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Sample records for efficient isotopic labeling

  1. 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.

  2. 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

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

    PubMed

    Lethu, Sébastien; Matsuoka, Shigeru; Murata, Michio

    2014-02-01

    An efficient synthesis involving two copper-catalyzed alkyl-alkyl coupling reactions has been designed to easily access doubly isotope-labeled fatty acids. Such NMR- and IR-active compounds were obtained in excellent overall yields and will be further used for determining the conformation of an alkyl chain of lipidic biomolecules upon interaction with proteins. PMID:24432759

  4. An economic approach to efficient isotope labeling in insect cells using homemade 15N-, 13C- and 2H-labeled yeast extracts.

    PubMed

    Opitz, Christian; Isogai, Shin; Grzesiek, Stephan

    2015-07-01

    Heterologous expression of proteins in insect cells is frequently used for crystallographic structural studies due to the high yields even for challenging proteins requiring the eukaryotic protein processing capabilities of the host. However for NMR studies, the need for isotope labeling poses extreme challenges in eukaryotic hosts. Here, we describe a robust method to achieve uniform protein (15)N and (13)C labeling of up to 90 % in baculovirus-infected insect cells. The approach is based on the production of labeled yeast extract, which is subsequently supplemented to insect cell growth media. The method also allows deuteration at levels of >60 % without decrease in expression yield. The economic implementation of the labeling procedures into a standard structural biology laboratory environment is described in a step-by-step protocol. Applications are demonstrated for a variety of NMR experiments using the Abelson kinase domain, GFP, and the beta-1 adrenergic receptor as examples. Deuterated expression of the latter provides spectra of very high quality of a eukaryotic G-protein coupled receptor. PMID:26070442

  5. 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

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

    DOEpatents

    Hooker, Jacob Matthew; Schonberger, Matthias; Schieferstein, Hanno; Fowler, Joanna S.

    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.

  7. Intrinsic isotopic 13C labelling of polyphenols.

    PubMed

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

    2013-12-01

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

  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. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  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. Isotope Labeling Study of Retinal Chromophore Fragmentation.

    PubMed

    Musbat, Lihi; Nihamkin, Maria; Ytzhak, Shany; Hirshfeld, Amiram; Friedman, Noga; Dilger, Jonathan M; Sheves, Mordechai; Toker, Yoni

    2016-04-28

    Previous studies have shown that the gas-phase fragmentation of the retinal chromophore after S0-S1 photoexcitation results in a prominent fragment of mass 248 which cannot be explained by the cleavage of any single bond along the polyene chain. It was therefore theorized that the fragmentation mechanism involves a series of isomerizations and cyclization processes, and two mechanisms for these processes were suggested. Here we used isotope labeling MS-MS to provide conclusive support for the fragmentation mechanism suggested by Coughlan et al. (J. Phys. Chem. Lett. 2014, 5, 3195). PMID:27046667

  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. 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

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

    PubMed Central

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

    2015-01-01

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

  17. Quantitative proteomics using reductive dimethylation for stable isotope labeling.

    PubMed

    Tolonen, Andrew C; Haas, Wilhelm

    2014-01-01

    Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample. PMID:25045933

  18. ICPLQuant - A software for non-isobaric isotopic labeling proteomics.

    PubMed

    Brunner, Achim; Keidel, Eva-Maria; Dosch, Dominik; Kellermann, Josef; Lottspeich, Friedrich

    2010-01-01

    The main goal of many proteomics experiments is an accurate and rapid quantification and identification of regulated proteins in complex biological samples. The bottleneck in quantitative proteomics remains the availability of efficient software to evaluate and quantify the tremendous amount of mass spectral data acquired during a proteomics project. A new software suite, ICPLQuant, has been developed to accurately quantify isotope-coded protein label (ICPL)-labeled peptides on the MS level during LC-MALDI and peptide mass fingerprint experiments. The tool is able to generate a list of differentially regulated peptide precursors for subsequent MS/MS experiments, minimizing time-consuming acquisition and interpretation of MS/MS data. ICPLQuant is based on two independent units. Unit 1 performs ICPL multiplex detection and quantification and proposes peptides to be identified by MS/MS. Unit 2 combines MASCOT MS/MS protein identification with the quantitative data and produces a protein/peptide list with all the relevant information accessible for further data mining. The accuracy of quantification, selection of peptides for MS/MS-identification and the automated output of a protein list of regulated proteins are demonstrated by the comparative analysis of four different mixtures of three proteins (Ovalbumin, Horseradish Peroxidase and Rabbit Albumin) spiked into the complex protein background of the DGPF Proteome Marker. PMID:19953540

  19. 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

  20. Synthesis and application of isotopically labeled flavin nucleotides.

    PubMed

    Mishanina, Tatiana V; Kohen, Amnon

    2015-07-01

    Flavin nucleotides, i.e. flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are utilized as prosthetic groups and/or substrates by a myriad of proteins, ranging from metabolic enzymes to light receptors. Isotopically labeled flavins have served as invaluable tools in probing the structure and function of these flavoproteins. Here we present an enzymatic synthesis of several radio- and stable-isotope labeled flavin nucleotides from commercially available labeled riboflavin and ATP. The synthetic procedure employs a bifunctional enzyme, Corynebacterium ammoniagenes FAD synthetase, that sequentially converts riboflavin to FMN and then to FAD. The final flavin product (FMN or FAD) is controlled by the concentration of ATP in the reaction. Utility of the synthesized labeled FAD cofactors is demonstrated in flavin-dependent thymidylate synthase. The described synthetic approach can be easily applied to the production of flavin nucleotide analogues from riboflavin precursors. PMID:26149960

  1. A rapid and robust method for selective isotope labeling of proteins.

    PubMed

    Lin, Myat T; Sperling, Lindsay J; Frericks Schmidt, Heather L; Tang, Ming; Samoilova, Rimma I; Kumasaka, Takashi; Iwasaki, Toshio; Dikanov, Sergei A; Rienstra, Chad M; Gennis, Robert B

    2011-12-01

    Amino-acid selective isotope labeling of proteins offers numerous advantages in mechanistic studies by revealing structural and functional information unattainable from a crystallographic approach. However, efficient labeling of proteins with selected amino acids necessitates auxotrophic hosts, which are often not available. We have constructed a set of auxotrophs in a commonly used Escherichia coli expression strain C43(DE3), a derivative of E. coli BL21(DE3), which can be used for isotopic labeling of individual amino acids or sets of amino acids. These strains have general applicability to either soluble or membrane proteins that can be expressed in E. coli. We present examples in which proteins are selectively labeled with (13)C- and (15)N-amino acids and studied using magic-angle spinning solid-state NMR and pulsed EPR, demonstrating the utility of these strains for biophysical characterization of membrane proteins, radical-generating enzymes and metalloproteins. PMID:21925267

  2. Segmental isotopic labeling of proteins for nuclear magnetic resonance.

    PubMed

    Liu, Dongsheng; Xu, Rong; Cowburn, David

    2009-01-01

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

  3. Efficient and facile synthesis of novel stable monodeuterium labeled ractopamine.

    PubMed

    Su, Feifei; Wu, Fulong; Tang, He; Wang, Zhonghua; Wu, Fanhong

    2015-01-01

    A novel synthetic route to stable deuterium labeled ractopamine was disclosed with 6.49% total yield and 97.7% isotopic abundance. Its structure and the isotope-abundance were confirmed according to (1)H-NMR and high-resolution mass spectrometry. PMID:26526706

  4. 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

  5. Efficient Thread Labeling for Monitoring Programs with Nested Parallelism

    NASA Astrophysics Data System (ADS)

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

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

  6. 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...

  7. 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

  8. 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

  9. 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

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

    PubMed Central

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

    2012-01-01

    Optimal accuracy and precision in small molecule profiling by mass spectrometry generally requires isotopically labeled standards chemically representative of all compounds of interest. However, preparation of mixed standards from commercially available pure compounds is often prohibitively expensive and time consuming, and many labeled compounds are not available in pure form. We used a single prototype uniformly labeled [U-13C]-compound to generate [U-13C]-volatile standards for use in subsequent experimental profiling studies. [U-13C]-α-linolenic acid (C18:3n-3, ALA) was thermally oxidized to produce labeled lipid degradation volatiles which were subsequently characterized qualitatively and quantitatively. Twenty-five [U-13C]-labeled volatiles were identified by headspace solid-phase microextraction-gas chromatography-time of flight-mass spectrometry (HS-SPME-GC-TOF-MS) by comparison of spectra with unlabeled volatiles. Using 250 μL starting sample, labeled volatiles were quantified by a reverse isotope dilution procedure. Using the [U-13C]-labeled standards, limits of detection comparable to or better than previous HS-SPME reports were achieved, 0.010–1.04 ng/g. The performance of the [U-13C]-volatile standards was evaluated using a commodity soybean oil (CSO) oxidized at 60°C from 0 to 15 d. Relative responses of n-decane, an unlabeled internal standard otherwise absent from the mixture, and [U-13C]-oxidation products changed by up to 8-fold as the CSO matrix was oxidized, demonstrating that reliance on a single standard in volatile profiling studies yields inaccurate results due to changing matrix effects. The [U-13C]-standard mixture was used to quantify 25 volatiles in oxidized CSO and low-ALA soybean oil with an average relative standard deviation of 8.5%. Extension of this approach to other labeled substrates, e.g., [U-13C]-sugars and amino acids, for profiling studies should be feasible and can dramatically improve quantitative results compared to use of a single standard. PMID:22662968

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. General statistical framework for quantitative proteomics by stable isotope labeling.

    PubMed

    Navarro, Pedro; Trevisan-Herraz, Marco; Bonzon-Kulichenko, Elena; Núñez, Estefanía; Martínez-Acedo, Pablo; Pérez-Hernández, Daniel; Jorge, Inmaculada; Mesa, Raquel; Calvo, Enrique; Carrascal, Montserrat; Hernáez, María Luisa; García, Fernando; Bárcena, José Antonio; Ashman, Keith; Abian, Joaquín; Gil, Concha; Redondo, Juan Miguel; Vázquez, Jesús

    2014-03-01

    The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including (18)O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H₂O₂ concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data. PMID:24512137

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

    DOEpatents

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

    2014-12-09

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

  19. 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 β-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

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

    SciTech Connect

    Lapham, J.; Crothers, D.M.

    1994-12-01

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

  1. 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, François; Hardouin, Julie; Ele Ekouna, Jean-Pierre; Cosette, Pascal; Lerouge, Patrice; Boitel-Conti, Michèle; Afonso, Carlos; Ségalas-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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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.

  7. 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

  8. 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)

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

    PubMed Central

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

    2012-01-01

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

  10. 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

  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. 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

  13. Sample-efficient learning with auxiliary class-label information

    PubMed Central

    Nguyen, Quang; Valizadegan, Hamed; Seybert, Amy; Hauskrecht, Milos

    2011-01-01

    Building classification models from clinical data collected for past patients often requires additional example labeling and annotation by a human expert. Since example labeling may require to review a complete electronic health record the process can be very time consuming and costly. To make the process more cost-efficient, the number of examples an expert needs to label should be reduced. We develop and test a new approach for the classification learning in which, in addition to class labels provided by an expert, the learner is provided with auxiliary information that reflects how strong the expert feels about the class label. We show that this information can be extremely useful for practical classification tasks based on human assessment and can lead to improved learning with a smaller number of examples. We develop a new classification approach based on the support vector machines and the learning to rank methodologies capable of utilizing the auxiliary information during the model learning process. We demonstrate the benefit of the approach on the problem of learning an alert model for Heparin Induced Thrombocytopenia (HIT) by showing an improved classification performance of the models that are trained on a smaller number of labeled examples. PMID:22195160

  14. A facile method for expression and purification of (15)N isotope-labeled human Alzheimer's β-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 β-amyloid peptides with Aβ42 peptide representing a major isoform in the senile plaques. Given the pathological significance of Aβ42 in the progression of AD, there is considerable interest in understanding the structural ensembles for soluble monomer and oligomeric forms of Aβ42. This report describes an efficient method to express and purify high quality (15)N isotope-labeled Aβ42 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 Aβ42 as a fusion with IFABP. After the over-expression of the (15)N isotope-labeled IFABP-Aβ42 fusion protein in the inclusion bodies, pure (15)N isotope-labeled Aβ42 peptide is obtained following a purification method that is streamlined and improved from the method originally developed for the isolation of unlabeled Aβ42 peptide (Garai et al., 2009). We obtain a final yield of ∼ 6 mg/L culture for (15)N isotope-labeled Aβ42 peptide. Mass spectrometry and (1)H-(15)N HSQC spectra of monomeric Aβ42 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 Aβ42 peptide as well as its other variants including any Aβ42 peptide mutants. PMID:26231074

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

    PubMed

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

    2014-03-30

    Knowledge of digesta passage kinetics in ruminants is essential to predict nutrient supply to the animal in relation to optimal animal performance, environmental pollution and animal health. Fractional passage rates (FPR) of feed are widely used in modern feed evaluation systems and mechanistic rumen models, but data on nutrient-specific FPR are scarce. Such models generally rely on conventional external marker techniques, which do not always describe digesta passage kinetics in a satisfactory manner. Here the use of stable isotope-labelled dietary nutrients as a promising novel tool to assess nutrient-specific passage kinetics is discussed. Some major limitations of this technique include a potential marker migration, a poor isotope distribution in the labelled feed and a differential disappearance rate of isotopes upon microbial fermentation in non-steady state conditions. Such limitations can often be circumvented by using intrinsically stable isotope-labelled plant material. Data are limited but indicate that external particulate markers overestimate rumen FPR of plant fibre compared with the internal stable isotope markers. Stable isotopes undergo the same digestive mechanism as the labelled feed components and are thus of particular interest to specifically measure passage kinetics of digestible dietary nutrients. PMID:24114801

  16. 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

  17. 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

  18. 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

  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. 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.

  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.; Rösch, F.

    2006-12-01

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

  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. Affordable uniform isotope labeling with (2)H, (13)C and (15)N in insect cells.

    PubMed

    Sitarska, Agnieszka; Skora, Lukasz; Klopp, Julia; Roest, Susan; Fernández, César; Shrestha, Binesh; Gossert, Alvar D

    2015-06-01

    For a wide range of proteins of high interest, the major obstacle for NMR studies is the lack of an affordable eukaryotic expression system for isotope labeling. Here, a simple and affordable protocol is presented to produce uniform labeled proteins in the most prevalent eukaryotic expression system for structural biology, namely Spodoptera frugiperda insect cells. Incorporation levels of 80% can be achieved for (15)N and (13)C with yields comparable to expression in full media. For (2)H,(15)N and (2)H,(13)C,(15)N labeling, incorporation is only slightly lower with 75 and 73%, respectively, and yields are typically twofold reduced. The media were optimized for isotope incorporation, reproducibility, simplicity and cost. High isotope incorporation levels for all labeling patterns are achieved by using labeled algal amino acid extracts and exploiting well-known biochemical pathways. The final formulation consists of just five commercially available components, at costs 12-fold lower than labeling media from vendors. The approach was applied to several cytosolic and secreted target proteins. PMID:25929326

  9. Enantioselective synthesis of isotopically labeled homocitric acid lactone.

    PubMed

    Moore, Jared T; Hanhan, Nadine V; Mahoney, Maximillian E; Cramer, Stephen P; Shaw, Jared T

    2013-11-15

    A concise synthesis of homocitric acid lactone was developed to accommodate systematic placement of carbon isotopes (specifically (13)C) for detailed studies of this cofactor. This new route uses a chiral allylic alcohol, available in multigram quantities from enzymatic resolution, as a starting material, which transposes asymmetry through an Ireland-Claisen rearrangement. PMID:24180620

  10. 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...

  11. An air-tolerant approach to the carbonylative Suzuki-Miyaura coupling: applications in isotope labeling.

    PubMed

    Ahlburg, Andreas; Lindhardt, Anders T; Taaning, Rolf H; Modvig, Amalie E; Skrydstrup, Troels

    2013-10-18

    Carbonylative Suzuki-Miyaura coupling conditions have been developed that proceed without the exclusion of oxygen and in the presence of nondegassed and nondried solvents. By adapting the method to a two-chamber setup, the direct handling of carbon monoxide, produced from stable CO precursors, is avoided. The protocol afforded the desired benzophenones with excellent functional group tolerance and in good yields. Substituting the CO precursor, in the CO-producing chamber, with its carbon-13 labeled version generated the corresponding carbon-13 labeled benzophenones. Finally, the developed system was applied in the synthesis and isotope labeling of two pharmaceuticals, nordazepam and Tricor. PMID:24004340

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

    SciTech Connect

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

    1986-12-01

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

  13. 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

  14. 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

  15. 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...

  16. 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.

  17. 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

  18. Stable-isotope-labeled carbohydrates and nucleosides: Synthesis and applications in chemistry and biology

    SciTech Connect

    Serianni, A.S.

    1994-12-01

    Carbohydrates play important roles in many key biochemical processes in living cells. For example, they are metabolized to produce energy, mediate cell-cell recognition, and play an indirect role (as constituents of DNA and RNA) in DNA replication, RNA transcription, and protein synthesis. These roles, and others of comparable biochemical significance, have been studied to varying extends with the use of stable isotopically labeled molecules, usually in conjunction with NMR spectroscopy and/or mass spectrometry. For example, carbohydrate metabolism has been monitored in vitro and in vivo with the use of isotopically labeled compounds. Molecular aspects of cell-cell recognition, mediated by cell-surface glycoproteins and glycolipids, have been probed through NMR studies of isotopically labeled oligosaccharides. More recently, the solution behavior of DNA and RNA has been examined through the use of labeled oligonucleotides. In all of these pursuits, the effort and expense to prepare labeled molecules, both of which can be substantial, are more than offset by the wealth of information derived from these studies. This information often cannot be accessed, or can be accessed only with great difficulty, using natural (unlabeled) compounds.

  19. 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

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

    NASA Astrophysics Data System (ADS)

    Chandra, Subhash

    2004-06-01

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

  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. 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

  3. 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

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

    PubMed

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

    2013-10-01

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

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

    PubMed Central

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

    2013-01-01

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

  6. Stable isotope labeling of glycoprotein expressed in silkworms using immunoglobulin G as a test molecule.

    PubMed

    Yagi, Hirokazu; Nakamura, Masatoshi; Yokoyama, Jun; Zhang, Ying; Yamaguchi, Takumi; Kondo, Sachiko; Kobayashi, Jun; Kato, Tatsuya; Park, Enoch Y; Nakazawa, Shiori; Hashii, Noritaka; Kawasaki, Nana; Kato, Koichi

    2015-06-01

    Silkworms serve as promising bioreactors for the production of recombinant proteins, including glycoproteins and membrane proteins, for structural and functional protein analyses. However, lack of methodology for stable isotope labeling has been a major deterrent to using this expression system for nuclear magnetic resonance (NMR) structural biology. Here we developed a metabolic isotope labeling technique using commercially available silkworm larvae. The fifth instar larvae were infected with baculoviruses for co-expression of recombinant human immunoglobulin G (IgG) as a test molecule, with calnexin as a chaperone. They were subsequently reared on an artificial diet containing (15)N-labeled yeast crude protein extract. We harvested 0.1 mg of IgG from larva with a (15)N-enrichment ratio of approximately 80%. This allowed us to compare NMR spectral data of the Fc fragment cleaved from the silkworm-produced IgG with those of an authentic Fc glycoprotein derived from mammalian cells. Therefore, we successfully demonstrated that our method enables production of isotopically labeled glycoproteins for NMR studies. PMID:25902760

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

    PubMed

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

    2015-01-01

    Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. PMID:25832445

  8. 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 δ13CO2 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 δ13C 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 δ13C 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. Efficient methods and practical guidelines for simulating isotope effects

    NASA Astrophysics Data System (ADS)

    Ceriotti, Michele; Markland, Thomas E.

    2013-01-01

    The shift in chemical equilibria due to isotope substitution is frequently exploited to obtain insight into a wide variety of chemical and physical processes. It is a purely quantum mechanical effect, which can be computed exactly using simulations based on the path integral formalism. Here we discuss how these techniques can be made dramatically more efficient, and how they ultimately outperform quasi-harmonic approximations to treat quantum liquids not only in terms of accuracy, but also in terms of computational cost. To achieve this goal we introduce path integral quantum mechanics estimators based on free energy perturbation, which enable the evaluation of isotope effects using only a single path integral molecular dynamics trajectory of the naturally abundant isotope. We use as an example the calculation of the free energy change associated with H/D and 16O/18O substitutions in liquid water, and of the fractionation of those isotopes between the liquid and the vapor phase. In doing so, we demonstrate and discuss quantitatively the relative benefits of each approach, thereby providing a set of guidelines that should facilitate the choice of the most appropriate method in different, commonly encountered scenarios. The efficiency of the estimators we introduce and the analysis that we perform should in particular facilitate accurate ab initio calculation of isotope effects in condensed phase systems.

  15. 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.

  16. Chemical Ligation and Isotope Labeling to Locate Dynamic Effects during Catalysis by Dihydrofolate Reductase.

    PubMed

    Luk, Louis Y P; Ruiz-Pernía, J Javier; Adesina, Aduragbemi S; Loveridge, E Joel; Tuñón, Iñaki; Moliner, Vincent; Allemann, Rudolf K

    2015-07-27

    Chemical ligation has been used to alter motions in specific regions of dihydrofolate reductase from E. coli and to investigate the effects of localized motional changes on enzyme catalysis. Two isotopic hybrids were prepared; one with the mobile N-terminal segment containing heavy isotopes ((2) H, (13) C, (15) N) and the remainder of the protein with natural isotopic abundance, and the other one with only the C-terminal segment isotopically labeled. Kinetic investigations indicated that isotopic substitution of the N-terminal segment affected only a physical step of catalysis, whereas the enzyme chemistry was affected by protein motions from the C-terminal segment. QM/MM studies support the idea that dynamic effects on catalysis mostly originate from the C-terminal segment. The use of isotope hybrids provides insights into the microscopic mechanism of dynamic coupling, which is difficult to obtain with other studies, and helps define the dynamic networks of intramolecular interactions central to enzyme catalysis. PMID:26079622

  17. 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.

  18. 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

  19. 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...

  20. 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.

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

    PubMed Central

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

    2011-01-01

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

  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. Pharmacokinetics of therapeutic doses of isotopically labeled platinum antitumor agents in the mouse and rat

    SciTech Connect

    Robins, A.B.; Leach, M.O.

    1983-03-01

    The pharmacokinetics of the antitumor agents ethylenediamine, platinum dichloride, and cisplatin have been investigated in mice bearing the ADJ/PC6 plasma cell tumor after therapeutic doses of these agents, labeled either with 191Pt or in the ligand with 14C. The distribution of the radioactive labels has been estimated in tumor, kidney, liver, and intestine in treated animals from 1 hour to 15 days after administration. The two labels, on ligand and on Pt, were equivalent only in some cases with respect to the total radioactivity within a tissue. Patterns of isotope distribution varied with time and tissue and suggested that the metal-ligand bond was dissociated as early as 1 hour after administration.

  4. 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

  5. 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; Bödi, 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

  6. 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.

  7. 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

  8. 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

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

    PubMed Central

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

    2012-01-01

    The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (TEM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by TEM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni2+ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of 15N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a Kd of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for TEM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of 15N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels. PMID:22659540

  10. 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

  11. 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.

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

    PubMed Central

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

    2012-01-01

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

  13. 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

  14. 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.

  15. Identification of ejaculated proteins in the house mouse (Mus domesticus) via isotopic labeling

    PubMed Central

    2011-01-01

    Background Seminal fluid plays an important role in successful fertilization, but knowledge of the full suite of proteins transferred from males to females during copulation is incomplete. The list of ejaculated proteins remains particularly scant in one of the best-studied mammalian systems, the house mouse (Mus domesticus), where artificial ejaculation techniques have proven inadequate. Here we investigate an alternative method for identifying ejaculated proteins, by isotopically labeling females with 15N and then mating them to unlabeled, vasectomized males. Proteins were then isolated from mated females and identified using mass spectrometry. In addition to gaining insights into possible functions and fates of ejaculated proteins, our study serves as proof of concept that isotopic labeling is a powerful means to study reproductive proteins. Results We identified 69 male-derived proteins from the female reproductive tract following copulation. More than a third of all spectra detected mapped to just seven genes known to be structurally important in the formation of the copulatory plug, a hard coagulum that forms shortly after mating. Seminal fluid is significantly enriched for proteins that function in protection from oxidative stress and endopeptidase inhibition. Females, on the other hand, produce endopeptidases in response to mating. The 69 ejaculated proteins evolve significantly more rapidly than other proteins that we previously identified directly from dissection of the male reproductive tract. Conclusion Our study attempts to comprehensively identify the proteins transferred from males to females during mating, expanding the application of isotopic labeling to mammalian reproductive genomics. This technique opens the way to the targeted monitoring of the fate of ejaculated proteins as they incubate in the female reproductive tract. PMID:21663664

  16. 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

  17. 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.

  18. 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

  19. Stable Carbon Isotopes As Indicators of Plant Water Use Efficiency

    NASA Astrophysics Data System (ADS)

    Powers, E. M.; Marshall, J. D.; Ubierna Lopez, N.

    2007-12-01

    Stable carbon isotopes have been utilized to better understand how environmental variables influence the efficiency of photosynthesis, specifically what factors limit the uptake and absorption of CO2 during photosynthesis. An understanding of the controls over both gas exchange and stomatal conductance can provide an explanation for the possible environmental influences on plant WUE. The δ13C of extractive-free wood was used as an index of plant water use efficiency at Mica Creek Experimental Watershed, Shoshone County, ID. The δ13C values of tree rings were used to determine the effects of clear cut and partial cut harvesting practices, the effect of elevation, and species differences in intrinsic water use efficiency (WUE) among coniferous species including: Thuja plicata, Larix occidentalis, Picea engelmannii, Pseudotsuga menziesii, Abies lasiocarpa, and Abies grandis. We found significant effects of harvest treatments (p=0.0197), elevation (p= 0.0268), and species (p<0.001) on tree δ13C. The significantly more enriched isotopic signatures observed in Thuja plicata (δ13C = -23.37 ±0.17‰), indicate that it is a more water use efficient species compared to Larix occidentalis (δ13C = -25.66 ±0.43‰), and Abies grandis (δ13C = -25.83 ±0.15‰). There was also an overall trend of δ13C enrichment with elevation. The isotopic composition of tree rings has been estimated to increase by 0.003 ‰ per meter of elevation gain, which may be related to a decrease in soil moisture with elevation. Finally, the mean δ13C values observed on partial cut (δ13C = -24.73 ±0.10‰) and clear cut treatments (δ13C = -24.45 ±0.29‰) were significantly more enriched than the mean value for the control treatment (δ13C = -25.25 ±0.19‰). The more enriched isotopic signatures observed on the harvested treatments indicate that the trees are more water use efficient, which may be a result of increased photosynthetic capacity with an increase in the availability of water, foliar nitrogen, and light to individual trees on the harvested treatments. The reduction of stand density through harvesting may reduce the transpirational water losses on a stand level, thus increasing the water availability for individual trees.

  20. Effect of acetaminophen on the leukocyte-labeling efficiency of indium oxine In 111

    SciTech Connect

    Augustine, S.C.; Schmelter, R.F.; Nelson, K.L.; Petersen, R.J.; Qualfe, M.A.

    1983-11-01

    The effect of acetaminophen on the labeling efficiency of leukocytes with indium oxine In 111 was studied. A blood sample was obtained from eight healthy men before and after they received acetaminophen 650 mg every four hours for 24 hours. After dividing the plasma from each sample into three portions, leukocytes were separated and labeled with indium oxine In 111. In an in vitro study, 200 ml of blood was obtained from one of the men, and the plasma was separated into four portions. Acetaminophen in 95% ethanol was added to three of the plasma fractions to produce acetaminophen concentrations of 4, 20, and 100 micrograms/ml; ethanol was added to the fourth fraction as a control. Each plasma fraction was then subdivided into three aliquots, and leukocytes were labeled as in the in vivo study. Mean leukocyte labeling efficiencies in both studies were calculated from the ratios of leukocyte radioactivity to initial radioactivity in the samples, expressed as percentages. Leukocyte labeling efficiencies before acetaminophen administration ranged from 79 to 85%; after administration, labeling efficiencies ranged from 70 to 87%. No significant differences in mean labeling efficiency before and after acetaminophen administration were noted in any of the subjects. Leukocyte labeling efficiencies in all in vitro plasma fractions were reduced, ranging from 54 to 63%, but no significant differences in labeling efficiency between any of the plasma fractions were found. Using the labeling procedures in this study, exposure of leukocytes from healthy men to acetaminophen in vivo or in vitro does not affect labeling efficiency with indium oxine In 111.

  1. 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

  2. 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.

  3. 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+.

  4. LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and (13)C-isotopic labeling of acyl-coenzyme A thioesters.

    PubMed

    Frey, Alexander J; Feldman, Daniel R; Trefely, Sophie; Worth, Andrew J; Basu, Sankha S; Snyder, Nathaniel W

    2016-05-01

    Acyl-coenzyme A (acyl-CoA) thioesters are evolutionarily conserved, compartmentalized, and energetically activated substrates for biochemical reactions. The ubiquitous involvement of acyl-CoA thioesters in metabolism, including the tricarboxylic acid cycle, fatty acid metabolism, amino acid degradation, and cholesterol metabolism highlights the broad applicability of applied measurements of acyl-CoA thioesters. However, quantitation of acyl-CoA levels provides only one dimension of metabolic information and a more complete description of metabolism requires the relative contribution of different precursors to individual substrates and pathways. Using two distinct stable isotope labeling approaches, acyl-CoA thioesters can be labeled with either a fixed [(13)C3 (15)N1] label derived from pantothenate into the CoA moiety or via variable [(13)C] labeling into the acyl chain from metabolic precursors. Liquid chromatography-hybrid quadrupole/Orbitrap high-resolution mass spectrometry using parallel reaction monitoring, but not single ion monitoring, allowed the simultaneous quantitation of acyl-CoA thioesters by stable isotope dilution using the [(13)C3 (15)N1] label and measurement of the incorporation of labeled carbon atoms derived from [(13)C6]-glucose, [(13)C5 (15)N2]-glutamine, and [(13)C3]-propionate. As a proof of principle, we applied this method to human B cell lymphoma (WSU-DLCL2) cells in culture to precisely describe the relative pool size and enrichment of isotopic tracers into acetyl-, succinyl-, and propionyl-CoA. This method will allow highly precise, multiplexed, and stable isotope-resolved determination of metabolism to refine metabolic models, characterize novel metabolism, and test modulators of metabolic pathways involving acyl-CoA thioesters. Graphical abstract LC-MS/HRMS allows resolution of variable stable isotopes incorporated into acyl-CoAs, enabling simultaneous quantitation and metabolic tracing. PMID:26968563

  5. Isotope labeling and theoretical study of the formation of a3* ions from protonated tetraglycine.

    PubMed

    Cooper, Travis; Talaty, Erach; Grove, Jerod; Van Stipdonk, Michael; Suhai, Sándor; Paizs, Béla

    2006-12-01

    Extensive 13C, 15N, and 2H labeling of tetraglycine was used to investigate the b3+ --> a3* reaction during low-energy collision-induced dissociation (CID) in a quadrupole ion-trap mass spectrometer. The patterns observed with respect to the retention or elimination of the isotope labels demonstrate that the reaction pathway involves elimination of CO and NH3. The ammonia molecule includes 2 H atoms from amide or amino positions, and one from an alpha-carbon position. The loss of NH3 does not involve elimination of the N-terminal amino group but, instead, the N atom of the presumed oxazolone ring in the b3+ ion. The CO molecule eliminated is the carbonyl group of the same oxazolone ring, and the alpha-carbon H atom is transferred from the amino acid adjacent to the oxazolone ring. Quantum chemical calculations indicate a multistep reaction cascade involving CO loss on the b3 --> a3 pathway and loss of NH=CH2 from the a3 ion to form b2. In the postreaction complex of b2 and NH=CH2, the latter can be attacked by the N-terminal amino group of the former. The product of this attack, an isomerized a3 ion, can eliminate NH3 from its N-terminus to form a3*. Calculations suggest that the ammonia and a3* species can form various ion-molecule complexes, and NH3 can initiate relay-type mobilization of the oxazolone H atoms from alpha-carbon positions to form a new oxazolone isomer. This multiple-step reaction scheme clearly explains the isotope labeling results, including unexpected scrambling of H atoms from alpha-carbon positions. PMID:16934997

  6. 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.

  7. 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

  8. Segmental Isotope Labelling of an Individual Bromodomain of a Tandem Domain BRD4 Using Sortase A

    PubMed Central

    Williams, Felix P.; Milbradt, Alexander G.; Embrey, Kevin J.

    2016-01-01

    Bromodomain and extra-terminal (BET) family of proteins are one of the major readers of epigenetic marks and an important target class in oncology and other disease areas. The importance of the BET family of proteins is manifested by the explosion in the number of inhibitors against these targets that have successfully entered clinical trials. One important BET family member is bromodomain containing protein 4 (BRD4). Structural and biophysical studies of BRD4 are complicated by its tertiary-structure consisting of two bromodomains connected by a flexible inter-domain linker of approximately 180 amino acids. A detailed understanding of the interplay of these bromodomains will be key to rational drug design in BRD4, yet there are no reported three-dimensional structures of the multi-domain BRD4 and NMR studies of the tandem domain are hampered by the size of the protein. Here, we present a method for rapid Sortase A-mediated segmental labelling of the individual bromodomains of BRD4 that provides a powerful strategy that will enable NMR studies of ligand-bromodomain interactions with atomic detail. In our labelling strategy, we have used U-[2H,15N]-isotope labelling on the C-terminal bromodomain with selective introduction of 13CH3 methyl groups on Ile (δ1), Val and Leu, whereas the N-terminal bromodomain remained unlabelled. This labelling scheme resulted in significantly simplified NMR spectra and will allow for high-resolution interaction, structure and dynamics studies in the presence of ligands. PMID:27128490

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

    PubMed

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

    2005-12-01

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

  10. 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

  11. 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.

  12. 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).

  13. 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.

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

    PubMed

    Gülsün, 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

  15. Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device.

    PubMed

    Morozov, Victor N; Gavryushkin, Alexander V; Deev, Alexander A

    2002-03-01

    A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10-50 microm in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried and placed face down onto the surface of a standard B/W video CCD chip with the protective window removed. We show here that such a simple inexpensive CCD detector can be used to quantify distribution of 14C and other radioactive isotopes on microarrays. PMID:11879919

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

    NASA Astrophysics Data System (ADS)

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

    2015-04-01

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

  17. Experimental investigation of rates and mechanisms of isotope exchange (O, H) between volcanic ash and isotopically-labeled water

    NASA Astrophysics Data System (ADS)

    Nolan, Gary S.; Bindeman, Ilya N.

    2013-06-01

    The hydrogen and oxygen isotope ratios in hydrous minerals and volcanic glass are routinely used as paleo-proxies to infer the isotopic values of meteoric waters and thus paleo-climatic conditions. We report a series of long-term exposure experiments of distal 7700 BP Mt. Mazama ash (-149‰ δ2H, +7‰ δ18O, 3.8 wt.% H2O) with isotopically-labeled water (+650‰ δ2H, +56‰ δ18O). Experiments were done at 70, 40 and 20 °C, and ranged in duration from 1 to 14454 h (˜20 months), to evaluate the rates of deuterium and 18O exchange, and investigate the relative role of exchange and diffusion. We also investigate the effect of drying on H2Otot and δ2H in native and reacted ash that can be used in defining the protocols for natural sample preparation. We employ Thermal Conversion Elemental Analyzer (TCEA) mass spectrometry, thermogravimetric analysis and a KBr pellet technique with infrared spectroscopy to measure the evolution of δ2H, total water, and OH water peaks in the course of exposure experiments, and in varying lengths of vacuum drying. Time series experiments aided by infrared measurements demonstrate the following new results: (i) It wasobserved that from 5 to >100‰ δ2H increases with time, with faster deuterium exchange at higher temperatures. Times at 15% of theoretical "full δ2H exchange" are: 15.8 years at 20 °C, 5.2 years at 40 °C, and 0.4 years at 70 °C. (ii) Even at extended exposure durations experiments show no net increase in water weight percent nor in δ18O in ash; water released from ash rapidly by thermal decomposition is not enriched in δ18O. This observation clearly suggests that it is hydrogen exchange, and not water addition or oxygen exchange that characterizes the process. (iii) Our time series drying, Fourier transform infrared (FTIR)-KBr and Thermogravimetric Analyzer (TGA) analyses collectively suggest a simple mechanistic view that there are three kinds of "water" in ash: water (mostly H2O) that is less strongly bonded on the surface of ash particles that are getting lost with 24-48 h of drying to up to 200-300 °C, bound water in glass in a range of combining proportions of SiOH to H2O, and a small reservoir of residual, tightly held water. Experimentation with vacuum drying at 130-220 °C, and with TGA up to 1000 °C provides a set of simple relationships and recommendations for users. Ash loses 1-1.2 wt.% water with weight loss stabilizing after 48 h of vacuum drying at 130 °C. This ash drying removes molecular water over the hydroxyl group in a proportion of ˜80:20% resulting in relatively constant δ2H values of the remaining total water in native ash. This study demonstrates that δ2H in ash can be rapidly changed by minor diagenesis even at relatively low temperatures of 20 °C. A diagenetic history of ash is needed to interpret the D/H ratio, but the δ18O values of water in ash are more robust.

  18. 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.

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

    PubMed

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

    2016-05-01

    The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein-by-protein basis. The ability to measure protein synthesis and degradation rates on a proteome-wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well-established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L-leucine was replaced with a stable isotope labelled analogue ([(2) H7 ]L-leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates. PMID:26929125

  20. Efficient magnetic cell labeling with protamine sulfate complexed to ferumoxides for cellular MRI.

    PubMed

    Arbab, Ali S; Yocum, Gene T; Kalish, Heather; Jordan, Elaine K; Anderson, Stasia A; Khakoo, Aarif Y; Read, Elizabeth J; Frank, Joseph A

    2004-08-15

    Recently, there have been several reports using various superparamagnetic iron oxide (SPIO) nanoparticles to label mammalian cells for monitoring their temporal and spatial migration in vivo by magnetic resonance imaging (MRI). The purpose of this study was to evaluate the efficiency and toxicity of labeling cells using 2 commercially available Food and Drug Administration (FDA)-approved agents, ferumoxides, a suspension of dextran-coated SPIO used as an MRI contrast agent, and protamine sulfate, conventionally used to reverse heparin anticoagulation but also used ex vivo as a cationic transfection agent. After labeling of human mesenchymal stem cells (MSCs) and hematopoietic (CD34+) stem cells and other mammalian cells with ferumoxides-protamine sulfate complexes (FE-Pro), cellular toxicity, functional capacity, and quantitative cellular iron incorporation were determined. FE-Pro-labeled cells demonstrated no short- or long-term toxicity, changes in differentiation capacity of the stem cells, or changes in phenotype when compared with unlabeled cells. Efficient labeling with FE-Pro was observed with iron content per cell varying between 2.01 +/- 0.1 pg for CD34+ cells and 10.94 +/- 1.86 pg for MSCs with 100% of cells labeled. Cell labeling using these agents should facilitate the translation of this method to clinical trials for evaluation of trafficking of infused or transplanted cells by MRI. PMID:15100158

  1. Tracking the metabolic pulse of plant lipid production with isotopic labeling and flux analyses: Past, present and future.

    PubMed

    Allen, Doug K; Bates, Philip D; Tjellström, Henrik

    2015-04-01

    Metabolism is comprised of networks of chemical transformations, organized into integrated biochemical pathways that are the basis of cellular operation, and function to sustain life. Metabolism, and thus life, is not static. The rate of metabolites transitioning through biochemical pathways (i.e., flux) determines cellular phenotypes, and is constantly changing in response to genetic or environmental perturbations. Each change evokes a response in metabolic pathway flow, and the quantification of fluxes under varied conditions helps to elucidate major and minor routes, and regulatory aspects of metabolism. To measure fluxes requires experimental methods that assess the movements and transformations of metabolites without creating artifacts. Isotopic labeling fills this role and is a long-standing experimental approach to identify pathways and quantify their metabolic relevance in different tissues or under different conditions. The application of labeling techniques to plant science is however far from reaching it potential. In light of advances in genetics and molecular biology that provide a means to alter metabolism, and given recent improvements in instrumentation, computational tools and available isotopes, the use of isotopic labeling to probe metabolism is becoming more and more powerful. We review the principal analytical methods for isotopic labeling with a focus on seminal studies of pathways and fluxes in lipid metabolism and carbon partitioning through central metabolism. Central carbon metabolic steps are directly linked to lipid production by serving to generate the precursors for fatty acid biosynthesis and lipid assembly. Additionally some of the ideas for labeling techniques that may be most applicable for lipid metabolism in the future were originally developed to investigate other aspects of central metabolism. We conclude by describing recent advances that will play an important future role in quantifying flux and metabolic operation in plant tissues. PMID:25773881

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

    PubMed

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

    2012-08-01

    Fluorescence analysis has proved to be a powerful detection technique for achieving single molecule analysis. However, it usually requires the labeling of targets with bright fluorescent tags since most chemicals and biomolecules lack fluorescence. Conventional fluorescence labeling methods require a considerable quantity of biomolecule samples, long reaction times and extensive chromatographic purification procedures. Herein, a micro/nanofluidics device integrating a nanochannel in a microfluidics chip has been designed and fabricated, which achieves rapid protein concentration, fluorescence labeling, and efficient purification of product in a miniaturized and continuous manner. As a demonstration, labeling of the proteins bovine serum albumin (BSA) and IgG with fluorescein isothiocyanate (FITC) is presented. Compared to conventional methods, the present micro/nanofluidics device performs about 10(4)-10(6) times faster BSA labeling with 1.6 times higher yields due to the efficient nanoconfinement effect, improved mass, and heat transfer in the chip device. The results demonstrate that the present micro/nanofluidics device promises rapid and facile fluorescence labeling of small amount of reagents such as proteins, nucleic acids and other biomolecules with high efficiency. PMID:22648530

  3. ­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).

  4. Comparison of transfection agents in forming complexes with ferumoxides, cell labeling efficiency, and cellular viability.

    PubMed

    Arbab, Ali Syed; Yocum, Gene Thomus; Wilson, Lindsey Bashaw; Parwana, Ashari; Jordan, Elaine Kay; Kalish, Heather; Frank, Joseph Alan

    2004-01-01

    By complexing ferumoxides or superparamagnetic iron oxide (SPIO) to transfection agents (TAs), it is possible to magnetically label mammalian cells. There has been no systematic study comparing TAs complexed to SPIO as far as cell labeling efficiency and viability. This study investigates the toxicity and labeling efficiency at various doses of FEs complexed to different TAs in mammalian cells. Different classes of TAs were used, such as polycationic amines, dendrimers, and lipid-based agents. Cellular toxicity was measured using doses of TAs from 1 to 50 microg/mL in incubation media. Iron incorporation efficiency was measured by combining various amounts of FEs and different doses of TAs. Lipofectamine2000 showed toxicity at lowest dose (1 microg/mL), whereas FuGENE6 and low molecular weight poly-L-lysine (PLL) showed the least toxicity. SPIO labeling efficiency was similar with high-molecular-weight PLL (388.1 kDa) and superfect, whereas FuGENE6 and low-molecular-weight PLL were inefficient in labeling cells. Concentrations of 25 to 50 microg/mL of FEs complexed to TAs in media resulted in sufficient endocytosis of the SPIO into endosomes to detect cells on cellular magnetic resonance imaging. PMID:15142409

  5. 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.

  6. 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

  7. 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  9. 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

  10. IDENTIFICATION AND QUANTITATIVE STUDIES OF PROTEIN IMMOBILIZATION SITES BY STABLE ISOTOPE LABELING AND MASS SPECTROMETRY

    PubMed Central

    Wa, Chunling; Cerny, Ron; Hage, David S.

    2008-01-01

    A method was developed for characterizing immobilization sites on a protein based on stable isotope labeling and MALDI-TOF mass spectrometry. The model for this work was human serum albumin (HSA) immobilized onto silica by the Schiff base method. The immobilized HSA was digested by various proteolytic enzymes in the presence of normal water, while soluble HSA was digested in 18O-enriched water for use as an internal standard. These two digests were mixed and analyzed, with the 18O/16O ratio for each detected peptide then being measured. Several peptides in the tryptic, Lys-C, and Glu-C digests gave significantly higher 18O/16O ratios than other peptides in the same digests, implying their involvement in immobilization. Analysis of these results led to identification of the N-terminus and several lysines as likely immobilization sites for HSA (e.g., K4, K41, K190, K225, K313 and K317). It was also possible from these results to quantitatively rank these sites in terms of the relative degree to which each might take part in immobilization. This method is not limited to HSA and silica but can be used with other proteins and supports. PMID:17134129

  11. Measuring the Composition and Stable-Isotope Labeling of Algal Biomass Carbohydrates via Gas Chromatography/Mass Spectrometry.

    PubMed

    McConnell, Brian O; Antoniewicz, Maciek R

    2016-05-01

    We have developed a method to measure carbohydrate composition and stable-isotope labeling in algal biomass using gas chromatography/mass spectrometry (GC/MS). The method consists of two-stage hydrochloric acid hydrolysis, followed by chemical derivatization of the released monomer sugars and quantification by GC/MS. Fully (13)C-labeled sugars are used as internal standards for composition analysis. This convenient, reliable, and accurate single-platform workflow offers advantages over existing methods and opens new opportunities to study carbohydrate metabolism of algae under autotrophic, mixotrophic, and heterotrophic conditions using metabolic flux analysis and isotopic tracers such as (2)H2O and (13)C-glucose. PMID:27042946

  12. 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

  13. 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.

  14. 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

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

    PubMed

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

    2011-11-01

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

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

    PubMed

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

    2016-04-01

    The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK). PMID:26991121

  17. 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

  18. 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

  19. 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.

  20. 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 β2-adrenergic receptor including experiments that characterize the functional conformational-changes associated with activation of distinct signaling pathways induced by different β-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

  1. The use of isotopic labels to probe the mechanism of DNA oxidation by iron bleomycin

    SciTech Connect

    Rabow, L.E.

    1990-01-01

    When the antitumor antibiotic bleomycin is activated anaerobically with Fe(III) and hydrogen peroxide or with Fe(II) and limiting oxygen, the DNA products are free nucleic acid base and an oxidatively damaged sugar lesion which undergoes strand scission when treated with alkali. Stabilization of the initial product by borohydride reduction and digestion by P{sub 1} nuclease and alkaline phosphatase afforded 2{double prime}-deoxypentitol-3{double prime}-O-5{prime}-phospho-2{prime}-deoxypurine nucleosides that accounted for 99, 81 and 48% of the pyrimidine base released from d(CGCGCG), poly(dA-dU) and poly(dG-dC), respectively. Further enzymatic degradation yielded 2-deoxy-D-erythro-pentitol and 2-deoxy-L-threo-pentitol which were identified by mass spectrometry. The 2{prime}-deoxypentos-4{prime}-ulose product arising from the interaction of d(CGCGCG) with bleomycin was 86 and 97% {sup 18}O-labeled at C-4{prime} at pH 9.0 and 7.8, respectively, when limiting {sup 16}O-labeled oxygen was used to activate bleomycin in {sup 18}O-water. Either complete isotopic exchange between solvent and a high-valent iron-oxo species of bleomycin or the equivalent of a 1e{sup {minus}} oxidation of the presumed 4 carbon-centered radical of DNA are required to account for these findings. When oxygen is supplied in excess of what is required to activate bleomycin, the C3{prime}-C4{prime} bond of DNA is ruptured to yield transbase propenals and oligonucleotides bearing 5-phosphate and 3-phosphoglycolate termini. Kinetics study of base propenal formation from a DNA-bound precursor and release of {sup 3}H from pro R and pro S poly(dA-(2-{sup 3}H)dU) showed that reported rapid release compared penal formation was the result of specific release from the pro R position and was not a consequence of the base release pathway nor nonstereospecific enolization of 2{prime} hydrogens.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. High-Performance Chemical Isotope Labeling Liquid Chromatography-Mass Spectrometry for Profiling the Metabolomic Reprogramming Elicited by Ammonium Limitation in Yeast.

    PubMed

    Luo, Xian; Zhao, Shuang; Huan, Tao; Sun, Difei; Friis, R Magnus N; Schultz, Michael C; Li, Liang

    2016-05-01

    Information about how yeast metabolism is rewired in response to internal and external cues can inform the development of metabolic engineering strategies for food, fuel, and chemical production in this organism. We report a new metabolomics workflow for the characterization of such metabolic rewiring. The workflow combines efficient cell lysis without using chemicals that may interfere with downstream sample analysis and differential chemical isotope labeling liquid chromatography mass spectrometry (CIL LC-MS) for in-depth yeast metabolome profiling. Using (12)C- and (13)C-dansylation (Dns) labeling to analyze the amine/phenol submetabolome, we detected and quantified a total of 5719 peak pairs or metabolites. Among them, 120 metabolites were positively identified using a library of 275 Dns-metabolite standards, and 2980 metabolites were putatively identified based on accurate mass matches to metabolome databases. We also applied (12)C- and (13)C-dimethylaminophenacyl (DmPA) labeling to profile the carboxylic acid submetabolome and detected over 2286 peak pairs, from which 33 metabolites were positively identified using a library of 188 DmPA-metabolite standards, and 1595 metabolites were putatively identified. Using this workflow for metabolomic profiling of cells challenged by ammonium limitation revealed unexpected links between ammonium assimilation and pantothenate accumulation that might be amenable to engineering for better acetyl-CoA production in yeast. We anticipate that efforts to improve other schemes of metabolic engineering will benefit from application of this workflow to multiple cell types. PMID:26947805

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

    PubMed

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

    2016-05-01

    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 Ca(2+) on calmodulin conformation alternation. This new cross-linking MS method is fast and would have high value in structural biology. Graphical Abstract ᅟ. PMID:26902947

  8. 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

    2016-05-01

    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.

  9. 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.

  10. A simple isotopic labeling method to study cysteine oxidation in Alzheimer's disease: oxidized cysteine-selective dimethylation (OxcysDML).

    PubMed

    Gu, Liqing; Robinson, Renã A S

    2016-04-01

    Cysteine is widely involved in redox signaling pathways through a number of reversible and irreversible modifications. Reversible modifications (e.g., S-glutathionylation, S-nitrosylation, disulfide bonds, and sulfenic acid) are used to protect proteins from oxidative attack and maintain cellular homeostasis, while irreversible oxidations (e.g., sulfinic acid and sulfonic acid) serve as hallmarks of oxidative stress. Proteomic analysis of cysteine-enriched peptides coupled with reduction of oxidized thiols can be used to measure the oxidation states of cysteine, which is helpful for elucidating the role that oxidative stress plays in biology and disease. As an extension of our previously reported cysDML method, we have developed oxidized cysteine-selective dimethylation (OxcysDML), to investigate the site-specific total oxidation of cysteine residues in biologically relevant samples. OxcysDML employs (1) blocking of free thiols by a cysteine-reactive reagent, (2) enrichment of peptides containing reversibly oxidized cysteine by a solid phase resin, and (3) isotopic labeling of peptide amino groups to quantify cysteine modifications arising from different biological conditions. On-resin enrichment and labeling minimizes sample handing time and improves efficiency in comparison with other redox proteomic methods. OxcysDML is also inexpensive and flexible, as it can accommodate the exploration of various cysteine modifications. Here, we applied the method to liver tissues from a late-stage Alzheimer's disease (AD) mouse model and wild-type (WT) controls. Because we have previously characterized this proteome using the cysDML approach, we are able here to probe deeper into the redox status of cysteine in AD. OxcysDML identified 1129 cysteine sites (from 527 proteins), among which 828 cysteine sites underwent oxidative modifications. Nineteen oxidized cysteine sites had significant alteration levels in AD and represent proteins involved in metabolic processes. Overall, we have demonstrated OxcysDML as a simple, rapid, robust, and inexpensive redox proteomic approach that is useful for gaining deeper insight into the proteome of AD. Graphical abstract OxcysDML enables the proteome comparision of cysteine reversible oxidations from two biological conditions. PMID:26800981

  11. 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

  12. 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).

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

    PubMed

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

    2014-08-01

    Bristol-Myers Squibb and others are developing drugs that target novel mechanisms to combat Alzheimer's disease. γ-Secretase inhibitors are one class of potential therapies that have received considerable attention. (R)-2-(4-Chloro-N-(2-fluoro-4-(1,2,4-oxadiazol-3-yl)benzyl)phenylsulfonamido)-5,5,5-trifluoropentanamide (Avagacestat) is a γ-secretase-inhibiting drug that has been investigated by Bristol-Myers Squibb in preclinical and clinical studies. An important step in the development process was the synthesis of a carbon-14-labeled analog for use in a human absorption, distribution, metabolism, and excretion study and a stable isotope labeled analog for use as a standard in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. Carbon-14 labeled Avagacestat was synthesized in seven steps in a 33% overall yield from carbon-14 labeled potassium cyanide. A total of 5.95 mCi was prepared with a specific activity of 0.81 μCi/mg and a radiochemical purity of 99.9%. (13) C6 -Labeled Avagacestat was synthesized in three steps in a 15% overall yield from 4-chloro[(13) C6 ]aniline. A total of 585 mg was prepared with a ultraviolet purity of 99.9%. PMID:25196195

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

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

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

    PubMed

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

    2015-01-01

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

  2. High-performance liquid chromatographic-tandem mass spectrometric evaluation and determination of stable isotope labeled analogs of rofecoxib in human plasma samples from oral bioavailability studies.

    PubMed

    Chavez-Eng, C M; Constanzer, M L; Matuszewski, B K

    2002-02-01

    A method for the simultaneous determination of a cyclooxygenase-2 inhibitor, 4-(4-methanesulfonylphenyl)-3-phenyl-5H-furan-2-one (rofecoxib, I) and [13C7]rofecoxib, (II), in human plasma has been developed to support the clinical oral bioavailability (BA) study of I. The method is based on high-performance liquid chromatography (HPLC) with atmospheric pressure chemical ionization tandem mass spectrometric (APCI-MS-MS) detection in the negative ionization mode using a heated nebulizer interface. Two different stable isotope labeled analogs of I were initially evaluated for their use as intravenous (i.v.) markers in the BA study. [13CD3]Rofecoxib was shown to be isotopically unstable in plasma and water containing solvent and an efficient deuterium exchange prevented its use in the study. On the other hand, the isotopic integrity of the subsequently synthesized [13C7]rofecoxib (II) was maintained, as expected, in plasma and other solvent systems. The results of these experiments clearly demonstrated the need for the careful evaluation of the isotopic integrity of the stable isotope labeled compound for the successful utilization of these compounds in BA studies and also as internal standards in the quantitative analysis of drugs in biological fluids. After liquid-liquid extraction of I, II, and internal standard (III) from plasma, the analytes were chromatographed on a narrow bore (100 mm x 3.0 mm) C18 analytical column, with mobile phase consisting of acetonitrile-water (1:1, v/v) at a flow-rate of 0.5 ml/min. The MS-MS detection was performed on a PE Sciex API III Plus tandem mass spectrometer operated in the selected reaction monitoring mode. The precursor-->product ion combinations of m/z 313-->257, 320-->292, and 327-->271 were used to quantify I, II, and III, respectively. The assay was validated in the concentration range of 0.1 to 100 ng/ml of plasma for both I and II. The precision of the assay (expressed as relative standard deviation) was less than 10% at all concentrations within the standard curve range, with adequate assay accuracy. The assay was utilized to support the clinical BA study in which oral doses of I were administered together with an i.v. dose of II to determine the oral BA of rofecoxib at 12.5- and 25-mg doses. PMID:11863283

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

    PubMed

    Becker, Harry; Walker, Richard J

    2003-09-11

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

  4. 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.

  5. Comparison of the labeling efficiency of BrdU, DiI and FISH labeling techniques in bone marrow stromal cells.

    PubMed

    Li, Na; Yang, Hui; Lu, Lingling; Duan, Chunli; Zhao, Chunli; Zhao, Huanying

    2008-06-18

    Cells are generally labeled during in vivo implantation studies enabling the cells to be traced. The relationship between the labeling efficiency and cellular proliferation after transplantation is critical for the interpretation of data obtained by detection of the signals on tissue sections. Here, we compare cellular labeling methods of rat marrow stromal cells that were labeled with 5-bromo-2-deoxyuridine (BrdU), 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) and fluorescence in situ hybridization (FISH). Our data show that (i) BrdU uniformly labeled the nuclei, (ii) DiI-labeled cells had many dots or stained clear and uniform when a longer exposure time was used during detection and (iii) FISH labeled the cells with dots along the edges of the nuclei. The labeling efficiency was 94.1+/-8.6%, 97.6+/-3.4% and 90.5+/-3.0%, in BrdU, DiI- and FISH-labeled cells, respectively. After sub-culturing of labeled cells, the percentage of BrdU-positive cells was found to be 71.9+/-18.0% and 18.4+/-6.9%, after the first and second passages, respectively. The percentage of DiI-labeled cells detected depended on the exposure time: a long exposure time (>10 s) resulted in identification of 95.1+/-4.0% and 94.5+/-3.9% DiI-positive cells after the first and second sub-cultures, respectively. The percentage of FISH-positive cells was found to be 87.0+/-3.0% and 89.1+/-9.7%. The BrdU labeling signal quickly decreased over time. Thus, BrdU should only be used to temporarily label dividing cells. In contrast, our data indicate that DiI and FISH labeling may be used to steadily trace cells during in vivo experiments. To our knowledge, this is the first time that the effects of different labeling methods over time have been examined during a cell transplantation study. PMID:18468584

  6. 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.

  7. Segmental isotope labeling of proteins for NMR structural study using a protein S tag for higher expression and solubility.

    PubMed

    Kobayashi, Hiroshi; Swapna, G V T; Wu, Kuen-Phon; Afinogenova, Yuliya; Conover, Kenith; Mao, Binchen; Montelione, Gaetano T; Inouye, Masayori

    2012-04-01

    A common obstacle to NMR studies of proteins is sample preparation. In many cases, proteins targeted for NMR studies are poorly expressed and/or expressed in insoluble forms. Here, we describe a novel approach to overcome these problems. In the protein S tag-intein (PSTI) technology, two tandem 92-residue N-terminal domains of protein S (PrS(2)) from Myxococcus xanthus is fused at the N-terminal end of a protein to enhance its expression and solubility. Using intein technology, the isotope-labeled PrS(2)-tag is replaced with non-isotope labeled PrS(2)-tag, silencing the NMR signals from PrS(2)-tag in isotope-filtered (1)H-detected NMR experiments. This method was applied to the E. coli ribosome binding factor A (RbfA), which aggregates and precipitates in the absence of a solubilization tag unless the C-terminal 25-residue segment is deleted (RbfAΔ25). Using the PrS(2)-tag, full-length well-behaved RbfA samples could be successfully prepared for NMR studies. PrS(2) (non-labeled)-tagged RbfA (isotope-labeled) was produced with the use of the intein approach. The well-resolved TROSY-HSQC spectrum of full-length PrS(2)-tagged RbfA superimposes with the TROSY-HSQC spectrum of RbfAΔ25, indicating that PrS(2)-tag does not affect the structure of the protein to which it is fused. Using a smaller PrS-tag, consisting of a single N-terminal domain of protein S, triple resonance experiments were performed, and most of the backbone (1)H, (15)N and (13)C resonance assignments for full-length E. coli RbfA were determined. Analysis of these chemical shift data with the Chemical Shift Index and heteronuclear (1)H-(15)N NOE measurements reveal the dynamic nature of the C-terminal segment of the full-length RbfA protein, which could not be inferred using the truncated RbfAΔ25 construct. CS-Rosetta calculations also demonstrate that the core structure of full-length RbfA is similar to that of the RbfAΔ25 construct. PMID:22389115

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

    PubMed

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

    2013-06-21

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

  9. Approach for identification and quantification of C-terminal peptides: incorporation of isotopic arginine labeling based on oxazolone chemistry.

    PubMed

    Liu, Minbo; Zhang, Lijuan; Zhang, Lei; Yao, Jun; Yang, Pengyuan; Lu, Haojie

    2013-11-19

    C-termini of proteins often play an important role in various biological processes. The determination of the protein C-terminus is crucial because it provides not only distinct functional annotation but also a way to monitor the proteolysis-modified proteins. In this study, an isotopic labeling approach based on oxazolone chemistry was developed to achieve the identification and quantification of C-termini. Aminolysis reagents such as arginine selectively react with the α-carboxyl group at the peptide C-terminus via an oxazolone-like intermediate. Side chain carboxyl groups do not participate in this reaction. When an isotopic mixture consisting of 50% arginine ((0)Arg) and 50% C6-arginine ((6)Arg) was introduced to react with C-terminus of protein and followed by proteolysis, the C-terminal peptide could be directly recognized in the mass spectrum due to its unique isotopic paired peaks, and the sequence could be interpreted in MS2. Besides, the incorporation of an additional basic amino acid in the C-terminal peptide greatly enhanced the signal intensity for C-termini detection. Moreover, the isotopic arginine labeling strategy could be applied for relative C-termini quantitation. Our method showed an excellent correlation of the measured ratios to theoretical ratios and high reproducibility within 2 orders of magnitude of the dynamic range. The correlation coefficients (R(2)) were higher than 0.99, with the coefficients of variation (CVs) ranging from 1.16 to 10.91%. Finally, the approach was used to analyze the C-termini from Thermoanaerobacter tengcongensis , which was cultured under different temperatures. As a result, 68 C-termini have been identified, and 53 of them were quantified in total using our strategy. In addition, 24 neo-C-terminal peptides have also been discovered. PMID:24147625

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

    PubMed Central

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

    2010-01-01

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

  11. 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 ...

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

    SciTech Connect

    Wiel, Stephen; Van Wie McGrory, Laura

    2003-08-04

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

  13. Absolute and relative protein quantification with the use of isotopically labeled p-hydroxymercuribenzoic acid and complementary MALDI-MS and ICPMS detection.

    PubMed

    Kutscher, Daniel J; Bettmer, Jörg

    2009-11-01

    Chemical labeling with subsequent mass spectrometric detection represents a common approach for protein quantification. Whereas most methods make use of stable isotope labels from natural elements such as (2)D, (13)C, (15)N, or (18)O, artificially introduced metals have gained interest as alternative markers. In this work we present the application of p-hydroxymercuribenzoic acid (pHMB) as a labeling reagent for cysteine-containing proteins. As a proof of concept, insulin was chosen as the model protein, and two different workflows were developed to its absolute and relative quantification with the use of complementary MALDI-MS and ICPMS. On the basis of the synthesis of isotopically labeled [(199)Hg]pHMB, and thus on the basis of the label-specific isotope dilution concept, a differential labeling procedure can be applied either to the comparative study of two different samples (relative quantification) or to the absolute quantification of insulin. In both cases, final detection by MALDI-MS followed by isotope pattern deconvolution was applied to extract the quantitative data from the mass spectra. Good agreement with the expected values was obtained for the relative insulin quantification, and the recovery for insulin applying the absolute quantification workflow was between 90% and 110% with an RSD of better than 5% in the low picomole range. PMID:19799408

  14. 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

  15. Isotopic labeling studies of the mechanism of dehydrogenation of methanol to methyl formate over copper-based catalysts

    SciTech Connect

    Cant, N.W.; Tonner, S.P.; Trimm, D.L.; Wainwright, M.S.

    1985-02-01

    Deuterium labeling has been used to study the processes occurring during the conversion of methanol to methyl formate over copper catalysts at 180-210/sup 0/C and pressures of 0.3 to 1 atm. Deuterium substitution has a dramatic effect on rate, which decreases in the ratio 8:4:2:1 in the series CH/sub 3/OH, CH/sub 3/OD, CD/sub 3/OH, CD/sub 3/OD. This can be interpreted as the product of a primary kinetic isotope effect of four for replacement of CH/sub 3/ by CD/sub 3/ and a separate thermodynamic isotope effect of two on the concentration of a surface methoxy intermediate when OH is replaced by OD. The isotope effect provides strong support for a mechanism in which the slow step is the conversion of the methoxy group to formaldehyde. Reversibility of the conversion of methanol to methoxy is reflected by hydroxyl group exchange with D/sub 2/ at a rate much in excess of methyl formate production. H/sub 2//HD/D/sub 2/ equilibration rates are still faster even though methanol coverages are high. The product distribution from CD/sub 3/OD/CH/sub 3/OH mixtures shows that methyl formate formation involves transfer of an H or a D with discrimination isotope effect of two. This rules out coupling by a methoxy plus CHO step but leaves unresolved the possibility that the formate is produced by a hemiacetal intermediate or by formaldehyde dimerization. This lack of resolution results from isotopic scrambling caused by concurrent transesterification reactions as demonstrated using CD/sub 3/OH/CH/sub 3/OCHO mixtures. 24 refs., 6 tabs.

  16. Research recommendations for applying vitamin A-labelled isotope dilution techniques to improve human vitamin A nutrition.

    PubMed

    Tanumihardjo, Sherry A; Kurpad, Anura V; Hunt, Janet R

    2014-01-01

    The current use of serum retinol concentrations as a measurement of subclinical vitamin A deficiency is unsatisfactory for many reasons. The best technique available for vitamin A status assessment in humans is the measurement of total body pool size. Pool size is measured by the administration of retinol labelled with stable isotopes of carbon or hydrogen that are safe for human subjects, with subsequent measurement of the dilution of the labelled retinol within the body pool. However, the isotope techniques are time-consuming, technically challenging, and relatively expensive. There is also a need to assess different types of tracers and doses, and to establish clear guidelines for the use and interpretation of this method in different populations. Field-friendly improvements are desirable to encourage the application of this technique in developing countries where the need is greatest for monitoring the risk of vitamin A deficiency, the effectiveness of public health interventions, and the potential of hypervitaminosis due to combined supplement and fortification programs. These techniques should be applied to validate other less technical methods of assessing vitamin A deficiency. Another area of public health relevance for this technique is to understand the bioconversion of β-carotene to vitamin A, and its relation to existing vitamin A status, for future dietary diversification programs. PMID:25537106

  17. Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling.

    PubMed

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

    2007-02-01

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

  18. 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.

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

  20. Stable isotope-labeled tracers for the investigation of fatty acid and triglyceride metabolism in humans in vivo

    PubMed Central

    Magkos, Faidon; Mittendorfer, Bettina

    2008-01-01

    Summary Understanding lipid metabolism and its regulation requires information on the rates at which lipids are produced within the body, absorbed (dietary lipids) into the body, transported within the body, and utilized by various tissues. This article focuses on the use of stable isotope-labeled tracers for the quantitative evaluation of major pathways of fatty acid and triglyceride metabolism in humans in vivo. Adipose tissue lipolysis and free fatty acid appearance in plasma, fatty acid tissue uptake and oxidation, and hepatic very low-density lipoprotein triglyceride secretion are among the metabolic pathways that can be studied by using stable isotope labeled tracers, and will be discussed in detail. The methodology has been in use for many years and is constantly being refined. A variety of tracers and analytical approaches are available and can be used; knowing the advantages, assumptions, and limitations of each is essential for the planning of studies and the interpretation of data, which can provide unique insights into human lipid metabolism. PMID:20161007

  1. 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

  2. Efficient transient genetic labeling of human CD34+ progenitor cells for in vivo application.

    PubMed

    Wiehe, Juliane M I; Niesler, Carola; Torzewski, Jan; Zimmermann, Oliver; Wiesneth, Markus; Schmitt, Michael; Schwarz, Klaus; Döhner, Hartmut; Hombach, Vinzenz; Greiner, Jochen

    2006-03-01

    Genetic labeling of human hematopoietic progenitor cells (HPC) and their consecutive fate-mapping in vivo is an approach to answer intriguing questions in stem cell biology. We recently reported efficient transient genetic labeling of human CD34+ HPC with the truncated low-affinity nerve growth factor receptor (DeltaLNGFR) for in vivo application. Here we investigate whether HPC labeling with DeltaLNGFR affects lineage-specific cell differentiation, whether DeltaLNGFR expression is maintained during lineage-specific cell differentiation and which leukemia cell line might be an appropriate cell culture model for human CD34+ HPC. Human CD34+ peripheral blood stem cells and various leukemia cell lines were characterized by immunophenotyping. Cells were transfected using nucleofection. Hematopoietic differentiation was studied by colony-forming assays. DeltaLNGFR expression was assessed using reverse transcription-PCR, immunofluorescence and flow cytometry. Nucleofection was efficient and did not significantly reduce hematopoietic cell differentiation. Mature myeloid cells (CD66b+) derived from human CD34+ HPC and Mutz2 cells maintained DeltaLNGFR expression at a high percentage (70 +/- 2% and 58 +/- 2%, respectively). Mutz2 cells may serve as an in vitro model for human myeloid HPC. The method described herein has been adopted to Good Manufacturing Practices (GMP) guidelines and is ready for in vivo application. PMID:17465806

  3. 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

  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. 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.

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

    PubMed

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

    2016-01-01

    Halogenated, labeled with tritium and doubly with deuterium and tritium, derivatives of L-tryptophan, i.e. 5'-bromo-[2-(3)H]-, 5'-bromo-[2-(2)H/(3)H]-, 5'-fluoro-[2-(3)H]-5'-fluoro-[2-(2)H/(3)H]-, 6'-fluoro-[2-(3)H]-, 6'-fluoro-[2-(2)H/(3)H]-L-tryptophan, as well as, L-tyrosine, i.e. 3'-fluoro-[2-(3)H]-, 3'-fluoro-[2-(2)H/(3)H]-, 3'-chloro-[2-(3)H]-, and 3'-chloro-[2-(2)H/(3)H]-L-tyrosine, and also L-phenylalanine, i.e. 2'-fluoro-[(3S)-(3)H]-, 2'-fluoro-[(3S)-(2)H/(3) H]-, 2'-chloro-[(3S)-(3)H]-, 2'-chloro-[(3S)-(2)H/(3)H]-, 4'-chloro-[(3S)-(3)H]-, and 4'-chloro-[(3S)-(2)H/(3)H]-L-phenylalanine were synthesized using enzymatic methods. Isotopomers of L-tryptophan were synthesized by coupling of halogenated indoles with S-methyl-L-cysteine carried out in deuteriated or tritiated incubation media. Labeled halogenated derivatives of L-tyrosine were obtained by the enzymatically supported exchange between halogenated L-tyrosine and isotopic water. Labeled halogenated isotopologues of L-Phe were synthesized by the enzymatic addition of ammonia to halogenated cinnamic acid. As a source of hydrogen tritiated water (HTO) and heavy water (D2O) with addition of HTO were used. PMID:26586485

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

    PubMed Central

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

    2012-01-01

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

  10. 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, Frédéric; 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

  11. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  13. 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.

  14. 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.

  15. 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

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

    NASA Astrophysics Data System (ADS)

    Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo

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

  18. 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

  19. Application of stable isotope labeled glutathione and rapid scanning mass spectrometers in detecting and characterizing reactive metabolites.

    PubMed

    Mutlib, Abdul; Lam, Wing; Atherton, Jim; Chen, Hao; Galatsis, Paul; Stolle, Wayne

    2005-01-01

    The formation of reactive metabolites from a number of compounds was studied in vitro using a mixture of non-labeled and stable isotope labeled glutathione (GSH) as a trapping agent. GSH was labeled by incorporating [1,2-(13)C(2),(15)N]glycine into the tripeptide to give an overall increase of 3 Da over the naturally occurring substance. Detection and characterization of reactive metabolites was greatly facilitated by using the data-dependent scanning features of the linear ion trap mass spectrometers to give complimentary and confirmatory data in a single analytical run. A comparison was made by analyzing the samples simultaneously on a triple-stage quadrupole mass spectrometer operated in the constant neutral loss mode. The compounds studied included 2-acetamidophenol, 3-acetamidophenol, 4-acetamidophenol (acetaminophen), and flufenamic acid. GSH adducts for each of these compounds produced a characteristic pattern of 'twin ions' separated by 3 Da in the mass spectral data. This greatly facilitated the detection and characterization of any GSH-related adducts present in the microsomal extracts. Furthermore, characterization of these adducts was greatly facilitated by the rapid scanning capability of linear ion trap instruments that provided full-scan, MS/MS and MS(3) data in one single analysis. This method of detecting and characterizing reactive metabolites generated in vitro was found to be far superior to any of the existing methods previously employed in this laboratory. The combination of two techniques, stable isotope labeled glutathione and linear ion traps, provided a very sensitive and specific method of identifying compounds capable of producing reactive metabolites in a discovery setting. The complimentary set of mass spectral data (including full-scan, MS/MS and MS(3) mass spectra), obtained rapidly in a single analysis with the linear ion trap instruments, greatly accelerated identification of metabolically bioactivated soft spots on the molecules. This in turn enabled chemists to rapidly design out the potential metabolic liability from the back-up compounds by making appropriate structural modifications. PMID:16261644

  20. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

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

    PubMed

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

    2014-07-29

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

  3. 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

  4. FTIR Study of the Photoinduced Processes of Plant Phytochrome Phya using Isotope-Labeled Bilins and Density Functional Theory Calculations

    PubMed Central

    Schwinté, Pascale; Foerstendorf, Harald; Hussain, Zakir; Gärtner, Wolfgang; Mroginski, Maria-Andrea; Hildebrandt, Peter; Siebert, Friedrich

    2008-01-01

    Fourier transform infrared spectroscopy was used to analyze the chromophore structure in the parent states Pr and Pfr of plant phytochrome phyA and the respective photoproducts lumi-R and lumi-F. The spectra were obtained from phyA adducts assembled with either uniformly or selectively isotope-labeled phytochromobilin and phycocyanobilin. The interpretation of the experimental spectra is based on the spectra of chromophore models calculated by density functional theory. Global 13C-labeling of the tetrapyrrole allows for the discrimination between chromophore and protein bands in the Fourier transform infrared difference spectra. All infrared difference spectra display a prominent difference band attributable to a stretching mode with large contributions from the methine bridge between the inner pyrrole rings (B-C stretching). Due to mode coupling, frequencies and isotopic shifts of this mode suggest that the Pr chromophore may adopt a distorted ZZZssa or ZZZasa geometry with a twisted A-B methine bridge. The transition to lumi-R is associated with only minor changes of the amide I bands indicating limited protein structural changes during the isomerization site of the C-D methine bridge. Major protein structural changes occur upon the transition to Pfr in which the chromophore adopts a ZZEssa or ZZEasa-like state. In addition, specific interactions with the protein alter the structure of the B-C methine bridge as concluded from the substantial downshift of the respective stretching mode. These interactions are removed during the photoreaction to lumi-F (ZZE→ZZZ), which involves only small protein structural changes. PMID:18390618

  5. Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya.

    PubMed

    Ocaña, Mireia Fernández; Fraser, Paul D; Patel, Raj K P; Halket, John M; Bramley, Peter M

    2009-02-16

    The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study. PMID:19154813

  6. 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/mL·h for Pro-Hyp-Gly to 163 ± 1 nmol/mL·h for Pro-Hyp after oral ingestion of 25 g of fish gelatin hydrolysate, and the coefficient of variation of three separate measurements was <7% for each peptide except for Glu-Hyp-Gly, which was near the detection limit. Our method is useful for absorption/metabolism studies of the Hyp-containing peptides and development of functionally characterized gelatin hydrolysate. PMID:25417748

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

    NASA Astrophysics Data System (ADS)

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

    2012-09-01

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

  8. 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

  9. Pre-malbrancheamide: Synthesis, Isotopic Labeling, Biosynthetic Incorporation, and Detection in Cultures of Malbranchea aurantiaca

    PubMed Central

    Ding, Yousong; Greshock, Thomas J.; Miller, Kenneth A.

    2009-01-01

    An advanced metabolite, named pre-malbrancheamide, involved in the biosynthesis of malbrancheamide (1) and malbrancheamide B (2) has been synthesized in double 13C-labeled form and was incorporated into the indole alkaloid 2 by Malbranchea aurantiaca. In addition, pre-malbrancheamide has been detected as a natural metabolite in cultures of M. aurantiaca. The biosynthetic implications of these experiments are discussed. PMID:18844365

  10. Direct biosynthetic cyclization of a distorted paracyclophane highlighted by double isotopic labelling of L-tyrosine.

    PubMed

    Ear, Alexandre; Amand, Séverine; Blanchard, Florent; Blond, Alain; Dubost, Lionel; Buisson, Didier; Nay, Bastien

    2015-03-28

    The biosynthesis of pyrrocidines, fungal PK-NRP compounds featuring a strained [9]paracyclophane, was investigated in Acremonium zeae. We used a synthetic L-tyrosine probe, labelled with oxygen 18 as a reporter of phenol reactivity and carbon 13 as a tracer of incorporation of this exogenous precursor. The ((18)O)phenolic oxygen was incorporated, suggesting that phenol behaves as a nucleophile during the formation of the bent aryl ether. PMID:25675395

  11. A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study

    PubMed Central

    2011-01-01

    Background Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up- or down-regulated proteins at a distinct time point but instead compare developmental stages of an organism or varying experimental conditions. Results Although the scientific questions behind these experiments are of course manifold, there are, nevertheless, two questions that commonly arise: 1) which proteins are differentially regulated regarding the selected experimental conditions, and 2) are there groups of proteins that show similar abundance ratios, indicating that they have a similar turnover? We give advice on how these two questions can be answered and comprehensively compare a variety of commonly applied computational methods and their outcomes. Conclusions This work provides guidance through the jungle of computational methods to analyze mass spectrometry-based isotope-labeled datasets and recommends an effective and easy-to-use evaluation strategy. We demonstrate our approach with three recently published datasets on Bacillus subtilis [1,2] and Corynebacterium glutamicum [3]. Special focus is placed on the application and validation of cluster analysis methods. All applied methods were implemented within the rich internet application QuPE [4]. Results can be found at http://qupe.cebitec.uni-bielefeld.de. PMID:21663690

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-02-01

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

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  20. 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

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

    NASA Astrophysics Data System (ADS)

    Claesen, Jürgen; 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.

  2. 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.

  3. Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone, and 6 beta-hydroxytestosterone.

    PubMed

    Furuta, Takashi; Suzuki, Atsushi; Matsuzawa, Mitsuhiro; Shibasaki, Hiromi; Kasuya, Yasuji

    2003-09-01

    A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions. PMID:12957675

  4. 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...

  5. 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

  6. 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.

  7. 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.

  8. Mechanism of tritium-atom-promoted isotope exchange in the benzene ring: application to tritium labeling of biologically important aryl compounds

    SciTech Connect

    Powell, M.F.; Morimoto, H.; Erwin, W.R.; Gordon, B.E.; Lemmon, R.M.

    1984-12-06

    Reaction of thermal tritium atoms, generated by microwave activation of T/sub 2/ gas, with benzene and biphenyl was studied at approx. -50 and -196/sup 0/C. The saturation reactions (i.e., benzene ..-->.. cyclohexane-t/sub 6/) predominated over isotope exchange (i.e. benzene ..-->.. benzene-t) at-196/sup 0/C. However, significant exchange labeling occurred at approx. -50/sup 0/C, with a concomitant reduction in the yields of saturated products. This reversal in labeled product yields at the different temperatures is due, in part, to the faster rate of H expulsion from the intermediate cyclohexadienyl radical at -50/sup 0/C and to the increased mobility of the warmer matrix that retards multiple T. reactions with the same aryl molecule by covering up singly tritiated intermediates. The less volatile aryl compound, biphenyl, was labeled in a diffusionally active matrix of either benzene or cyclohexane, whereas it could not be labeled otherwise.

  9. Synthesis of isotopically labeled daclatasvir for use in human clinical studies.

    PubMed

    Easter, John A; Burrell, Richard C; Bonacorsi, Samuel J

    2016-04-01

    Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 μCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps. PMID:26968868

  10. Combining capillary electrophoresis matrix-assisted laser desorption/ionization mass spectrometry and stable isotopic labeling techniques for comparative crustacean peptidomics.

    PubMed

    Wang, Junhua; Zhang, Yuzhuo; Xiang, Feng; Zhang, Zichuan; Li, Lingjun

    2010-06-25

    Herein we describe a sensitive and straightforward off-line capillary electrophoresis (CE) matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) interface in conjunction with stable isotopic labeling (SIL) technique for comparative neuropeptidomic analysis in crustacean model organisms. Two SIL schemes, including a binary H/D formaldehyde labeling technique and novel, laboratory-developed multiplexed dimethylated leucine-based isobaric tagging reagents, have been evaluated in these proof-of-concept experiments. We employ these isotopic labeling techniques in conjunction with CE-MALDI-MS for quantitative peptidomic analyses of the pericardial organs isolated from two crustacean species, the European green crab Carcinus maenas and the blue crab Callinectes sapidus. Isotopically labeled peptide pairs are found to co-migrate in CE fractions and quantitative changes in relative abundances of peptide pairs are obtained by comparing peak intensities of respective peptide pairs. Several neuropeptide families exhibit changes in response to salinity stress, suggesting potential physiological functions of these signaling peptides. PMID:20334868

  11. 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

  12. 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

  13. Cell-free synthesis of isotopically labelled peptide ligands for the functional characterization of G protein-coupled receptors.

    PubMed

    Joedicke, Lisa; Trenker, Raphael; Langer, Julian D; Michel, Hartmut; Preu, Julia

    2016-01-01

    Cell-free systems exploit the transcription and translation machinery of cells from different origins to produce proteins in a defined chemical environment. Due to its open nature, cell-free protein production is a versatile tool to introduce specific labels such as heavy isotopes, non-natural amino acids and tags into the protein while avoiding cell toxicity. In particular, radiolabelled peptides and proteins are valuable tools for the functional characterization of protein-protein interactions and for studying binding kinetics. In this study we evaluated cell-free protein production for the generation of radiolabelled ligands for G protein-coupled receptors (GPCRs). These receptors are seven-transmembrane-domain receptors activated by a plethora of extracellular stimuli including peptide ligands. Many GPCR peptide ligands contain disulphide bonds and are thus inherently difficult to produce in bacterial expression hosts or in Escherichia coli-based cell-free systems. Here, we established an adapted E. coli-based cell-free translation system for the production of disulphide bond-containing GPCR peptide ligands and specifically introduce tritium labels for detection. The bacterial oxidoreductase DsbA is used as a chaperone to favour the formation of disulphide bonds and to enhance the yield of correctly folded proteins and peptides. We demonstrate the correct folding and formation of disulphide bonds and show high-affinity ligand binding of the produced radio peptide ligands to the respective receptors. Thus, our system allows the fast, cost-effective and reliable synthesis of custom GPCR peptide ligands for functional and structural studies. PMID:27047736

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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, 30°C). 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.

  19. 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.

  20. 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.

  1. 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

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

    NASA Astrophysics Data System (ADS)

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

    2013-04-01

    Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the arctic will cause deeper permafrost thawing, followed by increased carbon mineralization and CH4 formation in water-saturated tundra soils, thus creating a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and δ13CH4 signatures were measured and the fractionation factors for the processes of oxidation (αox) and diffusion (αdiff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (such as landfill cover soils) have assumed a gas transport dominated by advection (αtrans = 1). In tundra soils, however, diffusion is the main gas transport mechanism and diffusive stable isotope fractionation should be considered alongside oxidative fractionation. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an αdiff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was αdiff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that αox differs widely between sites and horizons (mean αox = 1.017 ± 0.009) and needs to be determined on a case by case basis. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged, organic-rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost-affected ecosystems and their potential strengths in response to global warming.

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

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the Arctic will cause a deeper permafrost thawing followed by increased carbon mineralization and CH4 formation in water saturated tundra soils which might cause a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River Delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and δ13CH4-signatures were measured and the fractionation factors for the processes of oxidation (αox) and diffusion (αdiff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (e.g. landfill cover soils) have assumed a gas transport dominated by advection (αtrans = 1). In tundra soils, however, diffusion is the main gas transport mechanism, aside from ebullition. Hence, diffusive stable isotope fractionation has to be considered. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an αdiff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was αdiff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that αox differs widely between sites and horizons (mean αox, = 1.017 ± 0.009) and needs to be determined individually. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged organic rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost affected ecosystems and their potential strengths in response to global warming.

  4. Efficient 18F labeling of cysteine-containing peptides and proteins using tetrazine-trans-cyclooctene ligation.

    PubMed

    Liu, Shuanglong; Hassink, Matthew; Selvaraj, Ramajeyam; Yap, Li-Peng; Park, Ryan; Wang, Hui; Chen, Xiaoyuan; Fox, Joseph M; Li, Zibo; Conti, Peter S

    2013-01-01

    18F positron emission tomography (PET) has a number of attributes that make it clinically attractive, including nearly 100% positron efficiency, very high specific radioactivity, and a short half-life of ≈ 110 minutes. However, the short half-life of 18F and the poor nucleophilicity of fluoride introduce challenges for the incorporation of 18F into complex molecules. Recently, the tetrazine-trans-cyclooctene ligation was introduced as a novel 18F labeling method that proceeds with fast reaction rates without catalysis. Herein we report an efficient method for 18F labeling of free cysteines of peptides and proteins based on sequential ligation with a bifunctional tetrazinyl-maleimide and an 18F-labeled trans-cyclooctene. The newly developed method was tested for site-specific labeling of both c(RGDyC) peptide and vascular endothelial growth factor (VEGF)-SH protein. Starting with 4 mCi of 18F-trans-cyclooctene and only 10 μg of tetrazine-RGD (80-100 μM) or 15 μg of tetrazine-VEGF (6.0 μM), 18F-labeled RGD peptide and VEGF protein could be obtained within 5 minutes in 95% yield and 75% yield, respectively. The obtained tracers were then evaluated in mice. In conclusion, a highly efficient method has been developed for site-specific 18F labeling of cysteine-containing peptides and proteins. The special characteristics of the tetrazine-trans-cyclooctene ligation provide unprecedented opportunities to synthesize 18F-labeled probes with high specific activity for PET applications. PMID:23415400

  5. 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.

  6. Synthesis and Use of Stable-Isotope-Labeled Internal Standards for Quantification of Phosphorylated Metabolites by LC-MS/MS.

    PubMed

    Arrivault, Stéphanie; Guenther, Manuela; Fry, Stephen C; Fuenfgeld, Maximilian M F F; Veyel, Daniel; Mettler-Altmann, Tabea; Stitt, Mark; Lunn, John E

    2015-07-01

    Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a highly specific and sensitive technique for measuring metabolites. However, coeluting components in tissue extracts can interfere with ionization at the interface of the LC and MS/MS phases, causing under- or overestimation of metabolite concentrations. Spiking of samples with known amounts of stable-isotope-labeled internal standards (SIL-IS) allows measurements of the corresponding metabolites to be corrected for such matrix effects. We describe criteria for selection of suitable SIL-IS and report the enzymatic synthesis and purification of nine SIL-IS for hexose-, pentose-, and triose-phosphates, UDP-glucose, and adenosine monophosphate (AMP). Along with commercially available SIL-IS for seven other metabolites, these were validated by LC-MS/MS analyses of extracts from leaves, nonphotosynthetic plant tissues, mouse liver, and cells of Chlamydomonas reinhardtii, Escherichia coli and baker's yeast (Saccharomyces cerevisiae). With only a few exceptions, spiking with SIL-IS significantly improved the reproducibility of LC-MS/MS-based metabolite measurements across a wide range of extract dilutions, indicating effective correction for matrix effects by this approach. With use of SIL-IS to correct for matrix effects, LC-MS/MS offers unprecedented scope for reliable determination of photosynthetic and respiratory intermediates in a diverse range of organisms. PMID:26010726

  7. 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

  8. Quantitative analysis of global phosphorylation changes with high-resolution tandem mass spectrometry and stable isotopic labeling

    PubMed Central

    Kweon, Hye Kyong; Andrews, Philip C.

    2013-01-01

    Quantitative measurement of specific protein phosphorylation sites is a primary interest of biologists, as site-specific phosphorylation information provides insights into cell signaling networks and cellular dynamics at a system level. Over the last decade, selective phosphopeptide enrichment methods including IMAC and metal oxides (TiO2 and ZrO2) have been developed and greatly facilitate large scale phosphoproteome analysis of various cells, tissues and living organisms, in combination with modern mass spectrometers featuring high mass accuracy and high mass resolution. Various quantification strategies have been applied to detecting relative changes in expression of proteins, peptides, and specific modifications between samples. The combination of mass spectrometry-based phosphoproteome analysis with quantification strategies provides a straightforward and unbiased method to identify and quantify site-specific phosphorylation. We describe common strategies for mass spectrometric analysis of stable isotope labeled samples, as well as two widely applied phosphopeptide enrichment methods based on IMAC(NTA-Fe3+) and metal oxide (ZrO2). Instrumental configurations for on-line LC-tandem mass spectrometric analysis and parameters of conventional bioinformatic analysis of large data sets are also considered for confident identification, localization, and reliable quantification of site-specific phosphorylation. PMID:23611819

  9. Live-cell vibrational imaging of choline metabolites by stimulated Raman scattering coupled with isotope-based metabolic labeling

    PubMed Central

    Hu, Fanghao; Wei, Lu; Zheng, Chaogu; Shen, Yihui

    2014-01-01

    Choline is a small molecule that occupies a key position in the biochemistry of all living organisms. Recent studies have strongly implicated choline metabolites in cancer, atherosclerosis and nervous system development. To detect choline and its metabolites, existing physical methods such as magnetic resonance spectroscopy and positron emission tomography, are often limited by the poor spatial resolution and substantial radiation dose. Fluorescence imaging, although with submicrometer resolution, requires introduction of bulky fluorophores and thus is difficult in labeling the small choline molecule. By combining the emerging bond-selective stimulated Raman scattering microscopy with metabolic incorporation of deuterated choline, herein we have achieved high resolution imaging of choline-containing metabolites in living mammalian cell lines, primary hippocampal neurons and multicellular organism C. elegans. Different subcellular distributions of choline metabolites are observed between cancer cells and non-cancer cells, which may reveal functional difference in the choline metabolism and lipid-mediated signaling events. In neurons, choline incorporation is visualized within both soma and neurites, where choline metabolites are more evenly distributed compared to the protein. Furthermore, choline localization is also observed in the pharynx region of C. elegans larvae, consistent with its organogenesis mechanism. These applications demonstrate the potential of isotope-based stimulated Raman scattering microscopy for future choline-related disease detection and development monitoring in vivo. PMID:24555181

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

    PubMed

    Penttinen, Petri; Räsänen, Leena A; Lortet, Gilles; Lindström, 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

  11. 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

  12. Tracing nitrogenous disinfection byproducts after medium pressure UV water treatment by stable isotope labeling and high resolution mass spectrometry.

    PubMed

    Kolkman, Annemieke; Martijn, Bram J; Vughs, Dennis; Baken, Kirsten A; van Wezel, Annemarie P

    2015-04-01

    Advanced oxidation processes are important barriers for organic micropollutants (e.g., pharmaceuticals, pesticides) in (drinking) water treatment. Studies indicate that medium pressure (MP) UV/H2O2 treatment leads to a positive response in Ames mutagenicity tests, which is then removed after granulated activated carbon (GAC) filtration. The formed potentially mutagenic substances were hitherto not identified and may result from the reaction of photolysis products of nitrate with (photolysis products of) natural organic material (NOM). In this study we present an innovative approach to trace the formation of disinfection byproducts (DBPs) of MP UV water treatment, based on stable isotope labeled nitrate combined with high resolution mass spectrometry. It was shown that after MP UV treatment of artificial water containing NOM and nitrate, multiple nitrogen containing substances were formed. In total 84 N-DBPs were detected at individual concentrations between 1 to 135 ng/L bentazon-d6 equivalents, with a summed concentration of 1.2 μg/L bentazon-d6 equivalents. The chemical structures of three byproducts were confirmed. Screening for the 84 N-DBPs in water samples from a full-scale drinking water treatment plant based on MP UV/H2O2 treatment showed that 22 of the N-DBPs found in artificial water were also detected in real water samples. PMID:25760315

  13. 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

  14. 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

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

    PubMed Central

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

    2010-01-01

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

  16. 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

  17. Highly stable and efficient mRNA templates for mRNA–protein fusions and C-terminally labeled proteins

    PubMed Central

    Miyamoto-Sato, Etsuko; Takashima, Hideaki; Fuse, Shinichiro; Sue, Kaori; Ishizaka, Masamichi; Tateyama, Seiji; Horisawa, Kenichi; Sawasaki, Tatsuya; Endo, Yaeta; Yanagawa, Hiroshi

    2003-01-01

    For high-throughput in vitro protein selection using genotype (mRNA)–phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5′ untranslated region including SP6 promoter and Ω29 enhancer (a part of tobacco mosaic virus Ω), an A8 sequence (eight consecutive adenylate residues) at the 3′ end is suitable for fusion formation, while an XA8 sequence (XhoI and the A8 sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration. PMID:12888530

  18. 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

  19. 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

  20. Heavy isotope-labeling study of the metabolism of monomeric and tetrameric acetylcholinesterase forms in the murine neuronal-like T 28 hybrid cell line.

    PubMed

    Lazar, M; Salmeron, E; Vigny, M; Massoulié, J

    1984-03-25

    We have used the method of heavy isotope labeling to study the metabolic turnover of acetylcholinesterase forms in the neuroblastoma-derived T 28 hybrid cells in their differentiated state. These cells contain mostly G1 and G4 forms, together with a small proportion of G2, and secrete all these forms into the culture medium. The cells maintained constant and equal levels of acetylcholinesterase, with the same proportions of molecular forms, in a medium containing heavy isotope-labeled amino acids and in a control light medium of similar composition. In addition, they secreted acetylcholinesterase at the same rate in both media. After transfer of the cells into the heavy medium, heavy isotope-labeled acetylcholinesterase molecules progressively replace preexisting light molecules. We analyzed heavy and light components of acetylcholinesterase for each of the two major G1 and G4 forms, by reconstructing the pattern obtained in sucrose gradient differential sedimentation, using combinations of weighted elementary distributions. Heavy molecules were detected in cellular extracts after about 30 min for G1 and 3 h for G4. Both heavy forms also appeared in the medium after a lag of about 3 h. The cellular complement of G1 was renewed much faster than that of G4, the levels of the light forms being reduced to 50% of the original level after 3.5 and 40 h, respectively. Each of these forms appeared to consist of several metabolic pools, and we present simplified models which describe their possible relationships. PMID:6706975

  1. 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.

  2. Optimization of 13C dynamic nuclear polarization: isotopic labeling of free radicals

    NASA Astrophysics Data System (ADS)

    Niedbalski, Peter; Parish, Christopher; Kiswandi, Andhika; Lumata, Lloyd

    Dynamic nuclear polarization (DNP) is a physics technique that amplifies the nuclear magnetic resonance (NMR) signals by transferring the high polarization of the electrons to the nuclear spins. Thus, the choice of free radical is crucial in DNP as it can directly affect the NMR signal enhancement levels, typically on the order of several thousand-fold in the liquid-state. In this study, we have investigated the efficiency of four variants of the well-known 4-oxo-TEMPO radical (normal 4-oxo-TEMPO plus its 15N-enriched and/or perdeuterated variants) for use in DNP of an important metabolic tracer [1-13C]acetate. Though the variants have significant differences in electron paramagnetic resonance (EPR) spectra, we have found that changing the composition of the TEMPO radical through deuteration or 15N doping yields no significant difference in 13C DNP efficiency at 3.35 T and 1.2 K. On the other hand, deuteration of the solvent causes a significant increase of 13C polarization that is consistent over all the 4-oxo-TEMPO variants. These findings are consistent with the thermal mixing model of DNP. This work is supported by US Dept of Defense Award No. W81XWH-14-1-0048 and the Robert A. Welch Foundation Grant No. AT-1877.

  3. Efficient nano iron particle-labeling and noninvasive MR imaging of mouse bone marrow-derived endothelial progenitor cells

    PubMed Central

    Chen, Rong; Yu, Hui; Jia, Zhen-Yu; Yao, Qun-Li; Teng, Gao-Jun

    2011-01-01

    In this study, we sought to label mouse bone marrow-derived endothelial progenitor cells (EPCs) with Resovist® in vitro and to image them using 7.0 Tesla (T) magnetic resonance imaging (MRI). Mouse bone marrow-derived EPCs were cultured in endothelial basal medium with endothelial growth supplement. They were then characterized by immunocytochemistry, flow cytometry, and fluorescence quantitative polymerase chain reaction. Their functions were evaluated by measuring their uptake of 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (Dil-Ac-LDL), binding of fluorine isothiocyanate (FITC)-labeled Ulex europaeus agglutinin (UEA), and formation of capillary-like networks. EPCs were labeled with superparamagnetic iron oxide (SPIO) and their proliferation was then assessed in a water-soluble tetrazolium (WST-8)-based cell proliferation assay. Spin echo sequence (multislice, multiecho [MSME]) and gradient echo sequence (2D-FLASH) were used to detect differences in the numbers of labeled cells by 7.0 T MRI. The results showed that the cultured cells were of “cobblestone”-like shape and positive for CD133, CD34, CD31, von Willebrand factor, kinase domain receptor, and CD45, but negative for F4/80. They could take up Dil-Ac-LDL, bind FITC-UEA, and form capillary-like networks on Matrigel in vitro. Prussian-blue staining demonstrated that the cells were efficiently labeled with SPIO. The single-cell T2* effect was more obvious in the 2D-FLASH sequence than in the MSME sequence. Further, there were almost no adverse effects on cell vitality and proliferation. In conclusion, mouse bone marrow-derived EPCs can be efficiently labeled with SPIO and imaged with 7.0-T MRI. They may thus be traced by MRI following transplantation for blood vessel disorders and cancer treatment. PMID:21468354

  4. Stable isotope dilution analysis of small molecules with carboxylic acid functions using 18O labeling for HPLC-ESI-MS/MS: analysis of fumonisin B1.

    PubMed

    Bergmann, Dominik; Hübner, Florian; Humpf, Hans-Ulrich

    2013-08-21

    (18)O labeling is a well-known method for the stable isotope labeling of proteins and peptides. This study describes a modified procedure for using (18)O labeling on small molecules. Fumonisin B1, a worldwide occurring mycotoxin, which is routinely analyzed by HPLC-MS/MS, was chosen as model compound. (18)O labeling was achieved by acid-catalyzed oxygen exchange from H2(18)O. A mixture of different isotopologues was obtained from the exchange, which, however, could be used as an internal standard for HPLC-MS/MS analysis. The identity of the (18)O-labeled fumonisin B1 was confirmed by NMR and HRMS measurements. The applicability as internal standard has been verified by comparison of results obtained from the method described in this paper to results obtained by reference methods. The presented method is of special interest as the (18)O labeling can be generally applied to a large group of small molecules containing carboxylic groups. PMID:23895305

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

    PubMed

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

    2014-07-01

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

  6. 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

  7. NIR mega-Stokes fluorophores for bioorthogonal labeling and energy transfer systems--an efficient quencher for daunomycin.

    PubMed

    Cserép, Gergely B; Enyedi, Kata N; Demeter, Attila; Mező, Gábor; Kele, Péter

    2013-02-01

    A set of new azide- and alkyne-bearing lepidinium-based fluorophores were synthesized for bioorthogonal labeling schemes. These fluorescent dyes all show large Stokes-shifts with emission maxima in the near-infrared (NIR) region of the electromagnetic spectrum. The applicability of these dyes in the construction of energy-transfer systems was tested using one of these new fluorescent tags and daunomycin (Dau), an anticancer drug with fluorescent features. These daunomycin conjugates are the very first examples of fluorescently modulated constructs of this anticancer agent. The dually labeled architectures proved that the applied fluorescent dye can be utilized as an efficient quencher for daunomycin. Enzymatic cleavage of a dually labeled enzyme substrate resulted in full recovery of the fluorescence of daunomycin. Such fluorescently modulated Dau conjugates can provide useful information for the mechanism of action of Dau-regulated cell death processes. PMID:23225500

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

    PubMed

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

    2012-02-13

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

  9. 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

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

    PubMed

    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

  11. 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

  12. 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 ¹⁵N in animal proteins (δ¹⁵Nanimal) is greater than that in the diet consumed by the animals (δ¹⁵Ndiet), with a discrimination factor (Δ¹⁵N = δ¹⁵Nanimal - δ¹⁵Ndiet) that is known to vary according to nutritional conditions. The objectives of the present study were to test the hypothesis that Δ¹⁵N 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 Δ¹⁵N 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 Δ¹⁵N 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 Δ¹⁵N 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 Δ¹⁵N obtained from different N pools in dairy cows were all negatively correlated with ENU, the highest correlation was found when Δ¹⁵N was calculated from plasma proteins. Δ¹⁵N showed no correlation with urea-N recycling or rumen NH₃ 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

  13. 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

  14. 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

  15. BODIPY-labeled DC-SIGN-targeting glycodendrons efficiently internalize and route to lysosomes in human dendritic cells.

    PubMed

    Ribeiro-Viana, Renato; García-Vallejo, Juan J; Collado, Daniel; Pérez-Inestrosa, Ezequiel; Bloem, Karien; van Kooyk, Yvette; Rojo, Javier

    2012-10-01

    Glycodendrons bearing nine copies of mannoses or fucoses have been prepared by an efficient convergent strategy based on Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC). These glycodendrons present a well-defined structure and have an adequate size and shape to interact efficiently with the C-type lectin DC-SIGN. We have selected a BODIPY derivative to label these glycodendrons due to its interesting physical and chemical properties as chromophore. These BODIPY-labeled glycodendrons were internalized into dendritic cells by mean of DC-SIGN. The internalized mannosylated and fucosylated dendrons are colocalized with LAMP1, which suggests routing to lysosomes. The interaction of these glycodendrons with DC-SIGN at the surface of dendritic cells did not induce maturation of the cells. Signaling analysis by checking different cytokines indicated also the lack of induction the expression of inflammatory and noninflammatory cytokines by these second generation glycodendrons. PMID:22920925

  16. 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

  17. 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

  18. Simultaneous determination of α-, β- and γ-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 García; 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 α-, β- and γ-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 α-, β- and γ-HBCD in real water samples. The limits of quantification obtained in real samples for α-, β- and γ-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

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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

  4. 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

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

    SciTech Connect

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

    1985-02-04

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

  6. 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

  7. Static secondary-ion mass spectrometric investigation of the surface structure of organic plasma-deposited films prepared from stable-isotope-labeled precursors. 1. Carbonyl precursors.

    PubMed

    Chilkoti, A; Ratner, B D; Briggs, D

    1991-08-01

    Stable-isotope-labeled carbonyl precursors (acetaldehyde, acetone, and 2-butanone) were used to create plasma-deposited films (PDFs), which were then examined by positive- and negative-ion static SIMS. This allowed hydrocarbon (HC) fragments to be distinguished from oxygen-containing fragments in the static SIMS spectra of these PDFs. Both the positive- and negative-ion static SIMS fragmentation patterns of conventional HC and oxygen-containing polymers were qualitatively examined in order to assign structural units on the PDF surface that could account for the sallent features in the static SIMS fragmentation patterns of these PDFs. PMID:1952085

  8. Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations.

    PubMed

    Longhini, Andrew P; LeBlanc, Regan M; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H; Johnson, Bruce A; D'Souza, Victoria M; Kreutz, Christoph; Dayie, T Kwaku

    2016-04-01

    Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides usingin vitrotranscription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch fromBacillus anthracis(48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB. PMID:26657632

  9. Chemo-enzymatic synthesis of site-specific isotopically labeled nucleotides for use in NMR resonance assignment, dynamics and structural characterizations

    PubMed Central

    Longhini, Andrew P.; LeBlanc, Regan M.; Becette, Owen; Salguero, Carolina; Wunderlich, Christoph H.; Johnson, Bruce A.; D'Souza, Victoria M.; Kreutz, Christoph; Dayie, T. Kwaku

    2016-01-01

    Stable isotope labeling is central to NMR studies of nucleic acids. Development of methods that incorporate labels at specific atomic positions within each nucleotide promises to expand the size range of RNAs that can be studied by NMR. Using recombinantly expressed enzymes and chemically synthesized ribose and nucleobase, we have developed an inexpensive, rapid chemo-enzymatic method to label ATP and GTP site specifically and in high yields of up to 90%. We incorporated these nucleotides into RNAs with sizes ranging from 27 to 59 nucleotides using in vitro transcription: A-Site (27 nt), the iron responsive elements (29 nt), a fluoride riboswitch from Bacillus anthracis (48 nt), and a frame-shifting element from a human corona virus (59 nt). Finally, we showcase the improvement in spectral quality arising from reduced crowding and narrowed linewidths, and accurate analysis of NMR relaxation dispersion (CPMG) and TROSY-based CEST experiments to measure μs-ms time scale motions, and an improved NOESY strategy for resonance assignment. Applications of this selective labeling technology promises to reduce difficulties associated with chemical shift overlap and rapid signal decay that have made it challenging to study the structure and dynamics of large RNAs beyond the 50 nt median size found in the PDB. PMID:26657632

  10. 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.

  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

    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

  13. 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.; Sharamentov, S. I.; Vondrasek, R. C.; Zinkann, G. P.; Pikin, A. I.

    2015-08-15

    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.

  14. 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

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

    PubMed

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

    2014-08-01

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

  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. Determining the isotopic abundance of a labeled compound by mass spectrometry and how correcting for natural abundance distribution using analogous data from the unlabeled compound leads to a systematic error.

    PubMed

    Schenk, David J; Lockley, William J S; Elmore, Charles S; Hesk, Dave; Roberts, Drew

    2016-04-01

    When the isotopic abundance or specific activity of a labeled compound is determined by mass spectrometry (MS), it is necessary to correct the raw MS data to eliminate ion intensity contributions, which arise from the presence of heavy isotopes at natural abundance (e.g., a typical carbon compound contains ~1.1% (13) C per carbon atom). The most common approach is to employ a correction in which the mass-to-charge distribution of the corresponding unlabeled compound is used to subtract the natural abundance contributions from the raw mass-to-charge distribution pattern of the labeled compound. Following this correction, the residual intensities should be due to the presence of the newly introduced labeled atoms only. However, this will only be the case when the natural abundance mass isotopomer distribution of the unlabeled compound is the same as that of the labeled species. Although this may be a good approximation, it cannot be accurate in all cases. The implications of this approximation for the determination of isotopic abundance and specific activity have been examined in practice. Isotopically mixed stable-atom labeled valine batches were produced, and both these and [(14) C6 ]carbamazepine were analyzed by MS to determine the extent of the error introduced by the approach. Our studies revealed that significant errors are possible for small highly-labeled compounds, such as valine, under some circumstances. In the case with [(14) C6 ]carbamazepine, the errors introduced were minor but could be significant for (14) C-labeled compounds with particular isotopic distributions. This source of systematic error can be minimized, although not eliminated, by the selection of an appropriate isotopic correction pattern or by the use of a program that varies the natural abundance distribution throughout the correction. PMID:26916110

  19. 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

  20. Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing.

    PubMed

    Minato, Yuichi; Ueda, Takumi; Machiyama, Asako; Shimada, Ichio; Iwaï, Hideo

    2012-07-01

    Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been directly used with an identical protein. Here we applied both EPL and PTS methods to a 140 kDa dimeric multi-domain protein E. coli CheA, and successfully produced the ligated CheA dimer by both approaches. In EPL approach, extensive optimization of the ligation sites and the conditions were required to obtain sufficient amount for an NMR sample of CheA, because CheA contains a dimer forming domain and it was not possible to achieve high reactant concentrations (1-5 mM) of CheA fragments for the ideal EPL condition, thereby resulting in the low yield of segmentally labelled CheA dimer. PTS approach sufficiently produced segmentally labeled ligated CheA in vivo as well as in vitro without extensive optimizations. This is presumably because CheA has self-contained domains connected with long linkers, accommodating a seven-residue mutation without loss of the function, which was introduced by PTS to achieve the high yield. PTS approach was less laborious than EPL approach for the routine preparation of segmentally-isotope labeled CheA dimer. Both approaches remain to be further developed for facilitating preparations of segmental isotope-labelled samples without extensive optimizations for ligation. PMID:22740268

  1. 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.

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

    SciTech Connect

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

    2015-09-25

    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 {sup 15}N isotope tracer method was used in this study, whereby the {sup 15}N isotope was utilized as a tracer for nitrogen nutrient uptake. {sup 15}N isotope presence in the samples is determined by using emission spectrometer analysis and percentage of total nitrogen is determined by using Kjeldahl method. {sup 15}N 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. 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...

  4. 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

  5. Efficient methods for attaching non-radioactive labels to the 5' ends of synthetic oligodeoxyribonucleotides.

    PubMed Central

    Agrawal, S; Christodoulou, C; Gait, M J

    1986-01-01

    The syntheses are described of two types of linker molecule useful for the specific attachment of non-radioactive labels such as biotin and fluorophores to the 5' terminus of synthetic oligodeoxyribonucleotides. The linkers are designed such that they can be coupled to the oligonucleotide as a final step in solid-phase synthesis using commercial DNA synthesis machines. Increased sensitivity of biotin detection was possible using an anti-biotin hybridoma/peroxidase detection system. PMID:3748808

  6. 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

  7. Anaerobic central metabolic pathways in Shewanella oneidensis MR-1interpreted in the light of isotopic metabolite labeling, enzymeactivities and genome annotation

    SciTech Connect

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

    2006-06-27

    It has been proposed that during growth under anaerobic oroxygen-limited conditions Shewanella oneidensis MR-1 uses theserine-isocitrate lyase pathway common to many methylotrophic anaerobes,in which formaldehyde produced from pyruvate is condensed with glycine toform serine. The serine is then transformed through hydroxypyruvate andglycerate to enter central metabolism at phosphoglycerate. To examine itsuse of the serine-isocitrate lyase pathway under anaerobic conditions, wegrew S. oneidensis MR-1 on [1-13C]lactate as the sole carbon source witheither trimethylamine N-oxide (TMAO) or fumarate as an electron acceptor.Analysis of cellular metabolites indicates that a large percentage(>75 percent) of lactate was partially oxidized to either acetate orpyruvate. The 13C isotope distributions in amino acids and other keymetabolites indicate that, under anaerobic conditions, a complete serinepathway is not present, and lactate is oxidized via a highly reversibleserine degradation pathway. The labeling data also suggest significantactivity in the anaplerotic (malic enzyme and phosphoenolpyruvatecarboxylase) and glyoxylate shunt (isocitrate lyase and malate synthase)reactions. Although the tricarboxylic acid (TCA) cycle is often observedto be incomplete in many other anaerobes (absence of 2-oxoglutaratedehydrogenase activity), isotopic labeling supports the existence of acomplete TCA cycle in S. oneidensis MR-1 under TMAO reductioncondition.

  8. 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, Frédérique; Leroux, Cédric; 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 μm 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‰ (∆δ13C) when supplied with diatoms, + 1364‰ (∆δ15N) when supplied with bacteria, and + 24‰ (∆δ13C) and + 135‰ (∆δ15N) 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.

  9. 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

  10. 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.

  11. 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

  12. 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

  13. Thiazolidinedione bioactivation: a comparison of the bioactivation potentials of troglitazone, rosiglitazone, and pioglitazone using stable isotope-labeled analogues and liquid chromatography tandem mass spectrometry.

    PubMed

    Alvarez-Sanchez, Rubén; Montavon, François; Hartung, Thomas; Pähler, Axel

    2006-08-01

    Troglitazone, a thiazolidinedione (TZD) type insulin sensitizer for the treatment of diabetes, was withdrawn from the U.S. market after several fatal cases of hepatotoxicity. Although the mechanism(s) of these idiosyncratic adverse reactions are not completely understood, circumstantial evidence suggests at least a partial contribution of reactive metabolite formation. Despite isolated case reports of hepatotoxicity, the other TZD derivatives pioglitazone and rosiglitazone are comparatively safe. Herein, we report on the bioactivation potential of these drugs and their TZD ring isotope-labeled 2-(15)N-3,4,5-(13)C(3) analogues in rat and human liver microsomes supplemented with glutathione (GSH). Screening for GSH adducts as surrogate markers for reactive intermediate formation was performed by liquid chromatography tandem mass spectrometry. Chemical characterization of the GSH conjugates was conducted by acquisition of their respective product ion spectra and the comparison between unlabeled and stable isotope-labeled TZD derivatives. The data suggest that all drugs undergo bioactivation processes via a common metabolic activation on the TZD ring, yielding disulfide type GSH conjugates as evidenced by the loss of labeled positions in the TZD moiety. Additional bioactivation processes leading to GSH adducts not involving TZD ring scission were evident for troglitazone. In human liver microsomes at low substrate concentrations, only troglitazone yielded a predominant GSH adduct not involving TZD ring scission. This property may contribute, together with other factors such as the relatively high dose administered as well as its potential to induce hepatic cholestasis and oxidative stress, to the hepatotoxicity of this drug. PMID:16918252

  14. 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.

  15. 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; Rehkämper, 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

  16. 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

  17. Binding of misonidazole to hypoxic cells in monolayer and spheroid culture: evidence that a side-chain label is bound as efficiently as a ring label.

    PubMed Central

    Raleigh, J. A.; Franko, A. J.; Koch, C. J.; Born, J. L.

    1985-01-01

    The binding of ring-labelled and side-chain labelled misonidazole to hypoxic cells in monolayer and spheroid cultures of mammalian cells has been compared. The kinetics and patterns of binding for the two labelled compounds are indistinguishable. This finding has implications for the mechanism of binding and for the design of misonidazole analogues which might be used to identify hypoxic zones in tumours. PMID:3966980

  18. 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.

  19. Site-Specific Labeling of DNA and RNA Using an Efficiently Replicated and Transcribed Class of Unnatural Base Pairs

    PubMed Central

    Seo, Young Jun; Malyshev, Denis A.; Lavergne, Thomas; Ordoukhanian, Phillip; Romesberg, Floyd E.

    2014-01-01

    Site-specific labeling of enzymatically synthesized DNA or RNA has many potential uses in basic and applied research, ranging from facilitating biophysical studies to the in vitro evolution of functional nucleic acids, and the construction of various nanomaterials and biosensors. As part of our efforts to expand the genetic alphabet, we have developed a class of unnatural base pairs, exemplified by d5SICS-dMMO2 and d5SICS-dNaM, which are efficiently replicated and transcribed, and which may be ideal for the site-specific labeling of DNA and RNA. Here, we report the synthesis and analysis of the ribo- and deoxyribo-variants, (d)5SICS and (d)MMO2, modified with free or protected propargylamine linkers that allow for the site-specific modification of DNA or RNA during or after enzymatic synthesis. We also synthesized and evaluated the α-phosphorothioate variant of d5SICSTP, which provides a route to backbone thiolation and an additional strategy for the post-amplification site-specific labeling of DNA. The deoxynucleotides were characterized via steady-state kinetics and PCR, while the ribonucleosides were characterized by the transcription of both a short, model RNA as well as full length tRNA. The data reveal that while there are interesting nucleotide and polymerase-specific sensitivities to linker attachment, both (d)MMO2 and (d)5SICS may be used to produce DNA or RNA site-specifically modified with multiple, different functional groups with sufficient efficiency and fidelity for practical applications. PMID:21981600

  20. [Mode of action of chlorambucil. Initial metabolic studies using carbon 14 isotope labeling. Rheumatological implications of their results].

    PubMed

    Sauvezie, B; Rampon, S; Bussière, J L; Godenèche, D; Chollet, P; Plagne, R

    1975-05-01

    Mode of action of chlorambucil first metabolic studies by means of carbon-14 labelled isotpoes rheumatological implications of the results. Chlorambucil, gamma [rho-di(2-chloroethyl) aminophenyl] butyric acid, was labelled in either the carboxylic group (Ia), the mustard radical (Ib), or the carbon atom in the aplha position in the carboxylic group (Ic). The first studies by total autoradiography of mice, by organ fixation, by analysis of urine, faeces, and expired air of rats, and by microautoradiography of sections of organs and smears of tissues from rats and rabbits produced the following results: the metabolism of chlorambucil includes a long hepatic time; the molecule undergoes beta-oxidation almost certainly in the liver; thenthe oxidized form become localized in two potential target organs: the thymus and the bone marrow; there is no elective fixation in normal joints of any ot the three labelled compound studied; on the other hand, with all three molecules, there was elimination via the bile ducts, slight movement into the thoracic canal, and labeling ofHarder's glands (particularly with Ia and Ic); elimination of the three compounds in urine was complex. These facts suggest that chlorambucil undergoses activation in the liver, in the same way as cyclophosphamide; however, the similarity end there. The other steps in metabolism give rise to different molecules for these compounds. The high concentrations of chlorambucil in the thymus and the labelling of the marrow cells indicate that the action of this cytostatic compound is primarily immunodepressive. PMID:1145086

  1. 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.

  2. 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

  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. 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 ...

  5. 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.

  6. 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 ...

  7. Water-Use Efficiency and Stable Carbon Isotopes: Accounting for Photosynthetic Refixation

    NASA Astrophysics Data System (ADS)

    Ubierna Lopez, N.; Marshall, J. D.

    2007-12-01

    Three processes are performed by every green plant tissue: photosynthesis, respiration and refixation. Each of these affects the ratio of stable isotopes, 12C and 13C. Refixation allows plants to fix a portion of the CO2 produced via respiration prior to releasing the remaining CO2 back into the atmosphere. The process begins with a pool of CO2 already depleted in 13C and subsequently depletes it further, resulting in two simultaneous effects: enrichment of CO2 released into the atmosphere and depletion of biomass that is formed. Recently, considerable research has concentrated on identifying processes that determine the isotopic composition of a given plant tissue. A convincing explanation for the observed enrichment of stems versus leaves has still not been derived. We advocate that refixation can explain currently inexplicable patterns. We hypothesized that leaves re-fix carbon during their entire lifespan when light intensity is below the light compensation point and above total darkness. We grew Idaho hybrid poplars under controlled conditions in a growth chamber. Light intensity was regulated to create three different treatments: (1) Light (PAR=270 μmol/m2s), (2) Shade (PAR=89 μmol/m2s) and (3) Dark (PAR=0 μmol/m2s). For each treatment we modified respiration values by regulating the light environment between total darkness and the light compensation point. For the light treatment group, leaf respired CO2 was collected at 5% (PAR=14) and 22% (PAR=59) of the light growing environment. For the shade treatment group, leaf respired CO2 was collected at 22% (PAR=20) of the light growing environment. We estimated the amount of refixation as (Ddark- Dlight)/Ddark, where Ddark represents dark respiration (μmol/gs) and Dlight respiration during light periods (μmol/gs). Light treatments plants exhibited a maximum refixation level of 53% at PAR=59, with an associated enrichment of leaf respired C isotopic composition (δ13CLR) of 3.3‰. At PAR=14, refixation rate for Light plants decreased to 10% and the observed enrichment on δ13CLR was 1‰. Correspondingly, Shade plants showed a 37% level of refixation and 3.6‰ enrichment at PAR=20. Our findings support the hypothesis that leaves re-fix carbon under low-light intensity environments. The degree of refixation is proportional to light intensity, with higher refixation rates associated with higher light intensities and more depleted leaf biomass. The continuous refixation of the internal leaf carbon pool during leaf expansion together with diurnal refixation periods in mature leaves adds depleted biomass into the leaf that is likely to account for the patterns described in the literature (i.e. 2‰ depletion of leaf versus stem biomass). This can influence the interpretation of δ13C leaf biomass data of previous studies and can compromise the utility of δ13C from leaf tissue as a precise meter of water-use efficiency.

  8. Elucidating the two and three-body fragmentation channels on isotopically labeled nitrous oxide by a two-color asymmetric laser field

    NASA Astrophysics Data System (ADS)

    Kotsina, N.; Kaziannis, S.; Kosmidis, C.

    2016-05-01

    We report the interaction of isotopically labeled nitrous oxide (15N14N16O) time-of-flight mass spectroscopy. The phase dependence of two-body fragmentation channels for linear and circular polarization reveals that the nitrogen-nitrogen bond fission channel is in accordance with the 'selective ionization of oriented molecules' mechanism, while the nitrogen-oxygen bond rupture involves an electron recollision mechanism. For the three-body-fragmentation channel resulting in 14N3+ ions it is demonstrated that it may proceed via a concerted and/or a sequential manner. The phase dependence of the sequential channel is indicative of the following dissociation route: 15N14N16O → 15N14N + 16O → 15N + 14N + 16O.

  9. 99mTc-Labeled HYNIC-DAPI Causes Plasmid DNA Damage with High Efficiency

    PubMed Central

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

    2014-01-01

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

  10. Efficient labeling in vitro with non-ionic gadolinium magnetic resonance imaging contrast agent and fluorescent transfection agent in bone marrow stromal cells of neonatal rats.

    PubMed

    Li, Ying-Qin; Tang, Ying; Fu, Rao; Meng, Qiu-Hua; Zhou, Xue; Ling, Ze-Min; Cheng, Xiao; Tian, Su-Wei; Wang, Guo-Jie; Liu, Xue-Guo; Zhou, Li-Hua

    2015-07-01

    Although studies have been undertaken on gadolinium labeling-based molecular imaging in magnetic resonance imaging (MRI), the use of non-ionic gadolinium in the tracking of stem cells remains uncommon. To investigate the efficiency in tracking of stem cells with non-ionic gadolinium as an MRI contrast agent, a rhodamine-conjugated fluorescent reagent was used to label bone marrow stromal cells (BMSCs) of neonatal rats in vitro, and MRI scanning was undertaken. The fluorescent-conjugated cell uptake reagents were able to deliver gadodiamide into BMSCs, and cell uptake was verified using flow cytometry. In addition, the labeled stem cells with paramagnetic contrast medium remained detectable by an MRI monitor for a minimum of 28 days. The present study suggested that this method can be applied efficiently and safely for the labeling and tracking of bone marrow stromal cells in neonatal rats. PMID:25816076

  11. 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

  12. Use of differential isotopic labeling and mass spectrometry to analyze capacitation-associated changes in the phosphorylation status of mouse sperm proteins.

    PubMed

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

    2009-03-01

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

  13. Biosynthesis of seven carbon-13 labeled Alternaria toxins including altertoxins, alternariol, and alternariol methyl ether, and their application to a multiple stable isotope dilution assay.

    PubMed

    Liu, Yang; Rychlik, Michael

    2015-02-01

    An unprecedented stable isotope dilution assay for the genotoxic altertoxins along with exposure data of consumers is presented to enable a first risk assessment of these Alternaria toxins in foods. Altertoxins were produced as the most abundant Alternaria toxins in a modified Czapek-Dox medium with a low level of glucose as the carbon source and ammonium sulfate as the sole nitrogen source. Labeled altertoxins were synthesized in the same way using [(13)C6]glucose. Moreover, labeled alternariol, alternariol methyl ether, altenuene, and alternuisol were biosynthesized in another modified medium containing [(13)C6]glucose and sodium [(13)C2]acetate. A stable isotope dilution LC-MS/MS method was developed and used for food analysis. For altertoxin I, altertoxin II, alterperylenol, alternariol, and alternariol methyl ether, the limits of detection ranged from 0.09 to 0.53 μg kg(-1). The inter-/intra-day (n = 3 × 6) relative standard deviations of the method were below 13%, and the recoveries ranged between 96 and 109%. Among the various commercial food samples, some of the organic whole grains revealed low-level contamination with altertoxin I and alterperylenol, and paprika powder, which was heavily loaded with alternariol, alternariol methyl ether, and tentoxin, showed higher contamination level of altertoxin I and alterperylenol. Altertoxin II and III and stemphyltoxin III were not detectable. In addition, if the food was contaminated with altertoxins, it was likely to be co-contaminated with the other Alternaria toxins, but not necessarily vice versa. Maximum concentrations of altertoxin I and alterperylenol were detected in sorghum feed samples containing 43 and 58 μg kg(-1), respectively. This was significantly higher than that in the measured food samples. PMID:25577349

  14. 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...

  15. 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.; Fréchet, 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 μm 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 μm 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

  16. Lysine and protein metabolism in young women. Subdivision based on the novel use of multiple stable isotopic labels.

    PubMed Central

    Irving, C S; Thomas, M R; Malphus, E W; Marks, L; Wong, W W; Boutton, T W; Klein, P D

    1986-01-01

    A multitracer stable isotope study of lysine kinetics was carried out in fasted adult female volunteers to determine whether a multicompartmental model that partitions protein synthesis and breakdown into at least two types of tissue components can be constructed from plasma and breath data. Five female subjects, maintained on formula diets, received L-[13C1]lysine (27 mumol/kg) as an i.v. bolus and L-[15N2]lysine (27 mumol/kg) as an oral bolus 4 h postprandially. Plasma and breath samples were collected for 6 h. On an alternate day, subjects received NaH13CO3 (10 mumol/kg) as an i.v. bolus and breath samples were collected for 6 h. Plasma tracer lysine levels were determined by gas chromatography-mass spectrometry isotope ratiometry, and breath 13CO2 levels were measured by mass spectrometric gas isotope ratiometry. The tracer data could be fitted to a mammillary multicompartmental model that consisted of a lysine central compartment and slow- and fast-exchanging peripheral compartments containing 37, 38, and 324 mumol/kg, respectively. The rates of lysine oxidation, incorporation into protein, and release by protein breakdown were 21, 35, and 56 mmol/kg/h, respectively, in the fast-exchanging compartment, whereas the rates of protein synthesis and breakdown in the slow compartment were both 53 mmol/kg/min. These values corresponded to a whole-body lysine flux of 106 mmol/kg/h. The kinetic parameters were in excellent agreement with reported values obtained by constant-infusion methods. The measurements indicated that it will be possible to detect changes in amino acid pool sizes and protein synthesis and breakdown associated with the mobilization of protein stores from plasma and breath measurements in multitracer stable isotope experiments. PMID:3082937

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Methanol synthesis via CO2 hydrogenation over a Au/ZnO catalyst: an isotope labelling study on the role of CO in the reaction process.

    PubMed

    Hartadi, Yeusy; Widmann, Daniel; Behm, R Jürgen

    2016-04-20

    Methanol synthesis for chemical energy storage, via hydrogenation of CO2 with H2 produced by renewable energies, is usually accompanied by the undesired formation of CO via the reverse water-gas shift reaction. Aiming at a better mechanistic understanding of methanol formation from CO2/H2 on highly selective supported Au/ZnO catalysts we have investigated the role of CO in the reaction process using isotope labelling experiments. Using (13)C-labelled CO2, we found for reaction at 5 bar and 240 °C that (i) the methanol formation rate is significantly higher in CO2-containing gas mixtures than in a CO2-free mixture and (ii) in mixtures containing both CO2 and CO methanol formation from CO increases with the CO content up to 1% CO, and then remains at 20% of the total methanol formation up to a CO2/CO ratio of 1/1, making CO2 the preferred carbon source in these mixtures. A shift in the preferred carbon source for MeOH from CO2 towards CO is observed with increasing reaction temperatures between 240 °C and 300 °C. At even higher temperatures CO is expected to become the dominant carbon source. The consequences of these findings for the application of Au/ZnO catalysts for chemical storage of renewable energies are discussed. PMID:26923815

  2. 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.

  3. 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

  4. 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 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 (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

  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. Novel and efficient preparation of precursor [188Re(OH2)3(CO)3]+ for the labeling of biomolecules.

    PubMed

    Park, Sang Hyun; Seifert, Sepp; Pietzsch, Hans-Jurgen

    2006-01-01

    A novel and efficient method for preparing 188Re(I) tricarbonyl precursor [188Re(OH2)3(CO)3]+ has been developed by reacting [188Re]perrhenate with Schibli's kit in the presence of borohydride exchange resin (BER) as a reducing agent and an anion scavenger. The precursor was produced in more than 97% yield by reacting a solution of tetrahydroborate exchange resin (BER, 3 mg), borane-ammonia (BH3.NH3, 3 mg), and potassium boranocarbonate (K2[H3BCO2], 3 mg) in 0.9% saline with a solution of sodium perrhenate (Na188ReO4) with up to 50 MBq and concentrated phosphoric acid (85%, 7 microL) at 60 degrees C for 15 min. HPLC and TLC revealed 0% unreacted [188Re]perrhenate ion and <3% of colloidal 188ReO2. Since the precursor is produced with high radiochemical purity and labeling efficiency under the milder conditions than those required for the conventional reducing agents, the latter can be replaced. PMID:16417272

  7. 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-Muñiz, Alfredo; Plichta, Zdeněk; Horák, Daniel; Merkoçi, 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

  8. 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; Nozière, P; Ortigues-Marty, I

    2016-02-01

    Animal tissues are naturally 15N enriched relative to their diet and the extent of this difference (Δ15Nanimal-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 Δ15Nanimal-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 (Δ15Nanimal-diet) in these different body pools. The Δ15Nanimal-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 (Δ15Nduodenal digesta-diet mean value=0.42) compared with that observed in plasma proteins (Δ15Nplasma protein-diet mean value=2.41). These data support the idea that most of the isotopic N fractionation observed in ruminant proteins (Δ15Nplasma protein-diet) has a metabolic origin with very little direct impact of the overall digestion process on the existing relationship between Δ15Nplasma 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 (Δ15Nplasma protein-diet) could have a metabolic origin, but more studies are warranted to confirm this point with different diets and approaches. PMID:26776494

  9. Refinement of Isotopically Derived Fine Root Lifespans Using A Locally Released Radiocarbon Label in Oak Ridge, TN.

    NASA Astrophysics Data System (ADS)

    Gaudinski, J. B.; Riley, W. J.; Torn, M. S.; Joslin, J. D.

    2003-12-01

    Isotopic techniques (13C and 14C) are relative newcomers among the approaches used to quantify fine root (< 2 mm diameter) dynamics in a field setting. Direct measurements of the isotopic content of root tissues, used as a proxy for root age, have shown that at least some portion of the fine root system lives for 5-10 years or more. In this work we take advantage of a local radiocarbon (14C) release in Oak Ridge, TN in summer 1999, to examine (1) the influence of stored C in new root growth and (2) the lifespan of fine roots from a mature, temperate deciduous forest. This release provides a local 14C pulse of similar magnitude to the peak of the 14C bomb spike. However, since we have been able to make ecosystem wide measurements within one year of the local 14C release we have much greater time resolution than we do with the standard bomb-14C technique applied today (which is 1-2 years). We have constructed a new multi-compartment model of root growth and decay, whose structure was developed using data from field sampling at Oak Ridge, TN. Model results, constrained with a 14C time series of new root growth, show that fine roots are grown with 10% of their carbon coming from stored C sources. Additionally, a three-year time series of root cores shows that at least two pools are required to account for 14C changes in live and dead fine roots. Testing this 14C data set with our model shows that the shorter-lived root pool has a turnover time (mean lifetime) of a few months and the longer-lived pool has a turnover time of ~5 years.

  10. Dual, differential isotope labeling shows the preferential movement of labile plant constituents into mineral-bonded soil organic matter.

    PubMed

    Haddix, Michelle L; Paul, Eldor A; Cotrufo, M Francesca

    2016-06-01

    The formation and stabilization of soil organic matter (SOM) are major concerns in the context of global change for carbon sequestration and soil health. It is presently believed that lignin is not selectively preserved in soil and that chemically labile compounds bonding to minerals comprise a large fraction of the SOM. Labile plant inputs have been suggested to be the main precursor of the mineral-bonded SOM. Litter decomposition and SOM formation are expected to have temperature sensitivity varying with the lability of plant inputs. We tested this framework using dual (13) C and (15) N differentially labeled plant material to distinguish the metabolic and structural components within a single plant material. Big Bluestem (Andropogon gerardii) seedlings were grown in an enriched (13) C and (15) N environment and then prior to harvest, removed from the enriched environment and allowed to incorporate natural abundance (13) C-CO2 and (15) N fertilizer into the metabolic plant components. This enabled us to achieve a greater than one atom % difference in (13) C between the metabolic and structural components within the plant litter. This differentially labeled litter was incubated in soil at 15 and 35 °C, for 386 days with CO2 measured throughout the incubation. After 14, 28, 147, and 386 days of incubation, the soil was subsequently fractionated. There was no difference in temperature sensitivity of the metabolic and structural components with regard to how much was respired or in the amount of litter biomass stabilized. Only the metabolic litter component was found in the sand, silt, or clay fraction while the structural component was exclusively found in the light fraction. These results support the stabilization framework that labile plant components are the main precursor of mineral-associated organic matter. PMID:27142168

  11. 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

  12. 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

  13. Quantitative fingerprinting of O-linked glycans released from proteins using isotopic coded labeling with deuterated 1-phenyl-3-methyl-5-pyrazolone.

    PubMed

    Sić, Siniša; Maier, Norbert M; Rizzi, Andreas M

    2015-08-21

    Investigation of oligosaccharides attached to proteins as post-translational modification remains an important research field in the area of glycoproteomics as well as in biotechnology. The development of new tools for qualitative and quantitative analysis of glycans has gained high importance in recent years. This is particularly true with O-glycans for which quantitative data are still underrepresented in literature. This fact is probably due to the absence of an enzyme for general release of O-linked saccharides from glycoproteins and due to their low ionization yield in mass spectrometry (MS). In this paper, a method is established aimed at improved qualitative and quantitative analysis of mucin-type O-glycans. A chemical reaction combining release and derivatization of O-glycans in one step is combined here with mass spectrometric quantification. For the purpose of improved quantitative analysis, stable-isotope coded labeling by d0/d5 1-phenyl-3-methyl-5-pyrazolidone (PMP) was performed. The "heavy"-version of this label, penta-deutero (d5)-PMP, was synthesized for this purpose. Beneath improving the reproducibility of quantitation, PMP derivatization contributed to an enhancement of ionization yields in MS. By introducing an internal standard (e.g. GlcNAc3) the reproducibility for quantification can be improved. For higher abundant O-glycans a mean coefficient of variation (CV) less than 6% could be attained, for very low abundant CV values between 15 and 20%. For the determination of O-glycan profiles in mixtures, a HPLC separation was combined with a high resolution Qq-oaTOF instrument. RP-type stationary phases were successful in separating glycan species including some of isomeric ones. This separation step was particularly useful for removing of salts avoiding so the presence of various sodium clusters in the MS spectrum. PMID:26184710

  14. 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

  15. 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.

  16. Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman microscopy in combination with stable isotopic labeling.

    PubMed

    Matthäus, Christian; Krafft, Christoph; Dietzek, Benjamin; Brehm, Bernhard R; Lorkowski, Stefan; Popp, Jürgen

    2012-10-16

    Monocyte-derived macrophages play a key role in atherogenesis because their transformation into foam cells is responsible for deposition of lipids in plaques within arterial walls. The appearance of cytosolic lipid droplets is a hallmark of macrophage foam cell formation, and the molecular basics involved in this process are not well understood. Of particular interest is the intracellular fate of different individual lipid species, such as fatty acids or cholesterol. Here, we utilize Raman microscopy to image the metabolism of such lipids and to trace their subsequent storage patterns. The combination of microscopic information with Raman spectroscopy provides a powerful molecular imaging method, which allows visualization at the diffraction limit of the employed laser light and biochemical characterization through associated spectral information. In order to distinguish the molecules of interest from other naturally occurring lipids spectroscopically, deuterium labels were introduced. Intracellular distribution and metabolic changes were observed for serum albumin-complexed palmitic and oleic acid and cholesterol and quantitatively evaluated by monitoring the increase in CD scattering intensities at 0.5, 1, 3, 6, 24, 30, and 36 h. This approach may also allow for investigating the cellular trafficking of other molecules, such as nutrients, metabolites, and drugs. PMID:22954250

  17. 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

  18. 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.

  19. 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.

  20. Absolute quantification of Pru av 2 in sweet cherry fruit by liquid chromatography/tandem mass spectrometry with the use of a stable isotope-labelled peptide.

    PubMed

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

    2016-08-01

    Pru av 2, a pathogenesis-related (PR) protein present in the sweet cherry (Prunus avium L.) fruit, is the principal allergen of cherry and one of the chief causes of pollen food syndrome (oral allergy syndrome). In this study, a quantitative assay for this protein was developed with the use of the protein absolute quantification (AQUA) method, which consists of liquid chromatography/tandem mass spectrometry (LC/MS/MS) employing TGC[CAM]STDASGK[(13)C6,(15)N2], a stable isotope-labelled internal standard (SIIS) peptide. This assay gave a linear relationship (r(2)>0.99) in a concentration range (2.3-600fmol/μL), and the overall coefficient of variation (CV) for multiple tests was 14.6%. Thus, the contents of this allergenic protein in sweet cherry products could be determined using this assay. This assay should be valuable for allergological investigations of Pru av 2 in sweet cherry and detection of protein contamination in foods. PMID:26988485

  1. 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

  2. Photochemically Induced Dynamic Nuclear Polarization Observed by Solid-State NMR in a Uniformly (13)C-Isotope-Labeled Photosynthetic Reaction Center.

    PubMed

    Paul, Shubhajit; Bode, Bela E; Matysik, Jörg; Alia, A

    2015-10-29

    A sample of solubilized and quinone-depleted reaction centers from the purple bacterium Rhodobacter (R.) sphaeroides wild type has been prepared entirely (13)C and (15)N isotope labeled at all positions of the protein as well as of the cofactors. In this sample, the occurrence of the solid-state photo-CIDNP (photochemically induced dynamic nuclear polarization) effect has been probed by (13)C solid-state magic-angle spinning NMR under illumination. Under continuous illumination, signal intensities are modified by the three-spin mixing (TSM) mechanism. Time-resolved illumination experiments reveal the occurrence of light-induced nuclear polarization on the time scale of hundreds of microseconds, initially dominated by the transient polarization of the singlet branch of the radical-pair mechanism. A first kinetic analysis shows that the lifetime of the polarization from the singlet branch, indicated by the enhanced absorptive intensities of the signals from aliphatic carbons, is significantly extended. Upon arrival of the polarization from the triplet decay branch, emissive polarization caused by the TSM mechanism is observed. Also, this arrival is significantly delayed. The decay of TSM polarization occurs in two steps, assigned to intra- and intermolecular spin diffusion. PMID:26110356

  3. A study of the elimination of water from lithium-cationized tripeptide methyl esters by means of tandem mass spectrometry and isotope labeling.

    PubMed

    Talaty, Erach R; Cooper, Travis J; Piland, Debra L; Bateman, David J; Syed, Adeel; Stevenson, William; Van Stipdonk, Michael J

    2006-01-01

    Extensive isotope labeling (2H, 13C and 15N), collision-induced dissociation (CID) and multiple-stage tandem mass spectrometry were used to investigate the elimination of H2O from a series of model, metal-cationized tripeptide methyl esters. The present results corroborate our earlier suggestion that loss of water from lithiated peptides is initiated by a nucleophilic attack from the N-terminal side upon an amide carbonyl carbon atom to form a five-membered ring as an intermediate followed by 1,2-elimination of water. We show that the nucleophilic atom is the oxygen atom of the N-terminal amide group in the fragmentation of [AcGGGOMe+Li]+ as well as [GGGOMe+Li]+. However, the subsequent fragmentation is markedly different in the two cases as a result of the absence and presence of a free amino group. In particular, extensive scrambling of protons in the alpha-positions of GGGOMe is observed, presumably as a consequence of intervention of the basic amino group. PMID:16969769

  4. Identification of sinensetin metabolites in rat urine by an isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry.

    PubMed

    Wei, Guor-Jien; Sheen, Jenn-Feng; Lu, Wen-Chien; Hwang, Lucy Sun; Ho, Chi-Tang; Lin, Ching-I

    2013-05-29

    Sinensetin (SIN), one of the major polymethoxyflavones (PMFs) contained mainly in the citrus peels, has been reported to possess various bioactivities, including antifungal, antimutagenic, anticancer, and anti-inflammatory activities. Although the biotransformation of SIN in fungi and insects has been reported, the information about the metabolism of SIN in mammals is still unclear. In this study, formation of SIN metabolites in rats was investigated. Four isotope-labeled SINs ([4'-D3]SIN, [3'-D3]SIN, [5-D3]SIN, and [6-D3]SIN) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization mass spectrometry. The administered compound and four SIN metabolites were detected in rat urine. These metabolites were identified as 4'-hydroxy-5,6,7,3'-tetramethoxyflavone, 5-hydroxy-6,7,3',4'-tetramethoxyflavone, 6-hydroxy-5,7,3',4'-tetramethoxyflavone, and 7-hydroxy-5,6,3',4'-tetramethoxyflavone sulfate. PMID:23647150

  5. 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

  6. 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

  7. Hypophosphorylation of the architectural chromatin protein DEK in death-receptor-induced apoptosis revealed by the isotope coded protein label proteomic platform.

    PubMed

    Tabbert, Anja; Kappes, Ferdinand; Knippers, Rolf; Kellermann, Josef; Lottspeich, Friedrich; Ferrando-May, Elisa

    2006-11-01

    During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide. We exploited the ability of this system to produce nuclei arrested at different stages of apoptosis to analyze proteome alterations which occur prior to or at a low level of caspase activation. We show that the majority of proteins affected at the onset of apoptosis are involved in chromatin architecture and RNA metabolism. Among them is DEK, an architectural chromatin protein which is linked to autoimmune disorders. The proteomic analysis points to the occurrence of multiple PTMs in early apoptotic nuclei. This is confirmed by showing that the level of phosphorylation of DEK is decreased following apoptosis induction. These results suggest the unexpected existence of an early crosstalk between cytoplasm and nucleus during apoptosis. They further establish a previously unrecognized link between DEK and cell death, which will prove useful in the elucidation of the physiological function of this protein. PMID:17001602

  8. Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR-->M difference spectrum of bacteriorhodopsin.

    PubMed

    Liu, X M; Sonar, S; Lee, C P; Coleman, M; RajBhandary, U L; Rothschild, K J

    1995-01-01

    Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorthodopsin and other retinal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique. While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments. A new approach has recently been reported (Sonar et al., Nature Struct. Biol. 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL). We have now used this method to re-examine bands assigned in the bR-->M difference spectrum to tyrosine residues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins. PMID:7662870

  9. Identification of a Novel Neurotrophic Factor from Primary Retinal Müller Cells Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) *

    PubMed Central

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

    2014-01-01

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

  10. Stable isotope labeling by amino acids in cell culture and differential plasma membrane proteome quantitation identify new substrates for the MARCH9 transmembrane E3 ligase.

    PubMed

    Hör, Simon; Ziv, Tamar; Admon, Arie; Lehner, Paul J

    2009-08-01

    The regulation of cell surface receptor expression is essential for immune cell differentiation and function. At the plasma membrane ubiquitination is an important post-translational mechanism for regulating expression of a wide range of surface proteins. MARCH9, a member of the RING-CH family of transmembrane E3 ubiquitin ligases, down-regulates CD4, major histocompatibility complex-I (MHC), and ICAM-1 in lymphoid cells. To identify novel MARCH9 substrates, we used high throughput flow cytometry and quantitative mass spectrometry by stable isotope labeling by amino acids in cell culture (SILAC) to determine the differential expression of plasma membrane proteins in a MARCH9-expressing B cell line. This combined approach identified 13 potential new MARCH9 targets. All of the SILAC-identified targets for which antibodies were available were subsequently confirmed by flow cytometry, validating the proteomics results. A close correlation (r(2) = 0.93) between -fold down-regulation as determined by SILAC and flow cytometry was found, with no false positive hits detected. The potential new MARCH9 substrates cover a wide range of functions and include receptor-type protein-tyrosine phosphatases (e.g. PTPRJ/CD148) as well as Fc gamma receptor IIB (CD32B), HLA-DQ, signaling lymphocytic activation molecule (CD150), and polio virus receptor (CD155). The identification of plasma membrane targets by SILAC with confirmation by flow cytometry represents a novel and powerful approach to analyze changes in the plasma membrane proteome. PMID:19457934

  11. 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

  12. 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

  13. 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.

  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. 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

  16. 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, 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

  17. 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

  18. 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.

  19. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    PubMed Central

    Ren, Xiaomei; El-Sagheer, Afaf H.; Brown, Tom

    2016-01-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  20. Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes.

    PubMed

    Ren, Xiaomei; El-Sagheer, Afaf H; Brown, Tom

    2016-05-01

    A sterically undemanding azide analogue of dTTP (AHP dUTP) with an alkyl chain and ethynyl attachment to the nucleobase was designed and incorporated into DNA by primer extension, reverse transcription and polymerase chain reaction (PCR). An azide-modified 523 bp PCR amplicon with all 335 thymidines replaced by AHP dU was shown to be a perfect copy of the template from which it was amplified. Replacement of thymidine with AHP dU increases duplex stability, accounting in part for the high incorporation efficiency of the azide-modified triphosphate. Single-stranded azide-labelled DNA was conveniently prepared from PCR products by λ-exonuclease digestion and streptavidin magnetic bead isolation. Efficient fluorescent labelling of single and double-stranded DNA was carried out using dyes functionalized with bicyclo[6.1.0]non-4-yne (BCN) via the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction. This revealed that the degree of labelling must be carefully controlled to achieve optimum fluorescence and avoid fluorescence quenching. Dual-coloured probes were obtained in a single tube fluorescent labelling reaction; and varying the ratios of the two dyes provides a simple method to prepare DNA probes with unique fluorescent signatures. AHP dUTP is a versatile clickable nucleotide with potentially wide applications in biology and nanotechnology including single molecule studies and synthesis of modified aptamer libraries via SELEX. PMID:26819406

  1. Impact of clostridial glucosylating toxins on the proteome of colonic cells determined by isotope-coded protein labeling and LC-MALDI

    PubMed Central

    2011-01-01

    Background The anaerobe Clostridium difficile produces two major virulence factors toxin A and B that inactivate Rho proteins by glucosylation of a pivotal threonine residue. Purified toxins induce reorganization of the cytoskeleton and cell death in colonic cells. Whether all toxin effects on target cells depend on catalytic glucosyltransferase activity is unclear at present. Thus, we conducted a proteome approach to compare the protein profile of target cells treated either with wild type toxin A (rTcdA wt) or with a catalytically inactive mutant toxin A (mutant rTcdA). Relative protein quantification was feasible using isotope-coded protein labeling techniques (ICPL) and mass spectrometry (LC-MALDI). Results Altogether we found a significant differential expression of thirty proteins after treatment with rTcdA wt or mutant rTcdA. Mutant rTcdA caused up-regulation of seven proteins and sixteen proteins were responsive to rTcdA wt after 5 h. Long-term effect of rTcdA wt on protein expression was the down-regulation of eleven proteins. Up- or down-regulation of several proteins was verified by western blot analysis confirming the MS results. Conclusion Our results indicate incubation time-dependent effects of the clostridial glucosylating toxin A on colonic cells. The rTcdA wt impact more cellular functions than actin cytoskeleton reorganization and apoptosis. Furthermore, these data give insight into glucosyltransferase independent effects of clostridial glucosylating toxins on target cells after short incubation time. Additionally, our data reveal pro-inflammatory and proliferative effects of mutant rTcdA after short-term incubation. PMID:21849038

  2. 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 μg 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

  3. 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

  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. Changes of low-frequency vibrational modes induced by universal 15N- and 13C-isotope labeling in S2/S1 FTIR difference spectrum of oxygen-evolving complex.

    PubMed

    Kimura, Yukihiro; Mizusawa, Naoki; Ishii, Asako; Yamanari, Toshihiro; Ono, Taka-aki

    2003-11-18

    The effects of universal (15)N- and (13)C-isotope labeling on the low- (650-350 cm(-1)) and mid-frequency (1800-1200 cm(-1)) S(2)/S(1) Fourier transform infrared (FTIR) difference spectrum of the photosynthetic oxygen-evolving complex (OEC) were investigated in histidine-tagged photosystem (PS) II core particles from Synechocystis sp. PCC 6803. In the mid-frequency region, the amide II modes were predominantly affected by (15)N-labeling, whereas, in addition to the amide II, the amide I and carboxylate modes were markedly affected by (13)C-labeling. In the low-frequency region, by comparing a light-induced spectrum in the presence of ferricyanide as the electron acceptor, with the double difference S(2)/S(1) spectrum obtained by subtracting the Q(A)(-)/Q(A) from the S(2)Q(A)(-)/S(1)Q(A) spectrum, considerable numbers of bands found in the light-induced spectrum were assigned to the S(2)/S(1) vibrational modes in the unlabeled PS II core particles. Upon (13)C-labeling, changes were observed for most of the prominent bands in the S(2)/S(1) spectrum. Although (15)N-labeling also induced changes similar to those by (13)C-labeling, the bands at 616(-), 605(+), 561(+), 555(-), and 544(-) cm(-1) were scarcely affected by (15)N-labeling. These results indicated that most of the vibrational modes found in the low-frequency spectrum are derived from the coupling between the Mn-cluster and groups containing nitrogen and/or carbon atom(s) in a direct manner and/or through hydrogen bonding. Interestingly, an intensive band at 577(-) cm(-1) was not affected by (15)N- and (13)C-isotope labeling, indicating that this band arises from the mode that does not include either nitrogen or carbon atoms, such as the skeletal vibration of the Mn-cluster or stretching vibrational modes of the Mn-ligand. PMID:14609327

  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 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. 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, Eugénia; 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

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

    PubMed

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

    2014-01-01

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

  14. 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

    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.

  15. 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.

  16. 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 (Δ18OBL) and carbon (δ13C) 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 Δ18OBL 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 δ13C values decrease as gs decreases but the change in δ13C 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 Δ18OBL and δ13C could be used as proxies for E and gs, and be used to screen S. viridis for differences in WUE in breeding programs. The Δ18OBL and δ13C 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, Δ18OBL and δ13C can be useful proxies to screen genotypes for drought resistance by recording differences in E, gs, and WUE. Measuring Δ18OBL and δ13C 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.

  17. 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.

  18. 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

  19. 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.

  20. 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

  1. 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

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

    PubMed

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

    2015-05-01

    A reliable and sensitive isotope-labelled internal standard method for simultaneous determination of chlorantraniliprole and cyantraniliprole in fruits (apple and grape), vegetables (cucumber and tomato) and cereals (rice and wheat) using ultra-high-performance liquid chromatography-tandem mass spectrometry was developed. Isotope-labelled internal standards were effective in compensating for the loss in the pretreatment and overcoming the matrix effect. The analytes were extracted with acetonitrile and cleaned up with different kinds of sorbents. The determination of the target compounds was achieved in less than 4 min using a T3 column combined with an electrospray ionization source in positive mode. The overall average relative recoveries in all matrices at three spiking levels (10, 20 and 50 μg kg(-1)) ranged from 95.5 to 106.2 %, with all relative standard deviations being less than 14.4 % for all analytes. The limits of detection did not exceed 0.085 μg kg(-1) and the limits of quantification were below 0.28 μg kg(-1) in all matrices. The method was demonstrated to be convenient and accurate for the routine monitoring of chlorantraniliprole and cyantraniliprole in fruits, vegetables and cereals. PMID:25822158

  3. Identification of 5,7,3',4'-tetramethoxyflavone metabolites in rat urine by the isotope-labeling method and ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry.

    PubMed

    Lu, Wen-Chien; Sheen, Jenn-Feng; Hwang, Lucy Sun; Wei, Guor-Jien

    2012-08-22

    5,7,3',4'-Tetramethoxyflavone (TMF), one of the major polymethoxyflavones (PMFs) isolated from Kaempferia parviflor , has been reported possessing various bioactivities, including antifungal, antimalarial, antimycobacterial, and anti-inflammatory activities. Although several studies on the TMF have been reported, the information about the metabolism of TMF and the structures of TMF metabolites is still not yet clear. In this study, an isotope-labeling method was developed for the identification of TMF metabolites. Three isotope-labeled TMFs (5,7,3',4'-tetramethoxy[3'-D(3)]flavone, 5,7,3',4'-tetramethoxy[4'-D(3)]flavone, and 5,7,3',4'-tetramethoxy[5,4'-D(6)]flavone) were synthesized and administered to rats. The urine samples were collected, and the main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry. Five TMF metabolites were unambiguously identified as 3'-hydroxy-5,7,4'-trimethoxyflavone, 7-hydroxy-5,3',4'-trimethoxyflavone sulfate, 7-hydroxy-5,3',4'-trimethoxyflavone, 4'-hydroxy-5,7,3'-trimethoxyflavone, and 5-hydroxy-7,3',4'-trimethoxyflavone. PMID:22812915

  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. Triple-Label β Liquid Scintillation Counting

    PubMed Central

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

    2010-01-01

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

  6. 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

  7. 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 (δ13C) 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.

  8. Trypsin immobilization on hairy polymer chains hybrid magnetic nanoparticles for ultra fast, highly efficient proteome digestion, facile 18O labeling and absolute protein quantification.

    PubMed

    Qin, Weijie; Song, Zifeng; Fan, Chao; Zhang, Wanjun; Cai, Yun; Zhang, Yangjun; Qian, Xiaohong

    2012-04-01

    In recent years, quantitative proteomic research attracts great attention because of the urgent needs in biological and clinical research, such as biomarker discovery and verification. Currently, mass spectrometry (MS) based bottom up strategy has become the method of choice for proteomic quantification. In this strategy, the amount of proteins is determined by quantifying the corresponding proteolytic peptides of the proteins, therefore highly efficient and complete protein digestion is crucial for achieving accurate quantification results. However, the digestion efficiency and completeness obtained using conventional free protease digestion is not satisfactory for highly complex proteomic samples. In this work, we developed a new type of immobilized trypsin using hairy noncross-linked polymer chains hybrid magnetic nanoparticle as the matrix aiming at ultra fast, highly efficient proteomic digestion and facile (18)O labeling for absolution protein quantification. The hybrid nanoparticle is synthesized by in situ growth of hairy polymer chains from the magnetic nanoparticle surface using surface initiated atom transfer radical polymerization technique. The flexible noncross-linked polymer chains not only provide large amount of binding sites but also work as scaffolds to support three-dimensional trypsin immobilization which leads to increased loading amount and improved accessibility of the immobilized trypsin. For complex proteomic samples, obviously increased digestion efficiency and completeness was demonstrated by 27.2% and 40.8% increase in the number of identified proteins and peptides as well as remarkably reduced undigested proteins residues compared with that obtained using conventional free trypsin digestion. The successful application in absolute protein quantification of enolase from Thermoanaerobacter tengcongensis protein extracts using (18)O labeling and MRM strategy further demonstrated the potential of this hybrid nanoparticle immobilized trypsin for high throughput proteome quantification. PMID:22413971

  9. 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.

  10. Efficient utilization of the expanded criteria donor (ECD) deceased donor kidney pool: an analysis of the effect of labeling.

    PubMed

    Hirth, R A; Pan, Q; Schaubel, D E; Merion, R M

    2010-02-01

    We investigated the effect of the expanded criteria donor (ECD) label on (i) recovery of kidneys and (ii) acceptance for transplantation given recovery. An ECD is age > or = 60, or age 50-59 with > or = 2 of 3 specified comorbidities. Using data from the Scientific Registry of Transplant Recipients from 1999 to 2005, we modeled recovery rates through linear regression and transplantation probabilities via logistic regression, focusing on organs from donors just-younger versus just-older than the ECD age thresholds. We split the sample at July 1, 2002 to determine how decisions changed at the approximate time of implementation of the ECD definition. Before July 2002, the number of recovered kidneys with 0-1 comorbidities dropped at age 60, but transplantation probabilities given recovery did not. After July 2002, the number of recovered kidneys with 0-1 comorbidities rose at age 60, but transplantation probabilities contingent on recovery declined. No similar trends were observed at donor age 50 among donors with > or = 2 comorbidities. Overall, implementation of the ECD definition coincided with a reversal of an apparent reluctance to recover kidneys from donors over age 59, but increased selectiveness on the part of surgeons/centers with respect to these kidneys. PMID:20055795

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

    PubMed

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

    2012-07-20

    A novel and general approach for (13)C(2)- and (2)H-labeled phenethylamine derivatives has been developed, based on a highly convergent single-step assembly of the carbon skeleton. The efficient incorporation of two carbon-13 isotopes into phenethylamines was accomplished using a palladium-catalyzed double carbonylation of aryl iodides with near stoichiometric carbon monoxide. PMID:22725263

  12. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons

    PubMed Central

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I.; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the “in vivo” transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs. PMID:27047347

  13. A Simple and Efficient In Vivo Non-viral RNA Transfection Method for Labeling the Whole Axonal Tree of Individual Adult Long-Range Projection Neurons.

    PubMed

    Porrero, César; Rodríguez-Moreno, Javier; Quetglas, José I; Smerdou, Cristian; Furuta, Takahiro; Clascá, Francisco

    2016-01-01

    We report a highly efficient, simple, and non-infective method for labeling individual long-range projection neurons (LRPNs) in a specific location with enough sparseness and intensity to allow complete and unambiguous reconstructions of their entire axonal tree. The method is based on the "in vivo" transfection of a large RNA construct that drives the massive expression of green fluorescent protein. The method combines two components: injection of a small volume of a hyperosmolar NaCl solution containing the Pal-eGFP-Sindbis RNA construct (Furuta et al., 2001), followed by the application of high-frequency electric current pulses through the micropipette tip. We show that, although each component alone increases transfection efficacy, compared to simple volume injections of standard RNA solution, the highest efficacy (85.7%) is achieved by the combination of both components. In contrast with the infective viral Sindbis vector, RNA transfection occurs exclusively at the position of the injection micropipette tip. This method simplifies consistently labeling one or a few isolated neurons per brain, a strategy that allows unambiguously resolving and quantifying the brain-wide and often multi-branched monosynaptic circuits created by LRPNs. PMID:27047347

  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. 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

  16. Seasonal variations in photosynthesis, intrinsic water-use efficiency and stable isotope composition of poplar leaves in a short-rotation plantation.

    PubMed

    Broeckx, L S; Fichot, R; Verlinden, M S; Ceulemans, R

    2014-07-01

    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

  17. 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

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

    SciTech Connect

    Vanicek, Jiri; Miller, William H.

    2007-06-13

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

  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. Isotope effect in BEDT-TTF based organic superconductors

    SciTech Connect

    Kini, A.M.; Carlson, K.D.; Dudek, J.D.; Geiser, U.; Wang, H.H.; Williams, J.M.

    1996-10-01

    The results of the comprehensive isotope effect studies, in which seven different isotopically labeled (involving {sup 13}C, {sup 34}S and {sup 2}H labeling) BEDT-TTF derivatives and isotopically labeled anion [Cu({sup 15}N{sup 13}CS){sub 2}]{sup {minus}} were utilized, are summarized. For the first time, convincing evidence for a genuine BCS-like mass isotope effect in an organic superconductor is revealed in these studies.

  1. [18F]SiFA-isothiocyanate: a new highly effective radioactive labeling agent for lysine-containing proteins.

    PubMed

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

    2009-05-25

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

  2. 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

  3. 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

  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 homogeneou