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Sample records for efficient isotopic labeling

  1. Efficient and selective isotopic labeling of hemes to facilitate the study of multiheme proteins

    SciTech Connect

    Fonseca, Bruno M.; Tien, Ming; Rivera, Mario; Shi, Liang; Louro, Ricardo O.

    2012-04-02

    Specific isotopic labeling of hemes provides a unique opportunity to characterize the structure and function of heme-proteins. Unfortunately, present day methods do not allow efficient labeling in high yields of multiheme cytochromes c, which are of great biotechnological interest. Here, a method for production of recombinant multiheme cytochromes c in Escherichia coli with isotopically labeled hemes is reported. A small tetraheme cytochrome of 12 kDa from Shewanella oneidensis MR-1 was used to demonstrate the method, achieving a production of 4 mg of pure protein per liter. This method achieves, in a single step, efficient expression and incorporation of hemes isotopically labeled in specific atom positions adequate for spectroscopic characterization of these complex heme proteins. It is, furthermore, of general application to heme proteins opening new possibilities in the characterization of this important class of proteins.

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

    PubMed

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

    2014-02-01

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

  3. Isotope Labeling in Mammalian Cells

    PubMed Central

    Dutta, Arpana; Saxena, Krishna; Klein-Seetharaman, Judith

    2011-01-01

    Isotope labeling of proteins represents an important and often required tool for the application of nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and dynamics of proteins. Mammalian expression systems have conventionally been considered to be too weak and inefficient for protein expression. However, recent advances have significantly improved the expression levels of these systems. Here, we provide an overview of some of the recent developments in expression strategies for mammalian expression systems in view of NMR investigations. PMID:22167668

  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. Stable isotope labeling methods for DNA.

    PubMed

    Nelissen, Frank H T; Tessari, Marco; Wijmenga, Sybren S; Heus, Hans A

    2016-08-01

    NMR is a powerful method for studying proteins and nucleic acids in solution. The study of nucleic acids by NMR is far more challenging than for proteins, which is mainly due to the limited number of building blocks and unfavorable spectral properties. For NMR studies of DNA molecules, (site specific) isotope enrichment is required to facilitate specific NMR experiments and applications. Here, we provide a comprehensive review of isotope-labeling strategies for obtaining stable isotope labeled DNA as well as specifically stable isotope labeled building blocks required for enzymatic DNA synthesis. PMID:27573183

  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. Unsupervised Identification of Isotope-Labeled Peptides.

    PubMed

    Goldford, Joshua E; Libourel, Igor G L

    2016-06-01

    In vivo isotopic labeling coupled with high-resolution proteomics is used to investigate primary metabolism in techniques such as stable isotope probing (protein-SIP) and peptide-based metabolic flux analysis (PMFA). Isotopic enrichment of carbon substrates and intracellular metabolism determine the distribution of isotopes within amino acids. The resulting amino acid mass distributions (AMDs) are convoluted into peptide mass distributions (PMDs) during protein synthesis. With no a priori knowledge on metabolic fluxes, the PMDs are therefore unknown. This complicates labeled peptide identification because prior knowledge on PMDs is used in all available peptide identification software. An automated framework for the identification and quantification of PMDs for nonuniformly labeled samples is therefore lacking. To unlock the potential of peptide labeling experiments for high-throughput flux analysis and other complex labeling experiments, an unsupervised peptide identification and quantification method was developed that uses discrete deconvolution of mass distributions of identified peptides to inform on the mass distributions of otherwise unidentifiable peptides. Uniformly (13)C-labeled Escherichia coli protein was used to test the developed feature reconstruction and deconvolution algorithms. The peptide identification was validated by comparing MS(2)-identified peptides to peptides identified from PMDs using unlabeled E. coli protein. Nonuniformly labeled Glycine max protein was used to demonstrate the technology on a representative sample suitable for flux analysis. Overall, automatic peptide identification and quantification were comparable or superior to manual extraction, enabling proteomics-based technology for high-throughput flux analysis studies. PMID:27145348

  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. NMR studies of isotopically labeled RNA

    SciTech Connect

    Pardi, A.

    1994-12-01

    In summary, the ability to generate NMR quantities of {sup 15}N and {sup 13}C-labeled RNAs has led to the development of heteronuclear multi-dimensional NMR techniques for simplifying the resonance assignment and structure determination of RNAs. These methods for synthesizing isotopically labeled RNAs are only several years old, and thus there are still relatively few applications of heteronuclear multi-dimensional NMR techniques to RNA. However, given the critical role that RNAs play in cellular function, one can expect to see an increasing number of NMR structural studies of biologically active RNAs.

  10. Weaving a two-dimensional fishing net from titanoniobate nanosheets embedded with Fe₃O₄ 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 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. PMID:25757497

  11. The topology of metabolic isotope labeling networks

    PubMed Central

    Weitzel, Michael; Wiechert, Wolfgang; Nöh, 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  14. 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

  15. Benchmarking stable isotope labeling based quantitative proteomics.

    PubMed

    Altelaar, A F Maarten; Frese, Christian K; Preisinger, Christian; Hennrich, Marco L; Schram, Andree W; Timmers, H Th Marc; Heck, Albert J R; Mohammed, Shabaz

    2013-08-01

    Several quantitative mass spectrometry based technologies have recently evolved to interrogate the complexity, interconnectivity and dynamic nature of proteomes. Currently, the most popular methods use either metabolic or chemical isotope labeling with MS based quantification or chemical labeling using isobaric tags with MS/MS based quantification. Here, we assess the performance of three of the most popular approaches through systematic independent large scale quantitative proteomics experiments, comparing SILAC, dimethyl and TMT labeling strategies. Although all three methods have their strengths and weaknesses, our data indicate that all three can reach a similar depth in number of identified proteins using a classical (MS2 based) shotgun approach. TMT quantification using only MS2 is heavily affected by co-isolation leading to compromised precision and accuracy. This issue may be partly resolved by using an MS3 based acquisition; however, at the cost of a significant reduction in number of proteins quantified. Interestingly, SILAC and chemical labeling with MS based quantification produce almost indistinguishable results, independent of which database search algorithm used. PMID:23085607

  16. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards

    PubMed Central

    Basu, Sankha S; Blair, Ian A

    2013-01-01

    Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [13C315N]-pantothenate (vitamin B5), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2–3 weeks. PMID:22157971

  17. Catalytic gasification: Isotopic labeling and transient reaction

    SciTech Connect

    Saber, J.M.; Falconer, J.L.; Brown, L.F.

    1985-01-01

    Temperature-programmed reaction was used with labeled isotopes (/sup 13/C and /sup 18/O) to study interactions between carbon black and potassium carbonate in pure He and 10% CO/sub 2//90% He atmospheres. Catalytic gasification precursor complexes were observed. Carbon and oxygen-bearing carbon surface groups interacted with the carbonate above 500 K to form surface complexes. Between 500 K and 950 K, and in the presence of gaseous carbon dioxide, the complexes promoted carbon and oxygen exchange between the gas-phase CO/sub 2/ and the surface. Oxygen exchanged between the surface complexes; but carbon did not exchange between the carbonate and the carbon black. As the temperature rose, the complexes decomposed to produce carbon dioxide, and catalytic gasification then began. Elemental potassium formed, and the active catalyst appears to alternate between potassium metal and a potassium-oxygen-carbon complex.

  18. Synthesis of isotopically labelled 2-isopropylthioxanthone from 2,2'-dithiosalicylic acid and deuterium cumene.

    PubMed

    Fang, Chao; Yang, Weicheng; Yang, Chao; Wang, Haoran; Sun, Kai; Luo, Yong

    2016-06-30

    Two efficient synthetic routes of stable deuterium labelled 2-isopropylthioxanthone were presented with 98.1% and 98.8% isotopic abundance in acceptable yields and excellent chemical purities. Their structures and the isotope-abundance were confirmed according to proton nuclear magnetic resonance and liquid chromatography-mass spectrometry. PMID:27123759

  19. 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.

  20. Stable isotope labeling strategy based on coding theory.

    PubMed

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

    2015-10-01

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

  1. Stereoselective synthesis of stable-isotope-labeled amino acids

    SciTech Connect

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

    1994-12-01

    For magnetic resonance and vibrational spectroscopies to reach their full potential, they must be used in combination with sophisticated site-specific stable isotope labeling of biological macromolecules. Labeled amino acids are required for the study of the structure and function of enzymes and proteins. Because there are 20 common amino acids, each with its own distinguishing chemistry, they remain a synthetic challenge. The Oppolzer chiral auxiliary provides a general tool with which to approach the synthesis of labeled amino acids. By using the Oppolzer auxiliary, amino acids can be constructed from several small molecules, which is ideal for stable isotope labeling. In addition to directing the stereochemistry at the {alpha}-carbon, the camphorsultam can be used for stereo-specific isotope labeling at prochiral centers in amino acids. By using the camphorsultam auxiliary we have the potential to synthesize virtually any isotopomer of all of the common amino acids.

  2. Analysis of proteome dynamics in mice by isotopic labeling.

    PubMed

    Price, John C; Ghaemmaghami, Sina

    2014-01-01

    Recent advances in mass spectrometry and in vivo isotopic labeling have enabled proteome-wide analyses of protein turnover in complex organisms. Here, we describe a protocol for analyzing protein turnover rates in mouse tissues by comprehensive (15)N labeling. The procedure involves the complete isotopic labeling of blue green algae (Spirulina platensis) with (15)N and utilizing it as a source of dietary nitrogen for mice. We outline a detailed protocol for in-house production of (15)N-labeled algae, labeling of mice, and analysis of isotope incorporation kinetics by mass spectrometry. The methodology can be adapted to analyze proteome dynamics in most murine tissues and may be particularly useful in the analysis of proteostatic disruptions in mouse models of disease. PMID:24791984

  3. 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

  4. High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

    PubMed

    Laroche, Y; Storme, V; De Meutter, J; Messens, J; Lauwereys, M

    1994-11-01

    Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR. PMID:7765555

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

    PubMed

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

    2012-09-15

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

  6. Plant SILAC: Stable-Isotope Labelling with Amino Acids of Arabidopsis Seedlings for Quantitative Proteomics

    PubMed Central

    Lewandowska, Dominika; ten Have, Sara; Hodge, Kelly; Tillemans, Vinciane; Lamond, Angus I.; Brown, John W. S.

    2013-01-01

    Stable Isotope Labelling by Amino acids in Cell culture (SILAC) is a powerful technique for comparative quantitative proteomics, which has recently been applied to a number of different eukaryotic organisms. Inefficient incorporation of labelled amino acids in cell cultures of Arabidopsis thaliana has led to very limited use of SILAC in plant systems. We present a method allowing, for the first time, efficient labelling with stable isotope-containing arginine and lysine of whole Arabidopsis seedlings. To illustrate the utility of this method, we have combined the high labelling efficiency (>95%) with quantitative proteomics analyses of seedlings exposed to increased salt concentration. In plants treated for 7 days with 80 mM NaCl, a relatively mild salt stress, 215 proteins were identified whose expression levels changed significantly compared to untreated seedling controls. The 92 up-regulated proteins included proteins involved in abiotic stress responses and photosynthesis, while the 123 down-regulated proteins were enriched in proteins involved in reduction of oxidative stress and other stress responses, respectively. Efficient labelling of whole Arabidopsis seedlings by this modified SILAC method opens new opportunities to exploit the genetic resources of Arabidopsis and analyse the impact of mutations on quantitative protein dynamics in vivo. PMID:23977254

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

    PubMed

    Preston, Tom

    2014-01-01

    This paper discusses some of the recent improvements in instrumentation used for stable isotope tracer measurements in the context of measuring retinol stores, in vivo. Tracer costs, together with concerns that larger tracer doses may perturb the parameter under study, demand that ever more sensitive mass spectrometric techniques are developed. GCMS is the most widely used technique. It has high sensitivity in terms of sample amount and uses high resolution GC, yet its ability to detect low isotope ratios is limited by background noise. LCMSMS may become more accessible for tracer studies. Its ability to measure low level stable isotope tracers may prove superior to GCMS, but it is isotope ratio MS (IRMS) that has been designed specifically for low level stable isotope analysis through accurate analysis of tracer:tracee ratios (the tracee being the unlabelled species). Compound-specific isotope analysis, where GC is interfaced to IRMS, is gaining popularity. Here, individual 13C-labelled compounds are separated by GC, combusted to CO2 and transferred on-line for ratiometric analysis by IRMS at the ppm level. However, commercially-available 13C-labelled retinol tracers are 2 - 4 times more expensive than deuterated tracers. For 2H-labelled compounds, GC-pyrolysis-IRMS has now become more generally available as an operating mode on the same IRMS instrument. Here, individual compounds are separated by GC and pyrolysed to H2 at high temperature for analysis by IRMS. It is predicted that GC-pyrolysis-IRMS will facilitate low level tracer procedures to measure body retinol stores, as has been accomplished in the case of fatty acids and amino acids. Sample size requirements for GC-P-IRMS may exceed those of GCMS, but this paper discusses sample preparation procedures and predicts improvements, particularly in the efficiency of sample introduction. PMID:25537104

  8. Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin.

    PubMed

    Sonar, S; Lee, C P; Coleman, M; Patel, N; Liu, X; Marti, T; Khorana, H G; RajBhandary, U L; Rothschild, K J

    1994-08-01

    Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire. PMID:7664078

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

    PubMed

    Allen, Doug K

    2016-02-01

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

  10. Isotope labeling for NMR studies of macromolecular structure and interactions

    SciTech Connect

    Wright, P.E.

    1994-12-01

    Implementation of biosynthetic methods for uniform or specific isotope labeling of proteins, coupled with the recent development of powerful heteronuclear multidimensional NMR methods, has led to a dramatic increase in the size and complexity of macromolecular systems that are now amenable to NMR structural analysis. In recent years, a new technology has emerged that combines uniform {sup 13}C, {sup 15}N labeling with heteronuclear multidimensional NMR methods to allow NMR structural studies of systems approaching 25 to 30 kDa in molecular weight. In addition, with the introduction of specific {sup 13}C and {sup 15}N labels into ligands, meaningful NMR studies of complexes of even higher molecular weight have become feasible. These advances usher in a new era in which the earlier, rather stringent molecular weight limitations have been greatly surpassed and NMR can begin to address many central biological problems that involve macromolecular structure, dynamics, and interactions.

  11. 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

  12. 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

  13. 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...

  14. Determination of Phytochelatins in Rice by Stable Isotope Labeling Coupled with Liquid Chromatography-Mass Spectrometry.

    PubMed

    Liu, Ping; Cai, Wen-Jing; Yu, Lei; Yuan, Bi-Feng; Feng, Yu-Qi

    2015-07-01

    A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by stable isotope labeling coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry (IL-LC-ESI-MS/MS) analysis. A pair of isotope-labeling reagents [ω-bromoacetonylquinolinium bromide (BQB) and BQB-d(7)] were used to label PCs in plant sample and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled standards were used as the internal standards for quantitation to minimize the matrix and ion suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly enhanced through the introduction of a permanent charged moiety of quaternary ammonium of BQB into PCs. The detection sensitivities of PCs upon BQB labeling improved by 14-750-fold, and therefore, PCs can be quantitated using only 5 mg of plant tissue. Furthermore, under cadmium (Cd) stress, we found that the contents of PCs in rice dramatically increased with the increased concentrations and treatment time of Cd. It was worth noting that PC5 was first identified and quantitated in rice tissues under Cd stress in the current study. Taken together, this IL-LC-ESI-MS/MS method demonstrated to be a promising strategy in detection of PCs in plants with high sensitivity and reliability. PMID:26073168

  15. 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

  16. 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

  17. 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

  18. Simplified synthesis of isotopically labeled 5,5-dimethyl-pyrroline N-oxide.

    PubMed

    Leinisch, Fabian; Jiang, Jinjie; Deterding, Leesa J; Mason, Ronald P

    2011-01-01

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

  19. 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.

  20. 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).

  1. 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

  2. Stable isotope labeling of mammals (SILAM) for in vivo quantitative proteomic analysis.

    PubMed

    Rauniyar, Navin; McClatchy, Daniel B; Yates, John R

    2013-06-15

    Metabolic labeling of rodent proteins with ¹⁵N, a heavy stable isotope of nitrogen, provides an efficient way for relative quantitation of differentially expressed proteins. Here we describe a protocol for metabolic labeling of rats with an ¹⁵N-enriched spirulina diet. As a case study, we also demonstrate the application of ¹⁵N-enriched tissue as a common internal standard in quantitative analysis of differentially expressed proteins in neurodevelopment in rats at two different time points, postnatal day 1 and 45. We briefly discuss the bioinformatics tools, ProLucid and Census, which can easily be used in a sequential manner to identify and quantitate relative protein levels on a proteomic scale. PMID:23523555

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

    SciTech Connect

    Jewett, J.R., Fluor Daniel Hanford

    1997-02-24

    Suitable analytical methods must be tested and developed for monitoring the individual process steps within the fuel cycle of a fusion reactor and for tritium accountability. The utility of laser-Raman spectroscopy accompanied by mass spectrometry with an Omegatron was investigated using the analysis of all hydrogen isotopes and isotopically labeled methanes as an example. The Omegatron is useful for analyzing all hydrogen isotopes mixed with the stable helium isotopes. The application of this mass spectrometer were demonstrated by analyzing mixtures of deuterated methanes. In addition, it was employed to study the radiochemical Witzbach exchange reaction between tritium and methanes. A laser-Raman spectrometer was designed for analysis of tritium-containing gases and was built from individual components. A tritium-compatible, metal-sealed Raman cuvette having windows with good optical properties and additional means for measuring the stray light was first used successfully in this work. The Raman spectra of the hydrogen isotopes were acquired in the pure rotation mode and in the rotation-vibration mode and were used for on. The deuterated methanes were measured by Raman spectroscopy, the wavenumbers determined were assigned to the corresponding vibrations, and the wavenumbers for the rotational fine-structure were summarized in tables. The fundamental Vibrations of the deuterated methanes produced Witzbach reactions were detected and assigned. The fundamental vibrations of the molecules were obtained with Raman spectroscopy for the first time in this work. The @-Raman spectrometer assembled is well suited for the analysis of tritium- containing gases and is practical in combination with mass spectrometry using an Omegatron, for studying gases used in fusion.

  4. 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.

  5. Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

    PubMed

    Xian, Feng; Zi, Jin; Wang, Quanhui; Lou, Xiaomin; Sun, Haidan; Lin, Liang; Hou, Guixue; Rao, Weiqiao; Yin, Changcheng; Wu, Lin; Li, Shuwei; Liu, Siqi

    2016-08-01

    Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible. PMID:27234506

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

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  7. 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

  8. Isotope labelling to study molecular fragmentation during the dielectric barrier discharge wet reforming of methane

    NASA Astrophysics Data System (ADS)

    Montoro-Damas, Antonio M.; Gómez-Ramírez, Ana; Gonzalez-Elipe, Agustín R.; Cotrino, José

    2016-09-01

    Isotope labelling is used to study the wet plasma reforming of methane in a dielectric barrier discharge reactor using D2O and CH4 as reactants. Besides the formation of CO and hydrogen as main products, different partitions of H and D atoms are found in the hydrogen (i.e., H2, HD, D2), methane (i.e., CH4, CH3D and CH2D2) and water (D2O, DHO) molecules detected by mass spectrometry as outlet gases of the plasma process. The effect of operating parameters such as applied current, residence time and the addition of oxygen to the reaction mixture is correlated with the H/D distribution in these molecules, the overall reaction yield and the energetic efficiency of the process. The results prove the plasma formation of intermediate excited species that rendering water and methane instead of CO and hydrogen greatly contribute to decrease the overall energy efficiency of the reforming process.

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

    PubMed Central

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

    2014-01-01

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

  10. 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.

  11. 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

  12. 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

  13. Stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics.

    PubMed

    Hoedt, Esthelle; Zhang, Guoan; Neubert, Thomas A

    2014-01-01

    Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. In this chapter we review the methodology and application of SILAC, with an emphasis on three research areas: dynamics of posttranslational modifications, protein-protein interactions, and protein turnover. PMID:24952180

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

    DOEpatents

    Goldblatt, M.; McInteer, B.B.

    1974-01-29

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

  15. A convenient and efficient purification method for chemically labeled oligonucleotides.

    PubMed

    Hwang, Jihee; Kang, Junhee; Kim, Seong Keun; Kim, Younggyu

    2013-05-01

    We developed an efficient, cost-effective, and rapid purification method for chemically-labeled oligonucleotides that requires less time than conventional procedures such as ethanol precipitation or size-exclusion chromatography. Based on the hydrophilic and hydrophobic properties of DNA and amine-reactive fluorophores, we show that n-butanol saturated with distilled water may be used to remove unreacted fluorophores by sequestering them in the organic phase, while labeled DNA remains in the aqueous phase. This phase extraction method is simple, fast, and allows for processing multiple samples simultaneously, a necessity for high-throughput labeling strategies. PMID:23662899

  16. High efficiency labeling of glycoproteins on living cells

    PubMed Central

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

    2010-01-01

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

  17. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed Central

    Tao, Shujuan; Orlando, Ron

    2014-01-01

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

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

    PubMed Central

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

    2013-01-01

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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

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

    PubMed

    Fang, Mingchih; Cadwallader, Keith R

    2013-04-17

    Stable isotope dilution assays (SIDA) provide for accurate and precise quantitation of aroma components, such as alkylpyrazines, which are often present in low concentrations in complex food matrices. The unavailability of labeled standards is the main limitation to the widespread use of SIDA. This study describes the chlorination of several alkylpyrazines to form the corresponding chloroalkylpyrazine compounds, which are efficient starting materials for the synthesis of deuterium-labeled alkylpyrazines, namely [²H₃]-2-methylpyrazine (d-1), [²H₅]-2-ethylpyrazine (d-2), [²H₃]-2,3(or 6)-dimethylpyrazine (d-3A, d-3B), [²H₃]-2,[²H₃]-6-dimethylpyrazine (d-3C), [²H₅]-2,[²H₅]-6-diethylpyrazine (d-4), [²H₅]-2-ethyl-3(or 6)-methylpyrazine (d-5A, d-5B), 2,[²H₃]-3,5-trimethylpyrazine (d-6), [²H₅]-2-ethyl-3,6-dimethylpyrazine (d-7), [²H₅]-2-ethyl-3,5-dimethylpyrazine (d-8), and 2,3-diethyl-[²H₃]-5-methylpyrazine (d-9), which were obtained in good yields (57-100%) and high purities (86-98%). These stable isotopes were used as internal standards in SIDA to accurately and precisely determine selected alkylpyrazines in commercial peanut butter, cocoa powder, and instant coffee. 2,3-Diethyl-5-methylpyrazine (p-9) and 2-ethyl-3,5-dimethylpyrazine (p-8), despite their low abundance, had the highest odor-active values among the 13 pyrazines quantified in all products due to their very low odor thresholds. PMID:23528050

  7. X13CMS: Global Tracking of Isotopic Labels in Untargeted Metabolomics

    PubMed Central

    2015-01-01

    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 X13CMS, an extension of the widely used mass spectrometry-based metabolomic software package XCMS. X13CMS 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, X13CMS 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 X13CMS 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 X13CMS with an analysis of cultured rat astrocytes treated with uniformly

  8. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA.

    PubMed Central

    Batey, R T; Inada, M; Kujawinski, E; Puglisi, J D; Williamson, J R

    1992-01-01

    A general method for large scale preparation of uniformly isotopically labeled ribonucleotides and RNAs is described. Bacteria are grown on isotopic growth medium, and their nucleic acids are harvested and degraded to mononucleotides. These are enzymatically converted into ribonucleoside triphosphates, which are used in transcription reactions in vitro to prepare RNAs for NMR studies. For 15N-labeling, E.coli is grown on 15N-ammonium sulfate, whereas for 13C-labeling, Methylophilus methylotrophus is grown on 13C-methanol, which is more economical than 13C-glucose. To demonstrate the feasibility and utility of this method, uniformly 13C-labeled ribonucleotides were used to synthesize a 31 nucleotide HIV TAR RNA that was analyzed by 3D-NMR. This method should find widespread use in the structural analysis of RNA by NMR. Images PMID:1383928

  9. 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.

  10. 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.

  11. Origin of secondary nucleation as revealed by isotopic labelling

    NASA Astrophysics Data System (ADS)

    Shimizu, K.; Tsukamoto, K.; Horita, J.; Tadaki, T.

    1984-11-01

    Isotopic measurement by mass spectroscopy was for the first time applied to the secondary nucleation products formed in a supersaturated H 2O-alum solution in the presence of a seed crystal containing D 2O. It was shown that fine particles were chipped off from the seed into the solution and act as centres for secondary nucleation.

  12. Quantifying plant phenotypes with isotopic labeling and metabolic flux analysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Analyses of metabolic flux using stable isotopes in plants have traditionally been restricted to tissues with presumed homogeneous cell populations such as developing seeds, cell suspensions, or cultured roots and root tips. It is now possible to describe these and other more complex tissues such a...

  13. 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

  14. 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...

  15. 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

  16. 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

  17. 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.

  18. 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...

  19. 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

  20. 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.

  1. 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.

  2. Isotope Coded Labeling for Accelerated Protein Interaction Profiling using MS

    PubMed Central

    Venable, John D.; Steckler, Caitlin; Ou, Weijia; Grünewald, Jan; Agarwalla, Sanjay; Brock, Ansgar

    2015-01-01

    Protein interaction surface mapping using MS is widely applied but comparatively resource intensive. Here a workflow adaptation for use of isotope coded tandem mass tags for the purpose is reported. The key benefit of improved throughput derived from sample acquisition multiplexing and automated analysis is shown to be maintained in the new application. Mapping of the epitopes of two monoclonal antibodies on their respective targets serves to illustrate the novel approach. We conclude that the approach enables mapping of interactions by MS at significantly larger scales than hereto possible. PMID:26151661

  3. Metabolic Flux Elucidation for Large-Scale Models Using 13C Labeled Isotopes

    PubMed Central

    Suthers, Patrick F.; Burgard, Anthony P.; Dasika, Madhukar S.; Nowroozi, Farnaz; Van Dien, Stephen; Keasling, Jay D.; Maranas, Costas D.

    2007-01-01

    A key consideration in metabolic engineering is the determination of fluxes of the metabolites within the cell. This determination provides an unambiguous description of metabolism before and/or after engineering interventions. Here, we present a computational framework that combines a constraint-based modeling framework with isotopic label tracing on a large-scale. When cells are fed a growth substrate with certain carbon positions labeled with 13C, the distribution of this label in the intracellular metabolites can be calculated based on the known biochemistry of the participating pathways. Most labeling studies focus on skeletal representations of central metabolism and ignore many flux routes that could contribute to the observed isotopic labeling patterns. In contrast, our approach investigates the importance of carrying out isotopic labeling studies using a more comprehensive reaction network consisting of 350 fluxes and 184 metabolites in Escherichia coli including global metabolite balances on cofactors such as ATP, NADH, and NADPH. The proposed procedure is demonstrated on an E. coli strain engineered to produce amorphadiene, a precursor to the anti-malarial drug artemisinin. The cells were grown in continuous culture on glucose containing 20% [U-13C]glucose; the measurements are made using GC-MS performed on 13 amino acids extracted from the cells. We identify flux distributions for which the calculated labeling patterns agree well with the measurements alluding to the accuracy of the network reconstruction. Furthermore, we explore the robustness of the flux calculations to variability in the experimental MS measurements, as well as highlight the key experimental measurements necessary for flux determination. Finally, we discuss the effect of reducing the model, as well as shed light onto the customization of the developed computational framework to other systems. PMID:17632026

  4. Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme.

    PubMed

    Castañeda, Carlos; Liu, Jia; Chaturvedi, Apurva; Nowicka, Urszula; Cropp, T Ashton; Fushman, David

    2011-11-01

    Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals. PMID:21962295

  5. An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins.

    PubMed

    Hegeman, Adrian D; Harms, Amy C; Sussman, Michael R; Bunner, Anne E; Harper, Jeffrey F

    2004-05-01

    A procedure for determining the extent of phosphorylation at individual sites of multiply phosphorylated proteins was developed and applied to two polyphosphorylated proteins. The protocol, using simple chemical (Fischer methyl-esterification) and enzymatic (phosphatase) modification steps and an accessible isotopic labeling reagent (methyl alcohol-d(4)), is described in detail. Site-specific phosphorylation stoichiometries are derived from the comparison of chemically identical but isotopically distinct peptide species analyzed by microspray liquid chromatography-mass spectrometry (microLC-MS) using a Micromass Q-TOF2 mass spectrometer. Ten phosphorylation sites were unambiguously identified in tryptic digests of both proteins, and phosphorylation stoichiometries were determined for eight of the ten sites using the isotope-coded strategy. The extent of phosphorylation was also estimated from the mass spectral peak areas for the phosphorylated and unmodified peptides, and these estimates, when compared with stoichiometries determined using the isotope-coded technique, differed only marginally (within approximately 20%). PMID:15121193

  6. Mechanistic Insights into Catalytic Ethanol Steam Reforming Using Isotope-Labeled Reactants.

    PubMed

    Crowley, Stephen; Castaldi, Marco J

    2016-08-26

    The low-temperature ethanol steam reforming (ESR) reaction mechanism over a supported Rh/Pt catalyst has been investigated using isotope-labeled EtOH and H2 O. Through strategic isotope labeling, all nonhydrogen atoms were distinct from one another, and allowed an unprecedented level of understanding of the dominant reaction pathways. All combinations of isotope- and non-isotope-labeled atoms were detected in the products, thus there are multiple pathways involved in H2 , CO, CO2 , CH4 , C2 H4 , and C2 H6 product formation. Both the recombination of C species on the surface of the catalyst and preservation of the C-C bond within ethanol are responsible for C2 product formation. Ethylene is not detected until conversion drops below 100 % at t=1.25 h. Also, quantitatively, 57 % of the observed ethylene is formed directly through ethanol dehydration. Finally there is clear evidence to show that oxygen in the SiO2 -ZrO2 support constitutes 10 % of the CO formed during the reaction. PMID:27487203

  7. REDOR NMR of stable-isotope-labeled protein binding sites

    SciTech Connect

    Schaefer, J.

    1994-12-01

    Rotational-echo, double resonance (REDOR) NMR, a new analytical spectroscopic technique for solids spinning at the magic angle, has been developed over the last 5 years. REDOR provides a direct measure of heteronuclear dipolar coupling between isolated pairs of labeled nuclei. In a solid with a {sup 13}C-{sup 15}N labeled pair, for example, the {sup 13}C rotational echoes that form each rotor period following a{sup 1}H-{sup 13}C cross-polarization transfer can be prevented from reaching full intensity by insertion of a {sup 15}N {pi} pulse each half rotor period. The REDOR difference (the difference between a {sup 13}C NMR spectrum obtained under these conditions and one obtained with no {sup 15}N {pi} pulses) has a strong dependence on the {sup 13}C-{sup 15}N dipolar coupling, and hence, the {sup 13}C-{sup 15}N internuclear distance. REDOR is described as double-resonance even though three radio frequencies (typically {sup 1}H, {sup 13}C, and {sup 15}N) are used because the protons are removed from the important evolution part of the experiment by resonant decoupling. The dephasing of magnetization in REDOR arises from a local dipolar {sup 13}C-{sup 15}N field gradient and involves no polarization transfer. REDOR has no dependence on {sup 13}C or {sup 15}N chemical-shift tensors and does not require resolution of a {sup 13}C-{sup 15}N coupling in the chemical-shift dimension.

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

    PubMed

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

    2016-06-01

    Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. PMID:26913967

  9. 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

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

    NASA Astrophysics Data System (ADS)

    Wang, Shengnan; Suzuki, Satoru; Hibino, Hiroki

    2014-10-01

    Topological defects, such as point defects, dislocations and grain boundaries, have a dramatic influence on the chemical and physical properties of large-scale graphene grown by chemical vapor deposition (CVD) method. Here we demonstrate the Raman visualization of polycrystalline structures in an isotopically modified CVD graphene. By means of the reversible reaction of methane on a copper catalyst, the etching of 12C-lattice and surface deposition of 13C-atoms occur in CVD graphene by sequentially introducing hydrogen and isotopic methane after standard growth of graphene with full monolayer coverage. Spatial Raman spectroscopic mapping on labeled graphene reveals pronounced network-like 13C-rich regions, which are further identified to exist along the grain boundaries of graphene by low-energy electron microscopy. The structural defects inside the graphene grains are also targeted in the isotope labeling process. Our work opens a new way to investigate multiple grain structures in CVD graphene with a simple spectroscopic technique.Topological defects, such as point defects, dislocations and grain boundaries, have a dramatic influence on the chemical and physical properties of large-scale graphene grown by chemical vapor deposition (CVD) method. Here we demonstrate the Raman visualization of polycrystalline structures in an isotopically modified CVD graphene. By means of the reversible reaction of methane on a copper catalyst, the etching of 12C-lattice and surface deposition of 13C-atoms occur in CVD graphene by sequentially introducing hydrogen and isotopic methane after standard growth of graphene with full monolayer coverage. Spatial Raman spectroscopic mapping on labeled graphene reveals pronounced network-like 13C-rich regions, which are further identified to exist along the grain boundaries of graphene by low-energy electron microscopy. The structural defects inside the graphene grains are also targeted in the isotope labeling process. Our work opens a new

  11. 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.

  12. 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

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

    PubMed

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

    2016-06-30

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

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

    PubMed Central

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

    2016-01-01

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

  15. 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

  16. 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.

  17. Experiments for a systematic comparison between stable-isotope-(deuterium) labeling and radio-((14)C) labeling for the elucidation of the in vitro metabolic pattern of pharmaceutical drugs.

    PubMed

    Grunwald, Helge; Hargreaves, Patrick; Gebhardt, Klaus; Klauer, Dominique; Serafyn, Arnaud; Schmitt-Hoffmann, Anne; Schleimer, Michael; Schlotterbeck, Goetz; Wind, Mathias

    2013-11-01

    A systematic comparison between two labeling approaches for the investigation of the in vitro metabolic pattern of pharmaceutical drugs was performed by examining the use of (i) radiolabeled drugs analyzed with LC-MS-offline radiodetection and (ii) stable-isotope labeled drugs, used in a defined mixture with the unlabeled drug and analyzed by LC-MS with recognition of the specific isotopic pattern. (14)C was used for the radioisotope-approach and deuterium for the stable-isotope approach. Olanzapine, diclofenac and ketoconazole were chosen as model drugs, as they are commercially available in their non-, radio- and stable-isotope labeled forms. For all three model drugs, liver microsome- and hepatocyte-incubations (both from rat) were performed with various concentrations and incubation times for both, the radio- and the stable-isotope approaches. The metabolic pattern, including structure elucidation of all detected metabolites, was performed independently for all individual compounds and incubations. Subsequently, the metabolic patterns of the radio-, and the stable-isotope approaches were compared. In conclusion, all metabolites found with the radioisotope approach could also be found with the stable-isotope approach. Although the stable-isotope approach does not provide a quantitative result, it can be considered to be a highly suited analytical alternative for early in vitro metabolism investigations, especially when radiolabeled drug analogues are not yet available and quantitative results are not yet necessary. PMID:23933567

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

    PubMed

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

    2008-07-01

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

  19. Efficient methods and practical guidelines for simulating isotope effects.

    PubMed

    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 (16)O/(18)O 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. PMID:23298033

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

    PubMed

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

    2012-01-01

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

  1. Quantitative metabolomic profiling using dansylation isotope labeling and liquid chromatography mass spectrometry.

    PubMed

    Zhou, Ruokun; Li, Liang

    2014-01-01

    Differential chemical isotopic labeling (CIL) LC-MS has been used for quantifying a targeted metabolite in biological samples with high precision and accuracy. Herein we describe a high-performance CIL LC-MS method for generating quantitative and comprehensive profiles of the metabolome for metabolomics applications. After mixing two comparative samples separately labeled by light or heavy isotopic tags through chemical reactions, the peak intensity ratio of the labeled analyte pair can provide relative or absolute quantitative information on the metabolites. We describe the use of (12)C2- and (13)C2-dansyl chloride (DnsCl) as the isotope reagents to profile the metabolites containing amine and phenolic hydroxyl functional groups by LC-MS. This method can be used to compare the relative concentration changes of hundreds or thousands of amine- and phenol-containing metabolites among many comparative samples and generate absolute concentration information on metabolites for which the standards are available. Combined with statistical analysis and metabolite identification tools, this method can be used to identify key metabolites involved in differentiating comparative samples such as disease cases vs. healthy controls. PMID:25270927

  2. 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...

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

    PubMed

    Robinson, Renã A S; Evans, Adam R

    2012-06-01

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

  4. 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

  5. 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.

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

    PubMed

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

    2016-09-12

    Nitrogen-13 can be efficiently produced in biomedical cyclotrons in different chemical forms, and its stable isotopes are present in the majority of biologically active molecules. Hence, it may constitute a convenient alternative to Fluorine-18 and Carbon-11 for the preparation of positron-emitter-labelled radiotracers; however, its short half-life demands for the development of simple, fast, and efficient synthetic processes. Herein, we report the one-pot, enzymatic and non-carrier-added synthesis of the (13) N-labelled amino acids l-[(13) N]alanine, [(13) N]glycine, and l-[(13) N]serine by using l-alanine dehydrogenase from Bacillus subtilis, an enzyme that catalyses the reductive amination of α-keto acids by using nicotinamide adenine dinucleotide (NADH) as the redox cofactor and ammonia as the amine source. The integration of both l-alanine dehydrogenase and formate dehydrogenase from Candida boidinii in the same reaction vessel to facilitate the in situ regeneration of NADH during the radiochemical synthesis of the amino acids allowed a 50-fold decrease in the concentration of the cofactor without compromising reaction yields. After optimization of the experimental conditions, radiochemical yields were sufficient to carry out in vivo imaging studies in small rodents. PMID:27515007

  8. Pseudocontinuous arterial spin labeling with optimized tagging efficiency.

    PubMed

    Shin, David D; Liu, Thomas T; Wong, Eric C; Shankaranarayanan, Ajit; Jung, Youngkyoo

    2012-10-01

    The adiabatic inversion of blood in pseudocontinuous arterial spin labeling (PCASL) is highly sensitive to off-resonance effects and gradient imperfections and this sensitivity can lead to tagging efficiency loss and unpredictable variations in cerebral blood flow estimates. This efficiency loss is caused by a phase tracking error between the RF pulses and the flowing spins. This article introduces a new method, referred to as Optimized PCASL (OptPCASL), that minimizes the phase tracking error by applying an additional compensation RF phase term and in-plane gradients to the PCASL pulse train. The optimal RF phase and gradient amplitudes are determined using a prescan procedure, which consists of a series of short scans interleaved with automated postprocessing routines integrated to the scanner console. The prescan procedure is shown to minimize the phase tracking error in a robust and time efficient manner. As an example of its application, the use of OptPCASL for the improved detection of functional activation in the visual cortex is demonstrated and temporal signal-to-noise ratio (SNR), image SNR, and baseline cerebral blood flow measures are compared to those acquired from conventional PCASL. PMID:22234782

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

    PubMed Central

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

    1996-01-01

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

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

    PubMed

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

    2010-03-15

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

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

    PubMed

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

    2009-07-01

    An isotope tags for relative and absolute quantitation (iTRAQ)-based reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) method was developed for differential protein expression profiling in complex cellular extracts. The estrogen positive MCF-7 cell line, cultured in the presence of 17beta-estradiol (E2) and tamoxifen (Tam), was used as a model system. MS analysis was performed with a linear trap quadrupole (LTQ) instrument operated by using pulsed Q dissociation (PQD) detection. Optimization experiments were conducted to maximize the iTRAQ labeling efficiency and the number of quantified proteins. MS data filtering criteria were chosen to result in a false positive identification rate of <4%. The reproducibility of protein identifications was approximately 60%-67% between duplicate, and approximately 50% among triplicate LC-MS/MS runs, respectively. The run-to-run reproducibility, in terms of relative standard deviations (RSD) of global mean iTRAQ ratios, was better than 10%. The quantitation accuracy improved with the number of peptides used for protein identification. From a total of 530 identified proteins (P < 0.001) in the E2/Tam treated MCF-7 cells, a list of 255 proteins (quantified by at least two peptides) was generated for differential expression analysis. A method was developed for the selection, normalization, and statistical evaluation of such datasets. An approximate approximately 2-fold change in protein expression levels was necessary for a protein to be selected as a biomarker candidate. According to this data processing strategy, approximately 16 proteins involved in biological processes such as apoptosis, RNA processing/metabolism, DNA replication/transcription/repair, cell proliferation and metastasis, were found to be up- or down-regulated. PMID:19345114

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

    PubMed Central

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

    2008-01-01

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

  13. 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.

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

    PubMed

    Wang, Shengnan; Suzuki, Satoru; Hibino, Hiroki

    2014-11-21

    Topological defects, such as point defects, dislocations and grain boundaries, have a dramatic influence on the chemical and physical properties of large-scale graphene grown by chemical vapor deposition (CVD) method. Here we demonstrate the Raman visualization of polycrystalline structures in an isotopically modified CVD graphene. By means of the reversible reaction of methane on a copper catalyst, the etching of (12)C-lattice and surface deposition of (13)C-atoms occur in CVD graphene by sequentially introducing hydrogen and isotopic methane after standard growth of graphene with full monolayer coverage. Spatial Raman spectroscopic mapping on labeled graphene reveals pronounced network-like (13)C-rich regions, which are further identified to exist along the grain boundaries of graphene by low-energy electron microscopy. The structural defects inside the graphene grains are also targeted in the isotope labeling process. Our work opens a new way to investigate multiple grain structures in CVD graphene with a simple spectroscopic technique. PMID:25303722

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. Addressing Raman features of individual layers in isotopically labeled Bernal stacked bilayer graphene

    NASA Astrophysics Data System (ADS)

    Costa, Sara D.; Weis, Johan Ek; Frank, Otakar; Fridrichová, Michaela; Kalbac, Martin

    2016-06-01

    In this report important Raman modes for the evaluation of strain in graphene (the 2D and 2D‧) are analyzed. The isotope labeling is used to disentangle contribution of individual graphene layers of graphene bilayer to the studied Raman modes. It is shown that for Bernal-stacked bilayers, the 2D and the 2D‧ Raman modes have three distinct components that can be assigned to processes originating solely from the top graphene layer, bottom graphene layer, and from a combination of processes originating both from the top and bottom layers. The reported results thus enable addressing the properties of individual graphene layers in graphene bilayer by Raman spectroscopy.

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

    PubMed Central

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

    2014-01-01

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

  1. Multiplexed analysis of cage and cage free chicken egg fatty acids using stable isotope labeling and mass spectrometry.

    PubMed

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

    2013-01-01

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

  2. 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

  3. 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

  4. Performance of human mass balance studies with stable isotope-labeled drug and continuous flow-isotope ratio mass spectrometry: a progress report.

    PubMed

    Browne, T R; Szabo, G K; Ajami, A; Browne, D G

    1998-04-01

    We propose performing human mass balance studies by administering stable isotope labeled (13C or 15N) drug and quantitating excess (above background) 13C or 15N in urine, serum, and feces by continuous flow-isotope ratio mass spectrometry (CF-IRMS). Theoretical calculations and empirical data (dynamic range, linearity, sensitivity, precision, accuracy) are presented to establish that commercially available CF-IRMS instruments can quantitate stable isotope labeled (one or two 15N or 13C labels) drug concentrations of 1.0 microg/mL or greater in urine, serum (15N), or feces. More than two 13C labels may be necessary to quantitate 1.0 microg/mL of drug in serum. Three volunteers received 650 mg of 15N13C2-acetaminophen, and urine was collected for 72 hours. Percent of administered label recovered in urine from the three subjects was 97.4, 78.9, and 95.4 for 13C and 90.3, 77.0, and 90.6 for 15N. Fecal recovery of label for one subject was 0.9% (13C2) and 1.1% (15N). Serum pharmacokinetic values obtained by counting 13C or 15N in one subject were as expected for acetaminophen. This method appears to be promising, and further validation is ongoing. PMID:9590457

  5. 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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

    Huan, Tao; Li, Liang

    2015-07-21

    Generating precise and accurate quantitative information on metabolomic changes in comparative samples is important for metabolomics research where technical variations in the metabolomic data should be minimized in order to reveal biological changes. We report a method and software program, IsoMS-Quant, for extracting quantitative information from a metabolomic data set generated by chemical isotope labeling (CIL) liquid chromatography mass spectrometry (LC-MS). Unlike previous work of relying on mass spectral peak ratio of the highest intensity peak pair to measure relative quantity difference of a differentially labeled metabolite, this new program reconstructs the chromatographic peaks of the light- and heavy-labeled metabolite pair and then calculates the ratio of their peak areas to represent the relative concentration difference in two comparative samples. Using chromatographic peaks to perform relative quantification is shown to be more precise and accurate. IsoMS-Quant is integrated with IsoMS for picking peak pairs and Zero-fill for retrieving missing peak pairs in the initial peak pairs table generated by IsoMS to form a complete tool for processing CIL LC-MS data. This program can be freely downloaded from the www.MyCompoundID.org web site for noncommercial use. PMID:26086729

  8. Determination of Polychlorinated Biphenyls in Solid Samples by Isotope Dilution Mass Spectrometry Using ³⁷Cl-Labeled Analogues.

    PubMed

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

    2015-08-01

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

  9. 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

  10. Estimating plant water uptake source depths with optimized stable water isotope labeling

    NASA Astrophysics Data System (ADS)

    Seeger, Stefan; Weiler, Markus

    2016-04-01

    Depth profiles of pore water stable isotopes in soils in conjunction with measurements of stable water isotopes (SWI) in plant transpiration allow the estimation of the contributions of different soil depths to plant water uptake (PWU).
 However, SWI depth profiles that result from the variations of SWI in natural precipitation may lead to highly ambiguous results, i.e. the same SWI signature in transpiration could result from different PWU patterns or SWI depth profiles. The aim of this study was to find an optimal stable water isotope depth profile to estimate plant water uptake patterns and to compare different PWU source depth estimation methods. We used a new soil water transport model including fractionation effects of SWI and exchange between the vapor and liquid phase to simulate different irrigation scenarios. Different amounts of water with differing SWI signatures (glacier melt water, summer precipitation water, deuterated water) were applied in order to obtain a wide variety of SWI depth profiles. Based on these simulated SWI depth profiles and a set of hypothetical PWU patterns, the theoretical SWI signatures of the respective plant transpiration were computed. In the next step, two methods - Bayesian isotope mixing models (BIMs) and optimization of a parametric distribution function (beta function) - were used to estimate the PWU patterns from the different SWI depth profiles and their respective SWI signatures in the resulting transpiration. Eventually, the estimated and computed profiles were compared to find the best SWI depth profile and the best method. The results showed, that compared to naturally occurring SWI depth profiles, the application of multiple, in terms of SWI, distinct labeling pulses greatly improves the possible spatial resolution and at the same time reduces the uncertainty of PWU estimates.
 For the PWU patterns which were assumed for this study, PWU pattern estimates based on an optimized parametric distribution function

  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. Mass spectrometric studies of cocaine disposition in animals and humans using stable isotope-labeled analogues.

    PubMed

    Jindal, S P; Lutz, T

    1989-12-01

    Ion cluster technique in conjunction with gas chromatography-mass spectrometry (GC-MS) was used for the identification and quantitation of major metabolites of cocaine (1a) in rat and humans. In a typical experiment, a female rat weighing 250 gm was intraperitoneally administered a 20-mg/kg mixture of 1a, NCD3-cocaine (1b), OCD3-cocaine (1c), and 4T2-cocaine (1d). The urine was collected, extracted with organic solvents, and separated into several fractions using TLC and HPLC techniques. Tritium radioactivity in a metabolically stable position in 1d was useful in the separation of metabolites, while the deuterium labeled 1(b + c), creating an artificial isotopic cluster, provided specific identification of metabolites by mass spectrometric interpretation. Norcocaine (2), benzoylnorecgonine (3), N-hydroxynorcocaine (4), methylecgonidine (5), benzoylecgonine (11), ecgonine methyl ester (9), hydroxycocaine (7), hydroxymethoxycocaine (10), and dimethoxyhydroxycocaine (6) were found to be the major metabolites of 1a in the rat urine as well as in plasma. The whole brain analysis showed significant amounts of unmetabolized 1a and 2, and minor concentrations of 9, 5, 7, and 10, and traces of 6. Some of these metabolites have been reported earlier by us as well as other investigators and are unequivocally confirmed in this work. Unmetabolized 1a, its pharmacologically active metabolite 2, and other major metabolites were quantitated in the rat brain, plasma, and urine using stable isotope-labeled analogues as internal standards and selected ion monitoring (SIM) mass spectrometry. The pharmacokinetic profiles of 1a and 2 indicate half-lives of less than 20 min in the brain and plasma. These data are in good agreement with widely reported short-lived behavioral effects of cocaine.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2614690

  13. The effect of different meals on the absorption of stable isotope labelled phylloquinone

    PubMed Central

    Jones, Kerry S.; Bluck, Les J. C.; Wang, Laura Y.; Stephen, Alison M.; Prynne, Celia J.; Coward, W. Andy

    2010-01-01

    Few studies have investigated the absorption of phylloquinone (vitamin K1). We recruited twelve healthy, non-obese adults. On each study day, fasted subjects took a capsule containing 20 μg of 13C-labelled phylloquinone with one of three meals, defined as convenience, cosmopolitan and animal-oriented, in a three-way crossover design. The meals were formulated from the characteristics of clusters identified in dietary pattern analysis of data from the National Diet and Nutrition Survey conducted in 2000-2001. Plasma phylloquinone concentration and isotopic enrichment were measured over 8 h. Significantly more phylloquinone tracer was absorbed when consumed with the cosmopolitan and animal-oriented meals than with the convenience meal (P = 0.001 and P = 0.035, respectively). Estimates of the relative availability of phylloquinone from the meals were: convenience meal = 1.00, cosmopolitan meal = 0.31, and animal-oriented meal = 0.23. Combining the tracer data with availability estimates for phylloquinone from the meals provides overall relative bioavailability values of convenience = 1.00, cosmopolitan = 0.46 and animal-oriented = 0.29. Stable isotopes provide a useful tool to investigate further the bioavailability of low doses of phylloquinone. Different meals can affect the absorption of free phylloquinone. The meal-based study design used in the current work provides an approach that reflects more closely the way foods are eaten in a free-living population. PMID:19538810

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

    PubMed

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

    2013-12-01

    Secondary and tertiary amines have been reported to form [M-H](+) that correspond to dehydrogenation in matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this investigation, we studied the dehydrogenation of amines in MALDI-TOF MS by isotopic labeling. Aliphatic amines were labeled with deuterium on the methylene of an N-benzyl group, which resulted in the formation of [M-D](+) and [M-H](+) ions by dedeuteration and dehydrogenation, respectively. This method revealed the proton that was removed. The spectra of most tertiary amines with an N-benzyl group showed high-intensity [M-D](+) and [M-H](+) ion peaks, whereas those of secondary amines showed low-intensity ion peaks. Ratios between the peak intensities of [M-D](+) and [M-H](+) greater than 1 suggested chemoselective dehydrogenation at the N-benzyl groups. The presence of an electron donor group on the N-benzyl groups enhanced the selectivity. The dehalogenation of amines with an N-(4-halobenzyl) group was also observed alongside dehydrogenation. The amino ions from dehalogenation can undergo second dehydrogenation. These results provide the first direct evidence about the position at which dehydrogenation of an amine occurs and the first example of dehalogenation of haloaromatic compounds in MALDI-TOF MS. These results should be helpful in the structural identification and elucidation of synthetic and natural molecules. PMID:24338887

  15. Labeled trees and the efficient computation of derivations

    NASA Technical Reports Server (NTRS)

    Grossman, Robert; Larson, Richard G.

    1989-01-01

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

  16. 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

  17. 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

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2016-08-01

    Far-red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water-soluble lipid-polymer probes are synthesized harboring hydrophilic or hydrophobic far-red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid-polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses. PMID:27113918

  20. 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

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

    PubMed Central

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

    2013-01-01

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

  2. Isotope coded protein labeling coupled immunoprecipitation (ICPL-IP): a novel approach for quantitative protein complex analysis from native tissue.

    PubMed

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

    2013-05-01

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

  3. 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.

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

    PubMed Central

    2016-01-01

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

  5. Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling.

    PubMed

    Wei, Lu; Shen, Yihui; Xu, Fang; Hu, Fanghao; Harrington, Jamie K; Targoff, Kimara L; Min, Wei

    2015-03-20

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

  6. ­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

  7. Analysis of liposoluble carboxylic acids metabolome in human serum by stable isotope labeling coupled with liquid chromatography-mass spectrometry.

    PubMed

    Zhu, Quan-Fei; Zhang, Zheng; Liu, Ping; Zheng, Shu-Jian; Peng, Ke; Deng, Qian-Yun; Zheng, Fang; Yuan, Bi-Feng; Feng, Yu-Qi

    2016-08-19

    Fatty acids (FAs) are groups of liposoluble carboxylic acids (LCAs) and play important roles in various physiological processes. Abnormal contents or changes of FAs are associated with a series of diseases. Here we developed a strategy with stable isotope labeling combined with liquid chromatography-tandem mass spectrometry (IL-LC-MS) analysis for comprehensive profiling and relative quantitation of LCAs in human serum. In this strategy, a pair of isotope labeling reagents (2-dimethylaminoethylamine (DMED)) and d4-2-dimethylaminoethylamine (d4-DMED) were employed to selectively label carboxyl groups of LCAs. The DMED and d4-DMED labeled products can lose four characteristic neutral fragments of 45 and 49Da or 63 and 67Da in collision-induced dissociation. Therefore, quadruple neutral loss scan (QNLS) mode was established and used for non-targeted profiling of LCAs. The peak pairs of DMED and d4-DMED labeling with the same retention time, intensity and characteristic mass differences were extracted from the two NLS spectra respectively, and assigned as potential LCA candidates. Using this strategy, 241 LCA candidates were discovered in the human serum; 156 carboxylic acid compounds could be determined by searching HMDB and METLIN databases (FAs are over 90%) and 21 of these LCAs were successfully identified by standards. Subsequently, a modified pseudo-targeted method with multiple reaction monitoring (MRM) detection mode was developed and used for relative quantification of LCAs in human serum from type 2 diabetes mellitus (T2DM) patients and healthy controls. As a result, 81 LCAs were found to have significant difference between T2DM patients and healthy controls. Taken together, the isotope labeling combined with tandem mass spectrometry analysis demonstrated to be a powerful strategy for identification and quantification of LCA compounds in serum samples. PMID:27432792

  8. Effect of different magnetic nanoparticle coatings on the efficiency of stem cell labeling

    NASA Astrophysics Data System (ADS)

    Horák, Daniel; Babič, Michal; Jendelová, Pavla; Herynek, Vít; Trchová, Miroslava; Likavčanová, Katarina; Kapcalová, Miroslava; Hájek, Milan; Syková, Eva

    2009-05-01

    Maghemite nanoparticles with various coatings were prepared by the coprecipitation method and characterized by transmission electron microscopy, dynamic light scattering and IR in terms of morphology, size, polydispersity and surface coating. The labeling efficiency and the viability of both rat and human mesenchymal stem cells labeled with Endorem ®, poly( L-lysine) (PLL)-modified Endorem ®, uncoated γ-Fe 2O 3, D-mannose-, PLL- or poly( N,N-dimethylacrylamide) (PDMAAm)-coated γ-Fe 2O 3 nanoparticles were compared. Coated γ-Fe 2O 3 nanoparticles labeled cells better than did Endorem ®. High relaxation rates and in vitro magnetic resonance imaging of cells labeled with coated nanoparticles showed clearly visible contrast compared with unlabeled cells or cells labeled with Endorem ®.

  9. Efficient and Selective Chemical Labeling of Electrochemically Generated Peptides Based on Spirolactone Chemistry.

    PubMed

    Zhang, Tao; Niu, Xiaoyu; Yuan, Tao; Tessari, Marco; de Vries, Marcel P; Permentier, Hjalmar P; Bischoff, Rainer

    2016-06-21

    Specific digestion of proteins is an essential step for mass spectrometry-based proteomics, and the chemical labeling of the resulting peptides is often used for peptide enrichment or the introduction of desirable tags. Cleavage of the peptide bond following electrochemical oxidation of Tyr or Trp results in a spirolactone moiety at the newly formed C-terminus offering a handle for chemical labeling. In this work, we developed a highly efficient and selective chemical labeling approach based on spirolactone chemistry. Electrochemically generated peptide-spirolactones readily undergo an intramolecular rearrangement yielding isomeric diketopiperazines precluding further chemical labeling. A strategy was established to prevent intramolecular arrangement by acetylating the N-terminal amino group prior to electrochemical oxidation and cleavage allowing the complete and selective chemical labeling of the tripeptide LWL and the decapeptide ACTH 1-10 with amine-containing reagents. As examples, we show the successful introduction of a fluorescent label and biotin for detection or affinity enrichment. Electrochemical digestion of peptides and proteins followed by efficient chemical labeling constitutes a new, powerful tool in protein chemistry and protein analysis. PMID:27247048

  10. 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.

  11. 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.

  12. 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.

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

    PubMed

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

    2016-03-01

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

  14. Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope-assisted labeling.

    PubMed

    Haviland, Julia A; Tonelli, Marco; Haughey, Dermot T; Porter, Warren P; Assadi-Porter, Fariba M

    2012-08-01

    Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, type 2 diabetes mellitus, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy and cavity ring-down spectroscopy to analyze serial plasma samples and real-time breath measurements following selective (13)C-isotope-assisted labeling. These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. This novel diagnostics approach is fast, noninvasive, and sensitive for determining specific pathway utilization, and provides a direct translational application in the health care field. PMID:22304834

  15. Novel diagnostics of metabolic dysfunction detected in breath and plasma by selective isotope assisted labeling (SIAL)

    PubMed Central

    Haviland, Julia A.; Tonelli, Marco; Haughey, Dermot T.; Porter, Warren P.; Assadi-Porter, Fariba M.

    2012-01-01

    OBJECTIVE Metabolomics is the study of a unique fingerprint of small molecules present in biological systems under healthy and disease conditions. One of the major challenges in metabolomics is validation of fingerprint molecules to identify specifically perturbed pathways in metabolic aberrations. This step is crucial to the understanding of budding metabolic pathologies and the ability to identify early indicators of common diseases such as obesity, diabetes mellitus type II, metabolic syndrome, polycystic ovary syndrome, and cancer. We present a novel approach to diagnosing aberrations in glucose utilization including metabolic pathway switching in a disease state. METHODS We used a well-defined prenatally exposed glucocorticoid mouse model that results in adult females with metabolic dysfunction. We applied the complementary technologies of nuclear magnetic resonance spectroscopy, and cavity ringdown spectroscopy to analyze serial plasma samples and real-time breath measurements following selective 13C-isotope assisted labeling (SIAL). These platforms allowed us to trace metabolic markers in whole animals and identify key metabolic pathway switching in prenatally glucocorticoid-treated animals. RESULTS Total glucose flux is significantly proportionally increased through the major oxidative pathways of glycolysis and the pentose phosphate pathway in the prenatally glucocorticoid-treated animals relative to the control animals. CONCLUSION This novel diagnostics approach is fast, non-invasive and sensitive for determining specific pathway utilization, and provides a direct translational application in the healthcare field. PMID:22304834

  16. Expression of the Trichoderma reesei tyrosinase 2 in Pichia pastoris: isotopic labeling and physicochemical characterization.

    PubMed

    Westerholm-Parvinen, Ann; Selinheimo, Emilia; Boer, Harry; Kalkkinen, Nisse; Mattinen, Maija; Saloheimo, Markku

    2007-09-01

    Trichoderma reesei tyrosinase TYR2 has been demonstrated to be able to oxidize various phenolic compounds and also peptide and protein bound tyrosine, and thus is of great interest for different biotechnological applications. In order to understand the reaction mechanism of the enzyme it would be essential to solve its three dimensional structure. Pichia pastoris is a suitable expression system for the production of recombinant enzymes for NMR studies and therefore we expressed TYR2 in this host. As a result of extensive optimization, the production yield of active histidine tagged tyrosinase purified from P. pastoris shake flask cultures was increased from 2.5 to 24 mg/L. Correct copper concentration in the growth medium was critical for the expression of this copper containing enzyme. Our analysis showed that TYR2 expressed in P. pastoris is post-translationally modified; the C-terminal domain of 153 amino acids of the protein is proteolytically cleaved off from the catalytic domain and the only potential N-glycosylation site is glycosylated. The activities of TYR2 expressed in P. pastoris and T. reesei on diphenolic L-dopa and monophenolic L-tyrosine were rather similar. The TYR2 expressed in P. pastoris showed the same physicochemical properties in CD and unfolding assays as the native TYR2 enzyme. Uniform isotopic (15)N-labeling of TYR2 was carried out with (15)NH(4)SO(4) in minimal medium to assess the suitability of the expression system for investigation by NMR spectroscopy. PMID:17562370

  17. 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

  18. 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

  19. Stable isotope applications in biomolecular structure and mechanisms. A meeting to bring together producers and users of stable-isotope-labeled compounds to assess current and future needs

    SciTech Connect

    Trewhella, J.; Cross, T.A.; Unkefer, C.J.

    1994-12-01

    Knowledge of biomolecular structure is a prerequisite for understanding biomolecular function, and stable isotopes play an increasingly important role in structure determination of biological molecules. The first Conference on Stable Isotope Applications in Biomolecular Structure and Mechanisms was held in Santa Fe, New Mexico, March 27--31, 1994. More than 120 participants from 8 countries and 44 institutions reviewed significant developments, discussed the most promising applications for stable isotopes, and addressed future needs and challenges. Participants focused on applications of stable isotopes for studies of the structure and function of proteins, peptides, RNA, and DNA. Recent advances in NMR techniques neutron scattering, EPR, and vibrational spectroscopy were highlighted in addition to the production and synthesis of labeled compounds. This volume includes invited speaker and poster presentations as well as a set of reports from discussion panels that focused on the needs of the scientific community and the potential roles of private industry, the National Stable Isotope Resource, and the National High Magnetic Field Laboratory in serving those needs. This is the leading abstract. Individual papers are processed separately for the database.

  20. Identification and validation of protein-protein interactions by combining co-immunoprecipitation, antigen competition, and stable isotope labeling.

    PubMed

    Sommer, Frederik; Mühlhaus, Timo; Hemme, Dorothea; Veyel, Daniel; Schroda, Michael

    2014-01-01

    Co-immunoprecipitation (coIP) in combination with mass spectrometry (MS) is a powerful tool to identify potential protein-protein interactions. However, unspecifically precipitated proteins usually result in large numbers of false-positive identifications. Here we describe a detailed protocol particularly useful in plant sciences that is based on (15)N stable isotope labeling of cells, (14)N antigen titration, and coIP/MS to distinguish true from false protein-protein interactions. PMID:25059616

  1. Expression of the GM2-activator protein in the methylotrophic yeast Pichia pastoris, purification, isotopic labeling, and biophysical characterization.

    PubMed

    Wendeler, Michaela; Hoernschemeyer, Joerg; John, Michael; Werth, Norbert; Schoeniger, Maike; Lemm, Thorsten; Hartmann, Rudolf; Kessler, Horst; Sandhoff, Konrad

    2004-03-01

    The GM2-activator protein (GM2AP) belongs to a group of five small, nonenzymatic proteins that are essential cofactors for the degradation of glycosphingolipids in the lysosome. It mediates the interaction between the water-soluble enzyme beta-hexosaminidase A and its membrane-embedded substrate, ganglioside GM2, at the lipid-water interphase. Inherited defects in the gene encoding this glycoprotein cause a fatal neurological storage disorder, the AB variant of GM2 gangliosidosis. With the aim to establish a convenient eukaryotic system that allows the efficient production of functionally folded, glycosylated GM2AP and offers the potential of cost-efficient isotopic labeling for structural studies by NMR spectroscopy, we established the expression of recombinant GM2AP in the methylotrophic yeast Pichia pastoris. For the construction of expression plasmids, either the full cDNA encoding human GM2AP preproprotein was cloned in the expression vector pPIC3.5K, or the cDNA encoding only the mature form of GM2AP was inserted in the vector pPIC9K under control of the alcohol oxidase 1 promoter. Both plasmids led to the successful secretory expression of active, glycosylated GM2AP, which could easily be purified by Ni-NTA chromatography due to the hexahistidine tag introduced at the C-terminus. Remarkably, the expression of this membrane-active protein in P. pastoris was accompanied by two peculiarities which were not encountered in other expression systems for GM2AP: First, a significant fraction of the secreted protein existed in the form of aggregates, and second, considerable amounts of noncovalently bound lipids were associated with the recombinant protein. A three-step purification scheme was therefore devised consisting of Ni-NTA, reversed phase, and gel filtration chromatography, which finally yielded 10-12 mg of purified, monomeric GM2AP per liter of expression supernatant. MALDI- and ESI-TOF mass spectrometry were employed to assess the processing, homogeneity

  2. 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

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

    PubMed

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

    2008-10-01

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

  4. 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

  5. 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

  6. Efficient isotope separation by single-photon atomic sorting

    SciTech Connect

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

    2010-09-15

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

  7. 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.

  8. 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

  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

    2016-05-01

    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.

  10. 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.

  11. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

    We describe the use of carbon stable isotope (13C) labeled n-alkanes as a potential internal tracer to assess passage kinetics of ingested nutrients in ruminants. Plant cuticular n-alkanes originating from intrinsically 13C labeled ryegrass plants were pulse dosed intraruminally in four rumen-cannulated lactating dairy cows receiving four contrasting ryegrass silage treatments that differed in nitrogen fertilization level (45 or 90 kg nitrogen ha−1) and maturity (early or late). Passage kinetics through the gastrointestinal tract were derived from the δ13C (i.e. the ratio 13C:12C) in apparently undigested fecal material. Isotopic enrichment was observed in a wide range of long-chain n-alkanes (C27–C36) and passage kinetics were determined for the most abundant C29, C31 and C33 n-alkanes, for which a sufficiently high response signal was detected by combustion isotope ratio mass spectrometry. Basal diet treatment and carbon chain length of n-alkanes did not affect fractional passage rates from the rumen (K1) among individual n-alkanes (3.71–3.95%/h). Peak concentration time and transit time showed a quantitatively small, significant (p≤0.002) increase with carbon chain length. K1 estimates were comparable to those of the 13C labeled digestible dry matter fraction (3.38%/h; r = 0.61 to 0.71; p≤0.012). A literature review has shown that n-alkanes are not fermented by microorganisms in the rumen and affirms no preferential depletion of 13C versus 12C. Our results suggest that 13C labeled n-alkanes can be used as nutrient passage tracers and support the reliability of the δ13C signature of digestible feed nutrients as a tool to measure nutrient-specific passage kinetics. PMID:24124493

  14. 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

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

    PubMed

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

    2016-10-01

    Cenchritis muricatus protease inhibitor II (CmPI-II) is a tight-binding serine protease inhibitor of the Kazal family with an atypical broad specificity, being active against several proteases such as bovine pancreatic trypsin, human neutrophil elastase and subtilisin A. CmPI-II 3D structures are necessary for understanding the molecular basis of its activity. In the present work, we describe an efficient and straightforward recombinant expression strategy, as well as a cost-effective procedure for isotope labeling for NMR structure determination purposes. The vector pCM101 containing the CmPI-II gene, under the control of Pichia pastoris AOX1 promoter was constructed. Methylotrophic Pichia pastoris strain KM71H was then transformed with the plasmid and the recombinant protein (rCmPI-II) was expressed in benchtop fermenter in unlabeled or (15)N-labeled forms using ammonium chloride ((15)N, 99%) as the sole nitrogen source. Protein purification was accomplished by sequential cation exchange chromatography in STREAMLINE DirectHST, anion exchange chromatography on Hitrap Q-Sepharose FF and gel filtration on Superdex 75 10/30, yielding high quantities of pure rCmPI-II and (15)N rCmPI-II. Recombinant proteins displayed similar functional features as compared to the natural inhibitor and NMR spectra indicated folded and homogeneously labeled samples, suitable for further studies of structure and protease-inhibitor interactions. PMID:27353494

  16. 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

  17. 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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Mark E. Fuller; Tullis C. Onstott

    2003-12-17

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

  20. 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

  1. 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.

  2. 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.

  3. Phenotyping hepatocellular metabolism using uniformly labeled carbon-13 molecular probes and LC-HRMS stable isotope tracing.

    PubMed

    Meissen, John K; Pirman, David A; Wan, Min; Miller, Emily; Jatkar, Aditi; Miller, Russell; Steenwyk, Rick C; Blatnik, Matthew

    2016-09-01

    Metabolite stable isotope tracing is a powerful bioanalytical strategy that has the potential to unravel phenotypic markers of early pharmaceutical efficacy by monitoring enzymatic incorporation of carbon-13 atoms into targeted pathways over time. The practice of probing biological systems with carbon-13 labeled molecules using broad MS-based screens has been utilized for many years in academic laboratories but has had limited application in the pharmaceutical R&D environment. The goal of this work was to establish a LCMS analytical workflow that was capable of monitoring carbon-13 isotope changes in glycolysis, the TCA and urea cycles, and non-essential amino acid metabolism. This work applies a standardized protein precipitation with 80% cold methanol and two distinct reverse-phase ion-pair liquid chromatography methods coupled to either a positive- or negative-ion mode high-resolution accurate mass spectrometry screening method. The data herein combines thousands of single-point peak integrations into a novel metabolite network map as a visualization aid to probe and monitor stable isotope incorporation in murine hepatocytes using uniformly labeled (13)C6 glucose, (13)C3 lactate, and (13)C5 glutamine. This work also demonstrates that nitrogen metabolism may have a large influence on the TCA cycle and gluconeogenic carbon fluxes in hepatocyte cell culture. PMID:27343766

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

    PubMed

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

    2014-02-15

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

  5. 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

  6. 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

  7. Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy

    PubMed Central

    Greco, Todd M.; Guise, Amanda J.; Cristea, Ileana M.

    2016-01-01

    In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability. PMID:26867737

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

    PubMed Central

    2014-01-01

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

  9. 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.

  10. 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

  11. 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

  12. 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

  13. 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 ...

  14. An efficient climate model with water isotope physics: NEEMY

    NASA Astrophysics Data System (ADS)

    Hu, J.; Emile-Geay, J.

    2015-12-01

    This work describes the development of an isotope-enabled atmosphere-ocean global climate model, NEEMY. This is a model of intermediate complexity, which can run 100 model years in 30 hours using 33 CPUs. The atmospheric component is the SPEEDY-IER (Molteni et al. 2003; Dee et al. 2015a), which is a water isotope-enabled (with equilibrium and kinetic fractionation schemes in precipitation, evaporation and soil moisture) simplified atmospheric general circulation model, with T30 horizontal resolution and 8 vertical layers. The oceanic component is NEMO 3.4 (Madec 2008), a state-of-the-art oceanic model (~2° horizontal resolution and 31 vertical layers) with an oceanic isotope module (a passive tracer scheme). A 1000-year control run shows that NEEMY is stable and its energy is conserved. The mean state is comparable to that of CMIP3-era CGCMs, though much cheaper to run. Atmospheric teleconnections such as the NAO and PNA are simulated very well. NEEMY also simulates the oceanic meridional overturning circulation well. The tropical climate variability is weaker than observations, and the climatology exhibits a double ITCZ problem despite bias corrections. The standard deviation of the monthly mean Nino3.4 index is 0.61K, compared to 0.91K in observations (Reynolds et al. 2002). We document similarities and differences with a close cousin, SPEEDY-NEMO (Kucharski et al. 2015). With its fast speed and relatively complete physical processes, NEEMY is suitable for paleoclimate studies ; we will present some forced simulations of the past millennium and their use in forward-modeling climate proxies, via proxy system models (PSMs, Dee et al 2015b). References Dee, S., D. Noone, N. Buenning, J. Emile-Geay, and Y. Zhou, 2015a: SPEEDY-IER: A fast atmospheric GCM with water isotope physics. J. Geophys. Res. Atmos., 120: 73-91. doi:10.1002/2014JD022194. Dee, S. G., J. Emile-Geay, M. N. Evans, Allam, A., D. M. Thompson, and E. J. Steig, 2015b: PRYSM: an open-source framework

  15. 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

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

    SciTech Connect

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

    2011-09-26

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

  17. 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

  18. A Software Toolkit and Interface for Performing Stable Isotope Labeling and Top3 Quantification Using Progenesis LC-MS

    PubMed Central

    Brownridge, Philip; Xia, Dong; Mackay, Katherine; Gonzalez-Galarza, Faviel F.; Kenyani, Jenna; Harman, Victoria; Beynon, Robert J.; Jones, Andrew R.

    2012-01-01

    Abstract Numerous software packages exist to provide support for quantifying peptides and proteins from mass spectrometry (MS) data. However, many support only a subset of experimental methods or instrument types, meaning that laboratories often have to use multiple software packages. The Progenesis LC-MS software package from Nonlinear Dynamics is a software solution for label-free quantitation. However, many laboratories using Progenesis also wish to employ stable isotope-based methods that are not natively supported in Progenesis. We have developed a Java programming interface that can use the output files produced by Progenesis, allowing the basic MS features quantified across replicates to be used in a range of different experimental methods. We have developed post-processing software (the Progenesis Post-Processor) to embed Progenesis in the analysis of stable isotope labeling data and top3 pseudo-absolute quantitation. We have also created export ability to the new data standard, mzQuantML, produced by the Proteomics Standards Initiative to facilitate the development and standardization process. The software is provided to users with a simple graphical user interface for accessing the different features. The underlying programming interface may also be used by Java developers to develop other routines for analyzing data produced by Progenesis. PMID:22888986

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

    SciTech Connect

    Korzekwa, K.R.; Swinney, D.C.; Trager, W.F. )

    1989-11-14

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

  1. 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

  2. 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.

  3. 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

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

    NASA Astrophysics Data System (ADS)

    Dippold, Michaela; Kuzyakov, Yakov

    2015-04-01

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

  5. 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

  6. 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

  7. Quantitative Cross-linking/Mass Spectrometry Using Isotope-labeled Cross-linkers and MaxQuant.

    PubMed

    Chen, Zhuo A; Fischer, Lutz; Cox, Jürgen; Rappsilber, Juri

    2016-08-01

    The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS(3) and isotope-labeled BS(3)-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant. PMID:27302889

  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. Quantitative Cross-linking/Mass Spectrometry Using Isotope-labeled Cross-linkers and MaxQuant*

    PubMed Central

    Cox, Jürgen

    2016-01-01

    The conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS3 and isotope-labeled BS3-d4 revealed a number of observations: (1) Fully automated process using MaxQuant can quantify cross-links in our reference data set with 68% recall rate and 88% accuracy. (2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. (3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant. PMID:27302889

  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. 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

  12. Evaluation of five miniature chromatography systems for determining labeling efficiency of technetium Tc 99m pentetate.

    PubMed

    Robbins, P J

    1983-04-01

    The reliability and reproducibility of five miniature chromatography systems for the radiochemical purity of 99mTc-labeled technetium Tc 99m pentetate was evaluated. Radiochemical purity of technetium Tc 99m pentetate was determined 15-30 minutes after preparation once a month for nine consecutive months. A reference value was determined by gel filtration or by conventional-length paper chromatography and thin-layer chromatography. Radiochemical purity was determined simultaneously by five miniature chromatography systems. The miniature systems included an in-house system and commercial systems distributed by Ackerman Nuclear, Ashley Innovations, Atomic Products, and Technical Advancement. Each miniature system was tested in duplicate. A follow-up comparison of the Ackerman Nuclear and in-house systems was performed for an additional nine months. Labeling efficiency by the reference method was greater than 97% for all nine months. The miniature systems gave results that were comparable in most months. Very low labeling efficiency occurred with the Ackerman Nuclear system in month 9. The follow-up comparison produced only one unconfirmed result for the Ackerman Nuclear system that would have caused a laboratory to erroneously discard a batch of technetium Tc 99m pentetate. The miniature chromatography systems evaluated generally will give reliable and reproducible results for the radiochemical purity of technetium Tc 99m pentetate for nine months after receipt of the systems. PMID:6342380

  13. A simple and efficient method of labelling RBCs with 99mTc for spleen imaging.

    PubMed

    Ercan, M T; Bernay, I

    1990-01-01

    A new method of labelling human RBCs with 99mTc by the use of Sn-alpha-D-glucose 1-phosphate (GP) is presented. It was tested for spleen imaging in 16 normal volunteers. The labelling was carried out during the heating (30 min at 49.5 degrees C) to damage the cells and cooling (10 min at R.T.) steps. The labelling efficiency was 95.5 +/- 1.5% with Sn-GP and was better than Sn-PYP (61.5 +/- 25.0%). The radioactivity retention of RBCs was greater than 96% after 6 washings. The spleen was delineated very well in all the subjects. The spleen activity reached a plateau at 20 min post-injection. Spleen-to-liver and spleen-to-cardiac blood pool concentration ratios were high (10.4 +/- 2.2 and 10.1 +/- 3.2, respectively) calculated at 30 min. The method is simple, rapid and efficient. PMID:2341290

  14. 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

  15. (Bio)degradation of glyphosate in water-sediment microcosms - A stable isotope co-labeling approach.

    PubMed

    Wang, Shizong; Seiwert, Bettina; Kästner, Matthias; Miltner, Anja; Schäffer, Andreas; Reemtsma, Thorsten; Yang, Qi; Nowak, Karolina M

    2016-08-01

    Glyphosate and its metabolite aminomethylphosphonic acid (AMPA) are frequently detected in water and sediments. Up to date, there are no comprehensive studies on the fate of glyphosate in water-sediment microcosms according to OECD 308 guideline. Stable isotope co-labeled (13)C3(15)N-glyphosate was used to determine the turnover mass balance, formation of metabolites, and formation of residues over a period of 80 days. In the water-sediment system, 56% of the initial (13)C3-glyphosate equivalents was ultimately mineralized, whereas the mineralization in the water system (without sediment) was low, reaching only 2% of (13)C-glyphosate equivalents. This finding demonstrates the key role of sediments in its degradation. Glyphosate was detected below detection limit in the water compartment on day 40, but could still be detected in the sediments, ultimately reaching 5% of (13)C3(15)N-glyphosate equivalents. A rapid increase in (13)C(15)N-AMPA was noted after 10 days, and these transformation products ultimately constituted 26% of the (13)C3-glyphosate equivalents and 79% of the (15)N-glyphosate equivalents. In total, 10% of the (13)C label and 12% of the (15)N label were incorporated into amino acids, indicating no risk bearing biogenic residue formation from (13)C3(15)N-glyphosate. Initially, glyphosate was biodegraded via the sarcosine pathway related to microbial growth, as shown by co-labeled (13)C(15)N-glycine and biogenic residue formation. Later, degradation via AMPA dominated under starvation conditions, as shown by the contents of (13)C-glycine. The presented data provide the first evidence of the speciation of the non-extractable residues as well as the utilization of glyphosate as a carbon and nitrogen source in the water-sediment system. This study also highlights the contribution of both the sarcosine and the AMPA degradation pathways under these conditions. PMID:27140906

  16. 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

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

    ERIC Educational Resources Information Center

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

    2004-01-01

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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. Insight into the formation of molecular species in laser-induced plasma of isotopically labeled organic samples.

    PubMed

    Glaus, Reto; Riedel, Jens; Gornushkin, Igor

    2015-10-01

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

  3. 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, Stéphane; Croisier, Marie; Clerc-Rosset, Stéphanie; 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

  4. 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.

  5. 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

  6. 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

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

    PubMed

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

    2015-04-21

    There is no doubt that the contribution of microbially mediated bioprocesses toward maintenance of life on earth is vital. However, understanding these microbes in situ is currently a bottleneck, as most methods require culturing these microorganisms to suitable biomass levels so that their phenotype can be measured. The development of new culture-independent strategies such as stable isotope probing (SIP) coupled with molecular biology has been a breakthrough toward linking gene to function, while circumventing in vitro culturing. In this study, for the first time we have combined Raman spectroscopy and Fourier transform infrared (FT-IR) spectroscopy, as metabolic fingerprinting approaches, with SIP to demonstrate the quantitative labeling and differentiation of Escherichia coli cells. E. coli cells were grown in minimal medium with fixed final concentrations of carbon and nitrogen supply, but with different ratios and combinations of (13)C/(12)C glucose and (15)N/(14)N ammonium chloride, as the sole carbon and nitrogen sources, respectively. The cells were collected at stationary phase and examined by Raman and FT-IR spectroscopies. The multivariate analysis investigation of FT-IR and Raman data illustrated unique clustering patterns resulting from specific spectral shifts upon the incorporation of different isotopes, which were directly correlated with the ratio of the isotopically labeled content of the medium. Multivariate analysis results of single-cell Raman spectra followed the same trend, exhibiting a separation between E. coli cells labeled with different isotopes and multiple isotope levels of C and N. PMID:25831066

  8. 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.

  9. 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

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

    PubMed

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

    2016-03-15

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

  11. 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

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

    PubMed

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

    2013-07-01

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

  13. 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.

  14. 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.

  15. LC-quadrupole/Orbitrap high-resolution mass spectrometry enables stable isotope-resolved simultaneous quantification and ¹³C-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. PMID:26968563

  16. 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.

  17. 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

  18. 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...

  19. 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

  20. Comprehensive and Quantitative Profiling of the Human Sweat Submetabolome Using High-Performance Chemical Isotope Labeling LC-MS.

    PubMed

    Hooton, Kevin; Han, Wei; Li, Liang

    2016-07-19

    Human sweat can be noninvasively collected and used as a media for diagnosis of certain diseases as well as for drug detection. However, because of very low concentrations of endogenous metabolites present in sweat, metabolomic analysis of sweat with high coverage is difficult, making it less widely used for metabolomics research. In this work, a high-performance method for profiling the human sweat submetabolome based on chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) is reported. Sweat was collected using a gauze sponge style patch, extracted from the gauze by centrifugation, and then derivatized using CIL. Differential (12)C- and (13)C-dansylation labeling was used to target the amine/phenol submetabolome. Because of large variations in the total amount of sweat metabolites in individual samples, sample amount normalization was first performed using liquid chromatography with UV detection (LC-UV) after dansylation. The (12)C-labeled individual sample was then mixed with an equal amount of (13)C-labeled pooled sample. The mixture was subjected to LC-MS analysis. Over 2707 unique metabolites were detected across 54 sweat samples collected from six individuals with an average of 2002 ± 165 metabolites detected per sample from a total of 108 LC-MS runs. Using a dansyl standard library, we were able to identify 83 metabolites with high confidence; many of them have never been reported to be present in sweat. Using accurate mass search against human metabolome libraries, we putatively identified an additional 2411 metabolites. Uni- and multivariate analyses of these metabolites showed significant differences in the sweat submetabolomes between male and female, as well as between early and late exercise. These results demonstrate that the CIL LC-MS method described can be used to profile the human sweat submetabolome with high metabolomic coverage and high quantification accuracy to reveal metabolic differences in different sweat

  1. 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

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

    PubMed Central

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

    2011-01-01

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

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

    SciTech Connect

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

    2006-09-01

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

  4. Combining Capillary Electrophoresis Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry and Stable Isotopic Labeling Techniques for Comparative Crustacean Peptidomics

    PubMed Central

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

    2010-01-01

    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

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

    PubMed

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

    2016-01-20

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

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

    NASA Astrophysics Data System (ADS)

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

    2012-04-01

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

  7. A Coding Method for Efficient Subgraph Querying on Vertex- and Edge-Labeled Graphs

    PubMed Central

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

    2014-01-01

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

  8. International experiences in assessing vitamin A status and applying the vitamin A-labeled isotope dilution method.

    PubMed

    Lopez-Teros, Veronica; Chileshe, Justin; Idohou-Dossou, Nicole; Fajarwati, Tetra; Medoua Nama, Gabriel; Newton, Sam; Vinod Kumar, Malavika; Wang, Zhixu; Wasantwisut, Emorn; Hunt, Janet R

    2014-01-01

    Inadequate vitamin A (VA) nutrition continues to be a major problem worldwide, and many interventions being implemented to improve VA status in various populations need to be evaluated. The interpretation of results after an intervention depends greatly on the method selected to assess VA status. To evaluate the effect of an intervention on VA status, researchers in Cameroon, India, Indonesia, Mexico, Senegal and Zambia have used serum retinol as an indicator, and have not always found improvement in response to supplementation. One problem is that homeostatic control of serum retinol may mask positive effects of treatment in that changes in concentration are observed only when status is either moderately to severely depleted or excessive. Because VA is stored mainly in the liver, measurements of hepatic VA stores are the “gold standard” for assessing VA status. Dose response tests such as the relative dose response (RDR) and the modified relative dose response (MRDR), allow a qualitative assessment of VA liver stores. On the other hand, the use of the vitamin A-labeled isotope dilution (VALID) technique, (using 13C or 2H-labeled retinyl acetate) serves as an indirect method to quantitatively estimate total body and liver VA stores. Countries including Cameroon, China, Ghana, Mexico, Thailand and Zambia are now applying the VALID method to sensitively assess changes in VA status during interventions, or to estimate a population’s dietary requirement for VA. Transition to the use of more sensitive biochemical indicators of VA status such as the VALID technique is needed to effectively assess interventions in populations where mild to moderate VA deficiency is more prevalent than severe deficiency. PMID:25537105

  9. 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

  10. 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

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

    PubMed

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

    2015-11-28

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

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

    PubMed Central

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

    2015-01-01

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

  13. Simultaneous determination of seven β2-agonists in human and bovine urine by isotope dilution liquid chromatography-tandem mass spectrometry using compound-specific minimally (13)C-labelled analogues.

    PubMed

    González-Antuña, Ana; Rodríguez-González, Pablo; Centineo, Giuseppe; García Alonso, J Ignacio

    2014-10-29

    Seven β2-agonist (clenproperol, clenbuterol, salbutamol, bronbuterol, ractopamine, clenpenterol and clencyclohexerol) were determined simultaneously in human and bovine urine by isotope dilution LC-ESI-MS/MS in a triple quadrupole instrument. The method is based on the application of multiple linear regression in combination with compound-specific minimally (13)C-labelled analogues. Additionally, the increase of the bandpass of the first quadrupole during the selected reaction monitoring (SRM) measurement procedure allowed the simultaneous quantification of the seven compounds at sub ngg(-1) levels in a single chromatogram without resorting to a methodological calibration graph. Recovery values at concentration levels between 5.0 and 0.05ngg(-1) ranged from 95 to 110% in fortified bovine urine and from 91 to 108% in human urine, with relative standard deviations lower than 5% except for salbutamol and ractopamine. The proposed methodology was validated by analyzing the certified reference material BCR-503 (lyophilized bovine urine) certified for clenbuterol and salbutamol. The limits of detection (LOD) for a sample volume of 10mL of both human and bovine urine was found to be lower than 0.012ngg(-1) for all compounds, except to salbutamol in bovine urine which was of 0.029ngg(-1). The use of compound-specific isotopically labelled analogues minimally labelled in (13)C minimized the occurrence of isotope effects and corrected for matrix effects during ESI ionization and can be efficiently applied for the quantification of ultra-trace concentrations of β2-agonists in human and bovine urine. PMID:25468499

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

    SciTech Connect

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

    2012-03-26

    For the past decade, flux maps have provided researchers with an in-depth perspective on plant metabolism. As a rapidly developing field, significant headway has been made recently in computation, experimentation, and overall understanding of metabolic flux analysis. These advances are particularly applicable to the study of plant metabolism. New dynamic computational methods such as non-stationary metabolic flux analysis are finding their place in the toolbox of metabolic engineering, allowing more organisms to be studied and decreasing the time necessary for experimentation, thereby opening new avenues by which to explore the vast diversity of plant metabolism. Also, improved methods of metabolite detection and measurement have been developed, enabling increasingly greater resolution of flux measurements and the analysis of a greater number of the multitude of plant metabolic pathways. Methods to deconvolute organelle-specific metabolism are employed with increasing effectiveness, elucidating the compartmental specificity inherent in plant metabolism. Advances in metabolite measurements have also enabled new types of experiments, such as the calculation of metabolic fluxes based on (CO2)-C-13 dynamic labelling data, and will continue to direct plant metabolic engineering. Newly calculated metabolic flux maps reveal surprising and useful information about plant metabolism, guiding future genetic engineering of crops to higher yields. Due to the significant level of complexity in plants, these methods in combination with other systems biology measurements are necessary to guide plant metabolic engineering in the future.

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

    SciTech Connect

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

    2012-03-01

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

  16. 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

  17. 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

  18. 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

  19. Dual Label Stable Isotope Incubations Followed By Single Cell Nanosims Analyses To Investigate Microscale Phototroph-Heterotroph Interactions

    NASA Astrophysics Data System (ADS)

    Mayali, X.; Samo, T. J.; Nilson, D.; Arandia Gorostidi, N.; alonso Saez, L.; Moran, X. A.; Weber, P. K.

    2015-12-01

    In natural ecosystems such as lakes and oceans as well as human-engineered systems for sunlight-regulated biomass production (such as algal biofuel ponds), the interaction between autotrophic and heterotrophic processes are critical to determine whether such systems are net autotrophic or heterotrophic. Traditional methods to quantify autotrophy and heterotrophy include primary productivity and bacterial production measurements using radiolabeled substrates that quantify these processes on the bulk scale. To examine the microscale interactions between individual autotrophic and heterotrophic cells, we incubate mixed microbial assemblages with 13C-bicarbonate and 15N-leucine to label individual autotrophs and heterotrophs, respectively. We use nano imaging secondary ion mass spectrometry (with a Cameca NanoSIMS 50) to quantify the incorporation of the rare isotopes by single cells. We will present results from experiments examining the impact of warming on the exchange of C and N between algal and bacterial cells from the coastal Atlantic Ocean, which suggest that increased temperature may strengthen physical interactions and exchange. We will also present data from experiments examining the influence of attached bacteria on the cell-specific inorganic carbon fixation rates of biofuel-producing algal cultures which suggest that certain algal-attached bacterial groups grow faster than when free-living and influence algal growth. We conclude that the examination of individual cells uncover interactions that would be difficult, if not impossible, to investigate with bulk methods.

  20. 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

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

    PubMed

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

    2013-09-01

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

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

    PubMed Central

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

    2008-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-05-01

    We developed a new method (gas-phase stripping-derivatization coupled to LC-MS) to measure the 15N atom percent excess (APE) of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye by the well-known Griess reaction in the Long Path Absorption Photometer (LOPAP). The reaction solutions containing the dye are collected at the outflow of the LOPAP, purified by solid-phase extraction and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). The unlabeled azo dye (C18H19O2N5S) with a monoisotopic molecular mass of 369.41 g mol-1 can be detected as its protonated molecular ion ([M+H+], M) by HPLC-MS at a retention time of 2.8 min. Due to the natural isotope distribution M + 0, M + 1, M + 2, and M + 3 ions were considered for the calculation of the 15N APE. The optimal working range was found to be between 20 and 50% for the 15N/14N ratio. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method has been applied for the measurement of HO15NO emissions from soil in a dynamic chamber with and without spiking 15N labeled urea. Our results confirm biogenic HONO emissions from soil as HO15NO was measured after addition of 15N urea.

  4. 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

  5. Influence of 13C isotopic labeling location of 13C DNP of acetate using TEMPO free radical

    NASA Astrophysics Data System (ADS)

    Parish, Christopher; Niedbalski, Peter; Lumata, Lloyd

    2015-03-01

    Dynamic nuclear polarization (DNP) via the dissolution method enhances the liquid-state magnetic resonance (NMR or MRI) signals of insensitive nuclear spins by at least 10,000-fold. The basis for all these signal enhancements at room temperature is the polarization transfer from the electrons to nuclear spins at cryogenic temperature and high magnetic field. In this work, we have studied the influence of the location of 13C isotopic labeling on the DNP of sodium acetate at 3.35 T and 1.4 K using a wide ESR linewidth free radical 4-oxo-TEMPO. The carbonyl [1-13C]acetate spins produced a polarization level that is almost twice that of the methyl [2-13C]acetate spins. On the other hand, the polarization of the methyl 13C spins doubled to reach the level of [1-13C]acetate when the methyl group was deuterated. Meanwhile, the solid-state nuclear relaxation of these samples are the same and do not correlate with the polarization levels. These behavior implies that the nuclear relaxation for these samples is dominated by the contribution from the free radicals and the polarization levels can be explained by a thermodynamic picture of DNP.

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

    NASA Astrophysics Data System (ADS)

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

    2014-12-01

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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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

  17. Multi-Analytic Based Determination of Substrate Fate From in situ Stable Isotope Labeled Exposures of Natural Microbial Mats

    NASA Astrophysics Data System (ADS)

    Lipton, M. S.; Cory, A.; Riha, K. M.; Huang, E. L.; Boaro, A. A.; Metz, T. O.; Gritsenko, M. A.; Mobberley, J. M.; Nelson, W.; Kim, Y. M.; Moran, J.

    2015-12-01

    Microbial communities play impactful roles in almost every aspect of our society including the environment, climate, agriculture and human health, expanding the functional capacity of life on earth. The recent emergence of a suite of omics driven technologies offers powerful tools for investigating functionality of this community. However, these tools provide only a static snapshot of the community in space and time. The temporal nature of stable isotope probing (SIP) experiments expands the depth at which microbial communities can be investigated and understood. While selectively targeting only metabolically active organisms in a community, the labeled substrate can be tracked spatially, temporally and phylo-genetically and linked to active functions, organism interactions and exchanges. Single SIP technologies are limited in their ability to describe the biological system as a whole. However, integration of multiple SIP based analytics offers a more comprehensive description of substrate fate. The phototroph based microbial mat community resident in Hot Lake, a hypersaline lake located in Washington State, offers a tractable system for testing the multi analytic approach. We exposed the mat to three different 13C-labeled substrates (HCO3-, glucose and acetate) in situ at midday, and subsequently analyzed the mat 24 hours after incubation. The approach revealed different metabolic fates and organism specific uptake. When compared to acetate, glucose and HCO3- showed a greater incorporation into extracellular material, while acetate had a greater conversion to intracellular fatty acids, suggesting that HCO3- and glucose could be more readily shared as a community currency than acetate. All substrates were converted to amino acids and proteins, but while glucose and HCO3- demonstrated considerable incorporation into heterotrophic proteins, the conversion of acetate to these proteins was minimal, potentially implying that acetate derived intermediates are not a

  18. New untargeted metabolic profiling combining mass spectrometry and isotopic labeling: application on Aspergillus fumigatus grown on wheat.

    PubMed

    Cano, Patricia M; Jamin, Emilien L; Tadrist, Souria; Bourdaud'hui, Pascal; Péan, Michel; Debrauwer, Laurent; Oswald, Isabelle P; Delaforge, Marcel; Puel, Olivier

    2013-09-01

    Characterization of fungal secondary metabolomes has become a challenge due to the industrial applications of many of these molecules, and also due to the emergence of fungal threats to public health and natural ecosystems. Given that, the aim of the present study was to develop an untargeted method to analyze fungal secondary metabolomes by combining high-accuracy mass spectrometry and double isotopic labeling of fungal metabolomes. The strain NRRL 35693 of Aspergillus fumigatus , an important fungal pathogen, was grown on three wheat grain substrates: (1) naturally enriched grains (99% (12)C), (2) grains enriched 96.8% with (13)C, (3) grains enriched with 53.4% with (13)C and 96.8% with (15)N. Twenty-one secondary metabolites were unambiguously identified by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis. AntiBase 2012 was used to confirm the identity of these metabolites. Additionally, on the basis of tandem mass spectrometry (MS(n)) experiments, it was possible to identify for the first time the formula and the structure of fumigaclavine D, a new member of the fumigaclavines family. Post biosynthesis degradation of tryptoquivaline F by methanol was also identified during HPLC-HRMS analysis by the detection of a carbon atom of nonfungal origin. The interest of this method lies not only on the unambiguous determination of the exact chemical formulas of fungal secondary metabolites but also on the easy discrimination of nonfungal products. Validation of the method was thus successfully achieved in this study, and it can now be applied to other fungal metabolomes, offering great possibilities for the discovery of new drugs or toxins. PMID:23901908

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

    SciTech Connect

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

    1988-03-01

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

  20. 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

  1. Tracking amino acid's uptake into the protozoan Acanthamoeba castellanii by stable-isotope labelling and Raman spectral imaging

    NASA Astrophysics Data System (ADS)

    Naemat, Abida; Elsheikha, Hany M.; Notingher, Ioan

    2016-04-01

    The capacity of pathogens to acquire nutrients from their host cells is one of the most fundamental aspects of infection biology. Hence, measuring the patterns of nutrients' uptake by pathogens is essential for understanding the interactions of pathogens with eukaryotic host cells. In this study, we optimized a technique that allows fast and non-destructive measurement of the amino acid Phenylalanine (Phe) acquired by the trophozoite stage of the protozoan Acanthamoeba castellanii (A. castellanii) as they engage with individual human retinal pigment epithelial cells (ARPE-19). ARPE-19 host cells were pre-saturated with Deuterated Phe (L-Phe(D8)) to replace the native substrate Phe (L-Phe). The uptake of L-Phe(D8) by A. castellanii trophozoites was measured by Raman microspectroscopy. This approach allowed us to characterize the uptake patterns of this essential amino acid into A. castellanii trophozoites at a single cell level. At 24 hours post infection (PI) A. castellanii trophozoites are capable of salvaging L-Phe(D8) from host cells. The uptake pattern was time-dependent during the first 24 hours of infection and complete substitution with L-Phe(D8) in all parasites was detected at 48 hours PI. On the other hand, isolated A. castellanii trachyzoites (grown without host cells) did not show significant uptake for L-Phe(D8) from the media; only achieved an uptake ratio of 16-18% of L-Phe(D8) from the culture medium after 24 hours. These findings demonstrate the potential of combining Raman microspectroscopy and stable isotope labelling approaches to elucidate the role of metabolism in mediating A. castellanii interaction with host cells.

  2. 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

  3. 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

  4. 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

  5. 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

  6. Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Pettelkau, Jens; Thondorf, Iris; Theisgen, Stephan; Lilie, Hauke; Schröder, Thomas; Arlt, Christian; Ihling, Christian H.; Sinz, Andrea

    2013-12-01

    The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca2+. In-depth MS and MS/MS analysis of the cross-linked products was aided by 15 N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca2+-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca2+-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

  7. Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

    PubMed Central

    Masse, J E; Bortmann, P; Dieckmann, T; Feigon, J

    1998-01-01

    The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures. PMID:9592146

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

    PubMed

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

    2016-01-01

    Studies of pathological mechanisms and XOR inhibitor characterization, such as allopurinol, febuxostat, and topiroxostat, require accurate and sensitive measurements of XOR activity. However, the established assays have some disadvantages such as susceptibility to endogenous substances such as uric acid (UA), xanthine, or hypoxanthine. Here, we aimed to develop a novel XOR activity assay utilizing a combination of high-performance liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) for tissues such as the liver, kidney, and plasma. Stable isotope-labeled [(15)N2]-xanthine was utilized as substrate and the production of [(15)N2]-uric acid was determined. [(15)N2]-UA production by XOR was dependent on the amounts of [(15)N2]-xanthine and enzyme and the time of reaction. Because high concentrations of endogenous xanthine and hypoxanthine affect XOR activities, we employed a multi-component analysis using LC/HRMS to improve the accuracy of XOR activity assay. Quantification of [(15)N2]-UA was validated and showed good linearity, accuracy, and precision. We measured the XOR activities of retired ICR mice using [(15)N2]-xanthine and LC/MS. The XOR activities in plasma, kidney, and liver samples were 38.1±0.7, 158±5, 928±25pmol/min/mg of protein, respectively (mean±SD, n=5). Furthermore, we measured the XOR activities in the same samples using the LC/ultraviolet and LC/fluorescence (FL) methods. The level of [(15)N2]-xanthine oxidation by XOR was equal to that of xanthine oxidation and approximately 7.9-8.9 times higher than that of pterin oxidation. We found a good correlation between XOR activities examined using LC/MS assay with [(15)N2]-xanthine and those examined using LC/FL assay with pterin. This result suggested that although both the LC/MS assay with [(15)N2]-xanthine and the LC/FL assay with pterin were useful, the former provided information regarding XOR activities that more directly reflected the physiological condition than the latter

  9. 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

  10. 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

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

    PubMed Central

    Straus, Suzana K

    2004-01-01

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

  12. 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

  13. Measurement of Histone Methylation Dynamics by One-Carbon Metabolic Isotope Labeling and High-energy Collisional Dissociation Methylation Signature Ion Detection

    PubMed Central

    Tang, Hui; Tian, Bing; Brasier, Allan R.; Sowers, Lawrence C.; Zhang, Kangling

    2016-01-01

    Accumulating evidence suggests that cellular metabolites and nutrition levels control epigenetic modifications, including histone methylation. However, it is not currently possible to measure the metabolic control of histone methylation. Here we report a novel detection method to monitor methyl transfer from serine to histones through the one-carbon metabolic pathway, using stable-isotope labeling and detection of lysine methylation signature ions generated in high-energy-dissociation (HCD) tandem mass spectrometry. This method is a long-needed tool to study the metabolic control of histone methylation. PMID:27530234

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

    PubMed

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

    2016-02-18

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

  15. 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

  16. 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

  17. 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.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

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

  5. 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

  6. 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...

  7. 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.

  8. 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.

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

    PubMed

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

    2015-03-01

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

  11. 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.

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

    PubMed

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

    2014-01-01

    The preparative microbial synthesis of amino acids labelled with stable isotopes, including deuterium ( 2 H), suitable for biomedical applications by methylotrophic bacteria was studied using L-phenylalanine as example. This amino acid is secreted by Gram-negative aerobic facultative methylotrophic bacteria Brevibacterium methylicum, assimilating methanol via ribulose-5-monophosphate (RMP) cycle of assimilation of carbon, The data on adaptation of L-phenylalanine secreted by methylotrophic bacterium В. methylicum to the maximal concentration of deuterium in the growth medium with 98% 2 Н 2 O and 2% [ 2 Н]methanol, and biosynthesis of deuterium labelled L-phenylalanine With different levels of enrichment are presented. The strain was adapted by means of plating initial cells on firm (2% agarose) minimal growth media with an increasing gradient of 2 Н 2 O concentration from 0; 24.5; 49.0; 73.5 up to 98% 2 Н 2 O followed by subsequent selection of separate colonies stable to the action of 2 Н 2 O. These colonies were capable to produce L-phenylalanine. L-phenylalanine was extracted from growth medium by extraction with isopropanol with the subsequent crystallization in ethanol (output 0.65 g/l). The developed method of microbial synthesis allows to obtain deuterium labelled L-phenylalanine with different levels of isotopic enrichment, depending on concentration of 2 Н 2 O in growth media, from 17% (on growth medium with 24,5% 2 Н 2 O) up to 75% (on growth medium with 98% 2 Н 2 O) of deuterium in the molecule that is confirmed with the data of the electron impact (EI) mass- spectrometry analysis of methyl ethers of N-dimethylamino(naphthalene)-5-sulfochloride (dansyl) phenylalanine in these experimental conditions. PMID:25249528

  14. 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

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

    PubMed Central

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

    2013-01-01

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

  16. Stable isotopes as ecological tracers: an efficient method for assessing the contribution of multiple sources to mixtures

    NASA Astrophysics Data System (ADS)

    Bugalho, M. N.; Barcia, P.; Caldeira, M. C.; Cerdeira, J. O.

    2008-09-01

    Stable isotopes are increasingly being used as tracers of ecological processes potentially providing relevant information to environmental management issues. An application of the methodology consists in relating the stable isotopic composition of a sample mixture to that of sources. The number of stable isotopes, however, is usually lower than that of potential sources existing in an ecosystem, which creates mathematical difficulties in correctly tracing sources. We discuss a linear programming model which efficiently derives information on the contribution of sources to mixtures for any number of stable isotopes and any number of sources by addressing multiple sources simultaneously. The model identifies which sources are present in all, present in a subset of the samples or absent from all samples simultaneously and calculates minimum and maximum values of each source in the mixtures. We illustrate the model using a data set consisting of the isotopic signatures of different plant sources ingested by primary consumers in tropical riverine habitat in Asia. The model discussed may contribute to extend the scope of stable isotopes methodology to a range of new problems dealing with multiple sources and multiple tracers. For instance, in food web studies, if particular organic matter sources disappear or decrease in availability (e.g. climate change scenarios) the model allows simulation of alternative diets of the consumers providing potentially relevant information for managers and decision makers.

  17. Stable isotopes as ecological tracers: an efficient method for assessing the contribution of multiple sources to mixtures

    NASA Astrophysics Data System (ADS)

    Bugalho, M. N.; Barcia, P.; Caldeira, M. C.; Cerdeira, J. O.

    2008-06-01

    Stable isotopes are increasingly being used as tracers of ecological processes potentially providing relevant information to environmental management issues. An application of the methodology consists in relating the stable isotopic composition of a sample mixture to that of sources. The number of stable isotopes, however, is usually lower than that of potential sources existing in an ecosystem, which creates mathematical difficulties in correctly tracing sources. We discuss a linear programming model which efficiently derives information on the contribution of sources to mixtures for any number of stable isotopes and any number of sources by addressing multiple sources simultaneously. The model identifies which sources are present in all, present in a subset of the samples or absent from all samples simultaneously and calculates minimum and maximum values of each source in the mixtures. We illustrate the model using a data set consisting on the isotopic signatures of different plant sources ingested by primary consumers in tropical riverine habitat in Asia. The model discussed may contribute to extend the scope of stable isotopes methodology to a range of new problems dealing with multiple sources and multiple tracers. For instance, in food web studies, if particular organic matter sources disappear or decrease in availability (e.g. climate change scenarios) the model allows simulation of alternative diets of the consumers providing potentially relevant information for managers and decision makers.

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

    PubMed Central

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

    2014-01-01

    Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of 13C 90° pulses allows to either select or discriminate against 13C’ or 13Cα spins coupled to 15N. As a result, 1H-15N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns. PMID:25442777

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

    PubMed Central

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

    2015-01-01

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

  20. Nano-mole scale side-chain signal assignment by 1H-detected protein solid-state NMR by ultra-fast magic-angle spinning and stereo-array isotope labeling.

    PubMed

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

    2015-01-01

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

  1. 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

  2. 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.

  3. 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.

  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. Parallel β-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: Quantitative benchmark for the interpretation of amyloid and protein infrared spectra

    PubMed Central

    Woys, Ann Marie; Almeida, Aaron M.; Wang, Lu; Chiu, Chi Cheng; McGovern, Michael; de Pablo, Juan J.; Skinner, James L.; Gellman, Samuel H.; Zanni, Martin T.

    2012-01-01

    Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly 13C=18O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs. The frequen cies of the labels ranged from 1585 to 1595 cm−1, with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the linewidths and frequencies nearly quantitatively. Historically, the characteristic features of β-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel β-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm−1 linewidth. Narrower linewidths indicate that the amide I backbone is solvent protected

  6. Proteomic Analysis of Protein Turnover by Metabolic Whole Rodent Pulse-Chase Isotopic Labeling and Shotgun Mass Spectrometry Analysis.

    PubMed

    Savas, Jeffrey N; Park, Sung Kyu; Yates, John R

    2016-01-01

    The analysis of protein half-life and degradation dynamics has proven critically important to our understanding of a broad and diverse set of biological conditions ranging from cancer to neurodegeneration. Historically these protein turnover measures have been performed in cells by monitoring protein levels after "pulse" labeling of newly synthesized proteins and subsequent chase periods. Comparing the level of labeled protein remaining as a function of time to the initial level reveals the protein's half-life. In this method we provide a detailed description of the workflow required for the determination of protein turnover rates on a whole proteome scale in vivo. Our approach starts with the metabolic labeling of whole rodents by restricting all the nitrogen in their diet to exclusively nitrogen-15 in the form of spirulina algae. After near complete organismal labeling with nitrogen-15, the rodents are then switched to a normal nitrogen-14 rich diet for time periods of days to years. Tissues are harvested, the extracts are fractionated, and the proteins are digested to peptides. Peptides are separated by multidimensional liquid chromatography and analyzed by high resolution orbitrap mass spectrometry (MS). The nitrogen-15 containing proteins are then identified and measured by the bioinformatic proteome analysis tools Sequest, DTASelect2, and Census. In this way, our metabolic pulse-chase approach reveals in vivo protein decay rates proteome-wide. PMID:26867752

  7. In folio respiratory fluxomics revealed by 13C isotopic labeling and H/D isotope effects highlight the noncyclic nature of the tricarboxylic acid "cycle" in illuminated leaves.

    PubMed

    Tcherkez, Guillaume; Mahé, Aline; Gauthier, Paul; Mauve, Caroline; Gout, Elizabeth; Bligny, Richard; Cornic, Gabriel; Hodges, Michael

    2009-10-01

    While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, (13)C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA "cycle" does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation. PMID:19675152

  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. Carbon-proton scalar couplings in RNA. 3D heteronuclear and 2D isotope-edited NMR of a [sup 13]C-labeled extra-stable hairpin

    SciTech Connect

    Hines, J.V.; Landry, S.M.; Varani, G.; Tinoco, I. Jr. Lawrence Berkeley Lab., CA )

    1994-06-29

    Long range carbon-proton scalar couplings were measured for an RNA hairpin of 12 nucleotides using 3D and [sup 13]C-edited 2D NMR. The large one-bond carbon-proton scalar couplings ([sup 1]J[sub CH]) and small n-bond couplings ([sup 1]J[sub CH]) produce ECOSY type cross-peaks, thus facilitating the determination of the sign and magnitude of the smaller [sup 2]J[sub CH] or [sup 3]J[sub CH]. The UUCGRNA hairpin (5[prime]-rGGACUUCGGUCC-3[prime]), whose structure has been determined by our laboratory, was uniformly [sup 13]C-labeled at 30% isotopic enrichment. The observed [sup 1]J[sub CH] couplings were then correlated to the known structure. The signs of [sup 2]J[sub C4[prime]H5[prime

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

    PubMed Central

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

    2010-01-01

    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

  11. FTIR study of the photoinduced processes of plant phytochrome phyA using isotope-labeled bilins and density functional theory calculations.

    PubMed

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

    2008-08-01

    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 (13)C-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

  12. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling.

    PubMed

    Baker, Lindsay A; Daniëls, Mark; van der Cruijsen, Elwin A W; Folkers, Gert E; Baldus, Marc

    2015-06-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR. PMID:25956570

  13. An improved stable isotope N-terminal labeling approach with light/heavy TMPP to automate proteogenomics data validation: dN-TOP.

    PubMed

    Bertaccini, Diego; Vaca, Sebastian; Carapito, Christine; Arsène-Ploetze, Florence; Van Dorsselaer, Alain; Schaeffer-Reiss, Christine

    2013-06-01

    In silico gene prediction has proven to be prone to errors, especially regarding precise localization of start codons that spread in subsequent biological studies. Therefore, the high throughput characterization of protein N-termini is becoming an emerging challenge in the proteomics and especially proteogenomics fields. The trimethoxyphenyl phosphonium (TMPP) labeling approach (N-TOP) is an efficient N-terminomic approach that allows the characterization of both N-terminal and internal peptides in a single experiment. Due to its permanent positive charge, TMPP labeling strongly affects MS/MS fragmentation resulting in unadapted scoring of TMPP-derivatized peptide spectra by classical search engines. This behavior has led to difficulties in validating TMPP-derivatized peptide identifications with usual score filtering and thus to low/underestimated numbers of identified N-termini. We present herein a new strategy (dN-TOP) that overwhelmed the previous limitation allowing a confident and automated N-terminal peptide validation thanks to a combined labeling with light and heavy TMPP reagents. We show how this double labeling allows increasing the number of validated N-terminal peptides. This strategy represents a considerable improvement to the well-established N-TOP method with an enhanced and accelerated data processing making it now fully compatible with high-throughput proteogenomics studies. PMID:23641718

  14. 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

  15. 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

  16. Efficient 18F-Labeling of Large 37-Amino Acid pHLIP Peptide Analogues and their Biological Evaluation

    PubMed Central

    Daumar, Pierre; Wanger-Baumann, Cindy A.; Pillarsetty, NagaVaraKishore; Fabrizio, Laura; Carlin, Sean D.; Andreev, Oleg A.; Reshetnyak, Yana K.; Lewis, Jason S.

    2012-01-01

    Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP®) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pHe<7). Labeling of peptides with [18F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known “click” methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC “click chemistry” for the simple and efficient 18F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and a L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[18F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥ 98%. The subsequent CuI catalyzed “click” reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [18F]-D-WT-pHLIP and [18F]-L-K-pHLIP were obtained with total radiochemical yields of 5–20% after HPLC purification. The total reaction time was only 85 min including formulation. In vitro stability tests revealed high stability of the [18F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65 and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [18F]-D-WT-pHLIP and the negative

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

    PubMed

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

    2009-04-01

    Extensive X-ray crystallographic studies carried out on the catalytic-subunit of protein kinase A (PKA-C) enabled the atomic characterization of inhibitor and/or substrate peptide analogues trapped at its active site. Yet, the structural and dynamic transitions of these peptides from the free to the bound state are missing. These conformational transitions are central to understanding molecular recognition and the enzymatic cycle. NMR spectroscopy allows one to study these phenomena under functionally relevant conditions. However, the amounts of isotopically labeled peptides required for this technique present prohibitive costs for solid-phase peptide synthesis. To enable NMR studies, we have optimized both expression and purification of isotopically enriched substrate/inhibitor peptides using a recombinant fusion protein system. Three of these peptides correspond to the cytoplasmic regions of the wild-type and lethal mutants of the membrane protein phospholamban, while the fourth peptide correspond to the binding epitope of the heat-stable protein kinase inhibitor (PKI(5-24)). The target peptides were fused to the maltose binding protein (MBP), which is further purified using a His(6) tag approach. This convenient protocol allows for the purification of milligram amounts of peptides necessary for NMR analysis. PMID:19027069

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

    2013-02-20

    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. Regioselective synthesis of isotopically labeled Δ9-tetrahydrocannabinolic acid A (THCA-A-D3) by reaction of Δ9-tetrahydrocannabinol-D3 with magnesium methyl carbonate.

    PubMed

    Roth, Nadine; Wohlfarth, Ariane; Müller, Michael; Auwärter, Volker

    2012-10-10

    For the reliable quantification of Δ9-tetrahydrocannabinolic acid A (THCA-A), the biogenetic precursor of Δ9-tetrahydrocannabinol (THC), in biological matrices by LC-MS/MS and GC-MS(/MS), an isotopically labeled internal standard was synthesized starting from Δ9-tetrahydrocannabinol-D(3) (THC-D(3)). Synthesis strategy was based on a method reported by Mechoulam et al. in 1969 using magnesium methyl carbonate (MMC) as carboxylation reagent for the synthesis of cannabinoid acids. Preliminary experiments with THC to optimize yield of the product (THCA-A) resulted in the synthesis of the positional isomer tetrahydrocannabinolic acid B (THCA-B) as a byproduct. Using the optimized conditions for the desired isomer, THCA-A-D(3) was prepared and isolated with a yield of approx. 10% after two synthesis cycles. Isotope purity was estimated to be >99% by relative abundance of the molecular ions. The synthesized compound proved to be suitable as an internal standard for quantification of THCA-A in serum and hair samples of cannabis consumers. PMID:22921834

  20. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  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. 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.

  3. 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 (δ13C = 1346 ± 162‰) and the leaf water were strongly labelled (δ18O = -63 ± 8, δ2H = -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.

  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 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

  5. The use of stable isotope labeling and liquid chromatography/tandem mass spectrometry techniques to study the pharmacokinetics and bioavailability of the antimigraine drug, MK-0462 (rizatriptan) in dogs.

    PubMed

    Barrish, A; Olah, T V; Gatto, G J; Michel, K B; Dobrinska, M R; Gilbert, J D

    1996-01-01

    MK-0462 (rizatriptan) is a 5HT1D agonist being developed for the treatment of migraine. The assay for this substance in plasma and urine is based on HPLC with tandem mass spectrometry (MS/MS) detection. The procedure has been modified to include the simultaneous determination of the [triazole-13C2, 15N3-] stable-isotope-labelled analogue for which the lower quantifiable limit was 0.1 ng mL-1. The assay has been applied to study the pharmacokinetics of MK-0462 after simultaneous oral and intravenous administration of the drug and its stable-isotope-labelled analogue to dogs. The experiment afforded an estimate of plasma clearance concomitant with a precise measurement of the drug's oral bioavailability. The increasing use of LC-MS/MS in quantitative experiments may renew interest in stable isotopes as tools for pharmaceutical research. PMID:8755236

  6. 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

  7. 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

  8. 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 Central

    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

    2015-01-01

    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

  9. 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...

  10. 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

  11. Improved biocompatibility and efficient labeling of neural stem cells with poly(L-lysine)-coated maghemite nanoparticles

    PubMed Central

    Ahmed, Lada Brkić; Babič, Michal; Šlouf, Miroslav; Horák, Daniel

    2016-01-01

    Summary Background: Cell tracking is a powerful tool to understand cellular migration, dynamics, homing and function of stem cell transplants. Nanoparticles represent possible stem cell tracers, but they differ in cellular uptake and side effects. Their properties can be modified by coating with different biocompatible polymers. To test if a coating polymer, poly(L-lysine), can improve the biocompatibility of nanoparticles applied to neural stem cells, poly(L-lysine)-coated maghemite nanoparticles were prepared and characterized. We evaluated their cellular uptake, the mechanism of internalization, cytotoxicity, viability and proliferation of neural stem cells, and compared them to the commercially available dextran-coated nanomag®-D-spio nanoparticles. Results: Light microscopy of Prussian blue staining revealed a concentration-dependent intracellular uptake of iron oxide in neural stem cells. The methyl thiazolyl tetrazolium assay and the calcein acetoxymethyl ester/propidium iodide assay demonstrated that poly(L-lysine)-coated maghemite nanoparticles scored better than nanomag®-D-spio in cell labeling efficiency, viability and proliferation of neural stem cells. Cytochalasine D blocked the cellular uptake of nanoparticles indicating an actin-dependent process, such as macropinocytosis, to be the internalization mechanism for both nanoparticle types. Finally, immunocytochemistry analysis of neural stem cells after treatment with poly(L-lysine)-coated maghemite and nanomag®-D-spio nanoparticles showed that they preserve their identity as neural stem cells and their potential to differentiate into all three major neural cell types (neurons, astrocytes and oligodendrocytes). Conclusion: Improved biocompatibility and efficient cell labeling makes poly(L-lysine)-coated maghemite nanoparticles appropriate candidates for future neural stem cell in vivo tracking studies. PMID:27547609

  12. An efficient method for isolating individual long-chain alkenones for compound-specific hydrogen isotope analysis.

    PubMed

    D'Andrea, William J; Liu, Zhonghui; Da Rosa Alexandre, Marcelo; Wattley, Sarah; Herbert, Timothy D; Huang, Yongsong

    2007-05-01

    Hydrogen isotope ratios (2H/H or D/H) of long-chain unsaturated ketones (alkenones) preserved in lake and marine sediments hold great promise for paleoclimate studies. However, compound-specific hydrogen isotope analysis of individual alkenones has not been possible due to chromatographic coelution of alkenones with the same carbon chain length but different numbers of double bonds. Published studies have only reported the deltaD values of the mixture of coeluting alkenones. We developed an efficient procedure to isolate individual alkenones based on double-bond numbers using silica gel impregnated with silver nitrate. The chromatographic procedure is simple, inexpensive, and highly reproducible, offers 87-100% sample recovery, and allows for the first time hydrogen isotopic measurement on individual alkenones. deltaD values of specific di-, tri- and tetraunsaturated C37 alkenones produced by an Emiliania huxleyi culture, as well as those isolated from Greenland lake sediments, differ consecutively by 43-65 per thousand. These findings suggest that alkenones with different numbers of carbon-carbon double bonds express significantly different deltaD values and that coelution of different alkenones may lead to erroneous source water deltaD reconstructions. Our alkenone isolation approach opens a new avenue for paleoclimate reconstructions using hydrogen isotope ratios of individual alkenones. PMID:17391004

  13. 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.

  14. Monitoring the biodegradation of polycyclic aromatic hydrocarbons in a co-contaminated soil using stable isotope labeling

    NASA Astrophysics Data System (ADS)

    Wawra, Anna; Friesl-Hanl, Wolfgang; Watzinger, Andrea; Soja, Gerhard; Puschenreiter, Markus

    2016-04-01

    Conventional remediation techniques like "dig and dump" are costly and limited in scale. Plant- and microbe-based alternatives, e.g. phytoremediation options, offer a cheap and environmentally friendly approach that can be applied on larger areas. However, the application of phytoremediation techniques to co-contaminated sites may be hindered due to a potential inhibition of biodegradation processes by the presence of heavy metals in soil. Therefore, the objective of this study is to test the hypothesis that the degradation of organic pollutants can be enhanced by immobilising potentially toxic heavy metals. This study aims to identify the influence of heavy metal immobilisation on the degradation of organic pollutants, and to determine chemical, physical and biological measures further accelerating these processes. The influence of heavy metals on organic pollutant degradation dynamics is assessed using 13C-phospholipid fatty acid analysis (13C-PLFA). Application of 13C-labeled phenanthrene allows the identification of microbial groups responsible for the degradation process. For metal immobilisation and enhanced biodegradation, distinct mineral and organic soil amendments (iron oxides, gravel sludge, biochar) are deployed, partly in combination with fast-growing and pollution-tolerant woody plants (willow, black locust and alder). Results of an incubation batch experiment show a fast degradation of the phenanthrene label within the first two weeks by various microbial groups (gram negative bacteria as indicated by the cy17:0 peak) resulting in a decrease by up to 80% of the total PAH concentration (Σ 16 EPA PAHs) measured in soil. A similar trend was observed in the greenhouse pot experiment, whereby heavy metal accumulation in the woody plants growing on the co-contaminated soil significantly varied with plant species (willow > black locust, alder).

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  1. 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; 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

  2. Probing RNA-protein interactions using pyrene-labeled oligodeoxynucleotides: Qbeta replicase efficiently binds small RNAs by recognizing pyrimidine residues.

    PubMed

    Preuss, R; Dapprich, J; Walter, N G

    1997-10-31

    Binding of small RNAs by the RNA-dependent RNA polymerase of coliphage Qbeta was studied utilizing a fluorometric assay. A DNA oligonucleotide probe of sequence 5'-d(TTTTTCC) was 5'-end-labeled with pyrene. In this construct, the proximal thymine residues efficiently quench the fluorophore emission in solution. Upon stoichiometric binding of one probe per polymerase molecule, the pyrene steady-state fluorescence increases by two orders of magnitude, the fluorescence anisotropy increases, and a long fluorescence lifetime component of 140 ns appears. With addition of replicable RNA, steady-state fluorescence decreases in a concentration dependent manner and the long lifetime component is lost. This observation most likely reflects displacement of the pyrene-labeled probe from the proposed nucleic acid binding site II of Qbeta replicase. The effect was utilized to access binding affinities of different RNAs to this site in a reverse titration assay format. In 10 mM sodium phosphate (pH 7.0), 100 mM NaCl, at 16 degrees C, equilibrium dissociation constants for different template midi- and minivariant RNAs were calculated to be in the nanomolar range. In general, the minus and plus strands, concomitantly synthesized by Qbeta replicase during replication, exhibited discriminative affinities, while their hybrid bound less efficiently than either of the single strands. Different non-replicable tRNAs also bound to the polymerase with comparable dissociation constants. By titration with DNA homo-oligonucleotides it was shown that the probed site on Qbeta replicase does not require a 2' hydroxyl group for binding nucleic acids, but recognizes pyrimidine residues. Its interaction with thymine is lost in an A.T base-pair, while that with cytosine is retained after Watson-Crick base-pairing. These findings can explain the affinities of RNA-Qbeta replicase interactions reported here and in earlier investigations. The sensitivity of the described fluorometric assay allows

  3. 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

  4. 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

  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. 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

  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. Universal quantitative kinase assay based on diagonal SCX chromatography and stable isotope dimethyl labeling provides high-definition kinase consensus motifs for PKA and human Mps1.

    PubMed

    Hennrich, Marco L; Marino, Fabio; Groenewold, Vincent; Kops, Geert J P L; Mohammed, Shabaz; Heck, Albert J R

    2013-05-01

    In order to understand cellular signaling, a clear understanding of kinase-substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome. PMID:23510141

  10. Quantitative analysis of differential protein expression in cervical carcinoma cells after zeylenone treatment by stable isotope labeling with amino acids in cell culture.

    PubMed

    Zhang, Leilei; Jin, Jing; Zhang, Lijing; Hu, Ruifeng; Gao, Li; Huo, Xiaowei; Liu, Dongyu; Ma, Xiaoling; Wang, Canhong; Han, Jiayuan; Li, Liyong; Sun, Xiaobo; Cao, Li

    2015-08-01

    Cervical carcinoma is a malignant tumor that poses a serious threat to women's health and survival. Approximately 10-25% of cervical cancers are adenocarcinomas (ACs). AC has high rates of recurrence and mortality, while there is no effective treatment for now. Zeylenone (Zey), which is isolated from an ethanol extract of the leaves of Uvaria grandiflora Roxb. of the family Annonaceae, has shown potent inhibitory activity against various tumor cells, including cervical carcinoma cells. To gain insight into the molecular mechanism underlying the effect of Zey on AC, we quantified protein expression changes in AC cells treated with Zey. We used stable isotope labeling with amino acids in cell culture (SILAC) in combination with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) and bioinformatics analysis to compare protein expression profiles in HeLa cells before and after Zey treatment. Of 1805 differentially expressed proteins identified, 229 were screened as key protein molecules and classified into nine categories. Profiling of differentially-expressed proteins contributed to our understanding of the molecular mechanism by which Zey induces HeLa cell apoptosis. Using this method, candidate targets can be identified for developing new drugs against cervical carcinoma. PMID:26130516

  11. Novel tracer method to measure isotopic labeled gas-phase nitrous acid (HO15NO) in biogeochemical studies.

    PubMed

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

    2014-07-15

    Gaseous nitrous acid (HONO), the protonated form of nitrite, contributes up to ∼60% to the primary formation of hydroxyl radical (OH), which is a key oxidant in the degradation of most air pollutants. Field measurements and modeling studies indicate a large unknown source of HONO during daytime. Here, we developed a new tracer method based on gas-phase stripping-derivatization coupled to liquid chromatography-mass spectrometry (LC-MS) to measure the 15N relative exceedance, ψ(15N), of HONO in the gas-phase. Gaseous HONO is quantitatively collected and transferred to an azo dye, purified by solid phase extraction (SPE), and analyzed using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS). In the optimal working range of ψ(15N)=0.2-0.5, the relative standard deviation of ψ(15N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO15NO emissions from soil in a dynamic chamber with and without spiking 15) labeled urea. The identification of HO15NO from soil with 15N urea addition confirmed biogenic emissions of HONO from soil. The method enables a new approach of studying the formation pathways of HONO and its role for atmospheric chemistry (e.g., ozone formation) and environmental tracer studies on the formation and conversion of gaseous HONO or aqueous NO2- as part of the biogeochemical nitrogen cycle, e.g., in the investigation of fertilization effects on soil HONO emissions and microbiological conversion of NO2- in the hydrosphere. PMID:24954648

  12. 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

  13. Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

    PubMed Central

    Starkey, Jonathan M.; Zhao, Yingxin; Sadygov, Rovshan G.; Haidacher, Sigmund J.; LeJeune, Wanda S.; Dey, Nilay; Luxon, Bruce A.; Kane, Maureen A.; Napoli, Joseph L.; Denner, Larry; Tilton, Ronald G.

    2010-01-01

    Background Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes. Methodology/Principal Findings Total proteins were extracted from renal cortex of control and db/db mice at 20 weeks of age (after 12 weeks of hyperglycemia in the diabetic mice). Following trypsinization, 18O- and 16O-labeled control and diabetic peptides, respectively, were pooled and separated by two dimensional liquid chromatography (strong cation exchange creating 60 fractions further separated by nano-HPLC), followed by peptide identification and quantification using mass spectrometry. Proteomic analysis identified 53 proteins with fold change ≥1.5 and p≤0.05 after Benjamini-Hochberg adjustment (out of 1,806 proteins identified), including alcohol dehydrogenase (ADH) and retinaldehyde dehydrogenase (RALDH1/ALDH1A1). Ingenuity Pathway Analysis identified altered retinoic acid as a key signaling hub that was altered in the diabetic renal cortical proteome. Western blotting and real-time PCR confirmed diabetes-induced upregulation of RALDH1, which was localized by immunofluorescence predominantly to the proximal tubule in the diabetic renal cortex, while PCR confirmed the downregulation of ADH identified with mass spectrometry. Despite increased renal cortical tissue levels of retinol and RALDH1 in db/db versus control mice, all-trans-retinoic acid was significantly decreased in association with a significant decrease in PPARβ/δ mRNA. Conclusions/Significance Our results indicate that retinoic acid metabolism is significantly dysregulated in diabetic kidneys, and suggest that a shift in all-trans-retinoic acid metabolism is a novel feature in

  14. Drivers of Plant-Availability of Phosphorus from Thermally Conditioned Sewage Sludge as Assessed by Isotopic Labeling

    PubMed Central

    Andriamananjara, Andry; Rabeharisoa, Lilia; Prud’homme, Loïc; Morel, Christian

    2016-01-01

    Urban sewage sludge is a potential source of phosphorus (P) for agriculture and represents an alternative way to recycle P as fertilizer. However, the use of thermally conditioned sewage sludge (TCSS) required an accurate assessment of its value as P-fertilizer. This work aimed at assessing the plant-availability of P from TCSS. Uptake of P by a mixture of ryegrass and fescue from TCSS and triple super phosphate (TSP) fertilizers was studied using 32P-labeling technique in a greenhouse experiment. Phosphorus was applied at the rate of 50 mg P kg−1.We also conducted incubation experiments considering the same treatments to assess soil microbial respiration. Applications of TCSS and TSP increased plant P uptake that is related to the root P acquisition. The P taken up by plant from soil plant-available P was lower for control compared to TSP or TCSS that was attributed to the increase of root interception of soil P. The contribution of TSP to ryegrass nutrition (Pdff%) was 55% with 22% of the applied P which was taken up by plants (CPU%). The Pdff value for TCSS was 56% with 14% of fertilizer P recovery (CPU%). Shoot biomass and total P uptake from TCSS were lower than those from TSP. As a result, the agronomic effectiveness of TCSS calculated from Pdff value (in comparison with TSP treatment) was 102%, while the AE of TCSS estimated from CPU value (in % TSP) was 64%, which is attributed to microbial activity stimulation inducing P immobilization onto soil constituents and microbial biomass during plant growth. The high C/N ratio of TCSS stimulated soil microbial biomass that competes with plant roots to acquire nutrients, such as P. As a consequence, the P taken up from either native soil or TCSS decreased in similar proportions. The AE value calculated with Pdff% took into account these interactions between soil, plant, and microbial biomass, and is less dependent on operational conditions than the AE value calculated with %Precovery. PMID:27379240

  15. Drivers of Plant-Availability of Phosphorus from Thermally Conditioned Sewage Sludge as Assessed by Isotopic Labeling.

    PubMed

    Andriamananjara, Andry; Rabeharisoa, Lilia; Prud'homme, Loïc; Morel, Christian

    2016-01-01

    Urban sewage sludge is a potential source of phosphorus (P) for agriculture and represents an alternative way to recycle P as fertilizer. However, the use of thermally conditioned sewage sludge (TCSS) required an accurate assessment of its value as P-fertilizer. This work aimed at assessing the plant-availability of P from TCSS. Uptake of P by a mixture of ryegrass and fescue from TCSS and triple super phosphate (TSP) fertilizers was studied using (32)P-labeling technique in a greenhouse experiment. Phosphorus was applied at the rate of 50 mg P kg(-1).We also conducted incubation experiments considering the same treatments to assess soil microbial respiration. Applications of TCSS and TSP increased plant P uptake that is related to the root P acquisition. The P taken up by plant from soil plant-available P was lower for control compared to TSP or TCSS that was attributed to the increase of root interception of soil P. The contribution of TSP to ryegrass nutrition (Pdff%) was 55% with 22% of the applied P which was taken up by plants (CPU%). The Pdff value for TCSS was 56% with 14% of fertilizer P recovery (CPU%). Shoot biomass and total P uptake from TCSS were lower than those from TSP. As a result, the agronomic effectiveness of TCSS calculated from Pdff value (in comparison with TSP treatment) was 102%, while the AE of TCSS estimated from CPU value (in % TSP) was 64%, which is attributed to microbial activity stimulation inducing P immobilization onto soil constituents and microbial biomass during plant growth. The high C/N ratio of TCSS stimulated soil microbial biomass that competes with plant roots to acquire nutrients, such as P. As a consequence, the P taken up from either native soil or TCSS decreased in similar proportions. The AE value calculated with Pdff% took into account these interactions between soil, plant, and microbial biomass, and is less dependent on operational conditions than the AE value calculated with %Precovery. PMID:27379240

  16. A new strategy for sequential assignment of intrinsically unstructured proteins based on 15N single isotope labelling

    NASA Astrophysics Data System (ADS)

    Lopez, Juan; Ahuja, Puneet; Gerard, Melanie; Wieruszeski, Jean-Michel; Lippens, Guy

    2013-11-01

    We describe a new efficient strategy for the sequential assignment of amide resonances of a conventional 15N-1H HSQC spectrum of intrinsically unfolded proteins, based on composite NOESY-TOCSY and TOCSY-NOESY mixing times. These composite mixing times lead to a Hα-proton mediated unidirectional transfer of amide to amide proton. We have implemented the composite mixing times in an HSQC-NOESY-HSQC manner to obtain directional connectivity between amides of neighbouring residues. We experimentally determine the optimal mixing times for both transfer schemes, and demonstrate its use in the assignment for both a fragment of the neuronal tau protein and for α-synuclein.

  17. Plasma-treated polystyrene film that enhances binding efficiency for sensitive and label-free protein biosensing

    NASA Astrophysics Data System (ADS)

    Guo, Bihong; Li, Shaopeng; Song, Lusheng; Yang, Mo; Zhou, Wenfei; Tyagi, Deependra; Zhu, Jinsong

    2015-08-01

    A plasma-treated ultrathin polystyrene (PS) film surface was explored as a simple, robust, and low-cost surface chemistry solution for protein biosensing applications. This surface could dramatically improve the binding efficiency of the protein-protein interactions, which is defined as the binding signal per immobilized ligand. The PS-modified protein biosensor was readily fabricated by spin coating and plasma treatment. Various parameters for fabrication, including the concentration of the PS solution, rate of spin coating, and duration of plasma treatment, were systematically optimized based on the improvement of fluorescence signal yielded by the microfluidic network-aided fluorescence immunoassay. The performance of the label-free protein detection on the optimized surfaces was further evaluated by surface plasmon resonance imaging (SPRi). PS surfaces with optimal fabrication parameters exhibited up to an 620% enhancement of the protein binding response and approximately 210% of the protein binding per immobilized protein ligand compared with a self-assembled monolayer (SAM) surface of 11-mercapto undecanoic acid (MUA). The relationship between the fabrication parameters used and changes to the surface chemistry and the morphological properties were characterized with atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). It was revealed that the morphological changes observed in the plasma-treated PS film were the dominant factor for the improvement of the protein bioassay performance, rather than the chemical changes.

  18. Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS.

    PubMed

    Beverly, Michael; Dell, Amy; Parmar, Parul; Houghton, Leslie

    2016-07-01

    A label-free method for determining the 5'-end cap identity and orientation of a messenger RNA (mRNA) is described. Biotin-tagged probes that were complementary to the 5' end of target mRNA were used with RNase H to cleave the 5' end of the mRNA. The cleaved end sequence was isolated using streptavidin-coated magnetic beads and then analyzed by LC-MS. Quantitative and qualitative information on the 5' cap was determined from the unique mass of the isolated cleaved sequence. This approach, combined with the use of 5' RNA pyrophosphohydrolase, was also used to ascertain the orientation of the 5' cap. The assay showed low-picomole sensitivity for detecting capping reaction impurities. Uncapped triphosphate mRNA, spiked into 100 pmol of capped mRNA, could be detected over the tested range of 0.5 to 25 % with a linear response. The capping efficiency of several vaccinia-capped mRNA preparations was determined to be between 88 and 98 % depending on the modification type and length of the mRNA. mRNA of 2.2K and 9K nucleotides in length and containing the modified nucleotides pseudouridine and 5-methylcytidine were all successfully analyzed, demonstrating the utility of the technique to study mRNA capping. Graphical abstract mRNA 5' end analysis with RNAse H cleavage and capture probe. PMID:27193635

  19. 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.

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

    PubMed Central

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

    2013-01-01

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

  1. 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

  2. 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

  3. 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

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

    PubMed Central

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

    2012-01-01

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

  5. Comparing integrated stable isotope and eddy covariance estimates of water-use efficiency on a Mediterranean successional sequence.

    PubMed

    Scartazza, Andrea; Vaccari, Francesco Primo; Bertolini, Teresa; Di Tommasi, Paul; Lauteri, Marco; Miglietta, Franco; Brugnoli, Enrico

    2014-10-01

    Water-use efficiency (WUE), thought to be a relevant trait for productivity and adaptation to water-limited environments, was estimated for three different ecosystems on the Mediterranean island of Pianosa: Mediterranean macchia (SMM), transition (S(TR)) and abandoned agricultural (SAA) ecosystems, representing a successional series. Three independent approaches were used to study WUE: eddy covariance measurements, C isotope composition of ecosystem respired CO2, and C isotope discrimination (Δ) of leaf material (dry matter and soluble sugars). Seasonal variations in C-water relations and energy fluxes, compared in S(MM) and in SAA, were primarily dependent on the specific composition of each plant community. WUE of gross primary productivity was higher in SMM than in SAA at the beginning of the dry season. Both structural and fast-turnover leaf material were, on average, more enriched in (13)C in S(MM) than SAA, indicating relatively higher stomatal control and WUE for the long-lived macchia species. This pattern corresponded to (13)C-enriched respired CO2 in SMM compared to the other ecosystems. Conversely, most of the annual herbaceous SAA species (terophytes) showed a drought-escaping strategy, with relatively high stomatal conductance and low WUE. An ecosystem-integrated Δ value was weighted for each ecosystem on the abundance of different life forms, classified according to Raunkiar's system. Agreement was found between ecosystem WUE calculated using eddy covariance and those estimated using integrated Δ approaches. Comparing the isotopic methods, Δ of leaf soluble sugars provided the most reliable proxy for short-term changes in photosynthetic discrimination and associated shifts in integrated canopy-level WUE along the successional series. PMID:25085444

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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.

  11. 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

  12. Incorporation of 13C labelled root-shoot residues in soil in the presence of Lumbricus terrestris: An isotopic and molecular approach

    NASA Astrophysics Data System (ADS)

    Vidal, Alix; Alexis, Marie; Nguyen Tu, Thanh Tu; Anquetil, Christelle; Vaury, Véronique; Derenne, Sylvie; Quenea, Katell

    2016-04-01

    Litter from plant biomass deposited on soil surface can either be mineralized; releasing CO2 to the atmosphere, or transferred into the soil as organic compounds. Both pathways depend on biotic factors such as litter characteristics and the of soil organism activity. During the last decades, many studies have focused on the origin of organic matter, with a particular attention to the fate of root and shoot litter. It is generally admitted that roots decompose at a slower rate than shoots, resulting in a higher carbon sequestration in soil for compounds originating from roots. Earthworms play a central role in litter decomposition and carbon cycling, ingesting both organic and mineral compounds which are mixed, complexed and dejected in the form of casts at the soil surface or along earthworm burrows. The simultaneous impact of earthworms and root-shoot on soil carbon cycling is still poorly understood. This study aimed at (1) defining the rate of incorporation of root and shoot litter with or without earthworms and (2) characterizing the molecular composition of soil organic matter upon litter decomposition, after one year of experimentation. A mesocosm experiment was set up to follow the incorporation of 13C labelled Ryegrass root and shoot litter in the soil, in the presence of anecic earthworms (Lumbricus terrestris). Soil samples were collected at 0-20 and 40-60 cm, as well as surface casts, at the beginning and after 1, 2, 4, 8, 24 and 54 weeks of experiment. Organic carbon content and δ13C values were determined for all the samples with Elemental Analysis - Isotope Ratio Mass Spectrometry. Lipid-free soil and cast samples after 54 weeks of incubation were analyzed with Pyrolysis-Gas Chromatography-Mass Spectrometry. Pyrolysis products were grouped into six classes: polysaccharides, lignin derived compounds, phenols, N-compounds, aliphatic compounds and sterols. Each pyrolysis product was quantified thanks to its peak area, relative to the total area of the

  13. 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

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. Leaf oxygen and Carbon Isotopic Signatures Reflect Drought Resistance and Water Use Efficiency in the C4 Grass, Setaria viridis

    NASA Astrophysics Data System (ADS)

    Ellsworth, P.; Cousins, A. B.

    2014-12-01

    Low water availability is a major constraint in crop production, especially as agriculture is pushed to marginal lands. Therefore, improving drought resistance such as increasing water use efficiency (WUE) through plant breeding is needed to expand the range of soil water availability adequate for food production. With the goal of finding the genomic basis for WUE in C4 grasses, Setaria viridis makes an ideal model species because of its small size, short lifespan, and sequenced genome. Also it is part of the panicoid grass clade, which is one of the most important clades for food and biofuel production. In plant breeding programs, large numbers of genotypes must be quickly screened for drought resistance traits, but there is no well-defined method of screening for WUE in C4 grasses. However, bulk leaf oxygen (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

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

    PubMed

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2013-12-01

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

  2. 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.

  3. 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

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

    PubMed

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

    2013-01-01

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

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

    PubMed

    Cernusak, Lucas A; Winter, Klaus; Aranda, Jorge; Virgo, Aurelio; Garcia, Milton

    2009-09-01

    Variation in transpiration efficiency (TE) and its relationship with the stable carbon isotope ratio of wood was investigated in the saplings of three tropical tree species. Five individuals each of Platymiscium pinnatum (Jacq.) Dugand, Swietenia macrophylla King and Tectona grandis Linn. f. were grown individually in large (760 l) pots over 16 months in the Republic of Panama. Cumulative transpiration was determined by repeatedly weighing the pots with a pallet truck scale. Dry matter production was determined by destructive harvest. The TE, expressed as experiment-long dry matter production divided by cumulative water use, averaged 4.1, 4.3 and 2.9 g dry matter kg(-1) water for P. pinnatum, S. macrophylla and T. grandis, respectively. The TE of T. grandis was significantly lower than that of the other two species. Instantaneous measurements of the ratio of intercellular to ambient CO(2) partial pressures (c(i)/c(a)), taken near the end of the experiment, explained 66% of variation in TE. Stomatal conductance was lower in S. macrophylla than in T. grandis, whereas P. pinnatum had similar stomatal conductance to T. grandis, but with a higher photosynthetic rate. Thus, c(i)/c(a) and TE appeared to vary in response to both stomatal conductance and photosynthetic capacity. Stem-wood delta(13)C varied over a relatively narrow range of just 2.2 per thousand, but still explained 28% of variation in TE. The results suggest that leaf-level processes largely determined variation among the three tropical tree species in whole-plant water-use efficiency integrated over a full annual cycle. PMID:19661136

  6. Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies.

    PubMed

    Nikonowicz, E P; Sirr, A; Legault, P; Jucker, F M; Baer, L M; Pardi, A

    1992-09-11

    A procedure is described for the efficient preparation of isotopically enriched RNAs of defined sequence. Uniformly labelled nucleotide 5'triphosphates (NTPs) were prepared from E.coli grown on 13C and/or 15N isotopically enriched media. These procedures routinely yield 180 mumoles of labelled NTPs per gram of 13C enriched glucose. The labelled NTPs were then used to synthesize RNA oligomers by in vitro transcription. Several 13C and/or 15N labelled RNAs have been synthesized for the sequence r(GGCGCUUGCGUC). Under conditions of high salt or low salt, this RNA forms either a symmetrical duplex with two U.U base pairs or a hairpin containing a CUUG loop respectively. These procedures were used to synthesize uniformly labelled RNAs and a RNA labelled only on the G and C residues. The ability to generate milligram quantities of isotopically labelled RNAs allows application of multi-dimensional heteronuclear magnetic resonance experiments that enormously simplify the resonance assignment and solution structure determination of RNAs. Examples of several such heteronuclear NMR experiments are shown. PMID:1383927

  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

  8. 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

  9. 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*S⃞

    PubMed Central

    Prokhorova, Tatyana A.; Rigbolt, Kristoffer T. G.; Johansen, Pia T.; Henningsen, Jeanette; Kratchmarova, Irina; Kassem, Moustapha; Blagoev, Blagoy

    2009-01-01

    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 ζ (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

  10. Elementary metabolite units (EMU): a novel framework for modeling isotopic distributions.

    PubMed

    Antoniewicz, Maciek R; Kelleher, Joanne K; Stephanopoulos, Gregory

    2007-01-01

    Metabolic flux analysis (MFA) has emerged as a tool of great significance for metabolic engineering and mammalian physiology. An important limitation of MFA, as carried out via stable isotope labeling and GC/MS and nuclear magnetic resonance (NMR) measurements, is the large number of isotopomer or cumomer equations that need to be solved, especially when multiple isotopic tracers are used for the labeling of the system. This restriction reduces the ability of MFA to fully utilize the power of multiple isotopic tracers in elucidating the physiology of realistic situations comprising complex bioreaction networks. Here, we present a novel framework for the modeling of isotopic labeling systems that significantly reduces the number of system variables without any loss of information. The elementary metabolite unit (EMU) framework is based on a highly efficient decomposition method that identifies the minimum amount of information needed to simulate isotopic labeling within a reaction network using the knowledge of atomic transitions occurring in the network reactions. The functional units generated by the decomposition algorithm, called EMUs, form the new basis for generating system equations that describe the relationship between fluxes and stable isotope measurements. Isotopomer abundances simulated using the EMU framework are identical to those obtained using the isotopomer and cumomer methods, however, require significantly less computation time. For a typical (13)C-labeling system the total number of equations that needs to be solved is reduced by one order-of-magnitude (100s EMUs vs. 1000s isotopomers). As such, the EMU framework is most efficient for the analysis of labeling by multiple isotopic tracers. For example, analysis of the gluconeogenesis pathway with (2)H, (13)C, and (18)O tracers requires only 354 EMUs, compared to more than two million isotopomers. PMID:17088092

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

    PubMed Central

    Zudaire, Isabel; Ortiz-de-Solorzano, Carlos

    2013-01-01

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

  12. Distance-Dependent Emission from Dye-Labeled Oligonucleotides on Striped Au/Ag Nanowires: Effect of Secondary Structure and Hybridization Efficiency

    PubMed Central

    Stoermer, Rebecca L.; Keating, Christine D.

    2010-01-01

    When fluorescently tagged oligonucleotides are located near metal surfaces, their emission intensity is impacted by both electromagnetic effects (i.e., quenching and/or enhancement of emission) and the structure of the nucleic acids (e.g., random coil, hairpin, or duplex). We present experiments exploring the effect of label position and secondary structure in oligonucleotide probes as a function of hybridization buffer, which impacts the percentage of double-stranded probes on the surface after exposure to complementary DNA. Nanowires containing identifiable patterns of Au and Ag segments were used as the metal substrates in this work, which enabled us to directly compare different dye positions in a single multiplexed experiment and differences in emission for probes attached to the two metals. The observed metal–dye separation dependence for unstructured surface-bound oligonucleotides is highly sensitive to hybridization efficiency, due to substantial changes in DNA extension from the surface upon hybridization. In contrast, fluorophore labeled oligonucleotides designed to form hairpin secondary structures analogous to solution-phase molecular beacon probes are relatively insensitive to hybridization efficiency, since the folded form is quenched and therefore does not appreciably impact the observed distance-dependence of the response. Differences in fluorescence patterning on Au and Ag were noted as a function of not only chromophore identity but also metal–dye separation. For example, emission intensity for TAMRA-labeled oligonucleotides changed from brighter on Ag for 24-base probes to brighter on Au for 48-base probes. We also observed fluorescence enhancement at the ends of nanowires and at surface defects where heightened electromagnetic fields affect the fluorescence. PMID:17017805

  13. 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

  14. Synthesis and evaluation of an (125)I-labeled azide prosthetic group for efficient and bioorthogonal radiolabeling of cyclooctyne-group containing molecules using copper-free click reaction.

    PubMed

    Choi, Mi Hee; Shim, Ha Eun; Nam, You Ree; Kim, Hye Rim; Kang, Jung Ae; Lee, Dong-Eun; Park, Sang Hyun; Choi, Dae Seong; Jang, Beom-Su; Jeon, Jongho

    2016-02-01

    Herein we report the radiosynthesis of a pyridine derived azide prosthetic group for iodine radioisotope labeling of dibenzocyclooctyne (DBCO) conjugated molecules. The radiolabeling of the stannylated precursor 2 was conducted using [(125)I]NaI and chloramine-T to give (125)I-labeled azide ([(125)I]1) with high radiochemical yield (72±8%, n=4) and radiochemical purity (>99%). Using (125)I-labeled azide ([(125)I]1), cyclic RGD peptide and near infrared fluorescent molecule were efficiently labeled with modest to good radiochemical yields. The biodistribution study and SPECT/CT images showed that [(125)I]1 underwent rapid renal clearance. These results clearly demonstrated that [(125)I]1 could be used as an useful radiotracer for in vivo pre-targeted imaging as well as efficient in vitro radiolabeling of DBCO containing molecules. PMID:26748695

  15. 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

  16. 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

  17. 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

  18. SU-E-I-65: Estimation of Tagging Efficiency in Pseudo-Continuous Arterial Spin Labeling (pCASL) MRI

    SciTech Connect

    Jen, M; Yan, F; Tseng, Y; Chen, C; Lin, C; Liu, H

    2015-06-15

    Purpose: pCASL was recommended as a potent approach for absolute cerebral blood flow (CBF) quantification in clinical practice. However, uncertainties of tagging efficiency in pCASL remain an issue. This study aimed to estimate tagging efficiency by using short quantitative pulsed ASL scan (FAIR-QUIPSSII) and compare resultant CBF values with those calibrated by using 2D Phase Contrast (PC) MRI. Methods: Fourteen normal volunteers participated in this study. All images, including whole brain (WB) pCASL, WB FAIR-QUIPSSII and single-slice 2D PC, were collected on a 3T clinical MRI scanner with a 8-channel head coil. DeltaM map was calculated by averaging the subtraction of tag/control pairs in pCASL and FAIR-QUIPSSII images and used for CBF calculation. Tagging efficiency was then calculated by the ratio of mean gray matter CBF obtained from pCASL and FAIR-QUIPSSII. For comparison, tagging efficiency was also estimated with 2D PC, a previously established method, by contrast WB CBF in pCASL and 2D PC. Feasibility of estimation from a short FAIR-QUIPSSII scan was evaluated by number of averages required for obtaining a stable deltaM value. Setting deltaM calculated by maximum number of averaging (50 pairs) as reference, stable results were defined within ±10% variation. Results: Tagging efficiencies obtained by 2D PC MRI (0.732±0.092) were significantly lower than which obtained by FAIRQUIPPSSII (0.846±0.097) (P<0.05). Feasibility results revealed that four pairs of images in FAIR-QUIPPSSII scan were sufficient to obtain a robust calibration of less than 10% differences from using 50 pairs. Conclusion: This study found that reliable estimation of tagging efficiency could be obtained by a few pairs of FAIR-QUIPSSII images, which suggested that calibration scan in a short duration (within 30s) was feasible. Considering recent reports concerning variability of PC MRI-based calibration, this study proposed an effective alternative for CBF quantification with pCASL.

  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. 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

  1. 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.

  2. Efficiency of sex pre-selection of spermatozoa by albumin separation method evaluated by double-labelled fluorescence in-situ hybridization.

    PubMed

    Chen, M J; Guu, H F; Ho, E S

    1997-09-01

    To evaluate the separation efficiency of Ericsson's two- and three-layer albumin separation methods, semen samples from 21 healthy males were studied. Seven patients already had two or more sons, another seven had two or more daughters and the other seven had primary infertility due to female factors. The semen samples were divided into three aliquots: one remained unprocessed initially, the other two aliquots went through two- and three-layer albumin separation methods respectively. All samples were then stained with X-Y double staining probes. In each group, four or five samples were processed at room temperature, and two or three at body temperature (37 degrees C). The labelling efficiency of X-Y double staining probe was over 99%. The X:Y sperm ratios were even in the original samples. The ratios of the X and Y spermatozoa were altered slightly but significantly after the two-layer (P < or = 0.05) or the three-layer (P < or = 0.005) separation. The alterations occurred only at room temperature. The X spermatozoa increased and the Y spermatozoa decreased, both to a small degree of difference (1.4-3.5%). Double fluorescence in-situ hybridization analysis therefore showed that albumin separation methods do not enrich Y spermatozoa. PMID:9363707

  3. Carbon Isotope Composition of Carbohydrates and Polyols in Leaf and Phloem Sap of Phaseolus vulgaris L. Influences Predictions of Plant Water Use Efficiency.

    PubMed

    Smith, Millicent; Wild, Birgit; Richter, Andreas; Simonin, Kevin; Merchant, Andrew

    2016-08-01

    The use of carbon isotope abundance (δ(13)C) to assess plant carbon acquisition and water use has significant potential for use in crop management and plant improvement programs. Utilizing Phaseolus vulgaris L. as a model system, this study demonstrates the occurrence and sensitivity of carbon isotope fractionation during the onset of abiotic stresses between leaf and phloem carbon pools. In addition to gas exchange data, compound-specific measures of carbon isotope abundance and concentrations of soluble components of phloem sap were compared with major carbohydrate and sugar alcohol pools in leaf tissue. Differences in both δ(13)C and concentration of metabolites were found in leaf and phloem tissues, the magnitude of which responded to changing environmental conditions. These changes have inplications for the modeling of leaf-level gas exchange based upon δ(13)C natural abundance. Estimates of δ(13)C of low molecular weight carbohydrates and polyols increased the precision of predictions of water use efficiency compared with those based on bulk soluble carbon. The use of this technique requires consideration of the dynamics of the δ(13)C pool under investigation. Understanding the dynamics of changes in δ(13)C during movement and incorporation into heterotrophic tissues is vital for the continued development of tools that provide information on plant physiological performance relating to water use. PMID:27335348

  4. ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.

    SciTech Connect

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

    2000-12-01

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

  5. Efficient and direct identification of fructose fermenting and non-fermenting bacteria from calf gut microbiota using stable isotope probing and modified T-RFLP.

    PubMed

    Michinaka, Atsuko; Fujii, Toshio

    2012-01-01

    The aim of this study was the development of an efficient method to identify the prebiotics-assimilating-bacteria in gut microbiota using DNA-stable isotope probing (DNA-SIP) technology. For efficient probing of microbiota with stable isotopes, a small-scale repeated batch culture using a low-carbon-source-containing medium was developed. Fecal samples from cattle were inoculated and [U-(13)C]-fructose was applied to the culture after 24 h stabilization. Organic acid production, pH value of the period and the total diversity of microorganisms of the culture were successfully maintained during the chasing period. DNA samples were extracted from the culture and were subjected to isopycnic centrifugation and fractionation in order to separate fructose fermenters from non-fermenters. T-RFLP (Terminal Restriction Fragment Length Polymorphism) and the modified T-RFLP of each fraction suggested that Streptococcus bovis was the most dominant fructose fermenter in this culture. In addition, we improved the modified T-RFLP method and successfully identified Lactobacillus vitulinus and Megasphaella eldenii as minor fructose-fermenters and several species of Clostridium cluster IV as non-fermenters. From these results we concluded that the methods shown here provide a means for assessing the importance of individual prebiotics on gut microbiota. PMID:22990490

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

    NASA Astrophysics Data System (ADS)

    Dubrovskaya, Ekaterina; Turkovskaya, Olga

    2010-05-01

    Estimation of the efficiency of hydrocarbon mineralization in soil by measuring CO2-emission and variations in the isotope composition of carbon dioxide E. Dubrovskaya1, O. Turkovskaya1, A. Tiunov2, N. Pozdnyakova1, A. Muratova1 1 - Institute of Biochemistry and Physiology of Plants and Microorganisms, RAS, Saratov, 2 - A.N. Severtsov Institute of Ecology and Evolution, RAS, Moscow, Russian Federation Hydrocarbon mineralization in soil undergoing phytoremediation was investigated in a laboratory experiment by estimating the variation in the 13С/12С ratio in the respired СО2. Hexadecane (HD) was used as a model hydrocarbon pollutant. The polluted soil was planted with winter rye (Secale cereale) inoculated with Azospirillum brasilense strain SR80, which combines the abilities to promote plant growth and to degrade oil hydrocarbon. Each vegetated treatment was accompanied with a corresponding nonvegetated one, and uncontaminated treatments were used as controls. Emission of carbon dioxide, its isotopic composition, and the residual concentration of HD in the soil were examined after two and four weeks. At the beginning of the experiment, the CO2-emission level was higher in the uncontaminated than in the contaminated soil. After two weeks, the quantity of emitted carbon dioxide decreased by about three times and did not change significantly in all uncontaminated treatments. The presence of HD in the soil initially increased CO2 emission, but later the respiration was reduced. During the first two weeks, nonvegetated soil had the highest CO2-emission level. Subsequently, the maximum increase in respiration was recorded in the vegetated contaminated treatments. The isotope composition of plant material determines the isotope composition of soil. The soil used in our experiment had an isotopic signature typical of soils formed by C3 plants (δ13C,-22.4‰). Generally, there was no significant fractionation of the carbon isotopes of the substrates metabolized by the

  7. An Enhanced In Vivo Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Model for Quantification of Drug Metabolism Enzymes*

    PubMed Central

    MacLeod, A. Kenneth; Fallon, Padraic G.; Sharp, Sheila; Henderson, Colin J.; Wolf, C. Roland; Huang, Jeffrey T.-J.

    2015-01-01

    Many of the enzymes involved in xenobiotic metabolism are maintained at a low basal level and are only synthesized in response to activation of upstream sensor/effector proteins. This induction can have implications in a variety of contexts, particularly during the study of the pharmacokinetics, pharmacodynamics, and drug–drug interaction profile of a candidate therapeutic compound. Previously, we combined in vivo SILAC material with a targeted high resolution single ion monitoring (tHR/SIM) LC-MS/MS approach for quantification of 197 peptide pairs, representing 51 drug metabolism enzymes (DME), in mouse liver. However, as important enzymes (for example, cytochromes P450 (Cyp) of the 1a and 2b subfamilies) are maintained at low or undetectable levels in the liver of unstimulated metabolically labeled mice, quantification of these proteins was unreliable. In the present study, we induced DME expression in labeled mice through synchronous ligand-mediated activation of multiple upstream nuclear receptors, thereby enhancing signals for proteins including Cyps 1a, 2a, 2b, 2c, and 3a. With this enhancement, 115 unique, lysine-containing, Cyp-derived peptides were detected in the liver of a single animal, as opposed to 56 in a pooled sample from three uninduced animals. A total of 386 peptide pairs were quantified by tHR/SIM, representing 68 Phase I, 30 Phase II, and eight control proteins. This method was employed to quantify changes in DME expression in the hepatic cytochrome P450 reductase null (HRN) mouse. We observed compensatory induction of several enzymes, including Cyps 2b10, 2c29, 2c37, 2c54, 2c55, 2e1, 3a11, and 3a13, carboxylesterase (Ces) 2a, and glutathione S-transferases (Gst) m2 and m3, along with down-regulation of hydroxysteroid dehydrogenases (Hsd) 11b1 and 17b6. Using DME-enhanced in vivo SILAC material with tHR/SIM, therefore, permits the robust analysis of multiple DME of importance to xenobiotic metabolism, with improved utility for the study of

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

    We assemble a versatile molecular scaffold from simple building blocks to create binary and multiplexed stable isotope reagents for quantitative mass spectrometry. Termed Protected Amine Labels (PAL), these reagents offer multiple analytical figures of merit including, (1) robust targeting of peptide N-termini and lysyl side chains, (2) optimal mass spectrometry ionization efficiency through regeneration of primary amines on labeled peptides, (3) an amino acid-based mass tag that incorporates heavy isotopes of carbon, nitrogen, and oxygen to ensure matched physicochemical and MS/MS fragmentation behavior among labeled peptides, and (4) a molecularly efficient architecture, in which the majority of hetero-atom centers can be used to synthesize a variety of nominal mass and sub-Da isotopologue stable isotope reagents. We demonstrate the performance of these reagents in well-established strategies whereby up to four channels of peptide isotopomers, each separated by 4 Da, are quantified in MS-level scans with accuracies comparable to current commercial reagents. In addition, we utilize the PAL scaffold to create isotopologue reagents in which labeled peptide analogs differ in mass based on the binding energy in carbon and nitrogen nuclei, thereby allowing quantification based on MS or MS/MS spectra. We demonstrate accurate quantification for reagents that support 6-plex labeling and propose extension of this scheme to 9-channels based on a similar PAL scaffold. Finally, we provide exemplar data that extend the application of isotopologe-based quantification reagents to medium resolution, quadrupole time-of-flight mass spectrometers.

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

    PubMed Central

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

    2013-01-01

    Chemical cross-linking of proteins followed by proteolysis and mass spectrometric analysis of the resulting cross-linked peptides provides powerful insight into the quaternary structure of protein complexes. Mixed-isotope cross-linking (a method for distinguishing intermolecular cross-links) was coupled with liquid chromatography and ion mobility separations and mass spectrometry (LC-IMS-MS) to provide an additional separation dimension to the traditional cross-linking approach. This method produced multiplet m/z peaks that are aligned in the IMS drift time dimension and serve as signatures of intermolecular cross-linked peptides. We developed an informatics tool to use the amino acid sequence information inherent in the multiplet spacing for accurate identification of the cross-linked peptides. Because of the separation of peptides and cross-linked peptides in drift time, our LC-IMS-MS approach was able to confidently detect more intermolecular cross-linked peptides than LC-MS alone. PMID:23423792

  10. Synthesis of deuterium-labeled prochlorperazine

    SciTech Connect

    Hawes, E.M.; Gurnsey, T.S.; Shetty, H.U.; Midha, K.K.

    1983-06-01

    The propylpiperazine side chain of prochlorperazine was labeled with two, four, or six deuterium atoms by lithium aluminum deuteride reduction of the appropriate amide. The isotopic purity of the products after correcting for chlorine isotopes was greater than 95.7%.

  11. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics*

    PubMed Central

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-01-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  12. Detection of reactive metabolites using isotope-labeled glutathione trapping and simultaneous neutral loss and precursor ion scanning with ultra-high-pressure liquid chromatography triple quadruple mass spectrometry.

    PubMed

    Huang, Ke; Huang, Lingyi; van Breemen, Richard B

    2015-04-01

    Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in the United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advanced knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra, which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes. PMID:25774910

  13. Characterization of the Tyrosine Kinase-Regulated Proteome in Breast Cancer by Combined use of RNA interference (RNAi) and Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) Quantitative Proteomics.

    PubMed

    Stebbing, Justin; Zhang, Hua; Xu, Yichen; Grothey, Arnhild; Ajuh, Paul; Angelopoulos, Nicos; Giamas, Georgios

    2015-09-01

    Tyrosine kinases (TKs) are central regulators in cellular activities and perturbations of TK signaling contribute to oncogenesis. However, less than half of the TKs have been thoroughly studied and a global functional analysis of their proteomic portrait is lacking. Here we conducted a combined approach of RNA interference (RNAi) and stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics to decode the TK-regulated proteome and associated signaling dynamics. As a result, a broad proteomic repertoire modulated by TKs was revealed, upon silencing of the 65 TKs expressed in MCF7 breast cancer cells. This yielded 10 new distinctive TK clusters according to similarity in TK-regulated proteome, each characterized by a unique signaling signature in contrast to previous classifications. We provide functional analyses and identify critical pathways for each cluster based on their common downstream targets. Analysis of different breast cancer subtypes showed distinct correlations of each cluster with clinical outcome. From the significantly up- and down-regulated proteins, we identified a number of markers of drug sensitivity and resistance. These data supports the role of TKs in regulating major aspects of cellular activity, but also reveals redundancy in signaling, explaining why kinase inhibitors alone often fail to achieve their clinical aims. The TK-SILACepedia provides a comprehensive resource for studying the global function of TKs in cancer. PMID:26089344

  14. The utility of stable isotope labeled (SIL) analogues in the bioanalysis of endogenous compounds by LC-MS applied to the study of bile acids in a metabolomics assay.

    PubMed

    Zheng, Joanna J; Shields, Eric E; Snow, Kimberly J; Nelson, David M; Olah, Timothy V; Reily, Michael D; Robertson, Donald G; Shipkova, Petia A; Stryker, Steven A; Xin, Baomin; Drexler, Dieter M

    2016-06-15

    The growing field of biomarker bioanalysis by liquid chromatography mass spectrometry (LC-MS) is challenged with the selection of suitable matrices to construct relevant and valid calibration curves resulting in not only precise but also accurate data. Because surrogate matrices are often employed with the associated concerns about the accuracy of the obtained data, here we present an assay using surrogate analytes in naive biological matrices. This approach is illustrated with the analysis of endogenous bile acids (e-BAs) in serum and plasma using stable isotope-labeled (SIL) analogues as calibration standards to address the matrix concerns. Several deuterated BAs (d-BAs) were used as standards representing respectively grouped e-BAs with structural similarity allowing for the simultaneous bioanalysis of 16 e-BA. The utility of this LC-MS assay employing d-BAs is demonstrated with the analysis of samples resultant of a controlled metabolomics study where a cohort of rats was fed/fasted to investigate the change of e-BAs dependent on food consumption and fasting time. PMID:27033006

  15. Detection of Reactive Metabolites Using Isotope-Labeled Glutathione Trapping and Simultaneous Neutral Loss and Precursor Ion Scanning with Ultra-High-Pressure Liquid Chromatography Triple Quadrupole Mass Spectrometry

    PubMed Central

    Huang, Ke; Huang, Lingyi; van Breemen, Richard B.

    2015-01-01

    Metabolic activation of drugs to electrophilic species is responsible for over 60% of black box warnings and drug withdrawals from the market place in United States. Reactive metabolite trapping using glutathione (GSH) and analysis using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) or HPLC with high resolution mass spectrometry (mass defect filtering) have enabled screening for metabolic activation to become routine during drug development. However, current MS-based approaches cannot detect all GSH conjugates present in complex mixtures, especially those present in extracts of botanical dietary supplements. To overcome these limitations, a fast triple quadrupole mass spectrometer-based approach was developed that can detect positively and negatively charged GSH conjugates in a single analysis without the need for advance knowledge of the elemental compositions of potential conjugates and while avoiding false positives. This approach utilized UHPLC instead of HPLC to shorten separation time and enhance sensitivity, incorporated stable-isotope labeled GSH to avoid false positives, and used fast polarity switching electrospray MS/MS to detect GSH conjugates that form positive and/or negative ions. The general new method was then used to test the licorice dietary supplement Glycyrrhiza glabra which was found to form multiple GSH conjugates upon metabolic activation. Among the GSH conjugates found in the licorice assay were conjugates with isoliquiritigenin and glabridin, which is an irreversible inhibitor of cytochrome P450 enzymes. PMID:25774910

  16. An analysis of ash and isotopic carbon discrimination (delta13C) methods to evaluate water use efficiency in apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apple cultivars are selected for fruit quality, disease and insect resistance, not water use efficiency (WUE), however, the need for more water use efficient crops is accelerating due to climate change and increased competition for water resources. On a whole plant basis, calculation of water use e...

  17. Intact Protein Quantitation Using Pseudoisobaric Dimethyl Labeling.

    PubMed

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

    2016-07-19

    Protein structural and functional studies rely on complete qualitative and quantitative information on protein species (proteoforms); thus, it is important to quantify differentially expressed proteins at their molecular level. Here we report our development of universal pseudoisobaric dimethyl labeling (pIDL) of amino groups at both the N-terminal and lysine residues for relative quantitation of intact proteins. Initial proof-of-principle study was conducted on standard protein myoglobin and hepatocellular proteomes (HepG2 vs LO2). The amino groups from both the N-terminal and lysine were dimethylated with HXHO (X = (13)C or C) and NaBY3CN (Y = H or D). At the standard protein level, labeling efficiency, effect of product ion size, and mass resolution on quantitation accuracy were explored; and a good linear quantitation dynamic range up to 50-fold was obtained. For the hepatocellular proteome samples, 33 proteins were quantified with RSD ≤ 10% from one-dimensional reversed phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) analysis of the 1:1 mixed samples. The method in this study can be extended to quantitation of other intact proteome systems. The universal "one-pot" dimethyl labeling of all the amino groups in a protein without the need of preblocking of those on the lysine residues is made possible by protein identification and quantitation analysis using ProteinGoggle 2.0 with customized databases of both precursor and product ions containing heavy isotopes. PMID:27359340

  18. Nutrition Labeling

    NASA Astrophysics Data System (ADS)

    Metzger, Lloyd E.

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

  19. 18O Labeling over a Coffee Break: A Rapid Strategy for Quantitative Proteomics

    PubMed Central

    Mirza, Shama P.; Greene, Andrew S.; Olivier, Michael

    2009-01-01

    Proteomics-based quantification methods for differential protein expression measurements are among the most important and challenging techniques in the field of mass spectrometry. Though numerous quantification methods have been established, no method meets all the demands for measuring accurate protein expression levels. Of the various relative quantification methods by isotopic labeling, 18O labeling method has been shown to be simple, specific, cost-effective and applicable to a wide range of analyses. However, some researchers refrain from using the method due to long incubation periods required during the labeling process. To address this problem, we demonstrate a method by which the labeling procedure can be completed in 15 min. We digested and labeled samples using immobilized trypsin on micro-spin columns to speed up the enzyme-mediated oxygen substitution, thereby completing the labeling process within 15 min with high labeling efficiency. We demonstrate the efficiency and accuracy of the method using a four protein mixture and whole cell lysate from rat vascular endothelial cells. PMID:18510357

  20. Soil organic carbon can be up-taken by plant roots and stored in plant biosilica: NanoSIMS and isotopic labeling evidences

    NASA Astrophysics Data System (ADS)

    Alexandre, Anne; Santos, Guaciara M.; balesdent, Jerôme; Basile-Doelsch, Isabelle; Borschneck, Daniel; Cazevieille, Patrick; Chevassus-Rosset, Claire; Doelsch, Emmanuel; Harutyunyan, Araks; Lemee, Laurent; Mazur, Jean-Charles; Reyerson, Paul; Signoret, Patrick

    2015-04-01

    Plant biosilica particles called phytoliths contain occluded organic compounds (phytC). Over the last few years, phytC content, nature, origin, paleoenvironmental meaning and impact in the global C cycle has been the subject of increasing debate[1, 2]. Inconsistencies in phytC quantification were fed by the scarcity of in-situ characterization of phytC in phytoliths and by inadequate extraction methods[3]. Very recently, 14C-AMS analyses of soil organic matter (SOM), amendments, plant tissues, atmospheric CO2 and phytolith samples, evidenced that a small but significant pool of phytC is not photosynthetic but derived from old SOM[4,5]. From there, several investigations were started to go further into the characterization of phytC and the mechanisms in play behind old SOM absorption by plant roots and old SOM occlusion in plant biosilica. Here, we first reconstruct at high spatial resolution the 3-dimentional location of phytC and its C/N signature using 3D X-ray microscopy and Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS). A pool of phytC appears homogeneously distributed in the silica structure and its C:N estimate is in the range of amino acid signatures[6]. Then, we use 13C and 15N-labelled amino acids monitored from an hydroponic solution to grass roots, stems, leaves and phytoliths to evidence that amino acids are absorbed as such by the roots and are concentrated in phytC rather than in the plant tissues. These findings strengthen and complement the 14C evidences. Both of them dissuade attempts to use phytC as a proxy of plant C. Further, they open new avenues of investigation regarding the processes which drive SOM mobilization by plant uptake, for a better understanding of soil/plant interactions involved in the terrestrial C cycle. [1] Santos et al. 2010. Radiocarbon 52:113 [2] Santos et al. 2012. Biogeosci. 9:1873 [3] Corbineau et al. 2013 R. Paleobot. Palyn. 197: 179 [4] Reyerson et al. 2013 AGU Fall meeting 2013 (1803125) [5] Santos et al. 2014

  1. Tree-Ring Stable Isotopes Reveal Twentieth-Century Increases in Water-Use Efficiency of Fagus sylvatica and Nothofagus spp. in Italian and Chilean Mountains

    PubMed Central

    Tognetti, Roberto; Lombardi, Fabio; Lasserre, Bruno; Cherubini, Paolo; Marchetti, Marco

    2014-01-01

    Changes in intrinsic water use efficiency (iWUE) were investigated in Fagus sylvatica and Nothofagus spp. over the last century. We combined dendrochronological methods with dual-isotope analysis to investigate whether atmospheric changes enhanced iWUE of Fagus and Nothofagus and tree growth (basal area increment, BAI) along latitudinal gradients in Italy and Chile. Post-maturation phases of the trees presented different patterns in δ13C, Δ13C, δ18O, Ci (internal CO2 concentration), iWUE, and BAI. A continuous enhancement in isotope-derived iWUE was observed throughout the twentieth century, which was common to all sites and related to changes in Ca (ambient CO2 concentration) and secondarily to increases in temperature. In contrast to other studies, we observed a general increasing trend of BAI, with the exception of F. sylvatica in Aspromonte. Both iWUE and BAI were uncoupled with the estimated drought index, which is in agreement with the absence of enduring decline in tree growth. In general, δ13C and δ18O showed a weak relationship, suggesting the major influence of photosynthetic rate on Ci and δ13C, and the minor contribution of the regulation of stomatal conductance to iWUE. The substantial warming observed during the twentieth century did not result in a clear pattern of increased drought stress along these latitudinal transects, because of the variability in temporal trends of precipitation and in specific responses of populations. PMID:25398040

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

    SciTech Connect

    Battipaglia, Giovanna; Saurer, Matthias; Cherubini, Paulo; Califapietra, Carlo; McCarthy, Heather R; Norby, Richard J; Cotrufo, M. Francesca

    2013-01-01

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

  3. Tree-ring stable isotopes reveal twentieth-century increases in water-use efficiency of Fagus sylvatica and Nothofagus spp. in Italian and Chilean mountains.

    PubMed

    Tognetti, Roberto; Lombardi, Fabio; Lasserre, Bruno; Cherubini, Paolo; Marchetti, Marco

    2014-01-01

    Changes in intrinsic water use efficiency (iWUE) were investigated in Fagus sylvatica and Nothofagus spp. over the last century. We combined dendrochronological methods with dual-isotope analysis to investigate whether atmospheric changes enhanced iWUE of Fagus and Nothofagus and tree growth (basal area increment, BAI) along latitudinal gradients in Italy and Chile. Post-maturation phases of the trees presented different patterns in δ13C, Δ13C, δ18O, Ci (internal CO2 concentration), iWUE, and BAI. A continuous enhancement in isotope-derived iWUE was observed throughout the twentieth century, which was common to all sites and related to changes in Ca (ambient CO2 concentration) and secondarily to increases in temperature. In contrast to other studies, we observed a general increasing trend of BAI, with the exception of F. sylvatica in Aspromonte. Both iWUE and BAI were uncoupled with the estimated drought index, which is in agreement with the absence of enduring decline in tree growth. In general, δ13C and δ18O showed a weak relationship, suggesting the major influence of photosynthetic rate on Ci and δ13C, and the minor contribution of the regulation of stomatal conductance to iWUE. The substantial warming observed during the twentieth century did not result in a clear pattern of increased drought stress along these latitudinal transects, because of the variability in temporal trends of precipitation and in specific responses of populations. PMID:25398040

  4. Monte Carlo simulation of a PhosWatch detector using Geant4 for xenon isotope beta-gamma coincidence spectrum profile and detection efficiency calculations.

    PubMed

    Mekarski, P; Zhang, W; Ungar, K; Bean, M; Korpach, E

    2009-10-01

    A simulation tool has been developed using the Geant4 Toolkit to simulate a PhosWatch single channel beta-gamma coincidence detection system consisting of a CsI(Tl)/BC404 Phoswich well detector and pulse shape analysis algorithms implemented digital signal processor. The tool can be used to simulate the detector's response for all the gamma rays and beta particles emitted from (135)Xe, (133m)Xe, (133)Xe, (131m)Xe and (214)Pb. Two- and three-dimensional beta-gamma coincidence spectra from the PhosWatch detector can be produced using the simulation tool. The accurately simulated spectra could be used to calculate system coincidence detection efficiency for each xenon isotope, the corrections for the interference from the various spectral components from radon and xenon isotopes, and system gain calibration. Also, it can generate two- and three-dimensional xenon reference spectra to test beta-gamma coincidence spectral deconvolution analysis software. PMID:19647444

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

    PubMed

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

    2011-05-01

    5-bromo-2-deoxyurudine (BrdU) can be used as a methodological tool for in vivo investigations following in vitro prelabeling of isolated stem cells for subsequent cell tracking within the recipient host. The objective of this study was to determine how useful BrdU may be as a labeling modality for adipose derived stem cells (ASC) by examining BrdU toxicity, BrdU intracellular stability, and potential effects on ASC differentiation. Porcine and human ASC (pASC and hASC, respectively) were labeled with BrdU at 5 or 10 μM for 2, 6, 24, and 48 h. BrdU toxicity and stability over time in monolayer cultures, in 3-D collagen scaffolds implanted to a porcine model and after thawing from long-term storage were evaluated by MTT assays and immunohistochemistry. ASC differentiation was evaluated by Oil Red O staining. BrdU was not cytotoxic at all tested concentrations and incubation times. BrdU color intensity within each cell and the number of ASC labeled with BrdU decreased as a function of both incubation time and BrdU concentrations. Labeling intensities decreased over time and were undetectable after 6 passages for pASC and 4 passages for hASC. In 3-D scaffolds, BrdU-labeled ASC were identifiable after 90 days of in vitro cultures and for 30 days in a porcine model. BrdU did not prevent preadipocyte differentiation and BrdU labeling was still detectable after subsequent thawing after long-term storage of ASC. BrdU is an excellent candidate reagent to label and track ASC that will allow distinction between BrdU-labeled donor cells and host cells. The data provides a foundation for conducting future tissue engineering projects using BrdU-labeled ASC. PMID:21246262

  6. Rapid and efficient localization of depth electrodes and cortical labeling using free and open source medical software in epilepsy surgery candidates

    PubMed Central

    Princich, Juan Pablo; Wassermann, Demian; Latini, Facundo; Oddo, Silvia; Blenkmann, Alejandro Omar; Seifer, Gustavo; Kochen, Silvia

    2013-01-01

    Depth intracranial electrodes (IEs) placement is one of the most used procedures to identify the epileptogenic zone (EZ) in surgical treatment of drug resistant epilepsy patients, about 20–30% of this population. IEs localization is therefore a critical issue defining the EZ and its relation with eloquent functional areas. That information is then used to target the resective surgery and has great potential to affect outcome. We designed a methodological procedure intended to avoid the need for highly specialized medical resources and reduce time to identify the anatomical location of IEs, during the first instances of intracranial EEG recordings. This workflow is based on established open source software; 3D Slicer and Freesurfer that uses MRI and Post-implant CT fusion for the localization of IEs and its relation with automatic labeled surrounding cortex. To test this hypothesis we assessed the time elapsed between the surgical implantation process and the final anatomical localization of IEs by means of our proposed method compared against traditional visual analysis of raw post-implant imaging in two groups of patients. All IEs were identified in the first 24 H (6–24 H) of implantation using our method in 4 patients of the first group. For the control group; all IEs were identified by experts with an overall time range of 36 h to 3 days using traditional visual analysis. It included (7 patients), 3 patients implanted with IEs and the same 4 patients from the first group. Time to localization was restrained in this group by the specialized personnel and the image quality available. To validate our method; we trained two inexperienced operators to assess the position of IEs contacts on four patients (5 IEs) using the proposed method. We quantified the discrepancies between operators and we also assessed the efficiency of our method to define the EZ comparing the findings against the results of traditional analysis. PMID:24427112

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

    PubMed

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

    2001-01-01

    Monoclonal antibodies C215, reactive with colorectal carcinomas, and MOv18, reactive with most of the ovarian carcinomas, were radiohalogenated with [211At]astatine. The radiohalogen was conjugate coupled to antibodies via the intermediate labelling reagent N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE) in a two-step, single-pot reaction. Optimisation of the labelling of the reagent was achieved using N-iodosuccinimide, NIS, as the oxidising agent. The yields ranged from 69-95% in the labelling of 0.1-1.0 nmole of the m-MeATE precursor. Subsequent conjugation to antibodies resulted in yields of 58+/-7%. In vitro binding to tumour cells showed that the immunoreactivity of both antibodies was retained after astatine labelling. PMID:11182562

  8. Evidence of the chemical reaction of (18)O-labelled nitrite with CO2 in aqueous buffer of neutral pH and the formation of (18)OCO by isotope ratio mass spectrometry.

    PubMed

    Tsikas, Dimitrios; Böhmer, Anke; Gros, Gerolf; Endeward, Volker

    2016-05-01

    Inorganic nitrite (NO2(-), ON-O(-) ←→ (-)O-NO) is the autoxidation product of nitric oxide (NO). Nitrite can also be formed from inorganic nitrate (ONO2(-)), the major oxidation product of NO in erythrocytes, by the catalytic action of bacterial nitrate reductase in gut and oral microflora. Nitrite can be reduced to NO by certain cellular proteins and enzymes, as well as in the gastric juice under acidic conditions. Hemoglobin, xanthine oxidoreductase and carbonic anhydrase (CA) have been reported to convert nitrite to NO. Renal CA isoforms are involved in the reabsorption of nitrite and may, therefore, play an important role in NO homeostasis. Yet, the mechanisms underlying the action of CA on nitrite are incompletely understood. The nitrate/nitrite system is regarded as a reservoir of NO. We have recently shown that nitrite reacts chemically with carbon dioxide (CO2), the regular substrate of CA. The present communication reports a stable isotope ratio mass spectrometry (IRMS) study on the reaction of NO2(-) and CO2 performed in 50 mM HEPES buffer of pH 7.4 at 37 °C. By using (18)O-labelled nitrite ((18)ON-O(-)/(-18)O-NO) and CO2 we observed formation of (18)O-labelled CO2. This finding is an unequivocal evidence of the chemical reaction of (18)ON-O(-)/(-18)O-NO with CO2. The reaction is rapid and involves nucleophilic attack of the negatively charged nitrite via one of its oxygen atoms on the partially positively charged CO2 molecule to form the putative intermediate (18)ON-O-CO2(-)/(-)O2C-(18)O-NO. The by far largest fraction of this intermediate decomposes back to (18)ON-O(-)/(-18)O-NO and CO2. A very small fraction of the intermediate, however, rearranges and finally decomposes to form (18)OCO and nitrite. This reaction is slower in the presence of an isolated erythrocytic CA isoform II. In summary, NO2(-), CO2 and CA are ubiquitous. The chemical reaction of NO2(-) with CO2 and its modulation by CA isoforms may play important roles in the transport of

  9. The pharmacokinetic behavior of the soy isoflavone metabolite S-(−)equol and its diastereoisomer R-(+)equol in healthy adults determined by using stable-isotope-labeled tracers1234

    PubMed Central

    Zhao, Xueheng; Jha, Pinky; Heubi, James E; Brown, Nadine M

    2009-01-01

    Background: The nonsteroidal estrogen equol occurs as diastereoisomers, S-(−)equol and R-(+)equol, both of which have significant biological actions. S-(−)equol, the naturally occurring enantiomer produced by 20–30% of adults consuming soy foods, has selective affinity for estrogen receptor-β, whereas both enantiomers modulate androgen action. Little is known about the pharmacokinetics of the diastereoisomers, despite current interest in developing equol as a nutraceutical or pharmaceutical agent. Objective: The objective was to compare the pharmacokinetics of S-(−)equol and R-(+)equol by using [13C] stable-isotope-labeled tracers to facilitate the optimization of clinical studies aimed at evaluating the potential of these diastereoisomers in the prevention and treatment of estrogen- and androgen-dependent conditions. Design: A randomized, crossover, open-label study in 12 healthy adults (6 men and 6 women) compared the plasma and urinary pharmacokinetics of orally administered enantiomeric pure forms of S-(−)[2-13C]equol, R-(+)[2-13C]equol, and the racemic mixture. Plasma and urinary [13C]R-equol and [13C]S-equol concentrations were measured by tandem mass spectrometry. Results: Plasma [13C]equol concentration appearance and disappearance curves showed that both enantiomers were rapidly absorbed, attained high circulating concentrations, and had a similar terminal elimination half-life of 7–8 h. The systemic bioavailability and fractional absorption of R-(+)[2-13C]equol were higher than those of S-(−)[2-13C]equol or the racemate. The pharmacokinetics of racemic (±)[2-13C]equol were different from those of the individual enantiomers: slower absorption, lower peak plasma concentrations, and lower systemic bioavailability. Conclusions: The high bioavailability of both diastereoisomers contrasts with previous findings for the soy isoflavones daidzein and genistein, both of which have relatively poor bioavailability, and suggests that low doses of equol

  10. Phosphoprotein Isotope-Coded Solid-Phase Tag Approach for Enrichment and Quantitative Analysis of Phosphopeptides from Complex Mixtures

    SciTech Connect

    Qian, Weijun ); Goshe, Michael B.; Camp, David G. ); Yu, Li-Rong ); Tang, Keqi ); Smith, Richard D. )

    2003-10-15

    Many cellular processes are regulated by reversible protein phosphorylation and the ability to identify and quantify phosphoproteins from proteomes is essential for gaining a better understanding of these dynamic cellular processes. However, a sensitive, efficient and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a Phosphoprotein Isotope-coded Solid-phase Tag (PhIST) for isolating and measuring the relative abundance of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported Phosphoprotein Isotope-coded Affinity Tag (PhIAT)approach developed by our laboratory1-2, where the O-phosphate moiety on phosphoseryl or phosphothreonyl residues were derivatized by hydroxide ion-medated B-elimination followed by the addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary LC-MS/MS. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures was demonstrated using casein phosphoproteins. Its utility for proteomic applications is demonstrated by the labeling of soluble proteins from human breast cancer cell line.

  11. Integrated Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Quantitative Proteomic Analysis Identifies Galectin-1 as a Potential Biomarker for Predicting Sorafenib Resistance in Liver Cancer*

    PubMed Central

    Yeh, Chao-Chi; Hsu, Chih-Hung; Shao, Yu-Yun; Ho, Wen-Ching; Tsai, Mong-Hsun; Feng, Wen-Chi; Chow, Lu-Ping

    2015-01-01

    Sorafenib has become the standard therapy for patients with advanced hepatocellular carcinoma (HCC). Unfortunately, most patients eventually develop acquired resistance. Therefore, it is important to identify potential biomarkers that could predict the efficacy of sorafenib. To identify target proteins associated with the development of sorafenib resistance, we applied stable isotope labelling with amino acids in cell culture (SILAC)-based quantitative proteomic approach to analyze differences in protein expression levels between parental HuH-7 and sorafenib-acquired resistance HuH-7 (HuH-7R) cells in vitro, combined with an isobaric tags for relative and absolute quantitation (iTRAQ) quantitative analysis of HuH-7 and HuH-7R tumors in vivo. In total, 2,450 quantified proteins were identified in common in SILAC and iTRAQ experiments, with 81 showing increased expression (>2.0-fold) with sorafenib resistance and 75 showing decreased expression (<0.5-fold). In silico analyses of these differentially expressed proteins predicted that 10 proteins were related to cancer with involvements in cell adhesion, migration, and invasion. Knockdown of one of these candidate proteins, galectin-1, decreased cell proliferation and metastasis in HuH-7R cells and restored sensitivity to sorafenib. We verified galectin-1 as a predictive marker of sorafenib resistance and a downstream target of the AKT/mTOR/HIF-1α signaling pathway. In addition, increased galectin-1 expression in HCC patients' serum was associated with poor tumor control and low response rate. We also found that a high serum galectin-1 level was an independent factor associated with poor progression-free survival and overall survival. In conclusion, these results suggest that galectin-1 is a possible biomarker for predicting the response of HCC patients to treatment with sorafenib. As such, it may assist in the stratification of HCC and help direct personalized therapy. PMID:25850433

  12. 18O-Labeled Proteome Reference as Global Internal Standards for Targeted Quantification by Selected Reaction Monitoring-Mass Spectrometry

    SciTech Connect

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

    2011-10-11

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

  13. Spin labeling EPR.

    PubMed

    Klare, Johann P; Steinhoff, Heinz-Jürgen

    2009-01-01

    Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy has emerged as an efficient tool to elucidate the structure and conformational dynamics of biomolecules under native-like conditions. This article summarizes the basics as well as recent progress of site-directed spin labeling. Continuous wave EPR spectra analyses and pulse EPR techniques are reviewed with special emphasis on applications to the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis and the photosynthetic reaction center from Rhodobacter sphaeroides R26. PMID:19728138

  14. Quantitative Analysis of Isotope Distributions In Proteomic Mass Spectrometry Using Least-Squares Fourier Transform Convolution

    PubMed Central

    Sperling, Edit; Bunner, Anne E.; Sykes, Michael T.; Williamson, James R.

    2008-01-01

    Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic labeling and fractional residue labeling. We have developed a general and flexible analytical treatment of the complex isotope distributions that arise in these experiments, using Fourier transform convolution to calculate labeled isotope distributions and least-squares for quantitative comparison with experimental peaks. The degree of fractional atomic and fractional residue labeling can be determined from experimental peaks at the same time as the integrated intensity of all of the isotopomers in the isotope distribution. The approach is illustrated using data with fractional 15N-labeling and fractional 13C-isoleucine labeling. The least-squares Fourier transform convolution approach can be applied to many types of quantitive proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments. PMID:18522437

  15. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 3 2010-01-01 2010-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  16. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 10 Energy 3 2012-01-01 2012-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  17. 10 CFR 431.31 - Labeling requirements.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 10 Energy 3 2014-01-01 2014-01-01 false Labeling requirements. 431.31 Section 431.31 Energy DEPARTMENT OF ENERGY ENERGY CONSERVATION ENERGY EFFICIENCY PROGRAM FOR CERTAIN COMMERCIAL AND INDUSTRIAL EQUIPMENT Electric Motors Labeling § 431.31 Labeling requirements. (a) Electric motor nameplate—(1)...

  18. 10 CFR 431.31 - Labeling requirements.

    Code of Federal