determine the absolute bioavailability of I. The analytes and the stable-isotope labeled internal standard ([(13)C7(15)N(2)H7]-anacetrapib, III) were extracted from 100?L of human plasma by liquid-liquid extraction using 20/80 isopropyl alcohol/hexane (v/v). The chromatographic separation of the analytes was achieved using Waters BEH Shield RP 18 (502.1mm1.7?m) column and mobile phase gradient of 0.1% formic acid in water (Solvent A) and 0.1% formic acid in acetonitrile (Solvent B) at 0.6mL/min flow rate. The MS/MS detection was performed on AB Sciex 5000 or AB 5500 in positive electrospray ionization mode, operated in selected reaction monitoring mode. The assay was validated in the concentration range 1-2000ng/mL for I; and a lower curve range, 0.025-50ng/mL for II. In addition to the absolute bioavailability determination, it was desired to better elucidate the pharmacokinetic behavior of several hydroxylated metabolites of I. Toward this end, two exploratory assays for the hydroxy metabolites of I were qualified in the concentration range 0.5-500ng/mL. All metabolites were separated on a Supelco Ascentis Express Phenyl-Hexyl (502.1mm, 2.7?m) column. Metabolite M4 was analyzed in the negative mode with a mobile phase consisting of a gradient mixture of water (A) and acetonitrile (B). The other three metabolites, M1-M3 were analyzed in the positive mode using a mobile phase gradient of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The assays were utilized to support a clinical study in which a microdosing approach was used to determine the pharmacokinetics of anacetrapib and its metabolites. PMID:26773885

  10. Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor.

    PubMed

    Buffet, Pierre-Emmanuel; Amiard-Triquet, Claude; Dybowska, Agnieszka; Risso-de Faverney, Christine; Guibbolini, Marielle; Valsami-Jones, Eugnia; Mouneyrac, Catherine

    2012-10-01

    Although it is reported that metal and metal oxide nanoparticles, which are among the most rapidly commercialized materials, can cause toxicity to organisms, their fate in the environment and toxicity to marine organisms are not well understood. In this study, we used a stable isotope labelling approach to trace the fate of nanoparticles (NPs) in sediments and also investigated bio-uptake in two estuarine intra-sedimentary invertebrates Scrobicularia plana and Nereis diversicolor. We selected exposure to 3 mg kg(-1) sediment ZnO NPs since this level is a realistic prediction of the environmental concentration in sediments. 67ZnO NPs (DLS: 21-34 nm, positively charged: 31.3 mV) suspensions were synthesised in diethylene glycol (DEG). We explored the fate of 67ZnO NPs in sediment, 67Zn bioaccumulation and the biochemical (biomarkers of defence and damage) and behavioural (burrowing kinetics and feeding rates) biomarkers in both species to 67ZnO NPs and DEG on its own during a 16 d laboratory exposure. After exposure, 67Zn concentrations in sediment showed higher levels in the upper section (1cm: 2.59 mg kg(-1)) decreasing progressively (2 cm: 1.63 mg kg(-1), 3 cm: 0.90 mg kg(-1), 4 cm: 0.67 mg kg(-1)) to a minimum value at the bottom (5 cm: 0.31 mg kg(-1)). 67Zn bioaccumulation was observed in both organisms exposed to 67ZnO NPs in DEG but no major inter-species differences were found. At the biochemical level, 67ZnO NPs exposure significantly induced increased glutathione-S-transferase activity in worms and catalase activity in clams whereas superoxide dismutase activity and thiobarbituric acid reactive substance levels were not affected in any species. Exposure to DEG on its own leads to a significant increase of metallothionein-like protein levels in clams compared with those exposed to 67ZnO NPs or controls. Burrowing behaviour as well as feeding rate were significantly impaired in both species exposed to 67ZnO NPs. Concerning exposure to DEG on its own, burrowing behaviour impairments were also shown in both species and feeding rate was impaired in bivalves. At environmentally realistic concentration of 67ZnO NPs in sediment, there is no strong evidence for a severe nanoparticle effect since most effects were also observed in the presence of DEG alone. PMID:22858103

  11. Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation

    NASA Astrophysics Data System (ADS)

    Hendry, Katharine R.; Robinson, Laura F.; McManus, Jerry F.; Hays, James D.

    2014-01-01

    Todays Sargasso Sea is nutrient starved, except for episodic upwelling events caused by wind-driven winter mixing and eddies. Enhanced diatom opal burial in Sargasso Sea sediments indicates that silicic acid, a limiting nutrient today, may have been more available in subsurface waters during Heinrich Stadials, millennial-scale climate perturbations of the last glacial and deglaciation. Here we use the geochemistry of opal-forming organisms from different water depths to demonstrate changes in silicic acid supply and utilization during the most recent Heinrich Stadial. We suggest that during the early phase (17.5-18?ka), wind-driven upwelling replenished silicic acid to the subsurface, resulting in low Si utilization. By 17?ka, stratification reduced the surface silicic acid supply leading to increased Si utilization efficiency. This abrupt shift in Si cycling would have contributed to high regional carbon export efficiency during the recent Heinrich Stadial, despite being a period of increasing atmospheric CO2.

  12. Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation.

    PubMed

    Hendry, Katharine R; Robinson, Laura F; McManus, Jerry F; Hays, James D

    2014-01-01

    Today's Sargasso Sea is nutrient starved, except for episodic upwelling events caused by wind-driven winter mixing and eddies. Enhanced diatom opal burial in Sargasso Sea sediments indicates that silicic acid, a limiting nutrient today, may have been more available in subsurface waters during Heinrich Stadials, millennial-scale climate perturbations of the last glacial and deglaciation. Here we use the geochemistry of opal-forming organisms from different water depths to demonstrate changes in silicic acid supply and utilization during the most recent Heinrich Stadial. We suggest that during the early phase (17.5-18 ka), wind-driven upwelling replenished silicic acid to the subsurface, resulting in low Si utilization. By 17 ka, stratification reduced the surface silicic acid supply leading to increased Si utilization efficiency. This abrupt shift in Si cycling would have contributed to high regional carbon export efficiency during the recent Heinrich Stadial, despite being a period of increasing atmospheric CO2. PMID:24452197

  13. Nutritional efficiency of alpha-ketoisocaproate relative to leucine, assessed isotopically

    SciTech Connect

    Kang, C.W.; Walser, M.

    1985-10-01

    The efficiency of alpha-ketoisocaproate as a dietary substitute for leucine was assessed in rats by two techniques: first, the minimal dose of alpha-ketoisocaproate required, as a supplement to a leucine-free diet, to achieve a growth rate as great as animals receiving leucine was found to be between 2.2 and 4.4 times larger. Therefore the nutritional efficiency of alpha-ketoisocaproate lies between 0.23 and 0.46. Second, alpha-(1- UC)-ketoisocaproate and (TH)leucine were administered orally and the ratio of UC/TH incorporated into the leucine of whole-body protein and fibrin was measured. This ratio, divided by the ratio UC/TH injected, was the same in fibrin as in whole-body protein and averaged 0.39. Thus both techniques yield the same value, within the error of measurement, for the relative nutritional efficiency of alpha-ketoisocaproate. The authors also found that alpha-ketoisocaproate feeding at varying dosage did not alter this ratio in whole-body protein, suggesting that neither wide variations in growth rate nor exposure for 10 days to alpha-ketoisocaproate alters the relative rates of utilization (or oxidation) of alpha-ketoisocaproate vs. leucine.

  14. Identification of subunit-subunit interaction sites in ?A-WT crystallin and mutant ?A-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.

    PubMed

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

    2013-01-01

    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 (d? and d?) cross-linker-BSG (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

  15. Stable carbon isotope ratios and intrinsic water-use efficiency of Miocene fossil leaves compared to modern congeners

    SciTech Connect

    Marshall, J.D.; Zhang, J.; Rember, W.C.; Jennings, D.; Larson, P. )

    1994-06-01

    Miocene fossil leaves of forest trees were extracted from the Clarkia, Idaho fossil beds and their stable carbon isotope ratios were analyzed. Fossils had higher lignin concentrations and lower cellulose concentrations that modern leaves due to diagenesis and the HF used to extract the fossils. Therefore, [delta][sup 13]C of extracted fossil lignin was compared to that of modern lignin. Fossil lignin [delta][sup 13]C was significantly different from that of congeneric modern leaves (paired t-test, P<0.0001), but was 1.9% less negative. Gymnosperms (Metasequoia, Taxodium) were less negative than angiosperms (e.g., Magnolia, Quercus, Acer, Persea), but no difference between evergreen and deciduous species was detected. Using published estimates of the concentration and [delta][sup 13]C of atmospheric CO[sub 2] during the Miocene was estimated the CO[sub 2] partial pressure gradient across the stomata (intrinsic water-use efficiency). Intrinsic water-use efficiency was at least 70% higher during this past [open quotes]greenhouse[close quotes] period than at present.

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

    SciTech Connect

    Baker, Sheila N; Zhao, Hua; Pandey, Siddharth; Heller, William T; Bright, Frank; Baker, Gary A

    2011-01-01

    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.

  17. Prediction of equilibrium Li isotope fractionation between minerals and aqueous solutions at high P and T: An efficient ab initio approach

    NASA Astrophysics Data System (ADS)

    Kowalski, Piotr M.; Jahn, Sandro

    2011-10-01

    The mass-dependent equilibrium stable isotope fractionation between different materials is an important geochemical process. Here we present an efficient method to compute the isotope fractionation between complex minerals and fluids at high pressure, P, and temperature, T, representative for the Earth's crust and mantle. The method is tested by computation of the equilibrium fractionation of lithium isotopes between aqueous fluids and various Li bearing minerals such as staurolite, spodumene and mica. We are able to correctly predict the direction of the isotope fractionation as observed in the experiments. On the quantitative level the computed fractionation factors agree within 1.0 with the experimental values indicating predictive power of ab initio methods. We show that with ab initio methods we are able to investigate the underlying mechanisms driving the equilibrium isotope fractionation process, such as coordination of the fractionating elements, their bond strengths to the neighboring atoms, compression of fluids and thermal expansion of solids. This gives valuable insight into the processes governing the isotope fractionation mechanisms on the atomic scale. The method is applicable to any state and does not require different treatment of crystals and fluids.

  18. Introduction of a {sup 3}H label into the methyl and methylene positions of polyamines and sodium acetate by solid-state isotope exchange

    SciTech Connect

    Shevchenko, V.P.; Nagaev, I.Yu.; Potapova, A.V.

    1995-05-01

    The mechanisms of adding a {sup 3}H label to methyl and methylene positions of organic compounds are investigated. Labeled compounds with a high molar radioactivity can be prepared using a solid-state method. Extensive deamination and condensation of the starting compounds is observed during addition of {sup 3}H to ethylenediamine and its derivatives.

  19. Leaf oxygen and Carbon Isotopic Signatures Reflect Drought Resistance and Water Use Efficiency in the C4 Grass, Setaria viridis

    NASA Astrophysics Data System (ADS)

    Ellsworth, P.; Cousins, A. B.

    2014-12-01

    Low water availability is a major constraint in crop production, especially as agriculture is pushed to marginal lands. Therefore, improving drought resistance such as increasing water use efficiency (WUE) through plant breeding is needed to expand the range of soil water availability adequate for food production. With the goal of finding the genomic basis for WUE in C4 grasses, Setaria viridis makes an ideal model species because of its small size, short lifespan, and sequenced genome. Also it is part of the panicoid grass clade, which is one of the most important clades for food and biofuel production. In plant breeding programs, large numbers of genotypes must be quickly screened for drought resistance traits, but there is no well-defined method of screening for WUE in C4 grasses. However, bulk leaf oxygen (?18OBL) and carbon (?13C) isotopic signatures have shown potential as recorders of transpiration rate (E) and stomatal conductance (gs), and combined with biomass production potentially serve as a measure of WUE. Values of ?18OBL record differences in transpiration rate because leaf water becomes more enriched as transpiration rate decreases, and leaf tissue records the isotopic composition of leaf water in which it is synthesized. Additionally, in C4 plants ?13C values decrease as gs decreases but the change in ?13C in response to gs may not be adequate to tease apart differences in WUE. In this study, we grew S. viridis plants under well-watered and water-limited conditions to determine if ?18OBL and ?13C could be used as proxies for E and gs, and be used to screen S. viridis for differences in WUE in breeding programs. The ?18OBL and ?13C were significantly different between well-watered and water-limited plants and correlated with each other and with E, gs, and instantaneous water use efficiency (Anet/gs). Therefore, ?18OBL and ?13C can be useful proxies to screen genotypes for drought resistance by recording differences in E, gs, and WUE. Measuring ?18OBL and ?13C are relatively simple and quick, requiring the collection of a single leaf sample from each genotype instead of making laborious gas exchange measurements of E and gs.

  20. The Analysis on Influence of Main Factors on Theoretical Value of Energy Saving Rate for Energy Efficiency Labeling of Civil Buildings

    NASA Astrophysics Data System (ADS)

    Wang, Zhiwei; Wang, Zhenling; Jiang, Bo; Zhang, Fan; Li, Peng; Cao, Wei

    For typical residential buildings, no-large-scale and large-scale public buildings, according to China's Technical Guide for the Energy Efficiency Labeling of Civil Buildings, makes up missing data of the calculation benchmark and determines the boundary conditions for calculating the theoretical values of civil building energy efficiency. Based on equivalent full load hours method, develops a modular program and calculates building energy consumption for the demands of dynamic cooling and heating and lighting etc., finds out the corresponding relationship between star level's theoretical value of energy saving rate and specified-term limiting value in the Guide. With orthogonal experimental design and multiple linear regression, establishes the quantitative function of both the theoretical value of energy saving rate and main factors parameters, analyzes the impact of the control parameter on energy saving rate, and reveals the law of theoretical value of energy saving rate variation with the control parameter. For building energy efficiency labeling upgrade, presents technical measure need to be taken and analyses its feasibility. The results from the study can provide theoretical guidance for energy-saving design or retrofitting of civil buildings.

  1. SILAC yeast: from labeling to comprehensive proteome quantification.

    PubMed

    de Godoy, Lyris M F

    2014-01-01

    Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex mixtures, enabling characterization and comparison of cellular functional states in a proteome-wide scale. In this context, stable isotope labeling with amino acids in cell culture (SILAC) has emerged as a simple yet powerful approach, which has been applied to address different biological questions across a variety of systems, ranging from single cells to entire multicellular organisms. In this chapter, detailed instructions for SILAC labeling yeast are provided, including a series of quality checks for evaluating labeling efficiency and procedures for determining the optimal labeling parameters for a particular yeast strain. In addition, two different complete workflows for the comprehensive mass spectrometry-based SILAC quantification of close to the entire yeast proteome are described, which can be applied to assess any biological question of interest and, therefore, can be of broad use for the researchers in the field. PMID:24791983

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

    PubMed

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

    2015-01-01

    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 ?N before and after activated carbon treatment of laboratory-prepared ?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

  3. Long term changes in Intrinsic Water Use Efficiency, the palaoe record derived from stable carbon isotope measurements from tree rings.

    NASA Astrophysics Data System (ADS)

    Gagen, Mary; McCarroll, Danny; Loader, Neil; Young, Giles; Robertson, Iain

    2015-04-01

    Stable carbon isotope (?13C) measurements from the annual rings of trees are increasingly used to explore long term changes in plant-carbon-water relations, via changes in intrinsic water use efficiency (iWUE); the ratio of photosynthetic rate to stomatal conductance. Many studies report a significant increase in iWEU since industrialisation, which tracks rising global atmospheric CO2. Such changes are logical are trees are known to change their stomatal geometry, number and action in response to rising CO2. However, which increasing iWUE suggests physiological changes which should lead to increased growth increasing iWUE is rarely matched by enhanced tree growth when tree rings are measured, despite increases of up to 30% in iWUE over the recent past (van der Sleen et al 2015). Explanations for the mismatch between iWUE and tree growth records encompass questions over the veracity of ?13C records for recording physiological change (Silva and Howarth 2013), suggestions that moisture stress in warming climates becomes a limit to growth and prevents opportunistic use of rising CO2 by trees (Andreu-Hayles et al 2011) and questions regarding the use of tree ring width, which does not record tree height gain, to record growth. Here we present an extensive range of long term iWUE records, derived broadly from the temperate, high latitude and one tropical forest site to explore the palaeoclimatic perspective on the iWUE-fertilization conundrum in a spatio temporally extensive manner.

  4. Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer.

    PubMed

    Collier, Timothy S; Hawkridge, Adam M; Georgianna, D Ryan; Payne, Gary A; Muddiman, David C

    2008-07-01

    Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail. PMID:18512951

  5. The physiological basis for genetic variation in water use efficiency and carbon isotope composition in Arabidopsis thaliana.

    PubMed

    Easlon, Hsien Ming; Nemali, Krishna S; Richards, James H; Hanson, David T; Juenger, Thomas E; McKay, John K

    2014-02-01

    Ecologists and physiologists have documented extensive variation in water use efficiency (WUE) in Arabidopsis thaliana, as well as association of WUE with climatic variation. Here, we demonstrate correlations of whole-plant transpiration efficiency and carbon isotope composition (?(13)C) among life history classes of A. thaliana. We also use a whole-plant cuvette to examine patterns of co-variation in component traits of WUE and ?(13)C. We find that stomatal conductance (g s) explains more variation in WUE than does A. Overall, there was a strong genetic correlation between A and g s, consistent with selection acting on the ratio of these traits. At a more detailed level, genetic variation in A was due to underlying variation in both maximal rate of carboxylation (V cmax) and maximum electron transport rate (Jmax). We also found strong effects of leaf anatomy, where lines with lower WUE had higher leaf water content (LWC) and specific leaf area (SLA), suggesting a role for mesophyll conductance (g m) in variation of WUE. We hypothesize that this is due to an effect through g m, and test this hypothesis using the abi4 mutant. We show that mutants of ABI4 have higher SLA, LWC, and g m than wild-type, consistent with variation in leaf anatomy causing variation in g m and ?(13)C. These functional data also add further support to the central, integrative role of ABI4 in simultaneously altering ABA sensitivity, sugar signaling, and CO2 assimilation. Together our results highlight the need for a more holistic approach in functional studies, both for more accurate annotation of gene function and to understand co-limitations to plant growth and productivity. PMID:23893317

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

    PubMed

    Cernusak, Lucas A; Winter, Klaus; Aranda, Jorge; Virgo, Aurelio; Garcia, Milton

    2009-09-01

    Variation in transpiration efficiency (TE) and its relationship with the stable carbon isotope ratio of wood was investigated in the saplings of three tropical tree species. Five individuals each of Platymiscium pinnatum (Jacq.) Dugand, Swietenia macrophylla King and Tectona grandis Linn. f. were grown individually in large (760 l) pots over 16 months in the Republic of Panama. Cumulative transpiration was determined by repeatedly weighing the pots with a pallet truck scale. Dry matter production was determined by destructive harvest. The TE, expressed as experiment-long dry matter production divided by cumulative water use, averaged 4.1, 4.3 and 2.9 g dry matter kg(-1) water for P. pinnatum, S. macrophylla and T. grandis, respectively. The TE of T. grandis was significantly lower than that of the other two species. Instantaneous measurements of the ratio of intercellular to ambient CO(2) partial pressures (c(i)/c(a)), taken near the end of the experiment, explained 66% of variation in TE. Stomatal conductance was lower in S. macrophylla than in T. grandis, whereas P. pinnatum had similar stomatal conductance to T. grandis, but with a higher photosynthetic rate. Thus, c(i)/c(a) and TE appeared to vary in response to both stomatal conductance and photosynthetic capacity. Stem-wood delta(13)C varied over a relatively narrow range of just 2.2 per thousand, but still explained 28% of variation in TE. The results suggest that leaf-level processes largely determined variation among the three tropical tree species in whole-plant water-use efficiency integrated over a full annual cycle. PMID:19661136

  7. An Efficient and Cost-Effective Protocol for Selecting Transcription Factor Binding Sites that Reduces Isotope Usage

    PubMed Central

    Krystel, Joseph; Ayyanathan, Kasirajan

    2012-01-01

    To function, transcription factors must position themselves by binding to DNA in a sequence-specific manner. Knowing the binding sites of these factors is a necessary step in understanding their activity. The standard protocols used for selecting a consensus-binding sequence for a DNA binding domain often require the use of radioisotopes to attain the necessary level of power in the assay. Alternatives are often less sensitive and may require an expensive apparatus for visualizing. We have created a modified binding site selection (BSS) protocol to improve efficiency and decrease the use of radioisotope. A GST affinity-tagged DNA binding domain construct was immobilized on a GSH affinity column and used to select from a randomized oligonucleotide library identical to those typically used in a radiolabeled BSS protocol. This produced a library specifically pre-enriched for use in a standard sequential EMSA selection. Use of a pre-enriched library reduced the total number of labeled rounds required for selection, decreasing the use of radioisotope while maintaining efficacy. The protocol was used to select for the binding sequence for several Drosophila melanogaster transcription factors. The consensus sequence was then shown by competitive binding experiments to associate with the protein in a sequence-dependent manner. PMID:22951958

  8. Water use Efficiency in a Blue oak ( Quercus douglasii) Savanna - a Combined Analysis of Stable Isotopes and Eddy Covariance Measurements

    NASA Astrophysics Data System (ADS)

    Mambelli, S.; Tu, K. P.; Knohl, A.; Ma, S.; Baldocchi, D. D.; Dawson, T. E.

    2007-12-01

    Understanding the relationship between carbon assimilation and water consumption by natural vegetation is needed to assess how changes in climate will affect plant carbon and water exchange as well as the energy fluxes of ecosystems. While climate change is expected to cause significant warming, most models also suggest changes in the timing and amount of precipitation received; thus implications of this type of change are particularly acute in Mediterranean regions of the world. Blue oak savannas are already exposed to broad variation in water availability and to severe droughts during the summer months. Our objective was to evaluate the trade-off between carbon gain and water loss (Water Use Efficiency) in this ecosystem at both the leaf and at the ecosystem scales. We monitored the ratio of the partial pressures of CO2 inside the leaf (Ci) and in the outside air (Ca) or Ci/Ca, during the summer months of three subsequent years. This ratio is determined by the balance between photosynthetic capacity and stomatal conductance to water loss. Leaf-level estimates for individual trees were based on the carbon isotope composition (?13C) of bulk leaf tissue and of recently fixed carbohydrates (leaf soluble sugars). These leaf and individual tree based estimates were then compared with canopy-level estimates derived from continuous eddy covariance measurements of fluxes of CO2, water vapor and meteorological variables from two eddy covariance systems, one above (23m) and one below (2m) the tree canopy. We found that savanna Blue oak trees cope with severe drought through coordinated down-regulation of carbon and water fluxes, i.e. the ratio Ci/Ca remained stable over four summer months, despite decreasing soil water content and leaf water potentials. Stable C isotope composition of leaf soluble sugars is the most robust measure of Ci/Ca because it reflects the initial discrimination of photosynthetic products, without the confounding effects ascribed to storage, tissue chemical composition and time of tissue formation. Our findings at the leaf-level were confirmed at the ecosystem-level by using a two tower (above and below canopy) eddy covariance method.

  9. Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

    PubMed

    Gimnez, Estela; Balmaa, Meritxell; Figueras, Joan; Fort, Esther; de Bols, Carme; Sanz-Nebot, Victria; Peracaula, Rosa; Rizzi, Andreas

    2015-03-25

    In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (?ZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human ?1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and ?ZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers. PMID:25732693

  10. Allocation of atmospheric CO2 into labile sub-surface carbon pools: a stable isotope labelling approach in a tundra wetland

    NASA Astrophysics Data System (ADS)

    Rüggen, Norman; Knoblauch, Christian; Pfeiffer, Eva-Maria

    2015-04-01

    Greenhouse gas emissions from permafrost-affected wetlands are intensively studied due to their important role in the global carbon cycle. There are concerns of increasing methane and carbon dioxide fluxes from tundra wetlands due to permafrost degradation and hydrology changes in a warming Arctic. Understanding the sub-surface carbon pool interactions will improve the prediction on how trace gas fluxes from these ecosystems will respond to changing environmental conditions. Partitioning the sources of greenhouse gas fluxes will help to evaluate the quantitative role of recently produced plant photosynthates. Furthermore, partitioning allows separating respiration of long-term stored organic matter and freshly produced plant products. This knowledge is crucial for understanding the response of greenhouse gas fluxes in such wetlands to environmental changes. An in situ 13CO2 pulse-labelling experiment has been conducted in the northeast Siberian tundra (Samoylov island, Lena river delta) in August 2013 to quantify interactions among sub-surface carbon pools (DIC, DOC, CH4) in three depths (6, 16 and 36 cm) of the active layer. The experimental site was a low-centred polygon centre in a polygonal tundra landscape, with a sedge-moss (Carex-Scorpidium) plant association. The water table was at the soils' surface and the permafrost table in a depth of 50 cm. After the system has been 13CO2 pulse labelled, all three studied subsurface carbon pools (CH4, DIC and DOC) were clearly 13C-enriched, which accounts for atmospheric C incorporated into these pools. One day after the labelling, in 6 cm depth 1.5 percent of DIC and 0.1 percent of CH4were replaced by label C, which then steadily declined over a ten days period. The label C content of DOC increased gradually over the same period. In 16 cm depth, the label C increased gradually after labelling in both DIC and CH4. Label C was found in DIC and CH4 even in a depth of 36 cm, although in less pronounced concentrations. Carex material, exposed to the label, also substantially incorporated the label. Deduced from the results, we will present carbon exchange fluxes among sub-surface DIC, DOC and CH4 in a sedge-moss covered polygon-centre.

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

    PubMed Central

    Zudaire, Isabel; Ortiz-de-Solorzano, Carlos

    2013-01-01

    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

  12. Comparison of 64Cu-complexing bifunctional chelators for radioimmunoconjugation: labeling efficiency, specific activity and in vitro/in vivo stability

    PubMed Central

    Cooper, Maggie S.; Ma, Michelle T.; Sunassee, Kavitha; Shaw, Karen P.; Williams, Jennifer D.; Paul, Rowena L.; Donnelly, Paul S.; Blower, Philip J.

    2016-01-01

    High radiolabeling efficiency, preferably to high specific activity, and good stability of the radioimmunoconjugate are essential features for a successful immunoconjugate for imaging or therapy. In this study, the radiolabeling efficiency, in vitro stability and biodistribution of immunoconjugates with eight different bifunctional chelators labeled with 64Cu were compared. The anti-CD20 antibody, rituximab, was conjugated to four macrocyclic bifunctional chelators (p-SCN-Bn-DOTA, p-SCN-Bn-Oxo-DO3A, p-SCN-NOTA and p-SCN-PCTA), three DTPA derivatives (p-SCN-Bn-DTPA, p-SCN-CHX-A-DTPA and ITC-2B3M-DTPA) and a macrobicyclic hexamine (sarcophagine) chelator (sar-CO2H) = (1-NH2-8-NHCO(CH2)3CO2H)sar where sar = sarcophagine = 3,6,10,13,16,19-hexaazabicyclo[6.6.6]icosane). Radiolabeling efficiency under various conditions, in vitro stability in serum at 37C and in vivo biodistribution and imaging in normal mice over 48 h were studied. All chelators except sar-CO2H were conjugated to rituximab by thiourea bond formation with an average of 4.9 +/? 0.9 chelators per antibody molecule. Sar-CO2H was conjugated to rituximab by amide bond formation with 0.5 chelators per antibody molecule. Efficiencies of 64Cu radiolabeling were dependent on the concentration of immunoconjugate. Notably, the 64Cu-NOTA-rituximab conjugate demonstrated highest radiochemical yield (95%) under very dilute conditions (31 nM NOTA-rituximab conjugate). Similarly, sar-CO-rituximab, containing 1/10th the number of chelators per antibody compared to other conjugates retained high labeling efficiency (98 %) at an antibody concentration of 250 nM. In contrast to the radioimmunoconjugates containing DTPA derivatives, which demonstrated poor serum stability, all macrocyclic radioimmunoconjugates were very stable in serum with <6 % dissociation of 64Cu over 48 h. In vivo biodistribution profiles in normal female Balb/C mice were similar for all the macrocyclic radioimmunoconjugates with most of the activity remaining in the blood pool up to 48 h. Whilst all the macrocyclic bifunctional chelators are suitable for molecular imaging using 64Cu-labeled antibody conjugates, NOTA and sar-CO2H show significant advantages over the others in that they can be radiolabeled rapidly at room temperature, under dilute conditions resulting in high specific activity. PMID:22471317

  13. Synthesis and evaluation of an (125)I-labeled azide prosthetic group for efficient and bioorthogonal radiolabeling of cyclooctyne-group containing molecules using copper-free click reaction.

    PubMed

    Choi, Mi Hee; Shim, Ha Eun; Nam, You Ree; Kim, Hye Rim; Kang, Jung Ae; Lee, Dong-Eun; Park, Sang Hyun; Choi, Dae Seong; Jang, Beom-Su; Jeon, Jongho

    2016-02-01

    Herein we report the radiosynthesis of a pyridine derived azide prosthetic group for iodine radioisotope labeling of dibenzocyclooctyne (DBCO) conjugated molecules. The radiolabeling of the stannylated precursor 2 was conducted using [(125)I]NaI and chloramine-T to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (728%, n=4) and radiochemical purity (>99%). Using (125)I-labeled azide ([(125)I]1), cyclic RGD peptide and near infrared fluorescent molecule were efficiently labeled with modest to good radiochemical yields. The biodistribution study and SPECT/CT images showed that [(125)I]1 underwent rapid renal clearance. These results clearly demonstrated that [(125)I]1 could be used as an useful radiotracer for in vivo pre-targeted imaging as well as efficient in vitro radiolabeling of DBCO containing molecules. PMID:26748695

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

    PubMed

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

    2000-09-01

    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

  15. Labeling of Membrane Proteins by Cell-Free Expression.

    PubMed

    LaGuerre, Aisha; Lhr, Frank; Bernhard, Frank; Dtsch, Volker

    2015-01-01

    The particular advantage of the cell-free reaction is that it allows a plethora of supplementation during protein expression and offers complete control over the available amino acid pool in view of concentration and composition. In combination with the fast and reliable production efficiencies of cell-free systems, the labeling and subsequent structural evaluation of very challenging targets, such as membrane proteins, comes into focus. We describe current methods for the isotopic labeling of cell-free synthesized membrane proteins and we review techniques available to the practitioner pursuing structural studies by nuclear magnetic resonance spectroscopy. Though isotopic labeling of individual amino acid types appears to be relatively straightforward, an ongoing critical issue in most labeling schemes for structural approaches is the selective substitution of deuterons for protons. While few options are available, the continuous refinement of labeling schemes in combination with improved pulse sequences and optimized instrumentation gives promising perspectives for extended applications in the structural evaluation of cell-free synthesized membrane. PMID:26577739

  16. Identification of cargo proteins specific for the nucleocytoplasmic transport carrier transportin by combination of an in vitro transport system and stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics.

    PubMed

    Kimura, Makoto; Kose, Shingo; Okumura, Nobuaki; Imai, Kenichiro; Furuta, Maiko; Sakiyama, Noriyuki; Tomii, Kentaro; Horton, Paul; Takao, Toshifumi; Imamoto, Naoko

    2013-01-01

    The human importin-β family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-β family carriers and then supplemented with a particular importin-β family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-β family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-β. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals. PMID:23087160

  17. Investigation of therapeutic efficiency of bleomycin and bleomycin-glucuronide labeled with (131)I on the cancer cell lines.

    PubMed

    Ediz, Melis; Avc?ba??, U?ur; Unak, Perihan; Mftler, Fazilet Zmrt Biber; Medine, Emin ?lker; Yurt K?lar, Ayfer; Demiro?lu, Hasan; Gm?er, Fikriye Gl; Sakarya, Serhan

    2013-05-01

    The aim of this study is to determine the incorporations of radiolabeled bleomycin ((131)I-BLM) and bleomycin-glucuronide ((131)I-BLMGLU) on PC-3 (human prostate carcinoma cell line), Caco-2 (human colon adenocarcinoma cell line), Hutu-80 (Human Duodenum adenocarcinoma cell line), and A549 (Human lung adenocarcinoma epithelial cell line) cancerous cell lines. For this purpose, BLM and BLMGLU enyzmatically synthesized were labeled with (131)I, quality control studies were done and the incorporation yields of (131)I-BLM and (131)I-BLMGLU on these cell lines were measured. Quality-control studies showed that the radiolabeling yields were obtained as 95% and 90% for (131)I-BLM and (131)I-BLMGLU, respectively. Also, as a result of the cell culture studies, it was found that (131)I-BLM and (131)I-BLMGLU had higher incorporation on PC-3 cells than that of other cell lines. In addition to this, it was reported that the incorporation yield of (131)I-BLMGLU was higher than that (131)I-BLM. At the end of the study, cytotoxicities of BLM and BLMGLU on PC-3 cancerous cell line were inspected and fluorescent images of BLM and BLMGLU were taken on PC-3 cells by using fluorescein isothiocyanate. In conclusion, cell culture studies demonstrated that the incorporation values of (131)I-BLMGLU on the four cell lines were about five to six times higher than (131)I-BLM. Radiolabeled glucuronide derivatives can be used in cancer therapy and tumor imaging, depending on the properties of radioiodine for the ?-glucuronidase-rich tissues because glucuronidation leads to rapid and higher incorporation on adenocarcinoma cells. PMID:23350895

  18. Seasonal variations in photosynthesis, intrinsic water-use efficiency and stable isotope composition of poplar leaves in a short-rotation plantation

    PubMed Central

    Broeckx, L.S.; Fichot, R.; Verlinden, M.S.; Ceulemans, R.

    2014-01-01

    Photosynthetic carbon assimilation and transpirational water loss play an important role in the yield and the carbon sequestration potential of bioenergy-devoted cultures of fast-growing trees. For six poplar (Populus) genotypes in a short-rotation plantation, we observed significant seasonal and genotypic variation in photosynthetic parameters, intrinsic water-use efficiency (WUEi) and leaf stable isotope composition (δ13C and δ18O). The poplars maintained high photosynthetic rates (between 17.8 and 26.9 μmol m−2 s−1 depending on genotypes) until late in the season, in line with their fast-growth habit. Seasonal fluctuations were mainly explained by variations in soil water availability and by stomatal limitation upon photosynthesis. Stomatal rather than biochemical limitation was confirmed by the constant intrinsic photosynthetic capacity (Vcmax) during the growing season, closely related to leaf nitrogen (N) content. Intrinsic water-use efficiency scaled negatively with carbon isotope discrimination (Δ13Cbl) and positively with the ratio between mesophyll diffusion conductance (gm) and stomatal conductance. The WUEi – Δ13Cbl relationship was partly influenced by gm. There was a trade-off between WUEi and photosynthetic N-use efficiency, but only when soil water availability was limiting. Our results suggest that seasonal fluctuations in relation to soil water availability should be accounted for in future modelling studies assessing the carbon sequestration potential and the water-use efficiency of woody energy crops. PMID:25074859

  19. Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling1[W][OPEN

    PubMed Central

    Nelson, Clark J.; Alexova, Ralitza; Jacoby, Richard P.; Millar, A. Harvey

    2014-01-01

    Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis. PMID:25082890

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

    PubMed Central

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

    2010-01-01

    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

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

    PubMed Central

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

    2010-01-01

    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

  2. Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using ESEEM Spectroscopy, Isotopic Labeling, and DFT Calculations

    PubMed Central

    Klein, Eric L.; Astashkin, Andrei V.; Ganyushin, Dmitry; Riplinger, Christoph; Johnson-Winters, Kayunta; Neese, Frank; Enemark, John H.

    2009-01-01

    Electron spin echo envelope modulation (ESEEM) investigations were carried out on samples of the low-pH (lpH) form of vertebrate sulfite oxidase (SO) prepared with 35Cl- and 37Cl-enriched buffers as well as with buffer containing the natural abundance of Cl isotopes. The isotope-related changes observed in the ESEEM spectra provide direct and unequivocal evidence that Cl? is located in close proximity to the Mo(V) center of lpH SO. The measured isotropic hyperfine interaction constant of about 4 MHz (35Cl) suggests that the Cl? ion is either weakly coordinated to Mo(V) at its otherwise vacant axial position, trans to the oxo ligand, or is hydrogen-bonded to the equatorial exchangeable OH ligand. Scalar relativistic all-electron density functional theory (DFT) calculations of the hyperfine and nuclear quadrupole interaction parameters, along with steric and energetic arguments, strongly support the possibility that Cl? is hydrogen-bonded to the equatorial OH ligand rather than being directly coordinated to the Mo(V). PMID:19402624

  3. In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Noncyclic Nature of the Tricarboxylic Acid Cycle in Illuminated Leaves1[W

    PubMed Central

    Tcherkez, Guillaume; Mah, Aline; Gauthier, Paul; Mauve, Caroline; Gout, Elizabeth; Bligny, Richard; Cornic, Gabriel; Hodges, Michael

    2009-01-01

    While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, 13C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA cycle does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. PMID:19675152

  4. ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.

    SciTech Connect

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

    2000-12-01

    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.

  5. Adrenal androgen 11. beta. - and 19-hydroxylation: a study on kinetic isotope effect and metabolic switching of /sup 3/H and /sup 14/C labeled substrates

    SciTech Connect

    Osawa, Y.; Yarborough, C.

    1986-05-01

    Monooxygenations at 11..beta.. and 19 are two main metabolic pathways of androstenedione (A) by sheep and dog adrenals and could be catalyzed by a single cytochrome P-450 as observed for deoxycorticosterone metabolism with the bovine adrenal cytochrome P-450/sub 11..beta../. They found an unusually high apparent kinetic isotope effect (k/sub H//k/sub T/ = 11.7) on the 19-hydroxylation of (19-/sup 3/H/sub 3/, 4-/sup 14/C)A with sheep and dog adrenal homogenates. They therefore attempted to assess the inverse secondary kinetic isotope effect of the simultaneously occurring 11..beta..-hydroxylation. The substrate (16.1..mu..M and 14.7..mu..M, /sup 3/H//sup 14/C = 15.1 and 21.9 for sheep and dog, respectively) was incubated with adrenal tissue homogenate in the presence of NADPH for 1-10 min. The products were mixed with carrier standards and purified through TLC, acetylation, and countercurrent distribution to show constant /sup 3/H//sup 14/C ratio. The /sup 3/H//sup 14/C ratios of the major (19-/sup 3/H/sub 3/, 4-/sup 14/C)-11..beta..-OHA were found to be elevated 1.1-1.2 fold whereas those of (19-/sup 3/H/sub 2/, 4-/sup 14/C)-19-OHA were decreased to 0.06 and the recovered A showed 0.95-0.99 of the initial ratio. While they observed a metabolic switching between the 2..beta..- and 19-hydroxylations of (19-/sup 3/H/sub 3/, 4-/sup 14/C)A with human placental aromatase (Fronckowiak and Osawa), it is suggested that adrenal non-aromatizing androgen 19-hydroxylation operates a metabolic switching with the 11..beta..-hydroxylation.

  6. CAESARA high-efficiency CsI(Na) scintillator array for in-beam ?-ray spectroscopy with fast rare-isotope beams

    NASA Astrophysics Data System (ADS)

    Weisshaar, D.; Gade, A.; Glasmacher, T.; Grinyer, G. F.; Bazin, D.; Adrich, P.; Baugher, T.; Cook, J. M.; Diget, C. Aa.; McDaniel, S.; Ratkiewicz, A.; Siwek, K. P.; Walsh, K. A.

    2010-12-01

    We report on the construction and commissioning of the high-efficiency CAESium-iodide scintillator ARray CAESAR, a device designed for in-beam ?-ray spectroscopy experiments utilizing fast beams of rare isotopes at the National Superconducting Cyclotron Laboratory (NSCL) at Michigan State University (MSU). CAESAR consists of 192 CsI(Na) crystals, totaling 290 kg of active scintillator material. For 1 MeV ? rays, a full-energy-peak efficiency of 35% is achieved at an in-beam energy resolution of better than 10% FWHM after event-by-event Doppler reconstruction of the ? rays emitted by nuclei moving with velocities of v/c0.3-0.4. The spectral quality of the array allows for the identification of ?-ray transitions with intensities of several 10 counts in the full-energy peak and thus opens new avenues for the study of the most exotic nuclei available at the NSCL for in-beam spectroscopy.

  7. 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)

    Dubrovskaya, Ekaterina; Turkovskaya, Olga

    2010-05-01

    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 13С/12С ratio in the respired СО2. 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 (δ13C,-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 δ13C 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.

  8. An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes*

    PubMed Central

    MacLeod, A. Kenneth; Fallon, Padraic G.; Sharp, Sheila; Henderson, Colin J.; Wolf, C. Roland; Huang, Jeffrey T.-J.

    2015-01-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of drug pharmacokinetics, pharmacodynamics, and of chemically treated and genetically modified mouse models. PMID:25561501

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

    We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

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

    NASA Astrophysics Data System (ADS)

    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

    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.

  11. Quantitative proteomics using uniform (15)N-labeling, MASCOT, and the trans-proteomic pipeline.

    PubMed

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

    2007-10-01

    Stable isotope labeling combined with MS is a powerful method for measuring relative protein abundances, for instance, by differential metabolic labeling of some or all amino acids with (14)N and (15)N in cell culture or hydroponic media. These and most other types of quantitative proteomics experiments using high-throughput technologies, such as LC-MS/MS, generate large amounts of raw MS data. This data needs to be processed efficiently and automatically, from the mass spectrometer to statistically evaluated protein identifications and abundance ratios. This paper describes in detail an approach to the automated analysis of uniformly (14)N/(15)N-labeled proteins using MASCOT peptide identification in conjunction with the trans-proteomic pipeline (TPP) and a few scripts to integrate the analysis workflow. Two large proteomic datasets from uniformly labeled Arabidopsis thaliana were used to illustrate the analysis pipeline. The pipeline can be fully automated and uses only common or freely available software. PMID:17726679

  12. Synthesis of deuterium-labeled prochlorperazine.

    PubMed

    Hawes, E M; Gurnsey, T S; Shetty, H U; Midha, K K

    1983-06-01

    The propylpiperazine side chain of prochlorperazine was labeled with two, four, or six deuterium atoms by lithium aluminum deuteride reduction of the appropriate amide. The isotopic purity of the products after correcting for chlorine isotopes was greater than 95.7%. PMID:6875835

  13. An analysis of ash and isotopic carbon discrimination (delta13C) methods to evaluate water use efficiency in apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apple cultivars are selected for fruit quality, disease and insect resistance, not water use efficiency (WUE), however, the need for more water use efficient crops is accelerating due to climate change and increased competition for water resources. On a whole plant basis, calculation of water use e...

  14. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics.

    PubMed

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-09-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  15. Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

    PubMed

    Huang, Ke; Huang, Lingyi; van Breemen, Richard B

    2015-04-01

    Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes. PMID:25774910

  16. Isotopic labeling experiments that elucidate the mechanism of DNA strand cleavage by the hypoxia-selective antitumor agent 1,2,4-benzotriazine 1,4-di-N-oxide

    PubMed Central

    Shen, Xiulong; Rajapakse, Anuruddha; Gallazzi, Fabio; Junnotula, Venkatraman; Fuchs-Knotts, Tarra; Glaser, Rainer; Gates, Kent S.

    2014-01-01

    The 1,2,4-benzotriazine 1,4-dioxides are an important class of potential anticancer drugs that selectively kill the low-oxygen (hypoxic) cells found in solid tumors. These compounds undergo intracellular one-electron enzymatic reduction to yield an oxygen-sensitive drug radical intermediate that partitions forward, under hypoxic conditions, to generate a highly reactive secondary radical that causes cell killing DNA damage. Here we characterized bioreductively-activated, hypoxia-selective DNA-strand cleavage by 1,2,4-benzotriazine 1,4-dioxide. We found that one-electron enzymatic activation of 1,2,4-benzotriazine 1,4-dioxide under hypoxic conditions in the presence of the deuterium atom donor methanol-d4 produced non-deuterated mono-N-oxide metabolites. This and the results of other isotopic labeling studies provided evidence against the generation of atom-abstracting drug radical intermediates and are consistent with a DNA-damage mechanism involving release of hydroxyl radical from enzymatically-activated 1,2,4-benzotriazine 1,4-dioxides. PMID:24328261

  17. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics*

    PubMed Central

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-01-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  18. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

    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.

  19. Tree-Ring Stable Isotopes Reveal Twentieth-Century Increases in Water-Use Efficiency of Fagus sylvatica and Nothofagus spp. in Italian and Chilean Mountains

    PubMed Central

    Tognetti, Roberto; Lombardi, Fabio; Lasserre, Bruno; Cherubini, Paolo; Marchetti, Marco

    2014-01-01

    Changes in intrinsic water use efficiency (iWUE) were investigated in Fagus sylvatica and Nothofagus spp. over the last century. We combined dendrochronological methods with dual-isotope analysis to investigate whether atmospheric changes enhanced iWUE of Fagus and Nothofagus and tree growth (basal area increment, BAI) along latitudinal gradients in Italy and Chile. Post-maturation phases of the trees presented different patterns in δ13C, Δ13C, δ18O, Ci (internal CO2 concentration), iWUE, and BAI. A continuous enhancement in isotope-derived iWUE was observed throughout the twentieth century, which was common to all sites and related to changes in Ca (ambient CO2 concentration) and secondarily to increases in temperature. In contrast to other studies, we observed a general increasing trend of BAI, with the exception of F. sylvatica in Aspromonte. Both iWUE and BAI were uncoupled with the estimated drought index, which is in agreement with the absence of enduring decline in tree growth. In general, δ13C and δ18O showed a weak relationship, suggesting the major influence of photosynthetic rate on Ci and δ13C, and the minor contribution of the regulation of stomatal conductance to iWUE. The substantial warming observed during the twentieth century did not result in a clear pattern of increased drought stress along these latitudinal transects, because of the variability in temporal trends of precipitation and in specific responses of populations. PMID:25398040

  20. 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

    Battipaglia, Giovanna; Saurer, Matthias; Cherubini, Paulo; Califapietra, Carlo; McCarthy, Heather R; Norby, Richard J; Cotrufo, M. Francesca

    2013-01-01

    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.

  1. Soil organic carbon can be up-taken by plant roots and stored in plant biosilica: NanoSIMS and isotopic labeling evidences

    NASA Astrophysics Data System (ADS)

    Alexandre, Anne; Santos, Guaciara M.; balesdent, Jerme; Basile-Doelsch, Isabelle; Borschneck, Daniel; Cazevieille, Patrick; Chevassus-Rosset, Claire; Doelsch, Emmanuel; Harutyunyan, Araks; Lemee, Laurent; Mazur, Jean-Charles; Reyerson, Paul; Signoret, Patrick

    2015-04-01

    Plant biosilica particles called phytoliths contain occluded organic compounds (phytC). Over the last few years, phytC content, nature, origin, paleoenvironmental meaning and impact in the global C cycle has been the subject of increasing debate[1, 2]. Inconsistencies in phytC quantification were fed by the scarcity of in-situ characterization of phytC in phytoliths and by inadequate extraction methods[3]. Very recently, 14C-AMS analyses of soil organic matter (SOM), amendments, plant tissues, atmospheric CO2 and phytolith samples, evidenced that a small but significant pool of phytC is not photosynthetic but derived from old SOM[4,5]. From there, several investigations were started to go further into the characterization of phytC and the mechanisms in play behind old SOM absorption by plant roots and old SOM occlusion in plant biosilica. Here, we first reconstruct at high spatial resolution the 3-dimentional location of phytC and its C/N signature using 3D X-ray microscopy and Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS). A pool of phytC appears homogeneously distributed in the silica structure and its C:N estimate is in the range of amino acid signatures[6]. Then, we use 13C and 15N-labelled amino acids monitored from an hydroponic solution to grass roots, stems, leaves and phytoliths to evidence that amino acids are absorbed as such by the roots and are concentrated in phytC rather than in the plant tissues. These findings strengthen and complement the 14C evidences. Both of them dissuade attempts to use phytC as a proxy of plant C. Further, they open new avenues of investigation regarding the processes which drive SOM mobilization by plant uptake, for a better understanding of soil/plant interactions involved in the terrestrial C cycle. [1] Santos et al. 2010. Radiocarbon 52:113 [2] Santos et al. 2012. Biogeosci. 9:1873 [3] Corbineau et al. 2013 R. Paleobot. Palyn. 197: 179 [4] Reyerson et al. 2013 AGU Fall meeting 2013 (1803125) [5] Santos et al. 2014 AGU Fall meeting 2014 (B51A-0011) [6] Alexandre, et al., 2014. Biogeosci. Discuss. 11, 14699 :14727.

  2. Expression, purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies.

    PubMed

    Kessler, Naama; Zvi, Anat; Ji, Min; Sharon, Michal; Rosen, Osnat; Levy, Rina; Gorny, Miroslaw; Zolla-Pazner, Suzan; Anglister, Jacob

    2003-06-01

    The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible. PMID:12767822

  3. Rapid and efficient localization of depth electrodes and cortical labeling using free and open source medical software in epilepsy surgery candidates.

    PubMed

    Princich, Juan Pablo; Wassermann, Demian; Latini, Facundo; Oddo, Silvia; Blenkmann, Alejandro Omar; Seifer, Gustavo; Kochen, Silvia

    2013-01-01

    Depth intracranial electrodes (IEs) placement is one of the most used procedures to identify the epileptogenic zone (EZ) in surgical treatment of drug resistant epilepsy patients, about 20-30% of this population. IEs localization is therefore a critical issue defining the EZ and its relation with eloquent functional areas. That information is then used to target the resective surgery and has great potential to affect outcome. We designed a methodological procedure intended to avoid the need for highly specialized medical resources and reduce time to identify the anatomical location of IEs, during the first instances of intracranial EEG recordings. This workflow is based on established open source software; 3D Slicer and Freesurfer that uses MRI and Post-implant CT fusion for the localization of IEs and its relation with automatic labeled surrounding cortex. To test this hypothesis we assessed the time elapsed between the surgical implantation process and the final anatomical localization of IEs by means of our proposed method compared against traditional visual analysis of raw post-implant imaging in two groups of patients. All IEs were identified in the first 24 H (6-24 H) of implantation using our method in 4 patients of the first group. For the control group; all IEs were identified by experts with an overall time range of 36 h to 3 days using traditional visual analysis. It included (7 patients), 3 patients implanted with IEs and the same 4 patients from the first group. Time to localization was restrained in this group by the specialized personnel and the image quality available. To validate our method; we trained two inexperienced operators to assess the position of IEs contacts on four patients (5 IEs) using the proposed method. We quantified the discrepancies between operators and we also assessed the efficiency of our method to define the EZ comparing the findings against the results of traditional analysis. PMID:24427112

  4. The pharmacokinetic behavior of the soy isoflavone metabolite S-(−)equol and its diastereoisomer R-(+)equol in healthy adults determined by using stable-isotope-labeled tracers1234

    PubMed Central

    Zhao, Xueheng; Jha, Pinky; Heubi, James E; Brown, Nadine M

    2009-01-01

    Background: The nonsteroidal estrogen equol occurs as diastereoisomers, S-(−)equol and R-(+)equol, both of which have significant biological actions. S-(−)equol, the naturally occurring enantiomer produced by 20–30% of adults consuming soy foods, has selective affinity for estrogen receptor-β, whereas both enantiomers modulate androgen action. Little is known about the pharmacokinetics of the diastereoisomers, despite current interest in developing equol as a nutraceutical or pharmaceutical agent. Objective: The objective was to compare the pharmacokinetics of S-(−)equol and R-(+)equol by using [13C] stable-isotope-labeled tracers to facilitate the optimization of clinical studies aimed at evaluating the potential of these diastereoisomers in the prevention and treatment of estrogen- and androgen-dependent conditions. Design: A randomized, crossover, open-label study in 12 healthy adults (6 men and 6 women) compared the plasma and urinary pharmacokinetics of orally administered enantiomeric pure forms of S-(−)[2-13C]equol, R-(+)[2-13C]equol, and the racemic mixture. Plasma and urinary [13C]R-equol and [13C]S-equol concentrations were measured by tandem mass spectrometry. Results: Plasma [13C]equol concentration appearance and disappearance curves showed that both enantiomers were rapidly absorbed, attained high circulating concentrations, and had a similar terminal elimination half-life of 7–8 h. The systemic bioavailability and fractional absorption of R-(+)[2-13C]equol were higher than those of S-(−)[2-13C]equol or the racemate. The pharmacokinetics of racemic (±)[2-13C]equol were different from those of the individual enantiomers: slower absorption, lower peak plasma concentrations, and lower systemic bioavailability. Conclusions: The high bioavailability of both diastereoisomers contrasts with previous findings for the soy isoflavones daidzein and genistein, both of which have relatively poor bioavailability, and suggests that low doses of equol taken twice daily may be sufficient to achieve biological effects. PMID:19710188

  5. 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

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

    2010-09-01

    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 >or=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-kappaB- 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-kappaB-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

  6. Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer.

    PubMed

    Yeh, Chao-Chi; Hsu, Chih-Hung; Shao, Yu-Yun; Ho, Wen-Ching; Tsai, Mong-Hsun; Feng, Wen-Chi; Chow, Lu-Ping

    2015-06-01

    Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7(R)) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7(R) tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7(R) cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib