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1

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

NASA Astrophysics Data System (ADS)

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

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

2010-12-01

2

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

Microsoft Academic Search

The use of stable isotope enriched plant material can provide an unobstructed method of studying ecosystem nutrient dynamics between plants, soil, and atmosphere. However, the production of uniformly labeled perennial plant material is challenging due to plant physiological constraints and the mechanics of building and operating an isotope labeling system. In this study we present the design of a novel

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

2010-01-01

3

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

PubMed

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

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

2014-02-01

4

Isotope Labeling in Insect Cells  

PubMed Central

Recent years have seen remarkable progress in applying nuclear magnetic resonance (NMR) spectroscopy to proteins that have traditionally been difficult to study due to issues with folding, posttranslational modification, and expression levels or combinations thereof. In particular, insect cells have proved useful in allowing large quantities of isotope-labeled, functional proteins to be obtained and purified to homogeneity, allowing study of their structures and dynamics by using NMR. Here, we provide protocols that have proven successful in such endeavors.

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

2011-01-01

5

Carbon isotope labelling in graphene research.  

PubMed

The large scale production of graphene for any potential application relies on catalytic chemical vapour deposition (CVD). Despite much effort put into the graphene CVD research, there are still many challenges to be solved, not only concerning the growth itself, but also the subsequent treatment, i.e. transfer from the catalyst to a desired substrate. The need for fast progress naturally necessitates easy-to-use, versatile and efficient characterization methods. This perspective reviews the recent advances and potential of probably the most prospective one--Raman spectroscopy in connection with carbon isotope labelling of the CVD grown graphene layers. We discuss its use for the explanation and optimization of the growth process, followed by examples of employing isotope engineering in the studies of fundamental properties of graphene, not only by Raman spectroscopy. PMID:24817019

Frank, O; Kavan, L; Kalbac, M

2014-06-21

6

Carbon isotope labelling in graphene research  

NASA Astrophysics Data System (ADS)

The large scale production of graphene for any potential application relies on catalytic chemical vapour deposition (CVD). Despite much effort put into the graphene CVD research, there are still many challenges to be solved, not only concerning the growth itself, but also the subsequent treatment, i.e. transfer from the catalyst to a desired substrate. The need for fast progress naturally necessitates easy-to-use, versatile and efficient characterization methods. This perspective reviews the recent advances and potential of probably the most prospective one - Raman spectroscopy in connection with carbon isotope labelling of the CVD grown graphene layers. We discuss its use for the explanation and optimization of the growth process, followed by examples of employing isotope engineering in the studies of fundamental properties of graphene, not only by Raman spectroscopy.

Frank, O.; Kavan, L.; Kalbac, M.

2014-05-01

7

Isotope-labeled immunoassays without radiation waste  

PubMed Central

The practice of immunoassay has experienced a widespread transition from radioisotopic labeling to nonisotopic labeling over the last two decades. Radioisotope labels have drawbacks that hamper their applications: (i) perceived radiation hazards of reagents, (ii) regulatory requirements and disposal problems of working with radioactive materials, and (iii) short shelf-life of the labeled reagents. The advantage of isotopic labeling is the incorporation into analytes without altering structure or reactivity, as is often the case with ELISA or fluorescent detection systems. We developed a format for isotope label immunoassay with the long-life isotope 14C as the label and accelerator mass spectrometer (AMS) as the detection system. AMS quantifies attomole levels of several isotopes, including 14C. With this exquisite sensitivity, the sensitivity of an immunoassay is limited by the Kd of the antibody and not the detection system. The detection limit of the assays for atrazine and 2,3,7,8-tetrachlorodibenzo-p-dioxin was 2.0 × 10?10 M and 2.0 × 10?11 M, respectively, approximately an order of magnitude below the standard enzyme immunoassay. Notably, <1 dpm (0.45 pCi) of 14C-labeled compound was used in each assay, which is well below the limit of disposal (50 nCi per g) as nonradioactive waste. Thus, endogenous reporter ligands quantified by AMS provide the advantages of an RIA without the associated problems of radioactive waste.

Shan, Guomin; Huang, Wei; Gee, Shirley J.; Buchholz, Bruce A.; Vogel, John S.; Hammock, Bruce D.

2000-01-01

8

Simple, rapid method for the preparation of isotopically labeled formaldehyde  

DOEpatents

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.

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

2011-10-04

9

Intrinsic isotopic 13C labelling of polyphenols.  

PubMed

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

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

2013-12-01

10

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

PubMed Central

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.

Basu, Sankha S; Blair, Ian A

2013-01-01

11

Analysis of proteome dynamics in mice by isotopic labeling.  

PubMed

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

Price, John C; Ghaemmaghami, Sina

2014-01-01

12

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

Microsoft Academic Search

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

Fluor Daniel Hanford

1997-01-01

13

Visual exploration of isotope labeling networks in 3D.  

PubMed

Isotope labeling networks (ILNs) are graphs explaining the flow of isotope labeled molecules in a metabolic network. Moreover, they are the structural backbone of metabolic flux analysis (MFA) by isotopic tracers which has been established as a standard experimental tool in fluxomics. To configure an isotope labeling experiment (ILE) for MFA, the structure of the corresponding ILN must be understood to a certain extent even by a practitioner. Graph algorithms help to analyze the network structure but produce rather abstract results. Here, the major obstruction is the high dimension of these networks and the large number of network components which, consequently, are hard to figure out manually. At the interface between theory and experiment, the three-dimensional interactive visualization tool CumoVis has been developed for exploring the network structure in a step by step manner. Navigation and orientation within ILNs are supported by exploiting the natural 3D structure of an underlying metabolite network with stacked labeled particles on top of each metabolite node. Network exploration is facilitated by rotating, zooming, forward and backward path tracing and, most important, network component reduction. All features of CumoVis are explained with an educational example and a realistic network describing carbon flow in the citric acid cycle. PMID:18074156

Droste, P; Weitzel, M; Wiechert, W

2008-04-01

14

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

PubMed Central

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.

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

2013-01-01

15

Orientational ordering in liquid crystals: isotope labelling neutron diffraction experiments  

Microsoft Academic Search

Orientational order parameters for nematogens have been determined using the neutron diffraction isotope labelling technique, where the single molecule component of the scattering from mixtures of protonated and perdeuteriated molecules is extracted at low Q. Experiments were performed on the relatively rigid nematogens 4-methoxy-4?-cyanobiphenyl (1-OCB) and 4,4?-dimethoxyazoxybenzene (PAA). Legendre polynomial orientational order parameters -PL were extracted from the anisotropy of

R. W. Date; I. W. Hamley; G. R. Luckhurst; J. M. Seddon; R. M. Richardson

1992-01-01

16

Orientational ordering in liquid crystals: isotope labelling neutron diffraction experiments  

Microsoft Academic Search

Orientational order parameters for nematogens have been determined using the neutron diffraction isotope labelling technique, where the single molecule component of the scattering from mixtures of protonated and perdeuteriated molecules is extracted at low Q. Experiments were performed on the relatively rigid nematogens 4-methoxy-4'-cyanobiphenyl (1-OCB) and 4,4'-dimethoxyazoxybenzene (PAA). Legendre polynomial orientational order parameters -PL were extracted from the anisotropy of

R. W. Date; I. W. Hamley; G. R. Luckhurst; J. M. Seddon; R. M. Richardson

1992-01-01

17

GLYCAN REDUCTIVE ISOTOPE LABELING (GRIL) FOR QUANTITATIVE GLYCOMICS  

PubMed Central

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

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

2009-01-01

18

Quantitating isotopic molecular labels with accelerator mass spectrometry.  

PubMed

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

Vogel, John S; Love, Adam H

2005-01-01

19

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

PubMed

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 (13)C with (15)N, (18)O or (2)H 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 formation(1-4). 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 storage(5-7). 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 (13)C and (15)N 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 (13)C and (15)N 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%(13)C and 6.7 atom%(15)N uniform plant label, or material that is differentially labeled by up to 1.29 atom%(13)C and 0.56 atom%(15)N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration, and light levels in an airtight (13)C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling. PMID:24457314

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

2014-01-01

20

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

PubMed Central

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or 2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components. Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system's ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration, and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

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

2014-01-01

21

Relative Quantitation of Glycopeptides Based on Stable Isotope Labeling Using MALDI-TOF MS.  

PubMed

We have developed an effective, sensitive method for quantitative glycopeptide profiling using stable isotope labeling and MALDI-TOF mass spectrometry (MS). In this study, we synthesized benzoic acid-d0 N-succinimidyl ester (BzOSu) and benzoic acid-d5 N-succinimidyl ester (d-BzOSu) as light and heavy isotope reagents for stable isotope quantification for the comparative analysis of glycopeptides. Using this approach provided enhanced ionization efficiency in both positive and negative modes by MALDI-TOF MS. These reagents were quantitatively reacted with glycopeptides from human serum IgG (hIgG) at a wide range of concentrations; the labeling efficiency of the glycopeptides showed high reproducibility and a good calibration curve was obtained. To demonstrate the practical utility of this approach, we characterized the structures of glycopeptides from hIgG and from IgG1 produced by myeloma plasma. The glycopeptides were quantitatively analyzed by mixing Bz-labeled IgG1 glycopeptides with d-Bz-labeled hIgG glycopeptides. Glycan structural identification of the hIgG glycopeptides was demonstrated by combining the highly specific recognition of endo-?-N-acetyl glucosaminidases from Streptococcus pyogenes (endoS) or from Streptococcus pneumoniae (endo-D) with MALDI-TOF MS analysis. The obtained data revealed the glycan profile and the ratio of glycan structural isomers containing a galactosylated extension on IgG1, IgG2 and IgG3 glycopetides. PMID:25010467

Kurogochi, Masaki; Amano, Junko

2014-01-01

22

Mathematical modeling of isotope labeling experiments for metabolic flux analysis.  

PubMed

Isotope labeling experiments (ILEs) offer a powerful methodology to perform metabolic flux analysis. However, the task of interpreting data from these experiments to evaluate flux values requires significant mathematical modeling skills. Toward this, this chapter provides background information and examples to enable the reader to (1) model metabolic networks, (2) simulate ILEs, and (3) understand the optimization and statistical methods commonly used for flux evaluation. A compartmentalized model of plant glycolysis and pentose phosphate pathway illustrates the reconstruction of a typical metabolic network, whereas a simpler example network illustrates the underlying metabolite and isotopomer balancing techniques. We also discuss the salient features of commonly used flux estimation software 13CFLUX2, Metran, NMR2Flux+, FiatFlux, and OpenFLUX. Furthermore, we briefly discuss methods to improve flux estimates. A graphical checklist at the end of the chapter provides a reader a quick reference to the mathematical modeling concepts and resources. PMID:24218213

Nargund, Shilpa; Sriram, Ganesh

2014-01-01

23

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

Microsoft Academic Search

Whenever one wants to distinguish, recognize, and\\/or measure objects (connected components) in binary images, labeling is required. This paper presents two efficient label-equiv- alence-based connected-component labeling algorithms for 3-D binary images. One is voxel based and the other is run based. For the voxel-based one, we present an efficient method of deciding the order for checking voxels in the mask.

Lifeng He; Yuyan Chao; Kenji Suzuki

2011-01-01

24

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

PubMed Central

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.

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

2011-01-01

25

Hydrolysis of adenosine 5'-triphosphate: an isotope-labeling study  

SciTech Connect

A combination of /sup 18/O-labeling experiments and kinetic studies to clarify the nonenzymatic hydrolytic pathways of adenosine 5'-triphosphate (ATP) at pH values ranging from 0 to 8.3 has been used in this experiment. In 1 N and 0.1 N HCl, the data are consistent with the hypothesis that hydrolysis occurs by addition-elimination, with initial attack 93% ..gamma.. and 7% ..beta..; both lead only to ADP + P/sub i/. In the subsequent hydrolysis of the ADP to AMP + P/sub i/, attack is 83% ..beta.. and 17% ..cap alpha... At pH 8.3, the data are consistent with the hypothesis that hydrolsysis occurs by elimination-addition. Over the entire pH range studied, no oxygen exchange between water and ATP, ADP, or P/sub i/ was detected. Nonenzymatic hydrolysis and isotopic analysis of the resultant P/sub i/ comprise a preferred means of assaying the isotopic enrichment of (..gamma..-/sup 18/O)ATP to be used in studies of enzymatic processes.

Meyerson, S. (Standard Oil Co. (Indiana), Naperville, IL); Kuh, E.S.; Ramirez, F.; Marecek, J.F.

1982-12-15

26

General statistical framework for quantitative proteomics by stable isotope labeling.  

PubMed

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

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

2014-03-01

27

Who eats what, where and when? Isotope-labelling experiments are coming of age  

Microsoft Academic Search

Isotope-labelling experiments have changed the way microbial ecologists investigate the ecophysiology of microbial populations and cells in the environment. Insight into the ‘uncultivated majority’ accompanies methodology that involves the incorporation of stable isotopes or radioisotopes into sub-populations of environmental samples. Subsequent analysis of labelled biomarkers of sub-populations with stable-isotope probing (DNA-SIP, RNA-SIP, phospholipid-derived fatty acid-SIP) or individual cells with a

Josh D Neufeld; Michael Wagner; J Colin Murrell

2007-01-01

28

MALDI biotyper-based rapid resistance detection by stable-isotope labeling.  

PubMed

Against the background of increasing numbers of resistant microorganisms, the fast and cost-efficient detection of microbial resistance is an important clinical requirement for optimal therapeutic intervention. Current routine assays take at least 5 h, but in most cases an overnight incubation is necessary to identify resistant isolates. The usage of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) profiling in combination with growth media containing isotopically labeled amino acids facilitates the detection of resistant microorganisms after 3 h or less directly from the profile spectrum. Growing microorganisms incorporate isotopically labeled amino acids, increasing protein masses and thereby leading to mass shifts of their corresponding peaks in the profile spectra. In the presence of antibiotics, only resistant microorganisms are able to grow and to incorporate the labeled amino acids. This leads to a difference in the mass spectra of susceptible and resistant isolates, allowing their differentiation. In the presented study, we demonstrated the applicability of this novel approach for the detection of methicillin-resistant Staphylococcus aureus and tested different bioinformatics approaches for automated data interpretation. PMID:24006001

Sparbier, Katrin; Lange, Christoph; Jung, Jette; Wieser, Andreas; Schubert, Sören; Kostrzewa, Markus

2013-11-01

29

MALDI Biotyper-Based Rapid Resistance Detection by Stable-Isotope Labeling  

PubMed Central

Against the background of increasing numbers of resistant microorganisms, the fast and cost-efficient detection of microbial resistance is an important clinical requirement for optimal therapeutic intervention. Current routine assays take at least 5 h, but in most cases an overnight incubation is necessary to identify resistant isolates. The usage of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) profiling in combination with growth media containing isotopically labeled amino acids facilitates the detection of resistant microorganisms after 3 h or less directly from the profile spectrum. Growing microorganisms incorporate isotopically labeled amino acids, increasing protein masses and thereby leading to mass shifts of their corresponding peaks in the profile spectra. In the presence of antibiotics, only resistant microorganisms are able to grow and to incorporate the labeled amino acids. This leads to a difference in the mass spectra of susceptible and resistant isolates, allowing their differentiation. In the presented study, we demonstrated the applicability of this novel approach for the detection of methicillin-resistant Staphylococcus aureus and tested different bioinformatics approaches for automated data interpretation.

Sparbier, Katrin; Lange, Christoph; Jung, Jette; Wieser, Andreas; Schubert, Soren

2013-01-01

30

Quantification of isotopically overlapping deamidated and 18o-labeled peptides using isotopic envelope mixture modeling.  

PubMed

A robust peptide quantification method was developed where overlapping peptide isotopic distributions were fit with predicted peptide isotopic envelope mixture models (IEMMs). Application to two difficult quantitative problems was demonstrated. The first was the quantification of deamidation, where masses of isotopic peaks differ by 1 Da, and the second was (18)O labeling, where the isotopic peaks are shifted 2 and 4 Da. In both cases, peptide quantification cannot be performed by simple integration of extracted ion chromatograms, because the isotopic envelopes of mass-shifted peptides are normally not resolved. To test the methodology for quantification of deamidation, several synthetic peptides and their corresponding deamidated forms were mixed at various ratios (1:0, 1:2, 2:1, 4:1, 10:1, and 20:1) and analyzed using the IEMM method, resulting in a high correlation (R(2) = 0.96) between measured and known percentages of deamidation. The IEMM method was then incorporated into a workflow for deamidation quantification in a large-scale proteomics experiment. A series of normal (3 day, 2 year, 35 year, and 70 year) and cataractous (93 year) human lenses were analyzed using two-dimensional liquid chromatography tandem mass spectrometry, and deamidation quantities of several gammaS-crystallin peptides ([N14-Q16], N53, [Q63-Q70], and N143) were determined. Two peptides (N53 and [Q63-Q70]) had more extensive deamidation in the water-insoluble portions of normal lens samples, and deamidation at N143 was more extensive in the 93 year water-insoluble cataractous sample. The utility of the technique for analysis of (18)O-labeled peptides was examined using mixtures of labeled BSA peptides in known (16)O/(18)O ratios (10:1, 4:1, 1:1, 1:4, and 1:10). The methodology allowed for accurate measurements of ratios of (16)O/(18)O peptides over the wide range of relative abundances. PMID:19173613

Dasari, Surendra; Wilmarth, Phillip A; Reddy, Ashok P; Robertson, Lucinda J G; Nagalla, Srinivasa R; David, Larry L

2009-03-01

31

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

PubMed

Metabolic labeling of rodent proteins with ¹?N, 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 ¹?N-enriched spirulina diet. As a case study, we also demonstrate the application of ¹?N-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

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

2013-06-15

32

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

SciTech Connect

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.

Jewett, J.R., Fluor Daniel Hanford

1997-02-24

33

Dye-doped silica nanoparticles as efficient labels for glycans.  

PubMed

We report that dye-doped fluorescent silica nanoparticles (FSNPs) are highly efficient labels for glycans. Mono- and oligo-saccharides were conjugated to FSNPs using a general photocoupling chemistry. FSNP-labeled glycans were applied to image and detect bacteria, and to study carbohydrate-lectin interactions on a lectin microarray. PMID:21380421

Wang, Xin; Ramström, Olof; Yan, Mingdi

2011-04-14

34

Energy efficiency labelling in a Common Market (Australia).  

National Technical Information Service (NTIS)

The paper describes the compulsory and voluntary energy efficiency labelling schemes for household white goods (refrigerators, freezers, air-conditioners, dish-washers and heaters) as they were first applied in the Australian states of New South Wales and...

R. A. Kraemer

1991-01-01

35

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

PubMed

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 [²H?]-2-methylpyrazine (d-1), [²H?]-2-ethylpyrazine (d-2), [²H?]-2,3(or 6)-dimethylpyrazine (d-3A, d-3B), [²H?]-2,[²H?]-6-dimethylpyrazine (d-3C), [²H?]-2,[²H?]-6-diethylpyrazine (d-4), [²H?]-2-ethyl-3(or 6)-methylpyrazine (d-5A, d-5B), 2,[²H?]-3,5-trimethylpyrazine (d-6), [²H?]-2-ethyl-3,6-dimethylpyrazine (d-7), [²H?]-2-ethyl-3,5-dimethylpyrazine (d-8), and 2,3-diethyl-[²H?]-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

Fang, Mingchih; Cadwallader, Keith R

2013-04-17

36

A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)  

Microsoft Academic Search

Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural (''light'') amino acids are replaced by ''heavy'' SILAC amino acids. Cells grown in

Shao-En Ong; Matthias Mann

2006-01-01

37

A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)  

Microsoft Academic Search

Stable isotope labeling by amino acids in cell culture (SILAC) is a simple, robust, yet powerful approach in mass spectrometry (MS)-based quantitative proteomics. SILAC labels cellular proteomes through normal metabolic processes, incorporating non-radioactive, stable isotope-containing amino acids in newly synthesized proteins. Growth medium is prepared where natural (“light”) amino acids are replaced by “heavy” SILAC amino acids. Cells grown in

Matthias Mann; Shao-En Ong

2007-01-01

38

Kinetic isotope effects significantly influence intracellular metabolite (13) C labeling patterns and flux determination.  

PubMed

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from (13) C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of (13) C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the (13) C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the (13) C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in (13) C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC-MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in (13) C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions. PMID:23828762

Wasylenko, Thomas M; Stephanopoulos, Gregory

2013-09-01

39

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

PubMed Central

Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from 13C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of 13C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the 13C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the 13C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in 13C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in 13C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.

Wasylenko, Thomas M.; Stephanopoulos, Gregory

2014-01-01

40

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

PubMed Central

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

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

2014-01-01

41

An automated method for the analysis of stable isotope labeling data in proteomics.  

PubMed

An algorithm is presented for the generation of a reliable peptide component peak table from liquid chromatography-mass spectrometry (LC-MS) and subsequent quantitative analysis of stable isotope coded peptide samples. The method uses chemical noise filtering, charge state fitting, and deisotoping toward improved analysis of complex peptide samples. Overlapping peptide signals in mass spectra were deconvoluted by correlation with modeled peptide isotopic peak profiles. Isotopic peak profiles for peptides were generated in silico from a protein database producing reference model distributions. Doublets of heavy and light labeled peak clusters were identified and compared to provide differential quantification of pairs of stable isotope coded peptides. Algorithms were evaluated using peptides from digests of a single protein and a seven-protein mixture that had been differentially coded with stable isotope labeling agents and mixed in known ratios. The experimental results correlated well with known mixing ratios. PMID:15922621

Zhang, Xiang; Hines, Wade; Adamec, Jiri; Asara, John M; Naylor, Stephen; Regnier, Fred E

2005-07-01

42

The use of stable isotope labelling for the analytical chemistry of drugs.  

PubMed

This perspective reviews the potential for stable isotope labelling to examine the metabolic transformations of drugs. The increased sensitivity and widespread availability of modern nuclear magnetic resonance (NMR) and high-resolution mass spectrometers will increase the application of stable isotopes to study drug metabolism. Creating mass doublets by mixing a natural isotopic abundance compound with a labelled isotopomer and applying stable isotope filtering to high resolution mass spectrometry allows one to rapidly identify drug metabolites in very complex samples, such as blood or urine. Applying this approach to drug metabolism will require a significant synthesis effort. The relatively small number of (13) C, (15) N, or (17,18) O-labelled precursors exacerbates this problem, making the synthesis of the labelled drug often more difficult than that of the parent compound. We have developed new strategies for stable isotope labelling of complex molecules based on the rich chemistry of [(13) C]methyl phenyl sulfide, where the phenylthio group acts as a stable, non-volatile carrier for the valuable (13) C-label. For example we have used [(13) C]methyl phenyl sulfide to prepare the three possible (13) C-isotopomers ([1-(13) C]-, [2-(13) C]-, [1,2-(13) C(2) ]) of the two carbon precursors, ethyl 2-(phenylthio) acetate and ethyl N,N-dimethyl oxamate. In each case, these two-carbon labelling precursors are asymmetric and the differential reactivity of the carbons allows for either/or (13) C-labelling in the products. We demonstrate the utility of these two carbon precursors in the synthesis of aromatic ring-labelled N-(4-hydroxyphenyl)acetamide (acetaminophen or paracetamol). PMID:22170639

Unkefer, Clifford J; Martinez, Rodolfo A

2012-01-01

43

Side-chain specific isotopic labeling of proteins for infrared structural biology: the case of ring-D4-tyrosine isotope labeling of photoactive yellow protein.  

PubMed

An important bottleneck in the use of infrared spectroscopy as a powerful tool for obtaining detailed information on protein structure is the assignment of vibrational modes to specific amino acid residues. Side-chain specific isotopic labeling is a general approach towards obtaining such assignments. We report a method for high yield isotope editing of the bacterial blue light sensor photoactive yellow protein (PYP) containing ring-D(4)-Tyr. PYP was heterologously overproduced in Escherichia coli in minimal media containing ring-D(4)-Tyr in the presence of glyphosate, which inhibits endogenous biosynthesis of aromatic amino acids (Phe, Trp, and Tyr). Mass spectrometry of the intact protein and of tryptic peptides unambiguously demonstrated highly specific labeling of all five Tyr residues in PYP with 98% incorporation and undetectable isotopic scrambling. FTIR spectroscopy of the protein reveals a characteristic Tyr ring vibrational mode at 1515 cm(-1) that is shifted to 1436 cm(-1), consistent with that from ab initio calculations. PYP is a model system for protein structural dynamics and for receptor activation in biological signaling. The results described here open the way to the analysis of PYP using isotope-edited FTIR spectroscopy with side-chain specific labeling. PMID:22800658

Rathod, Rachana; Kang, Zhouyang; Hartson, Steven D; Kumauchi, Masato; Xie, Aihua; Hoff, Wouter D

2012-09-01

44

Radioactive labeling of antibody: a simple and efficient method  

Microsoft Academic Search

A simple and efficient method of covalently coupling the strong chelator diethylenetriaminepentaacetic acid to proteins was developed for radiolabeling immunoglobulin G antibodies. After being coupled and labeled with indium-111, a monoclonal antibody to carcinoembryonic antigen retained its ability to bind to its antigen in vitro and in vivo. In nude mice with a human colorectal xenograft, 41 percent of the

D. J. Hnatowich; W. W. Layne; R. L. Childs; D. Lanteigne; M. A. Davis; T. W. Griffin; P. W. Doherty

1983-01-01

45

Radioactive Labeling of Antibody: A Simple and Efficient Method  

Microsoft Academic Search

A simple and efficient method of covalently coupling the strong chelator diethylenetriaminepentaacetic acid to proteins was developed for radiolabeling immunoglobulin G antibodies. After being coupled and labeled with indium-111, a monoclonal antibody to carcinoembryonic antigen retained its ability to bind to its antigen in vitro and in vivo. In nude mice with a human colorectal xenograft, 41 percent of the

D. J. Hnatowich; W. W. Layne; R. L. Childs; D. Lanteigne; M. A. Davis; T. W. Griffin; P. W. Doherty

1983-01-01

46

Dye-Doped Silica Nanoparticles as Efficient Labels for Glycans  

PubMed Central

We report that dye-doped fluorescent silica nanoparticles (FSNPs) are highly efficient labels for glycans. Mono- and oligo-saccharides were conjugated to FSNPs using a general photocoupling chemistry. FSNP-labled glycans were applied to image and detect bacteria, and to study carbohydrate-lectin interactions on a lectin microarray.

Wang, Xin; Ramstrom, Olof; Yan, Mingdi

2014-01-01

47

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

PubMed

Knowledge of digesta passage kinetics in ruminants is essential to predict nutrient supply to the animal in relation to optimal animal performance, environmental pollution and animal health. Fractional passage rates (FPR) of feed are widely used in modern feed evaluation systems and mechanistic rumen models, but data on nutrient-specific FPR are scarce. Such models generally rely on conventional external marker techniques, which do not always describe digesta passage kinetics in a satisfactory manner. Here the use of stable isotope-labelled dietary nutrients as a promising novel tool to assess nutrient-specific passage kinetics is discussed. Some major limitations of this technique include a potential marker migration, a poor isotope distribution in the labelled feed and a differential disappearance rate of isotopes upon microbial fermentation in non-steady state conditions. Such limitations can often be circumvented by using intrinsically stable isotope-labelled plant material. Data are limited but indicate that external particulate markers overestimate rumen FPR of plant fibre compared with the internal stable isotope markers. Stable isotopes undergo the same digestive mechanism as the labelled feed components and are thus of particular interest to specifically measure passage kinetics of digestible dietary nutrients. © 2013 Society of Chemical Industry. PMID:24114801

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

2014-03-30

48

Determination of the enrichment of isotopically labelled molecules by mass spectrometry.  

PubMed

A general method for the determination of the enrichment of isotopically labelled molecules by mass spectrometry (MS) is described. In contrast to other published procedures, the method described here takes into account and corrects for measurement errors such as the contribution at M?-?1 due to loss of hydrogen or lack of spectral resolution and provides an uncertainty value for the determined enrichment. The general procedure requires the following steps: (1) evaluation of linearity in the mass spectrometer by injecting the natural abundance compound at different concentration levels, (2) determination of the purity of the mass cluster using the natural abundance analogue, (3) calculation of the theoretical isotope composition of the labelled compound using different tentative isotope enrichments, (4) calculation of 'convoluted' isotope distributions for the labelled compound taking into account the purity of the mass cluster determined with the natural abundance analogue and (5) comparison of the isotope distributions measured for the labelled compound with those calculated for different isotope enrichments using linear regression. The method was applied to a series of commercially available (13) C- and (2) H-labelled compounds and to a suite of singly (13) C-labelled ?2 -agonist prepared in-house both by gas chromatography (GC)-MS, GC-tandem MS (MS/MS) and liquid chromatography-MS/MS with satisfactory results. It was observed that the main uncertainty source for the isotope enrichment was the uncertainty in the purity of the measured cluster as determined with the natural abundance compound. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25044895

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

2014-08-01

49

Dynamic labeling strategy with 204Hg-isotopic methylmercurithiosalicylate for absolute peptide and protein quantification.  

PubMed

The methylmercury ion (CH(3)Hg(+)) demonstrated a high efficiency for directly labeling peptide/protein based on its specific and strong interaction with the sulfhydryl(s) in the peptide/protein and because of its smallest size among monofunctional organic mercurials studied, including methylmercury, ethylmercury, 4-(hydroxymercuric)benzoic acid, and 2,7-dibromo-4-hydroxymercurifluoresceine disodium. A simple 1:1 stoichiometry between CH(3)Hg(+) and sulfhydryl, confirmed with electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS) studies, made it easy to calibrate the stoichiometry of Hg in the peptide/protein. In order to avoid the direct use of the harmful CH(3)Hg(+), in this study a CH(3)Hg(+)-equivalent tag, methylmercurithiosalicylate (CH(3)Hg-THI), and its (204)Hg-enriched homologue (CH(3)(204)Hg-THI) were synthesized, and then CH(3)Hg(+) and/or CH(3)(204)Hg(+) released from CH(3)Hg-THI and/or CH(3)(204)Hg-THI in solution were utilized to demonstrate the dynamic labeling of glutathione (GSH) and two model proteins, beta-lactoglobulin (BLG) and ovalbumin (OVA), for the first time. Furthermore, the CH(3)(204)Hg-THI isotopical labeled GSH, BLG, and OVA standards (CH(3)(204)Hg-GSH, CH(3)(204)Hg-BLG, and CH(3)(204)Hg-OVA) were used to demonstrate the feasibility of absolute peptide/protein quantification using label-specific isotope dilution inductively coupled plasma mass spectrometry (ICPMS). On the basis of the accurate and sensitive determination of Hg using ICPMS, the detection limits of GSH, BLG, and OVA could reach 45.4, 45.4, and 15.1 pmol L(-1), respectively, suggesting the possibility for low-abundance peptide/protein quantification alongside the surefire quantification of moderate and highly abundant peptide/protein. PMID:20143794

Xu, Ming; Yan, Xiaowen; Xie, Qingqing; Yang, Limin; Wang, Qiuquan

2010-03-01

50

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

PubMed

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

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

2014-06-01

51

Isotope labeling for solution and solid-state NMR spectroscopy of membrane proteins.  

PubMed

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

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

2012-01-01

52

Enantioselective synthesis of isotopically labeled homocitric acid lactone.  

PubMed

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

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

2013-11-15

53

Fully automated isotopic dimethyl labeling and phosphopeptide enrichment using a microfluidic HPLC phosphochip.  

PubMed

Quantitative detection of phosphorylation levels is challenging and requires an expertise in both stable isotope labeling as well as enrichment of phosphorylated peptides. Recently, a microfluidic device incorporating a nanoliter flow rate reversed phase column as well as a titania (TiO(2)) enrichment column was released. This HPLC phosphochip allows excellent recovery and separation of phosphorylated peptides in a robust and reproducible manner with little user intervention. In this work, we have extended the abilities of this chip by defining the conditions required for on-chip stable isotope dimethyl labeling allowing for automated quantitation. The resulting approach will make quantitative phosphoproteomics more accessible. PMID:22975804

Polat, Ayse Nur; Kraiczek, Karsten; Heck, Albert J R; Raijmakers, Reinout; Mohammed, Shabaz

2012-11-01

54

Stable isotope labeling of oligosaccharide cell surface antigens  

SciTech Connect

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.

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

1998-12-31

55

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

Microsoft Academic Search

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

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

2007-01-01

56

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

NASA Astrophysics Data System (ADS)

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.

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

2006-12-01

57

[A novel method for absolute protein quantification using 18O isotope labeled concatamers of Q peptides combined with isotope dilution-multiple reaction monitoring mass spectrometry].  

PubMed

A method of concatamers of Q peptides (QconCATs) protein labeled with 18O-multiple reaction monitoring mass spectrometry for absolute quantification of proteins is established. The purity of the QconCAT recombinant protein was characterized by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and its purity was above 99%. The relative molecular mass was approximately 63.4 kDa. The peptides digested from the QconCAT recombinant protein and the extract of Thermoanaerobacter tengcongensis (TTE) were analyzed by mass spectrometry. The raw data were processed by pFind and pLabel softwares. The results showed that the efficiencies of protein digestion and the 18O labeling efficiency were able to meet the need of the protein quantification. The performance of the method was evaluated. The absolute contents of the selected proteins in TTE were determined with the relative standard deviations of less than 20% and the accuracy is high. The method not only avoid using the expensive reagent of stable isotope labeling with amino acids in cell culture (SILAC), but also provides an alternative way for the accurately absolute quantification of proteins in biological samples for quantitative proteomic research. PMID:24063190

Li, Nannan; Zhou, Lianqi; Mao, Xinli; Zhang, Jiao; Wei, Junying; Lin, Hongjun; Li, Jiabin; Tian, Fang; Zhang, Yangjun; Qian, Xiaohong

2013-06-01

58

Global Potential of Energy Efficiency Standards and Labeling Programs  

SciTech Connect

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

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

2008-06-15

59

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...

60

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

PubMed Central

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

de Jong, Felice A; Beecher, Chris

2013-01-01

61

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

PubMed

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

de Jong, Felice A; Beecher, Chris

2012-09-01

62

In-gel stable isotope labeling for relative quantification using mass spectrometry  

Microsoft Academic Search

Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts

Xiang Zhang; Bin Zheng; Lisa A Maroney; Heather R Christofk; Ning Wu; Lewis C Cantley; John M Asara

2006-01-01

63

Quantitative approaches for analysing fluxes through plant metabolic networks using NMR and stable isotope labelling  

Microsoft Academic Search

The quantitative analysis of metabolic networks is a prerequisite for understanding the integration and regulation of plant metabolism and for devising rational approaches for manipulating resource allocation in plants. The analysis of steady state stable isotope labelling experiments using nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful method for determining these fluxes in micro-organisms and its application to

N. J. Kruger; R. G. Ratcliffe; A. Roscher

2003-01-01

64

Negative ion ESI–MS analysis of natural yellow dye flavonoids—An isotopic labelling study  

Microsoft Academic Search

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

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

2009-01-01

65

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

Microsoft Academic Search

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

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

2009-01-01

66

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

PubMed

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker's yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine's medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only. PMID:19629476

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

2009-09-01

67

Stable isotopes' as trophic tracers: combining field sampling and manipulative labelling of food resources for macrobenthos  

Microsoft Academic Search

We combined 3 different approaches to determine the relative importance of microphytobenthos production as food for intertidal macrobenthic animals: (1) the natural abundance of stable-isotope ratios of carbon and nitrogen, (2) an in situ deliberate tracer addition of C-13-bicarbonate, which was transferred through the benthic food chain after its incorporation by benthic algae, and (3) a dual labelling experiment in

P. M. J. Herman; J. J. Middelburg; J. Widdows; C. M. Lucas; C. H. R. Heip

2000-01-01

68

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

PubMed Central

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

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

2008-01-01

69

Synthesis and stability of isotopically labeled p-chloro-m-xylenol (PCMX).  

PubMed

The synthesis, reaction kinetics, and pH stability of isotopically labeled p-chloro-m-xylenol (PCMX) were evaluated. While base catalysis was more rapid than acid catalysis, the latter allowed the use of a cosolvent for deuterium and tritium labeling using as little as 250 microL labeled water. Both acid and base catalysis were markedly more rapid than that reported previously for the deuteration of PCMX and related phenols. Isotopic labeling occurred only at the 2 and 6 ring positions, ortho to the phenolic group of PCMX. No deuterium loss was observed after storage for 21 days at 37 degrees C over a pH range of 2-14. Isotopic loss was observed only below pH 2. The prepared 3H-labeled PCMX had a specific activity of 1.18 mCi/mmol, a radiochemical purity of 99.0%, and a chemical purity exceeding 99.0%, with a high stability during prolonged cold storage. PMID:8272409

Reepmeyer, J C; Zielinski, W L; Leakey, J E; Si, Y

1993-10-01

70

Can ultrasonic energy efficiently speed (18)O-labeling of proteins?  

PubMed

We report in this work on the robustness of ultrasonic energy as a tool to speed the isotopic labeling of proteins using the (18)O-decoupling procedure. The first part of the decoupling procedure, comprising protein denaturation, reduction, alkylation and digestion, is done in 8 min under the effects of an ultrasonic field whilst the second part, the isotopic labeling, was assayed with and without the use of ultrasonic energy. Our results clearly demonstrate that the (18)O-isotopic labeling in a decoupling procedure cannot be accelerated using an ultrasonic field. PMID:19810034

Carreira, Ricardo J; Lodeiro, Carlos; Diniz, Mario S; Moura, Isabel; Capelo, José L

2009-11-01

71

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

PubMed

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

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

2004-05-01

72

Metabolic flux elucidation for large-scale models using 13C labeled isotopes.  

PubMed

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 (13)C, 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 antimalarial drug artemisinin. The cells were grown in continuous culture on glucose containing 20% [U-(13)C]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

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

2007-01-01

73

Development of a new isotopically labeled internal standard for ergosterol measurement by GC/MS.  

PubMed

Environmental exposure to fungi has been associated with a variety of adverse health effects. Ergosterol, a marker of total fungal biomass, can be used to quantify exposure to fungi. Unfortunately, environmental ergosterol measurement using published GC/MS/MS methods is prone to bias introduced by sample matrix effects, resulting in potential measurement inaccuracy. We developed an isotopically labeled internal standard ((13)C ergosterol) for ergosterol quantification by GC/MS/MS, to eliminate bias due to sample matrix effects and selective losses during preparation. To produce (13)C ergosterol, we grew Saccharomyces Cerevisiae on (13)C D-glucose under aerobic conditions at room temperature. The (13)C labeled ergosterol comprised 97.1% of the ergosterol in the dry yeast preparation. Ergosterol spike-recovery from house dust samples averaged 99.3% using the isotopically labeled yeast preparation as the internal standard (I.S.). By contrast, spike-recovery averaged 42.4% when 7-dehydrocholesterol (7-DHC) was the internal standard. Analysis of indoor house dust samples from a large epidemiologic study also showed the systematic underestimation of ergosterol when analyzed using 7-DHC as compared with using the yeast preparation containing the isotopically labeled authentic compound, (13)C-ergosterol. PMID:19657536

Sordillo, Joanne; Vespa, Donato; Haggerty, Linda; Youngs, Frederick; Gold, Diane; Milton, Donald

2009-08-01

74

Efficient Algorithms for Exact Inference in Sequence Labeling SVMs.  

PubMed

The task of structured output prediction deals with learning general functional dependencies between arbitrary input and output spaces. In this context, two loss-sensitive formulations for maximum-margin training have been proposed in the literature, which are referred to as margin and slack rescaling, respectively. The latter is believed to be more accurate and easier to handle. Nevertheless, it is not popular due to the lack of known efficient inference algorithms; therefore, margin rescaling - which requires a similar type of inference as normal structured prediction - is the most often used approach. Focusing on the task of label sequence learning, we here define a general framework that can handle a large class of inference problems based on Hamming-like loss functions and the concept of decomposability for the underlying joint feature map. In particular, we present an efficient generic algorithm that can handle both rescaling approaches and is guaranteed to find an optimal solution in polynomial time. PMID:24808034

Bauer, Alexander; Gornitz, Nico; Biegler, Franziska; Muller, Klaus-Robert; Kloft, Marius

2014-05-01

75

From isotope labeled CH?CN to N? inside single-walled carbon nanotubes.  

PubMed

The observation of one-dimensional N? inside single-walled carbon nanotubes raises the questions, how are the N? molecules formed and how do they manage to make their way to this peculiar place? We have used N(15) and C(13) isotope labeled acetonitrile during the synthesis of single-walled carbon nanotubes to investigate this process. The isotope shifts of phonons and vibrons are observed by Raman spectroscopy and X-ray absorption. We identify the catalytic decomposition of acetonitrile as the initial step in the reaction pathway to single-walled carbon nanotubes containing encapsulated N?. PMID:24322271

Kramberger, Christian; Thurakitseree, Theerapol; Einarsson, Erik; Takashima, Akito; Kinoshita, Toyohiko; Muro, Takayuki; Maruyama, Shigeo

2014-01-16

76

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

NASA Astrophysics Data System (ADS)

The observation of one-dimensional N2 inside single-walled carbon nanotubes raises the questions, how are the N2 molecules formed and how do they manage to make their way to this peculiar place? We have used N15 and C13 isotope labeled acetonitrile during the synthesis of single-walled carbon nanotubes to investigate this process. The isotope shifts of phonons and vibrons are observed by Raman spectroscopy and X-ray absorption. We identify the catalytic decomposition of acetonitrile as the initial step in the reaction pathway to single-walled carbon nanotubes containing encapsulated N2.

Kramberger, Christian; Thurakitseree, Theerapol; Einarsson, Erik; Takashima, Akito; Kinoshita, Toyohiko; Muro, Takayuki; Maruyama, Shigeo

2014-01-01

77

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

SciTech Connect

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.

de Veau, E.J.

1988-01-01

78

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

PubMed

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

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

2006-05-26

79

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

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

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

2013-11-01

80

125 IUDR labelling efficiency and cytotoxicity for murine tumours in vivo and in vitro  

Microsoft Academic Search

We have reviewed the literature on 125IUDR (5-iodo-2-deoxyuridine), the DNA label of choice for cell distribution studies. In previous studies, the cytotoxicity in relation to labelling efficiency has often been inadequately investigated or reported. We have studied four syngeneic mouse tumours and compared in vitro with in vivo labelling procedures. Ascites tumours could be effectively labelled in vivo by IP

Björn Hagmar; Walter Ryd

1982-01-01

81

Liquid-liquid extraction combined with differential isotope dimethylaminophenacyl labeling for improved metabolomic profiling of organic acids.  

PubMed

A large fraction of the known human metabolome belong to organic acids. However, comprehensive profiling of the organic acid sub-metabolome is a major analytical challenge. In this work, we report an improved method for detecting organic acid metabolites. This method is based on the use of liquid-liquid extraction (LLE) to selectively extract the organic acids, followed by using differential isotope p-dimethylaminophenacyl (DmPA) labeling of the acid metabolites. The (12)C-/(13)C-labeled samples are analyzed by liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS). It is shown that this LLE DmPA labeling method offers superior performance over the method of direct DmPA labeling of biofluids such as human urine. LLE of organic acids reduces the interference of amine-containing metabolites that may also react with DmPA. It can also remove water in a biofluid that can reduce the labeling efficiency. Using human urine as an example, it is demonstrated that about 2500 peak pairs or putative metabolites could be detected in a 30-min gradient LC-MS run, which is about 3 times more than that detected in a sample prepared using direct DmPA labeling. About 95% of the 1000 or so matched metabolites to the Human Metabolome Database (HMDB) are organic acids. It is further shown that this method can be used to handle as small as 10 ?L of urine. We believe that this method opens the possibility of generating a very comprehensive profile of the organic acid sub-metabolome that will be useful for comparative metabolomics applications for biological studies and disease biomarker discovery. PMID:24216202

Peng, Jun; Li, Liang

2013-11-25

82

Nuclear magnetic resonance measuring method using an isotope-labeled compound  

US Patent & Trademark Office Database

The present invention provides a method for measuring nuclear magnetic resonance that employs a compound in which a plurality of nuclei is labeled with isotopes as a probe agent, highly selectively and highly sensitively obtains a nuclear magnetic resonance signal of the above described probe agent, and can attach a spatial positional information to the above described nuclear magnetic resonance signal, and an apparatus therefore.

2014-07-08

83

Exchangeability of orthophosphate and pyrophosphate in soils: a double isotopic labelling study  

Microsoft Academic Search

Liquid polyphosphate fertilisers have shown advantages in field experiments as a phosphorus (P) source for crops grown on\\u000a calcareous soils. Polyphosphate fertilisers contain orthophosphate (oP), pyrophosphate (pP) and other condensed P species.\\u000a A double labelling technique was developed using ion chromatography for separation of oP and pP, the major P species in polyphosphate\\u000a fertilisers, in order to measure the isotopically

T. M. McBeath; E. Lombi; M. J. McLaughlin; E. K. Bünemann

2009-01-01

84

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

Microsoft Academic Search

We present the design and synthesis of a new quantitative strategy termed soluble polymer-based isotope labeling (SoPIL) and its application as a novel and inclusive method for the identification and relative quantification of individual proteins in complex snake venoms. The SoPIL reagent selectively captures and isolates cysteine-con- taining peptides, and the subsequent tagged peptides are released and analyzed using nanoflow

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

2007-01-01

85

Molecular and mass spectroscopic analysis of isotopically labeled organic residues  

NASA Technical Reports Server (NTRS)

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.

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

1989-01-01

86

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

PubMed

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

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

2012-01-01

87

Atlas encoding by randomized forests for efficient label propagation.  

PubMed

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

Zikic, Darko; Glocker, Ben; Criminisi, Antonio

2013-01-01

88

Pseudocontinuous Arterial Spin Labeling with Optimized Tagging Efficiency  

PubMed Central

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 (CBF) estimates. This efficiency loss is caused by a phase tracking error between the RF pulses and the flowing spins. This paper 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 pre-scan procedure, which consists of a series of short scans interleaved with automated post-processing routines integrated to the scanner console. The pre-scan 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 SNR, image SNR, and baseline CBF measures are compared to those acquired from conventional PCASL.

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

2011-01-01

89

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

PubMed Central

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

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

2011-01-01

90

Tests of isotopic separation efficiency of palladium packed columns  

SciTech Connect

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

Heung, L. K.; Staack, G. C.; Klein, J. E.; Jacobs, W. D. [Savannah River National Laboratory, 773-A, Savannah River Site, Aiken, SC 29808 (United States)

2008-07-15

91

Ligands of glutamate and dopamine receptors evenly labeled with hydrogen isotopes  

Microsoft Academic Search

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

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

2009-01-01

92

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

Microsoft Academic Search

A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10–50 ?m in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried

Victor N Morozov; Alexander V Gavryushkin; Alexander A Deev

2002-01-01

93

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

PubMed

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

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

2014-11-01

94

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

USGS Publications Warehouse

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

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

2007-01-01

95

Stable isotope labeling of lead compartments in rats with ultralow lead concentrations  

SciTech Connect

The role of the mammalian skeleton as an endogenous lead source is unclear. This is due in part to difficulties in distinguishing mobilized skeletal lead from other endogenous and exogenous lead sources. Therefore, the authors have applied ultraclean stable lead isotope techniques to label skeletal and soft tissue lead compartments within the rat with distinguishable lead isotopic signatures. Female Wistar rats were fed {sup 206}Pd-enriched drinking water and sacrificed after durations of 2, 4, 7, and 14 days. Blood, kidney, vertebra, and tibia tissues were analyzed for lead concentrations and stable isotopic compositions. The resulting isotopic ratios in soft and skeletal tissues differed by {approximately}40% after 2 days exposure to the {sup 206}Pb tracer. More than 90% of the tracer isotopic signature was contained in the soft tissues after 10 days exposure, while skeletal tissues acquired only {approximately}50% of the tracer by the end of the study. Because these animals were maintained under trace metal-clean conditions, they contained lead concentrations in whole blood, kidney, and bone tissues that are the lowest known reported for contemporary terrestrial mammals, and they are comparable to levels in preindustrial mammals. The elevated concentrations of lead in kidney relative to levels in blood are consistent with the presence of specific lead-binding sites in the kidney at very low levels of exposure.

Smith, D.R.; Flegal, A.R. (Univ. of California, Santa Cruz (United States)); Osterloh, J.D. (Univ. of California, San Francisco (United States)); Niemeyer, S. (Lawrence Livermore National Lab., CA (United States))

1992-04-01

96

Synthesis of isotopically labeled P-site substrates for the ribosomal peptidyl transferase reaction  

PubMed Central

Isotopomers of the ribosomal P-site substrate, the trinucleotide peptide conjugate CCA-pcb,1 have been designed and synthesized in 26–350020steps. These include individual isotopic substitution at the ?-proton, carbonyl carbon, and carbonyl oxygen of the amino acid, the O2' and O3' of the adenosine, and a remote label in the N3 and N4 of both cytidines. These isotopomers were synthesized by coupling cytidylyl-(3'5')-cytidine phosphoramidite isotopomers, as the common synthetic intermediates, with isotopically substituted A-Phe-cap-biotin (A-pcb). The isotopic enrichment is higher than 99% for 1-13C (Phe), 2-2H (Phe), and 3,4-15N2 (cytidine), 93% for 2'/3'- 18O (adenosine), and 64% for 1-18O (Phe). A new synthesis of highly enriched [1-18O2] phenylalanine has been developed. The synthesis of [3'-18O] adenosine was improved by Lewis acid aided regioselective ring opening of the epoxide and by an economical SN2-SN2 method with high isotopic enrichment (93%). Such substrates are valuable for studies of the ribosomal peptidyl transferase reaction by complete kinetic isotope effect analysis and of other biological processes catalyzed by nucleic acid related enzymes, including polymerases, reverse transcriptases, ligases, nucleases, and ribozymes.

Zhong, Minghong

2010-01-01

97

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

Microsoft Academic Search

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

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

2002-01-01

98

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

PubMed

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

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

2013-10-01

99

31P NMR correlation maps of 18O/16O chemical shift isotopic effects for phosphometabolite labeling studies  

PubMed Central

Intramolecular correlations among the 18O-labels of metabolic oligophosphates, mapped by J-decoupled 31P NMR 2D chemical shift correlation spectroscopy, impart stringent constraints to the 18O-isotope distributions over the whole oligophosphate moiety. The multiple deduced correlations of isotopic labels enable determination of site-specific fractional isotope enrichments and unravel the isotopologue statistics. This approach ensures accurate determination of 18O-labeling rates of phosphometabolites, critical in biochemical energy conversion and metabolic flux transmission. The biological usefulness of the J-decoupled 31P NMR 2D chemical shift correlation maps was validated on adenosine tri-phosphate fractionally 18O labeled in perfused mammalian hearts.

Nemutlu, Emirhan; Zhang, Song; Dzeja, Petras; Terzic, Andre; Macura, Slobodan

2011-01-01

100

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

SciTech Connect

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.

Yung, Yuk L. [California Inst. of Tech., Pasadena, CA (United States). Div. of Geological and Planetary Sciences; Thiemens, M.H. [California Univ., San Diego, CA (United States)

1993-11-01

101

Isotopically labeled crosslinking reagents: resolution of mass degeneracy in the identification of crosslinked peptides.  

PubMed

Mass spectrometry in three dimensions (MS3D) is a newly developed method for the determination of protein structures involving intramolecular chemical crosslinking of proteins, proteolytic digestion of the resulting adducts, identification of crosslinks by mass spectrometry (MS), peak assignment using theoretical mass lists, and computational reduction of crosslinks to a structure by distance geometry methods. To facilitate the unambiguous identification of crosslinked peptides from proteolytic digestion mixtures of crosslinked proteins by MS, we introduced double 18O isotopic labels into the crosslinking reagent to provide the crosslinked peptides with a characteristic isotope pattern. The presence of doublets separated by 4 Da in the mass spectra of these materials allowed ready discrimination between crosslinked and modified peptides, and uncrosslinked peptides using automated intelligent data acquisition (IDA) of MS/MS data. This should allow ready automation of the method for application to whole expressible proteomes. PMID:14592499

Collins, Christopher J; Schilling, Birgit; Young, Malin; Dollinger, Gavin; Guy, R Kiplin

2003-11-17

102

Quantitative cross-linking/mass spectrometry using isotope-labelled cross-linkers?  

PubMed Central

Dynamic proteins and multi-protein complexes govern most biological processes. Cross-linking/mass spectrometry (CLMS) is increasingly successful in providing residue-resolution data on static proteinaceous structures. Here we investigate the technical feasibility of recording dynamic processes using isotope-labelling for quantitation. We cross-linked human serum albumin (HSA) with the readily available cross-linker BS3-d0/4 in different heavy/light ratios. We found two limitations. First, isotope labelling reduced the number of identified cross-links. This is in line with similar findings when identifying proteins. Second, standard quantitative proteomics software was not suitable for work with cross-linking. To ameliorate this we wrote a basic open source application, XiQ. Using XiQ we could establish that quantitative CLMS was technically feasible. Biological significance Cross-linking/mass spectrometry (CLMS) has become a powerful tool for providing residue-resolution data on static proteinaceous structures. Adding quantitation to CLMS will extend its ability of recording dynamic processes. Here we introduce a cross-linking specific quantitation strategy by using isotope labelled cross-linkers. Using a model system, we demonstrate the principle and feasibility of quantifying cross-linking data and discuss challenges one may encounter while doing so. We then provide a basic open source application, XiQ, to carry out automated quantitation of CLMS data. Our work lays the foundations of studying the molecular details of biological processes at greater ease than this could be done so far. This article is part of a Special Issue entitled: New Horizons and Applications for Proteomics [EuPA 2012].

Fischer, Lutz; Chen, Zhuo Angel; Rappsilber, Juri

2013-01-01

103

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

PubMed Central

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

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

2013-01-01

104

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

PubMed

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

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

2013-01-01

105

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

NASA Astrophysics Data System (ADS)

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

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

2010-12-01

106

The department of commerce energy labeling and energy efficiency programs for household appliances  

Microsoft Academic Search

The development and present status of the Department of Commerce energy labeling and energy efficiency programs are described. Labels for room air conditioners, combination refrigerator-freezers, and water heaters are discussed in detail. Energy reduction goals under the energy efficiency programs are given. The possible effect of pending legislation is described.

B. J. McGuire Jr.

1975-01-01

107

Fluorophore-assisted carbohydrate electrophoresis (FACE) of oligosaccharides: efficiency of labelling and high-resolution separation  

Microsoft Academic Search

Reductive amination is a common technique for the derivatisation of reducing carbohydrates, thereby providing appropriate chromophores or fluorophores to overcome native detection deficiencies. Rarely, however, is the issue of labelling efficiency addressed for substrates larger than monosaccharides. Utilising a variety of radiolabelled synthetic maltooligosaccharides, we now present data on the APTS labelling efficiency for substrates up to an average degree

Michael G. O’Shea; Michael S. Samuel; Christine M. Konik; Matthew K. Morell

1998-01-01

108

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

PubMed

Chloropropanol (CP) esters are part of an emerging group of process-induced toxicants that are considered as potential health hazards particularly in palm oil. Mass spectrometry-based methodologies for identification of CP esters in food are critical in overcoming the challenges associated with direct detection methods. In the present study, a convenient strategy was employed to generate all possible CP acetates through reacting acetic anhydride with either glycerol in the presence of a chloride source or the corresponding CPs, such as 3-chloro-, 1,3-dichloro-, 2-chloro-, and 1,2-dichloropropanols, allowing for the identification of the individual CP acetates and assignment of their mass spectral fragmentations. Mass spectral fragmentations were confirmed through the use of the isotopic signature of chlorine in addition to the isotope labeling experiments performed using isotopically labeled precursors, such as [¹³C-U?] glycerol, [¹³C-U?] acetic anhydride, [¹³C-2,2'] acetic anhydride, and [d?] 3-monochloropropane-1,2-diol (3-MCPD) as reactants. Such studies have indicated that all CP esters can undergo two general fragmentations under electron impact (EI) conditions, one generating the acylium ion at m/z 45 and the other generating a chlorinated cyclic acyloxonium ion at m/z 135.6. Considering the fact that such ions can also be generated from any fatty acid containing CP esters after undergoing McLafferty rearrangement, the ion at m/z 135.6 can therefore be considered as a universal marker for the presence of CP esters undergoing EI fragmentation. Furthermore, these studies have also indicated the formation of ions characteristic of CP diesters, monoesters, and dichloro esters. PMID:23964824

Rahn, Anja K K; Yaylayan, Varoujan A

2013-09-18

109

In vivo investigation of homocysteine metabolism to polyamines by high-resolution accurate mass spectrometry and stable isotope labeling.  

PubMed

Polyamines are essential polycations, playing important roles in mammalian physiology. Theoretically, the involvement of homocysteine in polyamine synthesis via S-adenosylmethionine is possible; however, to our knowledge, it has not been established experimentally. Here, we propose an original approach for investigation of homocysteine metabolites in an animal model. The method is based on the combination of isotope-labeled homocysteine supplementation and high-resolution accurate mass spectrometry analysis. Structural identity of the isotope-labeled metabolites was confirmed by accurate mass measurements of molecular and fragment ions and comparison of the retention times and tandem mass spectrometry fragmentation patterns. Isotope-labeled methionine, spermidine, and spermine were detected in all investigated plasma and tissue samples. The induction of moderate hyperhomocysteinemia leads to an alteration in polyamine levels in a different manner. The involvement of homocysteine in polyamine synthesis and modulation of polyamine levels could contribute to a better understanding of the mechanisms connected with homocysteine toxicity. PMID:24736325

Ruseva, Silviya; Lozanov, Valentin; Markova, Petia; Girchev, Radoslav; Mitev, Vanio

2014-07-15

110

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

SciTech Connect

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

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

1983-11-01

111

Protein global fold determination using site-directed spin and isotope labeling.  

PubMed Central

We describe a simple experimental approach for the rapid determination of protein global folds. This strategy utilizes site-directed spin labeling (SDSL) in combination with isotope enrichment to determine long-range distance restraints between amide protons and the unpaired electron of a nitroxide spin label using the paramagnetic effect on relaxation rates. The precision and accuracy of calculating a protein global fold from only paramagnetic effects have been demonstrated on barnase, a well-characterized protein. Two monocysteine derivatives of barnase, (H102C) and (H102A/Q15C), were 15N enriched, and the paramagnetic nitroxide spin label, MTSSL, attached to the single Cys residue of each. Measurement of amide 1H longitudinal relaxation times, in both the oxidized and reduced states, allowed the determination of the paramagnetic contribution to the relaxation processes. Correlation times were obtained from the frequency dependence of these relaxation processes at 800, 600, and 500 MHz. Distances in the range of 8 to 35 A were calculated from the magnitude of the paramagnetic contribution to the relaxation processes and individual amide 1H correlation times. Distance restraints from the nitroxide spin to amide protons were used as restraints in structure calculations. Using nitroxide to amide 1H distances as long-range restraints and known secondary structure restraints, barnase global folds were calculated having backbone RMSDs <3 A from the crystal structure. This approach makes it possible to rapidly obtain the overall topology of a protein using a limited number of paramagnetic distance restraints.

Gaponenko, V.; Howarth, J. W.; Columbus, L.; Gasmi-Seabrook, G.; Yuan, J.; Hubbell, W. L.; Rosevear, P. R.

2000-01-01

112

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

PubMed Central

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

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

2011-01-01

113

Labeling  

MedlinePLUS

... Free" “Cosmeceutical” "Cruelty Free"/"Not Tested on Animals" "Hypoallergenic" "Organic" More on Labeling Claims Expiration Dating There are no regulations or requirements under current United States law that require cosmetic manufacturers to print expiration dates on the labels ...

114

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

NASA Astrophysics Data System (ADS)

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.

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

2009-07-01

115

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

NASA Astrophysics Data System (ADS)

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

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

2011-03-01

116

Isotope labelling study of CO oxidation-assisted epoxidation of propene. Implications for oxygen activation on Au catalysts.  

PubMed

(18)O isotope labelling studies of the CO oxidation-assisted epoxidation of propene, catalyzed by a mixture of Au/TiO(2) and TS-1, using a methanol-H(2)O solvent showed the O in the epoxide was exclusively from O(2) and not H(2)O or methanol. PMID:20393659

Jiang, Jian; Oxford, Sean M; Fu, Baosong; Kung, Mayfair C; Kung, Harold H; Ma, Jiantai

2010-06-01

117

Labeled trees and the efficient computation of derivations  

NASA Technical Reports Server (NTRS)

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.

Grossman, Robert; Larson, Richard G.

1989-01-01

118

Ultrasonic energy as a new tool for fast isotopic 18O labeling of proteins for mass spectrometry-based techniques: preliminary results.  

PubMed

Preliminary results regarding fast isotopic labeling of proteins with (18)O in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry technique are presented. Similar (16)O/(18)O isotopic labeling ratios were found for the overnight procedure (12h) and the new fast ultrasonic one (30 min) for the BSA, ovalbumin and alpha-lactalbumin proteins. The procedure, however, failed to promote double (18)O isotopic labeling for the proteins, ovalbumin and alpha-lactalbumin. Two different sonication frequencies, 35 and 130 kHz, were studied at two different sonication times of 15 and 30 min, being best results obtained with the procedure at 130 kHz of sonication frequency and 30 min of sonication time. For comparative purposes the overnight isotopic (18)O labeling procedure was done. In addition, the new fast isotopic labeling procedure was also studied without ultrasonication, in a water bath at 60 degrees C. PMID:18585297

Carreira, R J; Rial-Otero, R; López-Ferrer, D; Lodeiro, C; Capelo, J L

2008-07-15

119

Ultrasonic Energy as a New Tool for Fast Isotopic 18O Labeling of Proteins for Mass Spectrometry-Based Techniques: Preliminary Results  

SciTech Connect

Preliminary results regarding fast isotopic labeling of proteins with 18O in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry technique are presented. Similar 16O/18O isotopic labeling ratios were found for the overnight procedure (12 h) and the new fast ultrasonic one (30 min) for the BSA, ovalbumin and ?-lactalbumin proteins. The procedure, however, failed to promote double 18O isotopic labeling for the proteins ovalbumin and ?-lactalbumin. Two different sonication frequencies, 35 kHz and 130 kHz, were studied at two different sonication times of 15 min and 30 min, being best results obtained with the procedure at 130 kHz of sonication frequency and 30 min of sonication time. For comparative purposes the overnight isotopic 18O labeling procedure was done. In addition, the new fast isotopic labeling procedure was also studied without ultrasonication, in a water bath at 60 ºC.

Carreira, R.J.; Rial-Otero, R.; Lopez-Ferrer, Dani; Lodeiro, C.; Capelo, J.L.

2008-07-15

120

Multi-isotope labelling (13C, 18O, 2H) for studying organic matter cycling within plant-soil systems  

NASA Astrophysics Data System (ADS)

Carbon cycling has become of major interest for the understanding and mitigation of global climatic change. Terrestrial ecosystems have a large carbon sequestration potential, but many processes and fluxes of organic matter (OM) cycling within the plant-soil system are not yet well understood [1]. The dynamics of OM cycling within the plant soil-system are determined by environmental parameters, as well as chemical quality of OM input. A well-known technique to study OM dynamics is to label OM inputs with stable isotopes (e.g 13C). Changes in OM quality in the plant and in the soil can be assessed by compound specific isotopic analysis [2]. These techniques give a precise insight of the OM composition, but are laborious and expensive. Here we suggest a new multi-isotope labelling technique using stable 13C in combination with stable 18O and 2H isotopes, which provides information on OM quality by simple bulk material analysis. The method is based on the creation of an isotopic van Krevelen diagram, which is used to describe different compound groups by plotting the atomic ratios of O/C vs. H/C [3]. We could show that new assimilates can be labelled with 13C, 18O and 2H by adding the stable isotopes (continuously) in the gaseous phase (CO2 and water vapour) to the plants atmosphere. The label has been traced within the bulk material of different compartments of the plant-soil system (e.g. leaves, stems, roots, bulk soil). Our first results showed that after 2, 8 and 14 days of labelling the 18O/13C(new) ratio was notably different in leaf, stem and root tissue (0.0024, 0.0011 and 0.0007, respectively), suggesting a change in OM quality towards more C-rich compounds. d2H analysis will follow and an isotopic van Krevelen diagram will be produced (18O/13C(new) vs. 2H/13C(new)) to describe the changes in OM quality. The new multi-isotope labelling approach represent a powerful tool to address open questions in plant and soil research such as the allocation of organic molecules within the plant-soil system under changing environmental conditions or the influence of plant roots on soil organic matter stabilization and destabilization processes.

Studer, M. S.; Abiven, S.; Schmidt, M. W. I.; Siegwolf, R. T. W.

2012-04-01

121

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

PubMed

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

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

2002-03-01

122

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

NASA Astrophysics Data System (ADS)

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‰ ?2H, +7‰ ?18O, 3.8 wt.% H2O) with isotopically-labeled water (+650‰ ?2H, +56‰ ?18O). 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 ?2H 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 ?2H, 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‰ ?2H increases with time, with faster deuterium exchange at higher temperatures. Times at 15% of theoretical "full ?2H 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 ?18O in ash; water released from ash rapidly by thermal decomposition is not enriched in ?18O. This observation clearly suggests that it is hydrogen exchange, and not water addition or oxygen exchange that characterizes the process. (iii) Our time series drying, Fourier transform infrared (FTIR)-KBr and Thermogravimetric Analyzer (TGA) analyses collectively suggest a simple mechanistic view that there are three kinds of "water" in ash: water (mostly H2O) that is less strongly bonded on the surface of ash particles that are getting lost with 24-48 h of drying to up to 200-300 °C, bound water in glass in a range of combining proportions of SiOH to H2O, and a small reservoir of residual, tightly held water. Experimentation with vacuum drying at 130-220 °C, and with TGA up to 1000 °C provides a set of simple relationships and recommendations for users. Ash loses 1-1.2 wt.% water with weight loss stabilizing after 48 h of vacuum drying at 130 °C. This ash drying removes molecular water over the hydroxyl group in a proportion of ˜80:20% resulting in relatively constant ?2H values of the remaining total water in native ash. This study demonstrates that ?2H in ash can be rapidly changed by minor diagenesis even at relatively low temperatures of 20 °C. A diagenetic history of ash is needed to interpret the D/H ratio, but the ?18O values of water in ash are more robust.

Nolan, Gary S.; Bindeman, Ilya N.

2013-06-01

123

Electrolytic D\\/H isotope separation efficiency and electrocatalytic activity of Mo–Pt intermetallic phases  

Microsoft Academic Search

The aim of this work was to investigate efficiency of the electrolytic separation of hydrogen isotopes (D\\/H), from alkaline 6M KOH solution as standard electrolyte, on the Mo–Pt intermetallic phases as cathode materials. We measured the isotope separation factor (?) of the single-stage process, as a basic parameter determining the isotope separation efficiency. Furthermore, we have found that some combinations

Š?epan S. Miljani?; Aleksandar D. Maksi?; Nebojša I. Potkonjak; Milica P. Mar?eta Kaninski

2007-01-01

124

Split-field drift tube/mass spectrometry and isotopic labeling techniques for determination of single amino acid polymorphisms.  

PubMed

A combination of split-field drift tube/mass spectrometry and isotopic labeling techniques is evaluated as a means of identifying single amino acid polymorphisms (SAAPs) in proteins. The method is demonstrated using cytochromec (equine and bovine) and hemoglobin (bovine and sheep). For these studies, proteins from different species are digested with trypsin, and the peptides are labeled at primary amine groups [using either a light (H(3))- or heavy (D(3))-isotopic reagent]. SAAP analysis is carried out by mixing the light-labeled peptides of one species with the heavy-labeled peptides of the other and electrospraying the resulting mixture into a split-field drift tube/mass spectrometer. Peptides having the same sequence in both species appear as doublets in the mass spectrum [shifted in mass-to-charge (m/z) according to the number of incorporated labels]; additionally, these species have identical mobility distributions. Peptides having sequences that differ by one amino acid appear as peaks in the mass spectrum that are shifted in m/z according to the mass difference associated with the SAAP and the number of incorporated labels. The ion mobility distributions for these peptides (differing by only a single amino acid) can often be rationalized by their expected similarities or differences providing additional evidence that they are related. In all, 12 and 26 peptide variants (between species) corresponding to 5 and 11 amino acid polymorphisms have been identified for the cytochrome c and hemoglobin protein samples, respectively. PMID:16889409

Valentine, Stephen J; Sevugarajan, S; Kurulugama, Ruwan T; Koeniger, Stormy L; Merenbloom, Samuel I; Bohrer, Brian C; Clemmer, David E

2006-08-01

125

Isotope labelling - paired homologous double neutral loss scan-mass spectrometry for profiling of metabolites with a carboxyl group.  

PubMed

We developed a novel method for non-targeted screening of metabolites by high performance liquid chromatography-mass spectrometry with paired homologous double neutral loss scan mode after in vitro isotope labelling (IL-HPLC-PHDNL-MS). As a proof of concept, we investigated the carboxylic acid metabolite profiling in plant samples by the IL-HPLC-PHDNL-MS method. To this end, N,N-dimethylaminobutylamine (DMBA) and d(4)-N,N-dimethylaminobutylamine (d(4)-DMBA) were synthesized and utilized to label carboxylic acids. Our results show the MS response of carboxylic acids was enhanced by 20- to 40-fold after labelling. As for the IL-HPLC-PHDNL-MS analysis, DMBA and d(4)-DMBA labelled samples were mixed equally before MS analysis. Because the isotope labelled moieties (dimethylamino moiety, Me2N) of DMBA and d(4)-DMBA are easily ruptured and lost as neutral fragments (NL 45 and NL 49) under collision induced dissociation (CID), two neutral loss scans can be carried out simultaneously to record the signals of DMBA and d(4)-DMBA labelled samples, respectively. In this respect, the metabolites from two samples labelled with different isotope reagents are ionized at the same time but recorded separately by mass spectrometry, which can eliminate the MS response fluctuation and mutual interference. Using this method, six potential biomarkers involved in wounded tomato leaves were identified, and their structures were further elucidated by product ion scan and high resolution mass spectrometry analysis. Taken together, the IL-HPLC-PHDNL-MS method demonstrated good performance on the identification as well as relative quantification of metabolites with a carboxyl group in biological samples. PMID:24839964

Huang, Yun-Qing; Wang, Qiu-Yi; Liu, Jia-Qi; Hao, Yan-Hong; Yuan, Bi-Feng; Feng, Yu-Qi

2014-06-01

126

Identification and quantification of protein carbonylation using light and heavy isotope labeled Girard's P reagent.  

PubMed

Protein carbonyls are one of the most widely studied markers of oxidative stress. Determining increases in the concentration of protein carbonyls known to be associated with neurodegenerative diseases, heart disease, cancer and ageing. Identification of carbonylation sites in oxidized proteins has been a challenge. Even though recent advances in proteomics has facilitate the identification of carbonylation sites in oxidized proteins, confident identification remains a challenge due to the complicated nature of oxidative damage and the wide range of oxidative modifications. Here, we report the development of a multiplexing strategy that facilitates confident carbonylated peptide identification through a combination of heavy and light isotope coding and a multi-step filtering process. This procedure involves (1) labeling aliquots of oxidized proteins with heavy and light forms of Girard's reagent P (GPR) and combining them in a 1:1 ratio along with (2) LC/MS and MALDI-MS/MS analysis. The filtering process uses LC/MS and MALDI-MS/MS data to rule out false positives by rejecting peptide doublets that do not appear with the correct concentration ratio, retention time, tag number, or resolution. This strategy was used for the identification of heavily oxidized transferrin peptides and resulted in identification 13 distinct peptides. The competency of the method was validated in a complex mixture using oxidized transferrin in a yeast lysate as well as oxidized yeast. Twenty-five percent of the peptides identified in a pure oxidized sample of transferrin were successfully identified from the complex mixture. Analysis of yeast proteome stressed with hydrogen peroxide using this multiplexing strategy resulted in identification of 41 carbonylated peptides from 36 distinct proteins. Differential isotope coding of model peptides at different concentrations followed by mixing at different ratios was used to establish the linear dynamic range for quantification of carbonylated peptides using light and heavy forms of GPR. PMID:16996067

Mirzaei, Hamid; Regnier, Fred

2006-11-17

127

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

PubMed Central

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

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

2013-01-01

128

Trypsin-catalyzed N-terminal labeling of peptides with stable isotope-coded affinity tags for proteome analysis.  

PubMed

An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography-tandem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis. PMID:24354301

Pan, Yanbo; Ye, Mingliang; Zheng, Hao; Cheng, Kai; Sun, Zhen; Liu, Fangjie; Liu, Jing; Wang, Keyun; Qin, Hongqiang; Zou, Hanfa

2014-01-21

129

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

PubMed Central

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

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

1991-01-01

130

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

PubMed Central

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

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

2012-01-01

131

IsoMS: Automated Processing of LC-MS Data Generated by a Chemical Isotope Labeling Metabolomics Platform.  

PubMed

A chemical isotope labeling or isotope coded derivatization (ICD) metabolomics platform uses a chemical derivatization method to introduce a mass tag to all of the metabolites having a common functional group (e.g., amine), followed by LC-MS analysis of the labeled metabolites. To apply this platform to metabolomics studies involving quantitative analysis of different groups of samples, automated data processing is required. Herein, we report a data processing method based on the use of a mass spectral feature unique to the chemical labeling approach, i.e., any differential-isotope-labeled metabolites are detected as peak pairs with a fixed mass difference in a mass spectrum. A software tool, IsoMS, has been developed to process the raw data generated from one or multiple LC-MS runs by peak picking, peak pairing, peak-pair filtering, and peak-pair intensity ratio calculation. The same peak pairs detected from multiple samples are then aligned to produce a CSV file that contains the metabolite information and peak ratios relative to a control (e.g., a pooled sample). This file can be readily exported for further data and statistical analysis, which is illustrated in an example of comparing the metabolomes of human urine samples collected before and after drinking coffee. To demonstrate that this method is reliable for data processing, five (13)C2-/(12)C2-dansyl labeled metabolite standards were analyzed by LC-MS. IsoMS was able to detect these metabolites correctly. In addition, in the analysis of a (13)C2-/(12)C2-dansyl labeled human urine, IsoMS detected 2044 peak pairs, and manual inspection of these peak pairs found 90 false peak pairs, representing a false positive rate of 4.4%. IsoMS for Windows running R is freely available for noncommercial use from www.mycompoundid.org/IsoMS . PMID:24766305

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

2014-05-20

132

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

PubMed

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

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

2012-08-01

133

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

PubMed

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

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

2013-11-01

134

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

135

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

NASA Technical Reports Server (NTRS)

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.

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

2004-01-01

136

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

PubMed Central

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.

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

2012-01-01

137

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

NASA Astrophysics Data System (ADS)

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

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

2014-03-01

138

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

NASA Astrophysics Data System (ADS)

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

Loy, Alexander

2010-05-01

139

Modeling the temporal dynamics of monoterpene emission by isotopic labeling in Quercus ilex leaves  

NASA Astrophysics Data System (ADS)

A mathematical model to study the temporal dynamics of stable isotope 13C incorporation into monoterpene molecules, emitted from Mediterranean evergreen sclerophyll oak Quercus ilex L. leaves, was developed. The box model uses leaf level gas exchange and monoterpene emission data to assess biochemical and diffusional processes of the light-dependent monoterpene biosynthesis and emission within the leaf tissues. We estimated total leaf monoterpene pool exchange half-lifes against these processes. The slowest response took up to 38 h, while the fastest response occurred within 1 h, taking the sum of the lumped processes time constants into account. Separately, the turnover half-lives of the biochemical processes ranged between 26 min up to more than 4 h. The diffusional processes turnover times, driven by the physico-chemical properties of the monoterpene molecule, have been found to range between 32 min and 3 h, depending on the number of 13C-labeled carbon atoms. As a consequence, the steady-state assumption that is used in many larger scale emission models, may not hold in all cases and the application of process-based algorithms is beneficial to overcome such long transient pool dynamics of light-dependent monoterpene emission.

Noe, S. M.; Niinemets, Ü.; Schnitzler, J.-P.

2010-01-01

140

Isotope Coded Protein Labeling analysis of plasma specimens from acute severe dengue fever patients  

PubMed Central

Background Dengue fever is the most important arthropod born viral disease of public health significance. Although most patients suffer only from flu-like symptoms, a small group of patient experiences more severe forms of the disease. To contribute to a better understanding of its pathogenesis this study aims to identify proteins differentially expressed in a pool of five viremic plasma from severe dengue patients relative to a pool of five non-severe dengue patients. Results The use of Isotope Coded Protein Labeling (ICPLTM) to analyze plasma depleted of twenty high-abundance proteins allowed for the identification of 51 differentially expressed proteins, which were characterized by mass spectrometry. Using quantitative ELISA, three of these proteins (Leucine-rich glycoprotein 1, Vitamin D binding-protein and Ferritin) were confirmed as having an increased expression in a panel of severe dengue plasma. The proteins identified as overexpressed by ICPLTM in severe dengue plasma involve in clear up action after cell injury, tissue coherence and immune defense. Conclusion This ICPLTM study evaluating differences between acute severe dengue plasmas and acute non-severe dengue plasmas suggests that the three proteins identified are overexpressed early in the course of the disease. Their possible use as biomarkers for the prognostic of disease severity is discussed.

2012-01-01

141

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

PubMed

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

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

2004-08-15

142

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

PubMed

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, and glycosylation pattern of the recombinant protein. Surface plasmon resonance spectroscopy allowed the interaction of GM2AP with immobilized liposomes to be studied. A modified version of FM22 minimal medium was then used in the cost-effective (15)N-labeling of GM2AP to assess its amenability for the structural investigation by NMR spectroscopy. Initial (15)N,(1)H-HSQC experiments show a well-folded protein and provide evidence for extensive conformational exchange processes within the molecule. PMID:14766311

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

2004-03-01

143

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

Microsoft Academic Search

BackgroundMetabolic phenotyping has become an important ‘bird's-eye-view’ technology which can be applied to higher organisms, such as model plant and animal systems in the post-genomics and proteomics era. Although genotyping technology has expanded greatly over the past decade, metabolic phenotyping has languished due to the difficulty of ‘top-down’ chemical analyses. Here, we describe a systematic NMR methodology for stable isotope-labeling

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

2008-01-01

144

A SILAC compatible strain of Pichia pastoris for expression of isotopically labeled protein standards and quantitative proteomics  

PubMed Central

The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications, and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.

Austin, Ryan J.; Kuestner, Rolf E.; Chang, Debbie K.; Madden, Knut R.; Martin, Daniel B.

2012-01-01

145

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

PubMed

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

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

2004-01-01

146

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

SciTech Connect

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

Rabow, L.E.

1990-01-01

147

A stable isotope dual-labelling approach to detect multiple insemination in un-irradiated and irradiated Anopheles arabiensis mosquitoes  

PubMed Central

Background In the context of a Sterile Insect Technique programme, the occurrence of multiple insemination in the malaria mosquito Anopheles arabiensis Patton was studied using a novel labelling system with the stable isotopes 15N and 13C. The incidence of multiple insemination in the absence of radiation, and when males were irradiated in the pupal stage and competed against un-irradiated males were assessed. Males used in the experiments were labelled with either 15N or 13C and the label was applied to the larval rearing water. Males with either label and virgin females were caged at a 1:1:1 ratio. Males used in the radiation treatments were irradiated in the pupal stage with a partially or fully-sterilizing dose of 70 or 120 Gy, respectively. After mating, females were dissected and inseminated spermathecae analysed using mass spectrometry. Results The data indicate that about 25% of inseminated females had been inseminated multiply. The presence of irradiated males in the experiments did not affect the incidence of multiple insemination. In line with previous research, irradiated males were generally less competitive than un-irradiated males. Conclusion The implications of these findings for the Sterile Insect Technique are discussed, and further experiments recommended. The dual-labelling system used to determine paternity gave good results for 13C, however, for 15N it is recommended to increase the amount of label in future studies.

Helinski, Michelle EH; Hood, Rebecca C; Knols, Bart GJ

2008-01-01

148

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

PubMed

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

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

2011-06-01

149

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

PubMed Central

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

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

2011-01-01

150

Platelet labeling in plasma made simple and efficient: Preparation and evaluation  

SciTech Connect

Over the past few years the use of In-111 platelets (In-111-P) has continued to grow rapidly. The lack of efficient labeling in plasma have made the preparation procedures complex resulting into almost individualized methods which have used a variety of salt balance media, anticoagulants, and centrifugal forces, and thereby made the comparison of results from different laboratories impossible. The authors have developed a kit method for preparing In-111-P in plasma that restricts these parameters, uses 2 ..mu..g of dry Mercaptopyridine-N-oxide (Merc) or its Na-salt and a weak chelate of In-111 either in solution at pH 6-6.5 or in a dry form. Eighty to 95% labeling efficiency is achieved in 20 minutes incubation at an ambient temperature. Seventy percent radioactivity is incorporated within two minutes, 90% of which is bound to cytoplasmic components. The aggregability of In-111-P remains practically unchanged. In a caning model, In-111-P thus prepared, had 65% recovery and 7.5 days survival. Two - 50 ..mu..g of oxine or tropolone employed under identical conditions produced only 4%-28% and 7%-10% labeling efficiency. Similarly, with 2 ..mu..g Merc, labeling efficiency using Ga-67, Tc-99m, Tl-201, and I-131 was only 7.5%, 6.8%, 13.8% and 5.8% respectively. This new approach provides investigators a simple, efficient and uniform method for preparing viable In-111-P in plasma.

Thakur, M.L.; McKenney, S.; Park, C.H.; Philp, M.S.; Bruggman, T.; Werre, G.

1984-01-01

151

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

SciTech Connect

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.

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

2002-05-16

152

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

SciTech Connect

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

McMahon, James E.; Wiel, Stephen

2001-02-16

153

Phosphorus use efficiency by cotton measured through 32P isotope technique  

NASA Astrophysics Data System (ADS)

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

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

2012-04-01

154

Fluorescence-, isotope- or biotin-labeling of the 5 '-end of single-stranded DNA/RNA using T4 RNA ligase.  

PubMed Central

A rapid 5'-labeling method of single-stranded DNA/RNA was developed, which is based on the utilization of an adenylated intermediate in the reaction of T4 RNA ligase. This method is commonly useful for fluorescence-, isotope- or biotin-labeling of the 5'-ends of both oligo- and polynucleotides.

Kinoshita, Y; Nishigaki, K; Husimi, Y

1997-01-01

155

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

SciTech Connect

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

Mark E. Fuller; Tullis C. Onstott

2003-12-17

156

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

PubMed

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

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

2002-02-01

157

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napus Embryos1  

PubMed Central

Developing embryos of Brassica napus accumulate both triacylglycerols and proteins as major storage reserves. To evaluate metabolic fluxes during embryo development, we have established conditions for stable isotope labeling of cultured embryos under steady-state conditions. Sucrose supplied via the endosperm is considered to be the main carbon and energy source for seed metabolism. However, in addition to 220 to 270 mm carbohydrates (sucrose, glucose, and fructose), analysis of endosperm liquid revealed up to 70 mm amino acids as well as 6 to 15 mm malic acid. Therefore, a labeling approach with multiple carbon sources is a precondition to quantitatively reflect fluxes of central carbon metabolism in developing embryos. Mid-cotyledon stage B. napus embryos were dissected from plants and cultured for 15 d on a complex liquid medium containing 13C-labeled carbohydrates. The 13C enrichment of fatty acids and amino acids (after hydrolysis of the seed proteins) was determined by gas chromatography/mass spectrometry. Analysis of 13C isotope isomers of labeled fatty acids and plastid-derived amino acids indicated that direct glycolysis provides at least 90% of precursors of plastid acetyl-coenzyme A (CoA). Unlabeled amino acids, when added to the growth medium, did not reduce incorporation of 13C label into plastid-formed fatty acids, but substantially diluted 13C label in seed protein. Approximately 30% of carbon in seed protein was derived from exogenous amino acids and as a consequence, the use of amino acids as a carbon source may have significant influence on the total carbon and energy balance in seed metabolism. 13C label in the terminal acetate units of C20 and C22 fatty acids that derive from cytosolic acetyl-CoA was also significantly diluted by unlabeled amino acids. We conclude that cytosolic acetyl-CoA has a more complex biogenetic origin than plastidic acetyl-CoA. Malic acid in the growth medium did not dilute 13C label incorporation into fatty acids or proteins and can be ruled out as a source of carbon for the major storage components of B. napus embryos.

Schwender, Jorg; Ohlrogge, John B.

2002-01-01

158

Isotopically labeled CO(sub 2) from stratosphere: A tracer of carbon biogeochemistry.  

National Technical Information Service (NTIS)

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)...

Y. L. Yung M. H. Thiemens

1993-01-01

159

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

NASA Astrophysics Data System (ADS)

Tree-ring density and widths have been successfully used to reconstruct summer temperatures in high- northern latitudes, although a discrepancy between tree-growth and temperature has been found for recent decades. The so-called "reduced sensitivity" of tree rings to summer temperatures has been observed especially strongly in northern Siberia (Briffa et al., 1998) and drought stress (increased water use efficiency) arose from global warming and/or increasing CO2 are suggested as causes (Barber et al. 2000, Saurer et al. 2004). By using carbon isotope ratio as an indicator of drought stress and ring-width/density as indicators of growth, we can clarify how drought stress caused by recent global warming affects wood formation of Siberian trees. However, isotope dendroclimatology is still in its infancy and our understanding of basic physiological processes of isotope signal transfer from leaves to tree rings is insufficient. In order to understand translocation, storage, and allocation of photoassimilate to different organs of trees, we pulse- labeled ten L. gmelinii growing in a continuous permafrost zone with stable 13CO2. We studied seasonal course of carbon allocation patterns of photoassimilate among needles, branches, stem and roots and also how spring, summer, and autumn photoassimilate is later used for both earlywood and latewood formation. About half of the carbon in new needles was derived from stored material. The starch pool in non- needle parts, which can be used for xylem formation, drew about 43 percent of its carbon from previous year's photoassimilate, suggesting that carbon storage is the key mechanism behind autocorrelation in (isotope) dendroclimatology. Analysis of intra-annual 13C of the tree rings formed after the labeling revealed that earlywood contained photoassimilate from the previous summer and autumn as well as from the current spring. Latewood was mainly composed of photoassimilate from the current year's summer/autumn, although it also relied on stored material in some cases. Carbon isotope chronology of recent 100 years shows that the latewood 13C contains stronger climate signal than the earlywood and is significantly correlated to July temperature and July precipitation, corresponding to the timing of carbon incorporation that constitutes latewood. The results suggest the need for separating earlywood and latewood for isotope dendroclimatological study in Siberia. References: 1) Kagawa A., Sugimoto A., & Maximov, T.C. (2006) 13CO2 pulse-labelling of photoassimilates reveals carbon allocation within and between tree rings. Plant, Cell and Environment 29, 1571-1584. 2) Kagawa A., Sugimoto A., & Maximov, T. C. (2006) Seasonal course of translocation, storage, and remobilization of 13C pulse-labeled photoassimilate in naturally growing Larix gmelinii saplings. New Phytologist 171, 793-804. 3) Kagawa A., Naito D., Sugimoto A. & Maximov T. C. (2003) Effects of spatial and temporal variability in soil moisture on widths and 13C values of eastern Siberian tree rings. Journal of Geophysical Research 108 (D16), 4500, doi:10.1029/2002JD003019.

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

2006-12-01

160

Rapid Validation of Mascot Search Results via Stable Isotope Labeling, Pair Picking, and Deconvolution of Fragmentation Patterns*  

PubMed Central

Conventional LC-MS/MS data analysis matches each precursor ion and fragmentation pattern to their best fit within databases of theoretical spectra, yielding a peptide identification. Confidence is estimated by a score but can be validated by statistics, false discovery rates, and/or manual validation. A weakness is that each ion is evaluated independently, discarding potentially useful cross-correlations. In a classical approach to de novo sequence analysis, mixtures of peptides differing only in a carboxyl-terminal isotopic label yield fragmentation spectra with single, unlabeled b-type ions but pairs of isotope-labeled y-type ions, facilitating confident assignments. To apply this principle to identification by fragmentation pattern matching, we developed Validator, software that recognizes isotopic peptide pairs and compares their identifications and fragmentation patterns. Testing Validator 1 on a Mascot results file from FT-ICR LC-MS/MS of 16O/18O-labeled yeast cell lysate peptides yielded 2,775 peptide pairs sharing a common identification but differing in carboxyl-terminal label. Comparing observed b- and y-ions with the predicted fragmentation pattern improved the threshold Mascot score for 5% false discovery from 36 to 22, significantly increasing both sensitivity and specificity. Validator 2, which identifies pairs by precursor mass difference alone before comparing observed fragmentation with that predicted by Mascot, found 2,021 isotopic pairs, similarly achieving improved sensitivity and specificity. Finally Validator 3, which finds pairs based on mass difference alone and then deconvolutes fragmentation patterns independently of Mascot, found 964 predicted peptides. Validator 3 allowed raw mass spectrometry data to be mined not only to validate Mascot results but also to discover peptides missed by Mascot. Using standard desktop hardware, the Validator 1–3 software processed the 11,536 spectra in the 93-MB Mascot .DAT file in less than 6 min (32 spectra/s), revealing high confidence peptide identifications without regard to Mascot score, far faster than manual or other independent validation methods.

Volchenboum, Samuel L.; Kristjansdottir, Kolbrun; Wolfgeher, Donald; Kron, Stephen J.

2009-01-01

161

Integration of High Accuracy N-Terminus Identification in Peptide Sequencing and Comparative Protein Analysis Via Isothiocyanate-Based Isotope Labeling Reagent with ESI Ion-trap TOF MS  

NASA Astrophysics Data System (ADS)

A multifunctional isothiocyanate-based isotope labeling reagent, [ d 0]-/[ d 6]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b1 ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b1 ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsin-digested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research.

Leng, Jiapeng; Wang, Haoyang; Zhang, Li; Zhang, Jing; Wang, Hang; Cai, Tingting; Yao, Jinting; Guo, Yinlong

2011-07-01

162

A ROBUST ABSOLUTE DETECTION EFFICIENCY CALIBRATION METHOD UTILIZING BETA/GAMMA COINCIDENCE SIGNATURES AND ISOTOPICALLY PURIFIED NEUTRON ACTIVATED RADIOXENON ISOTOPES  

SciTech Connect

Efforts to calibrate the absolute efficiency of gas cell radiations detectors have utilized a number of methodologies which allow adequate calibration but are time consuming and prone to a host of difficult-to-determine uncertainties. A method that extrapolates the total source strength from the measured beta and gamma gated beta coincidence signal was developed in the 1960’s and 1970’s. It has become clear that it is possible to achieve more consistent results across a range of isotopes and a range of activities using this method. Even more compelling is the ease with which this process can be used on routine samples to determine the total activity present in the detector. Additionally, recent advances in the generation of isotopically pure radioxenon samples of Xe-131m, Xe-133, and Xe-135 have allowed these measurement techniques to achieve much better results than would have been possible before when using mixed isotopic radioxenon source. This paper will discuss the beta/gamma absolute detection efficiency technique that utilizes several of the beta-gamma decay signatures to more precisely determine the beta and gamma efficiencies. It will than compare these results with other methods using pure sources of Xe-133, Xe-131m, and Xe-135 and a Xe-133/Xe-133m mix.

McIntyre, Justin I.; Cooper, Matthew W.; Ely, James H.; Haas, Derek A.; Schrom, Brian T.

2012-09-21

163

Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy  

Microsoft Academic Search

The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to

Patrik Lundström; Pramodh Vallurupalli; D Flemming Hansen; Lewis E Kay

2009-01-01

164

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 ...

165

Rapid, high-efficiency labeling of leukocytes with In-111 after hemolytic removal of erythrocytes  

SciTech Connect

During the labeling of leukocytes with Indium-111, conventional methodology involves separation and washing to remove red cells. This technique results in the loss of a significant number of leukocytes. Citrated whole blood of ten normal volunteers was studied for an alternate labeling method following sedimentation for 30 to 45 minutes and low speed centrifugation of the leukocyte-rich plasma. The average labeling for these ten volunteers by Indium-111 was 90% versus 60% by the older technique. Viability as measured by the trypan blue exclusion test was greater than 95%, WBC losses were essentially zero, and no WBC clumping was observed. Eighteen patients referred for leukocyte imaging were studied by this method. In this patient population, there was 91% labeling with viability greater than 95% and no evidence of clumping. Less than 5% RBC's were noted in any lot. Indium-111 WBC activity 20 minutes post injection averaged 79% of whole blood activity. This modification results in decreased losses of white cells, reduces preparation time to less than 2 hours, and significantly improves the labeling efficiency of the final product. Liver/spleen ratios and image quality were unchanged from the original method.

Karesh, S.M.; Henkin, R.E.

1985-05-01

166

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

PubMed

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

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

2014-01-27

167

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

NASA Astrophysics Data System (ADS)

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

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

2013-06-01

168

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

PubMed Central

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

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

2013-01-01

169

A software toolkit and interface for performing stable isotope labeling and top3 quantification using Progenesis LC-MS.  

PubMed

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

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

2012-09-01

170

Efficient preparation and labeling of human induced pluripotent stem cells by nanotechnology  

PubMed Central

Efficient preparation and labeling of human induced pluripotent stem (iPS) cells is a great challenge in stem cell research and development. With the aim of investigating the feasibility of using nanotechnology to enhance the preparation efficiency of iPS cells and to label iPS cells for long-term tracing and imaging, in this paper, four transcription factor genes, ie, Oct4, Sox2, LIN28, and Nanog, and packaging plasmids such as PSPAX2 and PMD2.G were cotransfected into 293T cells using Generation 5.0 polyamidoamine dendrimer-modified magnetic nanoparticles (dMNPs) as a delivery system. The resultant supernatant liquids were incubated with human fibroblast cells at 37°C for 21 days, then the embryonic stem (ES) cell-like clones were screened, cultured, and identified. Finally, the prepared iPS cells were labeled with fluorescent magnetic nanoparticles (FMNPs). The results showed that dMNPs can efficiently deliver all vectors into 293T cells. The resultant lentiviruses’ titers were 10-fold more than those based on Lipofectamine™ 2000. Reverse transcription polymerase chain reaction analysis showed that four genes (Oct4, Sox2, LIN28, and Nanog) exhibited different expressions in iPS cells. Immunostaining analysis showed that specific surface markers of ES cells such as SSEA-3, SSEA-4, Tra-1-60, and Tra-1-81 were positive in iPS cells, and the terotomas were formed in NOD-SCID mice that were implanted with iPS cells. Red fluorescent signals could be observed in iPS cells labeled with FMNPs by fluorescent microscopy, and the magnetic signals were detected in labeled iPS cells by magnetic resonance imaging. In conclusion, human iPS cells can be efficiently generated using polyamidoamine dMNPs and lentivirus and labeled with FMNPs for long-term observation and tracking, which has great potential application in the research and development of stem cells in the near future.

Ruan, Jing; Shen, Jie; Wang, Zheng; Ji, Jiajia; Song, Hua; Wang, Kan; Liu, Bin; Li, Jinhui; Cui, Daxiang

2011-01-01

171

Examination of the rate of peptide biosynthesis in neuroendocrine cell lines using a stable isotopic label and mass spectrometry.  

PubMed

The biosynthesis of neuroendocrine peptides is typically examined by following the rate of appearance of a radioactive amino acid into mature forms of peptides. In the present study, we labeled cell lines with L-leucine containing 10 deuterium residues (d(10)-Leu) and used mass spectrometry to measure the biosynthetic rate of gamma-lipotropin in the AtT-20 cell line and insulin in the INS-1 cell line. After 3 h of labeling, both peptides show detectable levels of the d-labeled form in the cells and media. The relative levels of the d-labeled forms are greater in the media than in the cells, consistent with previous studies that found that newly synthesized peptides are secreted at a higher rate than older peptides under basal conditions. When AtT-20 cells were stimulated with KCl or forskolin, the ratio of d- to H-labeled gamma-lipotropin in the medium decreased, suggesting that the older peptide was in a compartment that could be released upon the appropriate stimulation. Overexpression of proSAAS in AtT-20 cells reduced the ratio of d- to H-labeled gamma-lipotropin, consistent with the proposed role of proSAAS as an endogenous inhibitor of prohormone convertase-1. Labeling with d10-Leu was also used to test whether altering the pH of the secretory pathway with chloroquine affected the rate of peptide biosynthesis. In AtT-20 cells, 30 microm chloroquine for 3 or 6 h significantly reduced the rate of formation of gamma-lipotropin in both cells and media. Similarly, INS-1 cells treated with 10, 30, or 60 microm chloroquine for 6 h showed a significant decrease in the rate of formation of insulin in both cells and media. These results are consistent with the acidic pH optima for peptide processing enzymes. Stable isotopic labeling with d10-Leu provides a sensitive method to examine the rate of peptide formation in neuroendocrine cell lines. PMID:15255936

Che, Fa-Yun; Yuan, Quan; Kalinina, Elena; Fricker, Lloyd D

2004-08-01

172

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

DOEpatents

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

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

2008-01-22

173

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

SciTech Connect

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

Wiel, Stephen; Van Wie McGrory, Laura

2003-08-04

174

Efficient calculation of exact fine structure isotope patterns via the multidimensional Fourier transform.  

PubMed

The isotope patterns of unknown analytes provide information that can be of great value in their identification as part of a mass spectrometry experiment. Determining the range of compounds that are consistent with an empirically observed isotope pattern requires, as an initial step, the calculation of the theoretical isotope patterns of all feasible candidate formulas, and this is not a trivial mathematical task. While algorithms based on the Fourier transform have been used for almost two decades to perform such calculation efficiently, they have hitherto not been able to provide the exact sets of masses and abundances that constitute the fundamental isotope pattern. This article presents a new approach to the treatment of such calculations, which involves arranging and manipulating the isotope patterns of distinct elements as multidimensional data structures. This enables the use of the multidimensional Fourier transform to calculate isotope patterns with an accuracy that is limited only by the errors of floating point arithmetic. The algorithm is both highly efficient and very easy to implement in many programming environments. An open-source implementation of the algorithm in the R programming language will be made publicly available and is also available upon request. PMID:24841326

Ipsen, Andreas

2014-06-01

175

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

PubMed Central

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

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

2012-01-01

176

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

Microsoft Academic Search

Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA\\u000a to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides\\u000a a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal\\u000a overlap, without information loss. Here we show that

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

2009-01-01

177

5-Diethylamino-naphthalene-1-sulfonyl chloride (DensCl): a novel triplex isotope labeling reagent for quantitative metabolome analysis by liquid chromatography mass spectrometry.  

PubMed

We describe a new set of isotope reagents, (12)C4-, (12)C2(13)C2-, and (13)C4-5-diethylamino-naphthalene-1-sulfonyl chloride (DensCl), in combination with liquid chromatography Fourier-transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS), for improved analysis of the amine- and phenol-containing submetabolome. The synthesis of the reagents is reported, and an optimized derivatization protocol for labeling amines and phenols is described. To demonstrate the utility of the triplex reagents for metabolome profiling of biological samples, urine samples collected daily from a healthy volunteer over a period of 14 days were analyzed. The overall workflow is straightforward, including differential isotope labeling of individual samples and a pooled sample that serves a global internal standard, mixing of the isotope differentially labeled samples and LC-MS analysis for relative metabolome quantification. Comparing to the dansyl chloride (DnsCl) duplex isotope reagents, the new triplex DensCl reagents offer the advantages of improved metabolite detectability due to enhanced sensitivity (i.e., about 1000 peak pairs detected by DensCl labeling vs about 600 peak pairs detected by DnsCl labeling) and analysis speed (i.e., simultaneous analysis of two comparative samples by DensCl vs only one comparative sample analyzed by DnsCl). PMID:24200037

Zhou, Ruokun; Guo, Kevin; Li, Liang

2013-12-01

178

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

PubMed Central

Deuterated styrene ([2H8]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [2H8]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [2H8]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.

Alexandrino, Maria; Knief, Claudia; Lipski, Andre

2001-01-01

179

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

Microsoft Academic Search

In an effort to shed additional light on the chemical mechanisms underlying the radiation–oxidation of polypropylene (PP), we are using samples having selective 13C isotopic labeling at the three unique sites within the macromolecular structure. After radiation exposure, we applied GC\\/MS, solid-state 13C NMR, and FTIR to evaluate the applicability of each technique in identifying the molecular labeling of the

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

2007-01-01

180

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

Microsoft Academic Search

In an effort to shed additional light on the chemical mechanisms underlying the radiation oxidation of polypropylene (PP), we are using samples having selective 13C isotopic labeling at the three unique sites within the macromolecular structure. After radiation exposure, we applied GC\\/MS, solid-state 13C NMR, and FTIR to evaluate the applicability of each technique in identifying the molecular labeling of

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

2007-01-01

181

Isotopically labeled sulfur compounds and synthetic selenium and tellurium analogues to study sulfur metabolism in marine bacteria  

PubMed Central

Summary Members of the marine Roseobacter clade can degrade dimethylsulfoniopropionate (DMSP) via competing pathways releasing either methanethiol (MeSH) or dimethyl sulfide (DMS). Deuterium-labeled [2H6]DMSP and the synthetic DMSP analogue dimethyltelluriopropionate (DMTeP) were used in feeding experiments with the Roseobacter clade members Phaeobacter gallaeciensis DSM 17395 and Ruegeria pomeroyi DSS-3, and their volatile metabolites were analyzed by closed-loop stripping and solid-phase microextraction coupled to GC–MS. Feeding experiments with [2H6]DMSP resulted in the incorporation of a deuterium label into MeSH and DMS. Knockout of relevant genes from the known DMSP demethylation pathway to MeSH showed in both species a residual production of [2H3]MeSH, suggesting that a second demethylation pathway is active. The role of DMSP degradation pathways for MeSH and DMS formation was further investigated by using the synthetic analogue DMTeP as a probe in feeding experiments with the wild-type strain and knockout mutants. Feeding of DMTeP to the R. pomeroyi knockout mutant resulted in a diminished, but not abolished production of demethylation pathway products. These results further corroborated the proposed second demethylation activity in R. pomeroyi. Isotopically labeled [2H3]methionine and 34SO4 2?, synthesized from elemental 34S8, were tested to identify alternative sulfur sources besides DMSP for the MeSH production in P. gallaeciensis. Methionine proved to be a viable sulfur source for the MeSH volatiles, whereas incorporation of labeling from sulfate was not observed. Moreover, the utilization of selenite and selenate salts by marine alphaproteobacteria for the production of methylated selenium volatiles was explored and resulted in the production of numerous methaneselenol-derived volatiles via reduction and methylation. The pathway of selenate/selenite reduction, however, proved to be strictly separated from sulfate reduction.

Brock, Nelson L; Citron, Christian A; Zell, Claudia; Berger, Martine; Wagner-Dobler, Irene; Petersen, Jorn; Brinkhoff, Thorsten; Simon, Meinhard

2013-01-01

182

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

Microsoft Academic Search

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

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

2005-01-01

183

Sulfonium ion derivatization, isobaric stable isotope labeling and data dependent CID- and ETD-MS/MS for enhanced phosphopeptide quantitation, identification and phosphorylation site characterization.  

PubMed

An amine specific peptide derivatization strategy involving the use of novel isobaric stable isotope encoded 'fixed charge' sulfonium ion reagents, coupled with an analysis strategy employing capillary HPLC, ESI-MS, and automated data dependent ion trap CID-MS/MS, -MS(3), and/or ETD-MS/MS, has been developed for the improved quantitative analysis of protein phosphorylation, and for identification and characterization of their site(s) of modification. Derivatization of 50 synthetic phosphopeptides with S,S'-dimethylthiobutanoylhydroxysuccinimide ester iodide (DMBNHS), followed by analysis using capillary HPLC-ESI-MS, yielded an average 2.5-fold increase in ionization efficiencies and a significant increase in the presence and/or abundance of higher charge state precursor ions compared to the non-derivatized phosphopeptides. Notably, 44% of the phosphopeptides (22 of 50) in their underivatized states yielded precursor ions whose maximum charge states corresponded to +2, while only 8% (4 of 50) remained at this maximum charge state following DMBNHS derivatization. Quantitative analysis was achieved by measuring the abundances of the diagnostic product ions corresponding to the neutral losses of 'light' (S(CH(3))(2)) and 'heavy' (S(CD(3))(2)) dimethylsulfide exclusively formed upon CID-MS/MS of isobaric stable isotope labeled forms of the DMBNHS derivatized phosphopeptides. Under these conditions, the phosphate group stayed intact. Access for a greater number of peptides to provide enhanced phosphopeptide sequence identification and phosphorylation site characterization was achieved via automated data-dependent CID-MS(3) or ETD-MS/MS analysis due to the formation of the higher charge state precursor ions. Importantly, improved sequence coverage was observed using ETD-MS/MS following introduction of the sulfonium ion fixed charge, but with no detrimental effects on ETD fragmentation efficiency. PMID:21952753

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

2012-04-01

184

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

ERIC Educational Resources Information Center

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

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

2004-01-01

185

Selective isotope labelling of leucine residues by using ?-ketoacid precursor compounds.  

PubMed

You can have one without the other: A new metabolic precursor compound can selectively introduce (13)C and (2)H patterns at leucine residues in proteins in cell-based expression systems without interfering with valine metabolism. The protocol provides selectively labelled macromolecules well suited for probing structure and dynamics in high-molecular-weight proteins by NMR spectroscopy. PMID:23564734

Lichtenecker, Roman J; Coudevylle, Nicolas; Konrat, Robert; Schmid, Walther

2013-05-10

186

Stable Isotope Labeled 4-(Dimethylamino)benzoic Acid Derivatives of Glycerophosphoethanolamine Lipids  

PubMed Central

A set of four (d0, d4, d6, and d10) deuterium enriched 4-(dimethylamino)benzoic acid (DMABA) N-hydroxysuccinimide (NHS) ester reagents was developed that react with the primary amine group of glycerophosphoethanolamine (PE) lipids to create derivatives where all subclasses of DMABA labeled PE are detected by a common precursor ion scan. The positive ion collision induced dissociation data from (d0, d4, d6, and d10)-DMABA labeled PE standards indicated that a precursor ion scan of m/z 191.1, 195.1, 197.1, and 201.1 could be used to selectively detect (d0, d4, d6, and d10)-DMABA modified PE, respectively, in a complex biological mixture. The PE lipids from a time course (0, 30, 60, and 300 min) of AAPH treatment of liposomes made of RAW 264.7 cell phospholipids were each labeled with the d0-, d4-, d10-, and d6-DMABA NHS ester reagents, respectively. The DMABA derivatives revealed loss of endogenous PE lipids and an increase in oxidized PE lipid throughout the time course of AAPH treatment. These DMABA NHS ester reagents provide a universal scan for diacyl, ether, and plasmalogen PE lipids that can not be readily observed otherwise, enable differential labeling, and provide an internal standard for each PE lipid.

Berry, Karin A. Zemski; Turner, William W.; VanNieuwenhze, Michael S.; Murphy, Robert C.

2010-01-01

187

Evapotranspiration partitioning of a winter wheat and summer maize double-cropping system using isotopic labeling  

NASA Astrophysics Data System (ADS)

The oxygen and hydrogen isotope compositions of ecosystem water pools and fluxes represent important tracers in the water cycle. Combining with eddy covariance technique, it is possible to partition evapotranspiration (ET) into evaporation (E) and transpiration (T) components based on ?18O and ?D measurements in the liquid and gas phases. The key challenge is to precisely determine the ?18O and ?D of ET (?ET), E (?E) and T (?T). In this study, we present high frequency 18O and D measurements of water pools (water vapor, precipitation, dew, groundwater, soil water, xylem water and leaf water) and water flux (evapotranspiration). We characterize, in conjunction with intensive field campaigns, the temporal dynamics of the ?18O and ?D signals during the growing season of a in a winter wheat and summer maize double-cropping system in North China in 2008. The three flux end member isotopic signals were determined as follows: continuous ?ET measurement was made with the flux-gradient method using a tunable diode laser analyzer, ?E was estimated with the Craig-Gordon model with a moisture-dependent soil resistance, and ?T was approximated by the ?18O and ?D of water in xylem (?x) in midday hours assuming ?T being equal to ?x under isotopic steady state, and by a non-steady state Craig-Gordon model for other times. We found that transpiration comprised ~90% of ET during peak wheat and corn growth periods. In a one-month period from wheat harvest to the establishment of a full corn canopy, transpiration contributed ~70% of ET. These results were consistent with soil lysimeter and eddy covariance measurements made during the same time period. Isotopic partitioning at sub-day time scales was however very scattered, due to large measurement noises in ?ET, uncertainties in the Craig-Gordon model predictions and spatial variability in ?x.

Sun, X.; Wen, X.; Yu, G.; Lee, X.

2010-12-01

188

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

PubMed Central

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

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

2012-01-01

189

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

NASA Astrophysics Data System (ADS)

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

Liang, Wenxin; Takahashi, Akihiro; Yokota, Haruo

190

Isotope labeling methods for studies of excited protein states by relaxation dispersion NMR spectroscopy.  

PubMed

The utility of nuclear magnetic resonance (NMR) spectroscopy as a tool for the study of biomolecular structure and dynamics has benefited from the development of facile labeling methods that incorporate NMR active probes at key positions in the molecule. Here we describe a protocol for the labeling of proteins that facilitates their study using a technique that is sensitive to millisecond conformational exchange processes. The samples necessary for an analysis of exchange dynamics are discussed, using the Abp1p SH3 domain from Saccharomyces cerevisiae as an example. For this system, the time frame for production of each sample, including in vitro refolding, is about 80 h. The samples so produced facilitate the measurement of accurate chemical shifts of low populated, invisible conformers that are part of the exchange pathway. The accuracy of the methodology has been established experimentally and the chemical shifts that are obtained provide important restraints in structure calculations of the excited state. PMID:19876024

Lundström, Patrik; Vallurupalli, Pramodh; Hansen, D Flemming; Kay, Lewis E

2009-01-01

191

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

Microsoft Academic Search

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

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

2002-01-01

192

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

SciTech Connect

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

Carney, P.L.; Rogers, P.E.; Johnson, D.K. (Abbott Laboratories, Abbott Park, IL (USA))

1989-03-01

193

Method for comparative analysis of ribonucleic acids using isotope labeling and mass spectrometry.  

PubMed

Here, we describe a method for the comparative analysis of ribonucleic acids (RNAs). This method allows sequence or modification information from a previously uncharacterized RNA to be obtained by direct comparison with a reference RNA, whose sequence or modification information is known. This simple and rapid method is enabled by the differential labeling of two RNA samples. One sample, the reference RNA, is labeled with (16)O during enzymatic digestion. The second sample, the candidate or unknown RNA, is labeled with (18)O. By combining the two digests, digestion products that share the same sequence or post-transcriptional modification(s) between the reference and candidate will appear as doublets separated by 2 Da. Sequence or modification differences between the two will generate singlets that can be further characterized to identify how the candidate sequence differs from the reference. We illustrate the application of this approach for sequencing individual RNAs and demonstrate how this method can be used to identify sequence-specific differences in RNA modification. This comparative analysis of RNA digests (CARD) approach is scalable to multiple candidate RNAs using one or multiple reference RNAs and is compatible with existing methods for quantitative analysis of RNAs. PMID:22985222

Li, Siwei; Limbach, Patrick A

2012-10-16

194

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

NASA Astrophysics Data System (ADS)

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

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

2012-09-01

195

High efficiency Hall effect micro-biosensor platform for detection of magnetically labeled biomolecules.  

PubMed

Detection of magnetically labeled biomolecules using micro-Hall biosensors is a promising method for monitoring biomolecular recognition processes. The measurement efficiency of standard systems is limited by the time taken for magnetic beads to reach the sensing area of the Hall devices. Here, micro-current lines were integrated with Hall effect structures to manipulate the position of magnetic beads via field gradients generated by localized currents flowing in the current lines. Beads were accumulated onto the sensor surface within seconds of passing currents through the current lines. Real-time detection of magnetic beads using current lines integrated with Hall biosensors was achieved. These results are promising in establishing Hall biosensor platforms as efficient and inexpensive means of monitoring biomolecular reactions for medical applications. PMID:17055242

Sandhu, Adarsh; Kumagai, Yoshimichi; Lapicki, Adam; Sakamoto, Satoshi; Abe, Masanori; Handa, Hiroshi

2007-04-15

196

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

NASA Astrophysics Data System (ADS)

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

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

2014-06-01

197

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

SciTech Connect

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

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

1987-10-01

198

An efficient method to calculate the aggregated isotopic distribution and exact center-masses.  

PubMed

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. PMID:22351289

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

2012-04-01

199

An Optimized Method for Computing 18O/16O Ratios of Differentially Stable-isotope Labeled Peptides in the Context of Post-digestion 18O Exchange/Labeling  

PubMed Central

Differential 18O/16O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H218O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs, has made trypsin-catalyzed 18O post-digestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed 18O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the 18O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual 18O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then 18O/16O ratios derived via evolutionary programming. The algorithm is tested using trypsin–catalyzed 18O post-digestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both, accuracy and precision are improved utilizing this rigorous mathematical approach. Utilizing this algorithmic technique, we demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios for differentially labeled BSA peptides, by accounting for artifacts caused by a variable degree of post-digestion 18O exchange. We further demonstrate the effectiveness of this method to accurately calculate 18O/16O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its non myristylated mutant.

Ye, Xiaoying; Luke, Brian T.; Johann, Donald J.; Ono, Akira; Chan, King C.; Prieto, DaRue A.; Issaq, Haleem J.; Veenstra, Timothy D.; Blonder, Josip

2012-01-01

200

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

PubMed Central

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

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

2014-01-01

201

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

NASA Astrophysics Data System (ADS)

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 = (?E - ?P)/ (?ox - ?trans) where fox is the fraction of CH4 oxidized; ?E is ?13C of emitted CH4; ?P is ?13C of produced CH4; ?ox is the isotopic fractionation factor of oxidation; ?trans 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 ?ox and ?trans 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 (?trans = 1), other CH4 transport mechanisms as diffusion have to be considered in peatlands and ?trans exceeds a value of 1. At our study we determined ?trans = 1.013 ± 0.003 for CH4 when diffusion is the predominant transport mechanism. Furthermore, results showed that ?ox differs widely between sites and horizons (?ox = 1.013 ± 0.012) and has to be determined for each case. The impact of both fractionation factors on the quantification of CH4 oxidation was estimated by considering both the potential diffusion rate at different water contents and potential oxidation rates. Calculations for a water saturated tundra soil indicated a CH4 oxidation efficiency of 88% in the upper horizon. Using carbon isotope fractionation improves the in situ quantification of CH4 oxidation in wetlands and thus the assessment of current and potential CH4 sources and sinks in these ecosystems.

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

2012-04-01

202

Identification of metabolites of honokiol in rat urine using 13C stable isotope labeling and liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.  

PubMed

A general approach based on stable isotope labeling and UPLC/Q-TOF-MS analysis of in vivo novel metabolites of honokiol has been developed in our study. In this method, urine samples were collected after intravenous administration of mixture of regular and [(13)C6]-labeled honokiol at 1:1 ratio to healthy rats. The metabolites could be easily recognized by the determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to that between isotope-labeled and non-isotope-labeled honokiol. A total of 51 metabolites were detected, 37 of which were tentatively identified based on mass accuracy (<5 ppm). Among them, 33 of honokiol metabolites were first reported with 5 metabolites belonging to phase I and other 32 metabolites belonging to phase II metabolites. Our results highlighted that the main phase I metabolic pathways of honokiol in rats were oxidation, and the phase II metabolic pathways were sulfation, glucuronidation, acetylation as well as amino acids conjugation. This was the first research focused on the biotransformation of honokiol in rats, and the identification of these metabolites might provide us essential information for further pharmacological and clinical studies of honokiol. PMID:23618226

Liu, Juan; Tang, Minghai; Lai, Huijun; Dong, Yinfeng; Xie, Caifeng; Ye, Haoyu; Ma, Liang; Qiu, Neng; Li, Yanfang; Cai, Lulu; Chen, Lijuan

2013-06-21

203

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

PubMed Central

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.

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

2014-01-01

204

iMS2Flux - a high-throughput processing tool for stable isotope labeled mass spectrometric data used for metabolic flux analysis  

PubMed Central

Background Metabolic flux analysis has become an established method in systems biology and functional genomics. The most common approach for determining intracellular metabolic fluxes is to utilize mass spectrometry in combination with stable isotope labeling experiments. However, before the mass spectrometric data can be used it has to be corrected for biases caused by naturally occurring stable isotopes, by the analytical technique(s) employed, or by the biological sample itself. Finally the MS data and the labeling information it contains have to be assembled into a data format usable by flux analysis software (of which several dedicated packages exist). Currently the processing of mass spectrometric data is time-consuming and error-prone requiring peak by peak cut-and-paste analysis and manual curation. In order to facilitate high-throughput metabolic flux analysis, the automation of multiple steps in the analytical workflow is necessary. Results Here we describe iMS2Flux, software developed to automate, standardize and connect the data flow between mass spectrometric measurements and flux analysis programs. This tool streamlines the transfer of data from extraction via correction tools to 13C-Flux software by processing MS data from stable isotope labeling experiments. It allows the correction of large and heterogeneous MS datasets for the presence of naturally occurring stable isotopes, initial biomass and several mass spectrometry effects. Before and after data correction, several checks can be performed to ensure accurate data. The corrected data may be returned in a variety of formats including those used by metabolic flux analysis software such as 13CFLUX, OpenFLUX and 13CFLUX2. Conclusion iMS2Flux is a versatile, easy to use tool for the automated processing of mass spectrometric data containing isotope labeling information. It represents the core framework for a standardized workflow and data processing. Due to its flexibility it facilitates the inclusion of different experimental datasets and thus can contribute to the expansion of flux analysis applications.

2012-01-01

205

Expression and isotopic labeling of structural domains of the human protein DEK  

Microsoft Academic Search

The 375 amino acid human protein DEK has been expressed in two functional, structured domains. DEK is an abundant nuclear protein that associates with chromatin and alters its topology by introducing positive supercoiling in DNA, which results in lower replication efficiency. DEK has clinical importance as transfection of the cDNA of the C-terminal region of DEK can partially reverse the

Matthew Devany; N. Prasad Kotharu; Hiroshi Matsuo

2005-01-01

206

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

NASA Astrophysics Data System (ADS)

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

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

207

Gram-scale synthesis and efficient purification of 13C-labeled levoglucosan from 13C glucose.  

PubMed

(13)C-Labeled levoglucosan has been synthesized and purified in good yield, and on the gram scale in one step from commercially available (13)C glucose. This one-step protocol uses 2-chloro-1,3-dimethylimidazolinium chloride that serves to selectively activate the anomeric carbon toward substitution reactions. The labeled glucose is then smoothly converted to the anhydroglucose. Purification is efficiently achieved on large scale by chromatography on silica gel. PMID:24285138

Alexander, Lisa; Hoyt, Caroline; Michalczyk, Ryszard; Wu, Ruilian; Thorn, Dave L; Silks, L A Pete

2013-01-01

208

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

SciTech Connect

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.

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

2006-09-01

209

Latest developments in sample treatment for 18O-isotopic labeling for proteomics mass spectrometry-based approaches: a critical review.  

PubMed

Nowadays isotopic (18)O-labeling of peptides has recalled the attention of researchers due to its simplicity of application and high versatility for proteomics studies. Protein quantification, differential peptide mass mapping, studies regarding proteins overexpressed or underexpressed, or the searching of biomarkers can be accomplished by using (18)O-labeling. In this critical review we comment on the different ways in which (18)O-labeling can be done, highlighting the key parameters of the different sample treatments to obtain a reliable and reproducible labeling. In addition we describe and compare the latest improvement in terms of sample treatment that allows to reduce the handling and to increase the throughput for this sample treatment. Finally, we hypothesize on the future trends of these methods under the light of the new technological advances to speed protein cleavage. PMID:20082805

Capelo, J L; Carreira, R J; Fernandes, L; Lodeiro, C; Santos, H M; Simal-Gandara, J

2010-02-15

210

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

SciTech Connect

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

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

1989-05-02

211

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

SciTech Connect

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

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

2012-03-01

212

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

PubMed

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 (13)CO(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. PMID:22371075

O'Grady, John; Schwender, Jörg; Shachar-Hill, Yair; Morgan, John A

2012-03-01

213

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

NASA Astrophysics Data System (ADS)

Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the arctic will cause deeper permafrost thawing, followed by increased carbon mineralization and CH4 formation in water-saturated tundra soils, thus creating a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and ?13CH4 signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (such as landfill cover soils) have assumed a gas transport dominated by advection (?trans = 1). In tundra soils, however, diffusion is the main gas transport mechanism and diffusive stable isotope fractionation should be considered alongside oxidative fractionation. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox = 1.017 ± 0.009) and needs to be determined on a case by case basis. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged, organic-rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost-affected ecosystems and their potential strengths in response to global warming.

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

2013-04-01

214

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

NASA Astrophysics Data System (ADS)

Permafrost-affected tundra soils are significant sources of the climate-relevant trace gas methane (CH4). The observed accelerated warming of the Arctic will cause a deeper permafrost thawing followed by increased carbon mineralization and CH4 formation in water saturated tundra soils which might cause a positive feedback to climate change. Aerobic CH4 oxidation is regarded as the key process reducing CH4 emissions from wetlands, but quantification of turnover rates has remained difficult so far. The application of carbon stable isotope fractionation enables the in situ quantification of CH4 oxidation efficiency in arctic wetland soils. The aim of the current study is to quantify CH4 oxidation efficiency in permafrost-affected tundra soils in Russia's Lena River Delta based on stable isotope signatures of CH4. Therefore, depth profiles of CH4 concentrations and ?13CH4-signatures were measured and the fractionation factors for the processes of oxidation (?ox) and diffusion (?diff) were determined. Most previous studies employing stable isotope fractionation for the quantification of CH4 oxidation in soils of other habitats (e.g. landfill cover soils) have assumed a gas transport dominated by advection (?trans = 1). In tundra soils, however, diffusion is the main gas transport mechanism, aside from ebullition. Hence, diffusive stable isotope fractionation has to be considered. For the first time, the stable isotope fractionation of CH4 diffusion through water-saturated soils was determined with an ?diff = 1.001 ± 0.000 (n = 3). CH4 stable isotope fractionation during diffusion through air-filled pores of the investigated polygonal tundra soils was ?diff = 1.013 ± 0.003 (n = 18). Furthermore, it was found that ?ox differs widely between sites and horizons (mean ?ox, = 1.017 ± 0.009) and needs to be determined individually. The impact of both fractionation factors on the quantification of CH4 oxidation was analyzed by considering both the potential diffusion rate under saturated and unsaturated conditions and potential oxidation rates. For a submerged organic rich soil, the data indicate a CH4 oxidation efficiency of 50% at the anaerobic-aerobic interface in the upper horizon. The improved in situ quantification of CH4 oxidation in wetlands enables a better assessment of current and potential CH4 sources and sinks in permafrost affected ecosystems and their potential strengths in response to global warming.

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

2012-12-01

215

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

NASA Astrophysics Data System (ADS)

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

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

2014-05-01

216

Characterization of isotopically labeled coal liquefaction solvents and products by deuterium and carbon-13 nuclear magnetic resonance spectrometry  

SciTech Connect

Primary and secondary deuterium isotope shifts are observed in the carbon-13 NMR spectrum of partially deuterated tetralin and naphthalene. These shifts are used to quantitate the extent of deuteration at each carbon site. Deuterium exchange at the tetralin ..cap alpha.. position is faster than coal liquefaction. A deuterium NMR method, using the natural abundance signal of the solvent as an internal standard, shows not only the relative distribution of deuterium in coal liquid products produced from deuterated tetralin as the donor solvent but also the absolute amount of deuterium in nonvolatile products. Some carbon-carbon coupling constants are reported for (1-/sup 13/C)tetralin, (1-/sup 13/C)octahydrophenathrene, and (4-/sup 13/C)-2,6-dimethyltetralin. The carbon-13 compounds verify that thermal ring opening is a negligible process under coal liquefaction conditions unless a catalyst is present. Ring contraction remains constant at about 5% of the thermal products with or without a catalyst. Deuterium NMR is used to follow the deuterium exchange between partially deuterated tetralin and mestylene. The deuterium label goes to the mesitylene methyls as expected. 13 references, 8 figures, 6 tables.

Young, D.C.; McNeil, R.I.; Cronauer, D.C.; Ruberto, R.G.; Galya, L.G.

1984-03-01

217

Investigation of synergy effects in selective oxidation catalysts through in situ laser Raman spectroscopy/isotopic labeling technique  

SciTech Connect

Previous studies over two-phase catalysts consisting of a simple molybdate (MnMoO{sub 4}, CdMoO{sub 4}) in close contact with molybdenum oxide (MoO{sub 3}) have shown the existence of a strong synergy effect in partial oxidation of C{sub 4} hydrocarbons to maleic anhydride. In this paper, the authors present results of the investigation of this synergy effect using an isotopic labeling technique coupled with in situ laser Raman spectroscopy. Results from these studies, when integrated with their previous work, provide complementary evidence for the catalytic job distribution of the two phases, in which the MoO{sub 3} phase incorporates its lattice oxygen into the hydrocarbon molecule, while the simple molybdate phase provides the oxygen necessary to regenerate MoO{sub 3} sites through an oxygen spillover mechanism. The in situ Raman spectroscopy experiments combined with structural specificity studies provide further clues about the catalytic sites responsible for selective versus complete oxidation.

Ozkan, U.S.; Smith, M.R.; Driscoll, S.A. (Ohio State Univ., Columbus (United States))

1992-03-01

218

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

PubMed

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 the multicellular organism C. elegans. Different subcellular distributions of choline metabolites are observed between cancer cells and non-cancer cells, which may reveal a 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 proteins. 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

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

2014-05-21

219

Relative quantitation of protein nitration by liquid chromatography-mass spectrometry using isotope-coded dimethyl labeling and chemoprecipitation  

PubMed Central

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

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

2012-01-01

220

Investigation of intramolecular proton migration in a series of model, metal-cationized tripeptides using in situ generation of an isotope label.  

PubMed

In this study we used an isotope label, generated in situ, to investigate intramolecular proton migration or scrambling during formation of [b(2)+17+Li](+) products by collision-induced dissociation (CID) of Li(+)-cationized tripeptides. To generate the isotope label, we used a McLafferty-type rearrangement of N-terminally acetylated, C-terminal peptide tert-butyl esters in which all amide positions were exchanged with deuterium. Using a set of small, model peptides, we show that intramolecular proton scrambling occurs during CID, particularly amongst adjacent sites along a peptide backbone, on the time scales employed for low-energy collisional activation in an ion-trap mass spectrometer. PMID:16353129

Bulleigh, Kellis; Howard, Angela; Do, Trang; Wu, Qun; Anbalagan, Victor; Stipdonk, Michael Van

2006-01-01

221

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

PubMed Central

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

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

2010-01-01

222

Relative quantitation of glycans using stable isotopic labels 1-(d0/d5) phenyl-3-methyl-5-pyrazolone by mass spectrometry.  

PubMed

A deuterium reagent, 1-(d5) phenyl-3-methyl-5-pyrazolone (d5-PMP), has been synthesized and used for relative quantitative analysis of oligosaccharides by mass spectrometry (MS) using d0/d5-PMP stable isotopic labeling. Previously reported permethylation-based isotopic labels generate variable mass differences, and reductive amination-based isotopic labels cause a loss of some acid-labile groups in carbohydrates. In contrast, d0/d5-PMP stable isotopic labeling is performed at the reducing end of glycans under basic conditions without desialylation, and the mass difference (?m=10 Da) between the heavy form (d5-PMP derivative) and light form (d0-PMP derivative) of each glycan is invariable. When the two derivative forms of a glycan are mixed in equimolar amounts, a pair of peaks with a 10-Da mass differences is observed in the MS profile. The difference at relative intensity between the d0- and d5-PMP derivatives reflects the difference in quantity of glycans in two samples, making it possible to carry out both qualitative and relative quantitative analyses of glycans in glycomic studies. Application of this method on DP(2) to DP(6) maltodextrin oligosaccharides and N-linked glycans released from ribonuclease B and bovine fetuin demonstrates a 10-fold relative quantitative dynamic range, a satisfying reproducibility (coefficient of variation [CV] ? 8.34%), and good accuracy (relative error [RE] ? 5.1%) of the method. The suggested technique has been successfully applied for comparative quantitative analysis of free oligosaccharides in human and bovine milk. PMID:21803021

Zhang, Ping; Zhang, Ying; Xue, Xiangdong; Wang, Chenjian; Wang, Zhongfu; Huang, Linjuan

2011-11-01

223

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

SciTech Connect

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

Bouhelal, R.; Bockaert, J.; Mermet-Bouvier, R.; Guillon, G.; Homburger, V.

1987-06-25

224

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

Microsoft Academic Search

During apoptosis nuclear morphology changes dramatically due to alterations of chromatin architecture and cleavage of structural nuclear proteins. To characterize early events in apoptotic nuclear dismantling we have performed a proteomic study of apoptotic nuclei. To this end we have combined a cell-free apoptosis system with a proteomic platform based on the differential isotopic labeling of primary amines with N-nicotinoyloxy-succinimide.

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

2006-01-01

225

Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment  

Microsoft Academic Search

Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging.\\u000a On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications\\u000a frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive.\\u000a One of the viable alternatives, successfully used for producing proteins for solution NMR studies, is

Ying Fan; Lichi Shi; Vladimir Ladizhansky; Leonid S. Brown

2011-01-01

226

Stable isotope labeling for matrix-assisted laser desorption/ionization mass spectrometry and post-source decay analysis of ribonucleic acids.  

PubMed

Matrix-assisted laser desorption/ionization mass spectrometry is a powerful analytical tool for the structural characterization of oligonucleotides and nucleic acids. Here we report the application of stable isotope labeling for the simplified characterization of ribonucleic acids (RNAs). An (18)O label is incorporated at the 3'-phosphate of oligoribonucleotides during the enzymatic processing of intact RNAs. As implemented, a buffer solution containing a 50 : 50 mixture of H(2)O and (18)O-labeled H(2)O is used during endonuclease digestion. Upon digestion, characteristic doublets representative of the isotopic distribution of oxygen are noted for those products that contain 3'-phosphate groups. This approach is used to distinguish readily endonuclease digestion products from incomplete digestion products and non-specific cleavage products. In addition, RNase digestion products containing the characteristic isotopic doublet can be selected for further characterization by post-source decay (PSD) analysis. PSD products carrying the 3'-phosphate group will appear as a doublet, thereby simplifying fragment ion assignment. PMID:12938108

Berhane, Beniam T; Limbach, Patrick A

2003-08-01

227

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

PubMed

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

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

2013-01-01

228

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

SciTech Connect

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

Wiel, Stephen; McMahon, James E.

2005-04-28

229

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

PubMed

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

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

2014-07-01

230

Memory-efficient calculation of the isotopic mass states of a molecule.  

PubMed

Our previous work postulated a transition concept among different isotopic mass states (i.e., isotopic species) of a molecule, and developed a hierarchical algorithm for accurately calculating their masses and abundances. A theoretical mass spectrum can be generated by convoluting a peak shape function to these discrete mass states. This approach suffers from limited memory if a level in the hierarchical structure has too many mass states. Here we present a memory efficient divide-and-recursively-combine algorithm to do the calculation, which also improves the truncation method used in the previous hierarchical algorithm. Instead of treating all of the elements in a molecule as a whole, the new algorithm first 'strips' each element one by one. For the mass states of each element, a hierarchical structure is established and kept in the memory. This process reduces the memory usage by orders of magnitude (e.g., for bovine insulin, memory can be reduced from gigabytes to kilobytes). Next, a recursive algorithm is applied to combine mass states of elements to mass states of the whole molecule. The algorithm described above has been implemented as a computer program called Isotope Calculator, which was written in C++. It is freely available under the GNU Lesser General Public License from http://www.cs.brandeis.edu/~hong/software.html or http://people.brandeis.edu/~agar. PMID:20814974

Li, Long; Karabacak, N Murat; Cobb, Jennifer S; Wang, Qi; Hong, Pengyu; Agar, Jeffrey N

2010-09-01

231

High-efficiency liquid-scintillation counting of 14C-labelled material in aqueous solution and determination of specific activity of labelled proteins  

PubMed Central

1. Inexpensive scintillation mixtures are described which enable the detection of as little as 40??c of 14C in aqueous solution with an efficiency of counting of over 80%. 2. A rapid method for the counting of alkaline, acidic and neutral aqueous solutions of up to 1ml. volume is described. Ethanol or 2-ethoxyethanol is used as blending agent. 3. The scintillation counting of alkaline solutions is applied to the accurate determination of the specific activity of 14C-labelled proteins from plant tissues. 4. Attention has been paid to the importance of a standardized washing procedure for the removal of all traces of radioactive material from glassware.

Hall, T. C.; Cocking, E. C.

1965-01-01

232

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

Microsoft Academic Search

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

Zhou

2008-01-01

233

Identification of a novel biomarker for oxidative stress induced by hydrogen peroxide in primary human hepatocytes using the 2-nitrobenzenesulfenyl chloride isotope labeling method.  

PubMed

Aim: Oxidative stress is involved in the progression of non-alcoholic steatohepatitis (NASH). However, there are few biomarkers that are easily measured and accurately reflect the disease states. The aim of this study was to identify novel oxidative stress markers using the 2-nitrobenzenesulfenyl (NBS) stable isotope labeling method and to examine the clinical utility of these diagnostic markers for NASH. Methods: Proteins extracted from phosphate buffered saline- and hydrogen peroxide-loaded human primary hepatocyte were labeled with the [(12)C]- and [(13)C]-NBS reagents, respectively. Pairs of peaks with 6-Da differences in which the [(13)C]-NBS labeling was more intense than the [(12)C]-NBS labeling were detected by MALDI-TOF/MS and identified by MS/MS ion searching. Results: Four pairs of peaks, m/z 1705-1711, m/z 1783-1789, m/z 1902-1908 and m/z 2790-2796, were identified as cytochrome c oxidase VIb (COX6B), liver carboxylesterase 1 (CES1), carbamoyl-phosphate synthase 1 (CPS1) and superoxide dismutase (MnSOD), respectively. Furthermore, serum MnSOD protein levels were significantly higher in NASH patients than in simple steatosis (SS) patients. The serum MnSOD levels tended to increase in parallel with the stage of fibrosis. Conclusion: The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH. PMID:20236361

Takami, Yoichiro; Uto, Hirofumi; Tamai, Tsutomu; Sato, Yuko; Ishida, Yo-Ichi; Morinaga, Hiroyuki; Sakakibara, Yoichi; Moriuchi, Akihiro; Oketani, Makoto; Ido, Akio; Nakajima, Tomoaki; Okanoue, Takeshi; Tsubouchi, Hirohito

2010-04-01

234

Measurement of 13C and 15N isotope labeling by gas chromatography/combustion/isotope ratio mass spectrometry to study amino acid fluxes in a plant-microbe symbiotic association.  

PubMed

We have developed a method based on a double labeling with stable isotopes and gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) analyses to study amino acid exchange in a symbiotic plant-microbe association. Isotopic precision was studied for 21 standards including 15 amino acid derivatives, three N-protected amino acid methyl esters, three amines and one international standard. High correlations were observed between the ?(13)C and ?(15)N values obtained by GC/C/IRMS and those obtained by an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (R(2) = 0.9868 and 0.9992, respectively). The mean precision measured was 0.04‰ for ?(13)C and 0.28‰ for ?(15)N (n = 15). This method was applied in vivo to the symbiotic relationship between alfalfa (Medicago sativa L.) and N(2)-fixing bacteria. Plants were simultaneously labeled over 10 days with (13)C-depleted CO(2) ((12)CO(2)), which was assimilated through photosynthesis by leaves, and (15)N(2) fixed via nodules. Subsequently, the C and N isotope compositions (i.e. ?(13)C and ?(15)N) of free amino acids were analyzed in leaves and nodules by GC/C/IRMS. The method revealed the pattern of C and N exchange between leaves and nodules, highlighting that ?-aminobutanoic acid and glycine may represent an important form of C transport from leaves to the nodules. The results confirmed the validity, reliability and accuracy of the method for assessing C and N fluxes between plants and symbiotic bacteria and support the use of this technique in a broad range of metabolic and fluxomic studies. PMID:21290446

Molero, Gemma; Aranjuelo, Iker; Teixidor, Pilar; Araus, José Luis; Nogués, Salvador

2011-03-15

235

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)

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.

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

2013-12-01

236

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

PubMed

Fluorescent sensing systems based on the quenching of fluorophores have found wide applications in bioassays. An efficient quencher will endow the sensing system a high sensitivity. The frequently used quenchers are based on organic molecules or nanomaterials, which usually need tedious synthesizing and modifying steps, and exhibit different quenching efficiencies to different fluorophores. In this work, we for the first time report that aggregated perylene derivative can serve as a broad-spectrum and label-free quencher that is able to efficiently quench a variety of fluorophores, such as green, red and far red dyes labeled on DNA. By choosing nucleases as model biomolecules, such a broad-spectrum quencher was then employed to construct a multiplexed bioassay platform through a label-free manner. Due to the high quenching efficiency of the aggregated perylene, the proposed platform could detect nuclease with high sensitivity, with a detection limit of 0.03U/mL for EcoRV, and 0.05U/mL for EcoRI. The perylene quencher does not affect the activity of nuclease, which makes it possible to design post-addition type bioassay platform. Moreover, the proposed platform allows simultaneous and multicolor analysis of nucleases in homogeneous solution, demonstrating its value of potential application in rapid screening of multiple bio-targets. PMID:24662061

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

2014-08-15

237

Isotope coded protein label quantification of serum proteins—Comparison with the label-free LC–MS and validation using the MRM approach  

Microsoft Academic Search

Protein quantification based upon mass spectrometry is gaining ground in diverse applications of biological and clinical relevance. The present article focuses on one of the most complex biological fluids – serum – and provides a novel ICPL based quantification protocol. The results are compared to a label-free (data independent alternate scanning) absolute quantification method. The validation is performed using MRM

A. Turtoi; G. D. Mazzucchelli; E. De Pauw

2010-01-01

238

Isotope labeling studies on the formation of 5-(hydroxymethyl)-2-furaldehyde (HMF) from sucrose by pyrolysis-GC/MS.  

PubMed

Although it is generally assumed that the reactivity of sucrose, a nonreducing sugar, in the Maillard reaction is due to its hydrolysis into free glucose and fructose, however, no direct evidence has been provided for this pathway, especially in dry and high temperature systems. Using specifically (13)C-labeled sucrose at C-1 of the fructose moiety, HMF formation was studied at different temperatures. Under dry pyrolytic conditions and at temperatures above 250 degrees C, 90% of HMF originated from fructose moiety and only 10% originated from glucose. Alternatively, when sucrose was refluxed in acidic methanol at 65 degrees C, 100% of HMF was generated from the glucose moiety. Moreover, the relative efficiency of the known HMF precursor 3-deoxyglucosone to generate HMF was compared to that of glucose, fructose and sucrose. Glucose exhibited a much lower conversion rate than 3-deoxyglucosone, however, both fructose and sucrose showed much higher conversion rates than 3-deoxyglucosone thus precluding it as a major precursor of HMF in fructose and sucrose solutions. Based on the data generated, a mechanism of HMF formation from sucrose is proposed. According to this proposal sucrose degrades into glucose and a very reactive fructofuranosyl cation. In dry systems this cation can be effectively converted directly into HMF. PMID:18611024

Perez Locas, Carolina; Yaylayan, Varoujan A

2008-08-13

239

Quantification of free sialic acid in human plasma through a robust quinoxalinone derivatization and LC-MS/MS using isotope-labeled standard calibration.  

PubMed

We report an accurate quantification of free sialic acid (SA) in human plasma using LC-MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC-MS/MS analysis of free SA. In addition, the use of (13)C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring (MRM) mode for sensitive and selective detection. We have achieved a lower limit of quantification (LLOQ) of 20ng/mL and a total running time of 4.2min, which is the most sensitive and quick measurement for free SA in biomatrices. PMID:24291723

Wang, Dan; Zhou, Xiang; Wang, Lin; Wang, Sihe; Sun, Xue-Long

2014-01-01

240

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

PubMed

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

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

2014-05-01

241

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

SciTech Connect

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.

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

1984-01-01

242

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

PubMed Central

We isolated aminoacyl-tRNA (60-70% yield) from human and rat tissues and measured, by GC/MS, its labeling in vivo by [15N]- and [13C]leucine. Tracer dilution artifacts seemed unlikely since, after infusion of L-[1-13C,15N]leucine into rats, (i) muscle leucyl-tRNA labeling exceeded tissue free leucine labeling, (ii) values were largely unaffected by storing over 5 min at 22 degrees C, and (iii) L-[2,4,5-methyl-13C]leucine was not incorporated into leucyl-tRNA during homogenization. Leucyl-tRNA labeling in liver and muscle suggested charging from extra- and intracellular pools: e.g., after infusing L-[1-13C,15N]leucine, rat muscle tissue free leucine 13C labeling (8.97 +/- 0.30 atom % excess) exceeded that by 15N (3.37 +/- 0.33 atom % excess), and both were significantly lower (P less than 0.02) than venous plasma (13C, 12.1 +/- 1.8; 15N, 5.54 +/- 0.6 atom % excess) indicating tracer dilution by transamination and by proteolysis; however, leucyl-tRNA labeling by either isotope (13C, 10.26 +/- 0.50; 15N, 4.72 +/- 0.72 atom % excess) was significantly above mixed tissue free leucine (P less than 0.05). Labeling of leucyl-tRNA in human erector spinae muscle (obtained after preoperative L-[1-13C]leucine infusion) was, at 4.98 +/- 0.43 atom % excess, lower (27%) than venous plasma leucine (P less than 0.05) and intermediate between muscle free leucine (9% lower; P less than 0.01) and venous alpha-ketoisocaproate (11% higher; P less than 0.02). Human placental leucyl-tRNA labeling (after predelivery tracer infusion) was 37% lower (P less than 0.05) than maternal uterine vein labeling but not significantly different from placental free leucine or umbilical arterial leucine. Images

Watt, P W; Lindsay, Y; Scrimgeour, C M; Chien, P A; Gibson, J N; Taylor, D J; Rennie, M J

1991-01-01

243

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

PubMed Central

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

2013-01-01

244

Efficient access to 3'-terminal azide-modified RNA for inverse click-labeling patterns.  

PubMed

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

Santner, Tobias; Hartl, Markus; Bister, Klaus; Micura, Ronald

2014-01-15

245

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

SciTech Connect

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

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

2006-06-27

246

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...

247

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

PubMed

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

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

2013-01-01

248

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

PubMed

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

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

2014-06-01

249

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

PubMed

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

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

2006-08-01

250

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

PubMed

Glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline was used for relative quantitation of N-glycans. In a first step, the labeling method by reductive amination was optimized for this reagent. It could be demonstrated that selecting aniline as limiting reactant and using the reductant in excess is critical for achieving high derivatization yields (over 95 %) and good reproducibility (relative standard deviations ?1-5 % for major and ?5-10 % for minor N-glycans). In a second step, zwitterionic-hydrophilic interaction liquid chromatography in capillary columns coupled to electrospray mass spectrometry with time-of-flight analyzer (?ZIC-HILIC-ESI-TOF-MS) was applied for the analysis of labeled N-glycans released from intact glycoproteins. Ovalbumin, bovine ?1-acid-glycoprotein and bovine fetuin were used as test glycoproteins to establish and evaluate the methodology. Excellent separation of isomeric N-glycans and reproducible quantitation via the extracted ion chromatograms indicate a great potential of the proposed methodology for glycoproteomic analysis and for reliable relative quantitation of glycosylation variants in biological samples. PMID:23846592

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

2013-09-01

251

Combined Chemical and Enzymatic Stable Isotope Labeling for Quantitative Profiling of Detergent-insoluble Membrane Proteins Isolated Using Triton X-100 and Brij-96  

PubMed Central

Effective quantitative profiling of detergent-insoluble membrane proteins using high-throughput mass spectrometry (MS)-based proteomics would allow a better understanding of physiological and pathological processes that take place at the cell surface. To increase the coverage of proteins present in detergent-resistant membrane microdomains (DRMMs), a combination of 16O/18O and isotope coded affinity tags (ICAT) labeling was used in a comparative analysis of detergent-insoluble membrane proteins isolated from rat basophilic leukemia cells (RBL-2H3), with either Triton X-100 or Brij-96. The analysis resulted in the quantification of 738 unique proteins from Triton X-100 and Brij-96 isolated DRMMs, significantly exceeding the number of proteins quantified from either single labeling technique. Twenty-five non-cysteine-containing proteins were quantified, as well as 32 cysteine-containing proteins that would have been missed if either 16O/18O or ICAT labeling had been used exclusively, which illustrate better proteome coverage and enhanced ability to quantitate. The comparative analysis revealed that proteins were more readily extracted using Triton X-100 than Brij-96; however, Triton X-100 also extracted larger quantities of non-DRMMs-associated proteins. This result confirms previous, targeted studies suggesting that DRMMs isolated using Triton X-100 and Brij-96 differ in their protein content.

Blonder, Josip; Yu, Li-Rong; Radeva, Galina; Chan, King C.; Lucas, David A.; Waybright, Timothy J.; Issaq, Haleem J.; Sharom, Frances J.; Veenstra, Timothy D.

2011-01-01

252

Highly efficient and isotope selective photo-ionization of barium atoms using diode laser and LED light.  

PubMed

We demonstrated a simple method to photo-ionize barium atoms using 791 nm diode laser together with 310 nm UV LED. It solved the bottle-neck problem of previous method using 791 nm diode laser and 337 nm N(2) laser, whose ionization rate was limited by the repetition rate of N(2) laser. Compared with previous method, it has advantages of high efficiency together with simple and cheap setups. By tuning the frequency of 791 nm laser to be resonant with the desired isotope, isotope selective photo-ionization has been realized. PMID:21935008

Wang, B; Zhang, J W; Gao, C; Wang, L J

2011-08-15

253

Strategy combining separation of isotope-labeled unfolded proteins and matrix-assisted laser desorption\\/ionization mass spectrometry analysis enables quantification of a wide range of serum proteins  

Microsoft Academic Search

A novel strategy for the quantitative profiling of serum proteome is described. It includes an ammonium sulfate depletion of the serum, an affordable stable isotope labeling chemistry for samples with a large amount of protein, separation of the unfolded proteins, and relative quantification by matrix-assisted laser desorption\\/ionization (MALDI) mass spectrometry (MS). Labeling of unfolded proteins was performed using normal (D0)

Wei-Li Liao; Illarion V. Turko

2008-01-01

254

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

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

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

2009-10-01

255

Water use efficiency and carbon isotope composition of plants in a cold desert environment.  

PubMed

The effects of the availabilities of water and nitrogen on water use efficiency (WUE) of plants were investigated in a sagebrush steppe. The four species studied wereArtemisia tridentata (shrub),Ceratoides lanata (suffrutescent shrub),Elymus lanceolatus (rhizomatous grass), andElymus elymoides (tussock grass). Water and nitrogen levels were manipulated in a two-by-two factorial design resulting in four treatments: control (no additions), added water, added nitrogen, and added water and nitrogen. One instantaneous and two long-term indicators of WUE were used to testa priori predictions of the ranking of WUE among treatments. The short-term indicator was the instantaneous ratio of assimilation to transpiration (A/E). The long-term measures were 1) the slope of the relationship between conductance to water vapor and maximum assimilation and 2) the carbon isotope composition (?(13)C) of plant material. Additional water decreased WUE, whereas additional nitrogen increased WUE. For both A/E and ?(13)C, the mean for added nitrogen alone was significantly greater than the mean for added water alone, and means for the control and added water and nitrogen fell in between. This ranking of WUE supported the hypothesis that both water and nitrogen limit plant gas exchange in this semiarid environment. The short- and long-term indicators were in agreement, providing evidence in support of theoretical models concerning the water cost of carbon assimilation. PMID:23494339

Toft, N L; Anderson, J E; Nowak, R S

1989-03-01

256

The origins of volatile oxidation products in the thermal degradation of polypropylene, identified by selective isotopic labeling  

Microsoft Academic Search

Making use of polypropylene samples that are selectively labeled with carbon-13 at each of the three unique positions within the repeating unit, we are conducting mass spectral analyses of the volatile organic oxidation products that are produced when the polymer is subjected to elevated temperature in the presence of air. By examination of both the parent and fragmentation ion peaks

Robert Bernstein; Steven M. Thornberg; Roger A. Assink; Adriane N. Irwin; James M. Hochrein; Jason R. Brown; Dora K. Derzon; Sara B. Klamo; Roger L. Clough

2007-01-01

257

Isotopic composition of H2 from wood burning: Dependency on combustion efficiency, moisture content, and ?D of local precipitation  

NASA Astrophysics Data System (ADS)

Differences in isotopic composition between the various sources of H2 are large, but only a few measurements have been carried out to constrain them. Two conflicting values have been published for H2 from biomass burning. Both rely on the assumption that the isotopic composition of H2 should scale with the isotopic composition of the precipitation at the location where the biomass grew. Here we test this hypothesis using 18 wood samples collected from various locations around the globe. The sample locations cover a range of ?D content of H2 in precipitation, from below -120‰ in Siberia and Canada to -15‰ in Zimbabwe. The results confirm the predicted dependence of the H2 isotopic composition on the precipitation in the sampling region. The water content itself is found to at most slightly affect the results. Furthermore, ?D of H2 depends strongly on combustion efficiency. Thus, the isotopic composition of H2 from biomass burning shows a strong variability around the globe and between different stages of a fire. It is suggested that, rather than a global bulk number, global models that attempt to reproduce the spatial and temporal distribution of ?D in H2 should incorporate explicitly the variability of ?D(H2) from biomass burning on ?D in precipitation.

RöCkmann, Thomas; Gómez ÁLvarez, Catalina X.; Walter, Sylvia; van der Veen, Carina; Wollny, Adam G.; Gunthe, Sachin S.; Helas, Günther; PöSchl, Ulrich; Keppler, Frank; Greule, Markus; Brand, Willi A.

2010-09-01

258

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

NASA Astrophysics Data System (ADS)

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

Fujinuma, R.; Balster, N. J.

2009-12-01

259

Site-specific labeling of DNA and RNA using an efficiently replicated and transcribed class of unnatural base pairs.  

PubMed

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

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

2011-12-14

260

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

Microsoft Academic Search

Background  Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome\\u000a of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance\\u000a values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up-

Stefan P Albaum; Hannes Hahne; Andreas Otto; Ute Haußmann; Dörte Becher; Ansgar Poetsch; Alexander Goesmann; Tim W Nattkemper

2011-01-01

261

Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry  

Microsoft Academic Search

Quantitative proteome profiling using stable isotope protein tagging and automated tandem mass spectrometry (MS\\/MS) is an emerging technology with great potential for the functional analysis of biological systems and for the detection of clinical diagnostic or prognostic marker proteins. Owing to the enormous complexity of proteomes, their comprehensive analysis is an as-yet-unresolved technical challenge. However, biologically or clinically important information

Hui Zhang; Xiao-jun Li; Daniel B Martin; Ruedi Aebersold

2003-01-01

262

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

SciTech Connect

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

Hines, J.V.; Landry, S.M.; Varani, G.; Tinoco, I. Jr. (Univ. of California, Berkeley, CA (United States) Lawrence Berkeley Lab., CA (United States))

1994-06-29

263

Elucidation of functions of human cytochrome P450 enzymes: identification of endogenous substrates in tissue extracts using metabolomic and isotopic labeling approaches.  

PubMed

One of the central problems in biochemistry in the postgenomic era is the elucidation of functions of proteins, including "orphan" human cytochromes P450 (P450s), when the substrates are unknown. A general strategy for identification of endogenous substrates of P450s in tissue extracts using metabolomic and isotopic labeling approaches is described, involving four main steps: (1) In vitro incubation of a P450 enzyme system with cofactor and tissue extract is done under a mixture of (18)O(2)/(16)O(2) (1:1). (2) Liquid chromatography/mass spectrometry (LC/MS) assay of an organic extract of the reaction mixture is performed. (3) The isotopic labeling products appearing as M/M + 2 doublets can be directly identified using the program DoGEX (Sanchez-Ponce, R. and Guengerich, F. P. Anal. Chem. 2007, 79, 3355-3362). (4) Characterization of potential candidates is done. Validation of the strategy was established using human P450 7A1 as an initial model to identify its known product, 7alpha-hydroxycholesterol, in liver extracts. The strategy was then applied to human P450s 1A2, 2C8, and 2C9 in untargeted substrate searches with human liver extracts. A total of seven fatty acids were identified and verified as substrates of these three hepatic P450s. The products were subsequently characterized as hydroxylation and epoxidation derivatives of fatty acids, using gas chromatography/mass spectrometry (GC/MS) analysis. Finally, kinetic studies were performed to confirm that the fatty acids are oxidized by P450s 1A2, 2C8, and 2C9. Thus, this strategy has been demonstrated to be useful in identifying reactions in tissue extracts with orphan human P450s. PMID:19301915

Tang, Zhongmei; Martin, Martha V; Guengerich, F Peter

2009-04-15

264

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

PubMed Central

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

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

2012-01-01

265

Use of a stable carbon isotope to assess the efficiency of a drinking water treatment method with CO?.  

PubMed

CO? gas with a special isotopic signature (?¹³C = -35.2‰ vs. VPDB) was used as a marker to evaluate the efficiency of a drinking water treatment method and the effect of an ultrasonic (US) stirrer. This treatment was developed to prevent precipitation and corrosion effects in water-supply systems. The research work was performed using a laboratory-scale pilot plant that was filled with tap water. The stable isotope analyses of ?¹³C-DIC (Dissolved Inorganic Carbon) in the water samples indicated that the maximum content of added CO? gas in DIC was in the range of 35 to 45%. The use of the US stirrer during the entire experiment decreased the method's overall efficiency by 10%, due to degassing at a late stage of the experiment but accelerated the dissolution process in the early experimental stage. PMID:22377992

Poberžnik, M; Leis, A; Lobnik, A

2012-01-01

266

Thermal desorption studies of isotopically-labeled oxygen-induced superconductivity in La sub 2 CuO sub 4+. delta  

SciTech Connect

Isotopically-labeled oxygen enrichment and thermal desorption mass spectroscopy (TDMS) have been combined to study interstitial oxygen desorption from superconducting La{sub 2}CuO{sub 4+{delta}} ({delta} {le} 0.032). Single crystal samples of magnetic insulating La{sub 2}CuO{sub 4.00} were annealed at 860K under 1--3 kbar oxygen pressure for 12--100 hours to yield hole-doped, superconducting La{sub 2}CuO{sub 4+{delta}} samples with 35K < {Tc} < 40K. Whereas no TDMS signals were observed for the insulator, rapid bursts (FWHM < 0.5 sec) of molecular oxygen were observed above 350K while heating the superconductor at less than 1 K sec{sup {minus}1} in high vacuum. A kinetic model is proposed in which the interstitial oxygen diffuses to internal grain boundaries and defects during heating, thereby inducing stress in the lattice as it attempts to revert to the LaCuO{sub 4.00} crystal structure. This stress is relieved by lattice fracture at grain boundaries during the TDMS experiment, releasing the trapped oxygen from the sample as micro-cracks are formed. In addition, the facile oxygen exchange between interstitial and lattice oxygen sites has been discovered by TDMS and weight gain measurements from isotopically-enriched crystals, supporting the structural model of Chaillout, et al. in which the interstitial oxygen atom dimerizes with a lattice oxygen ion.

Shinn, N.D.; Bartram, M.E.; Schirber, J.E.; Overmyer, D.L.; Rogers, J.W. Jr. (Sandia National Labs., Albuquerque, NM (USA)); Fisk, Z.; Cheong, S.W. (Los Alamos National Lab., NM (USA))

1990-01-01

267

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

SciTech Connect

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

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

2013-02-20

268

Combined Gel Probe and Isotope Labeling Technique for Measuring Dissimilatory Nitrate Reduction to Ammonium in Sediments at Millimeter-Level Resolution?  

PubMed Central

Dissimilatory NO3? reduction in sediments is often measured in bulk incubations that destroy in situ gradients of controlling factors such as sulfide and oxygen. Additionally, the use of unnaturally high NO3? concentrations yields potential rather than actual activities of dissimilatory NO3? reduction. We developed a technique to determine the vertical distribution of the net rates of dissimilatory nitrate reduction to ammonium (DNRA) with minimal physical disturbance in intact sediment cores at millimeter-level resolution. This allows DNRA activity to be directly linked to the microenvironmental conditions in the layer of NO3? consumption. The water column of the sediment core is amended with 15NO3? at the in situ 14NO3? concentration. A gel probe is deployed in the sediment and is retrieved after complete diffusive equilibration between the gel and the sediment pore water. The gel is then sliced and the NH4+ dissolved in the gel slices is chemically converted by hypobromite to N2 in reaction vials. The isotopic composition of N2 is determined by mass spectrometry. We used the combined gel probe and isotopic labeling technique with freshwater and marine sediment cores and with sterile quartz sand with artificial gradients of 15NH4+. The results were compared to the NH4+ microsensor profiles measured in freshwater sediment and quartz sand and to the N2O microsensor profiles measured in acetylene-amended sediments to trace denitrification.

Stief, Peter; Behrendt, Anna; Lavik, Gaute; De Beer, Dirk

2010-01-01

269

Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.  

PubMed

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ?98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers. PMID:22784215

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

2012-08-15

270

The effect of FISH and CARD-FISH on the isotopic composition of (13)C- and (15)N-labeled Pseudomonas putida cells measured by nanoSIMS.  

PubMed

The use of nanoSIMS for the exploration of microbial activities in natural habitats often implies that stable isotope tracer experiments are combined with in situ hybridization techniques (i.e. fluorescence in situ hybridization (FISH) or catalyzed reporter deposition (CARD)-FISH). In this study, Pseudomonas putida grown on (13)C- and (15)N-labeled carbon and nitrogen, collected in exponential growth and stationary phases, was hybridized and analyzed by nanoSIMS. It was shown that (13)C and (15)N fractions decreased after FISH and CARD-FISH in comparison to chemically untreated cells. However, the fractions were influenced differently by various treatments. After paraformaldehyde fixation of exponentially growing cells, a reduction of the (13)C and (15)N fractions was measured from 94±1.2% and 89.5±3.8% to 90.2±0.8% and 64±4.6%, respectively, indicating that nitrogen isotopic composition was most influenced. A further decrease of the (13)C and (15)N fractions to 80.7±6.5 and 59.5±4.1%, respectively, was measured after FISH, while CARD-FISH decreased the fractions to 57.4±3.0% and 47.1±4.1%, respectively. The analysis of cells collected in different growth phases revealed that the effect of various treatments seemed to be dependent on the cell's physiological state. In addition, a mathematical model that can be used in further studies was developed in order to calculate the amount of carbon introduced into the cells by chemical treatments. These results can be valuable for environmental FISH-nanoSIMS studies where the isotopic composition of single cells will be used to quantitatively assess the importance of specific populations to certain biochemical processes and determine budget estimations. PMID:24702905

Musat, Niculina; Stryhanyuk, Hryhoriy; Bombach, Petra; Adrian, Lorenz; Audinot, Jean-Nicolas; Richnow, Hans H

2014-06-01

271

Efficient Solid-Phase Synthesis of pppRNA by Using Product-Specific Labeling.  

PubMed

A novel solid-phase synthesis and purification strategy for 5'-triphosphate oligonucleotides by using lipophilic tagging of the triphosphate moiety is reported. This is based on triphosphate synthesis with 5'-O-cyclotriphosphate intermediates, whereby a lipophilic tag, such as decylamine, is introduced during the ring-opening reaction to give a linear gamma-phosphate-tagged species. This method enables the highly efficient synthesis of 5'-triphosphorylated RNA derivatives and their gamma-phosphate-substituted analogues and will especially facilitate the advancement of therapeutic approaches that make use of 5'-triphosphate oligonucleotides as potent activators of the cytosolic immune sensor RIG-I. PMID:24668741

Goldeck, M; Tuschl, T; Hartmann, G; Ludwig, J

2014-04-25

272

An Efficient Method for Site-specific 18F-Labeling of Biomolecules Using the Rapid Condensation Reaction between 2-Cyanobenzothiazole and Cysteine  

PubMed Central

An efficient method based on a rapid condensation reaction between 2–cyanobenzothiazole (CBT) and cysteine has been developed for 18F–labeling of N–terminal cysteine–bearing peptides and proteins. An 18F–labeled dimeric cRGD ([18F]CBTRGD2) has been synthesized with an excellent radiochemical yield (92% based on radio–HPLC conversion, 80% decay–corrected and isolated yield) and radiochemical purity (>99%) under mild conditions using 18F–CBT, and shown good in vivo tumor targeting efficiency for PET imaging. The labeling strategy was also applied to the site–specific 18F–labeling of a protein, Renilla lucifierase (RLuc8) with a cysteine residue at its N–terminus. The protein labeling was achieved with 12% of decay–corrected radiochemical yield and more than 99% radiochemical purity. This strategy should provide a general approach for an efficient and site–specific 18F–labeling of various peptides and proteins for in vivo molecular imaging applications.

Jeon, Jongho; Shen, Bin; Xiong, Liqin; Miao, Zheng; Lee, Kyung Hyun; Rao, Jianghong; Chin, Frederick T.

2012-01-01

273

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

PubMed

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

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

2013-11-01

274

Mass measurements using isotopically labeled solvents reveal the extent of solvent transport during redox in thin films on electrodes  

SciTech Connect

Transport of solvent during the redox reactions of thin films on electrodes has been identified as a possible influence on both the thermodynamic and kinetic aspects of their electrochemical responses. A variety of methods has been used in attempts to measure solvent content of these films, including ellipsometry and profilimetry. However, those techniques which rely on measurement of thickness suffer from inability to deconvolute the contributions to swelling (or deswelling) from ion and solvent transport. Thus, the situation remains one in which speculation abounds, but accurate measurements are unavailable. In this communication, the authors report on the application of the quartz crystal microbalance (QCM) technique to the determination of solvent transport during redox in thin films of nickel ferrocyanide (the nickel analogue of Prussian Blue) by comparing the difference in the total mass change (comprised of contributions from both ion and solvent transport) which results from use of isotopically substituted solvent. To their knowledge, these experiments represent the first accurate, unambiguous measurements of solvent transport in thin films on electrodes. It is especially significant that these measurements are made in the presence of simultaneous ion transport.

Lasky, S.J.; Buttry, D.A.

1988-08-31

275

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

PubMed

Identification of the fragment ion species associated with the ion reaction mechanism of triacetone triperoxide (TATP), a homemade peroxide-based explosive, is presented. Ion mobility spectrometry (IMS) has proven to be a key analytical technique in the detection of trace explosive material. Unfortunately, IMS alone does not provide chemical identification of the ions detected; therefore, it is unknown what ion species are actually formed and separated by the IMS. In IMS, ions are primarily characterized by their drift time, which is dependent on the ion?s mass and molecular cross-section; thus, IMS as a standalone technique does not provide structural signatures, which is in sharp contrast to the chemical and molecular information that is generally obtained from other customary analytical techniques, such as NMR, Raman and IR spectroscopy and mass spectrometry. To help study the ion chemistry that gives rise to the peaks observed in IMS, the hardware of two different commercial IMS instruments has been directly coupled to triple quadrupole (QQQ) mass spectrometers, in order to ascertain each ion?s corresponding mass/charge (m/z) ratios with different dopants at two temperatures. Isotope labeling was then used to help identify and confirm the molecular identity of the explosive fragment and adduct ions of TATP. The m/z values and isotope labeling experiments were used to help propose probable molecular formulas for the ion fragments. In this report, the fragment and adduct ions m/z 58 and 240 of TATP have been confirmed to be [C3H6NHH](+) and [TATPNH4](+), respectively; while the fragment ions m/z 73 and 89 of TATP are identified as having the molecular formulas [C4H9NH2](+) and [C4H9O2](+), respectively. It is anticipated that the work in this area will not only help to facilitate improvements in mobility-based detection (IMS and MS), but also aid in the development and optimization of MS-based detection algorithms for TATP. PMID:24913870

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

2014-09-01

276

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

PubMed

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 ?-chain: ?K191, ?K208, ?K224, ?K429, ?K457, ?K539, ?K562, in the ?-chain: ?K233, and in the ?-chain: ?K170 and ?K273. Glycations were found at ?K133 and ?K75, alternatively ?K85. Notably, the lysine 539 is a site involved in FXIII-mediated cross-linking of fibrin. With isotope labeling in vitro, using [(14)C-acetyl]salicylic acid and [(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 ?M aspirin) and physiologically (2-8 mM glucose) relevant plasma concentrations. No competition between acetylation and glycation could be demonstrated. Thus, fibrinogen is acetylated at several lysine residues, some of which are involved in the cross-linking of fibrinogen. This may mechanistically explain why aspirin facilitates fibrin degradation. We find no support for the idea that glycation of fibrin(ogen) interferes with acetylation of fibrinogen. PMID:22507986

Svensson, Jan; Bergman, Ann-Charlotte; Adamson, Ulf; Blombäck, Margareta; Wallén, Håkan; Jörneskog, Gun

2012-05-01

277

Intraoperative beta probe: A device for detecting tissue labeled with positron or electron emitting isotopes during surgery  

SciTech Connect

An intraoperative beta probe was designed, built, and tested for detection of radio-labeled malignant tissues that has the advantage of being selectively sensitive to beta while insensitive to gamma radiation. Since beta radiation (electrons or positrons) has a short range in tissue, this probe is ideal for detecting tracers in tumors at the surface of the surgical field. This probe contains a plastic scintillation detector sensitive to beta rays and to a lesser degree some background gamma rays. A second detector counts spurious gamma rays and allows for their subtraction from the activity measured by the first detector. Sensitivity of the dual probe for I-131 and F-18 was measured to be 108 counts/s/kBq (4000 counts/s/[mu]Ci). The dual-detector probe faithfully measured the 10:1 tumor'' to background ratio of radioactivity concentrations in a simulated environment of a tumor in the presence of intense background 511 keV photons. In another phantom experiment, simulating abdominal tumor deposits with various realistic I-131 radioactive concentrations, the probe was able to accurately identify tumors of approximately 50 mg with a tumor/normal radioactivity concentration of 3/1 in 10 s.

Daghighian, F. (Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 (United States)); Mazziotta, J.C. (Division of Brain Mapping of Neuropsychiatric Institute, Department of Neurology, Department of Pharmacology, Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California 90024 (United States)); Hoffman, E.J. (Department of Pharmacology, Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California 90024 (United States)); Shenderov, P.; Eshaghian, B. (Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 (United States)); Siegel, S.; Phelps, M.E. (Department of Pharmacology, Department of Radiological Sciences, UCLA School of Medicine, Los Angeles, California 90024 (United States))

1994-01-01

278

Comprehensive and highly sensitive urinary steroid hormone profiling method based on stable isotope-labeling liquid chromatography-mass spectrometry.  

PubMed

Steroid hormones are crucial substances that mediate a wide range of vital physiological functions of the body. Determination of the levels of steroid hormones plays an important role in understanding the mechanism of the steroid hormone-related diseases. In this study, we present a novel targeted metabolic profiling method based on the introduction of an easily protonated stable isotope tag to a hydroxyl-containing steroid hormone with a synthesized derivatization reagent, deuterium 4-(dimethylamino)-benzoic acid (d(4)-DMBA), and liquid chromatography-mass spectrometry (LC-MS). Different from other reported derivatization reagents that have been used to enhance the sensitivities for estrogens or androgens, our method is comprehensive with the capability of covering hydroxyl-containing androgens, estrogens, corticoids, and progestogens. Furthermore, the nonderivatized steroid hormones (e.g., 17?-hydroxyprogesterone, progesterone, and androstenedione) were not destroyed during the derivatization process, and their levels could still be obtained in one LC-MS run. We were able to detect 24 steroid hormones at subng/mL levels (the lower limit of detection could reach 5 pg/mL for estrone and 16?-hydroxy estrone, which is equivalent to 0.1 pg on column) with maximum sensitivity enhancement factors of more than 10(3)- to 10(4)-fold after derivatization. The method was successfully applied to the measurement of free (unconjugated) steroid hormones in urine samples of males, females, and pregnant women. Because the significant role the steroid hormone pathway plays in humans, a comprehensive, sensitive, specific, and accurate method for profiling the steroid hormone metabolome shall offer new insights into hormone-related diseases. PMID:23110480

Dai, Weidong; Huang, Qiang; Yin, Peiyuan; Li, Jia; Zhou, Jia; Kong, Hongwei; Zhao, Chunxia; Lu, Xin; Xu, Guowang

2012-12-01

279

Inter-residue coupling and equilibrium unfolding of PPII helical peptides. Vibrational spectra enhanced with (13)C isotopic labeling.  

PubMed

Unordered proteins, unfolded peptides, and several "random coil" models have been shown to have local conformations similar to that of polyproline II (PPII). Inter-residue coupling of selected residues in a series of related peptides having predominantly PPII conformations were measured using IR, VCD, and Raman spectra of selected variants that were doubly C(1)-labeled with (13)C on the amide C?O. The characteristics of the (13)C?O component of the IR, VCD, and Raman amide I' bands and their sensitivity to the local structure of the peptide are compared to predictions based on DFT level calculations for related structures and used to estimate coupling interactions between pairs of C?O groups along the backbone of this helical structure. In the PPII case, the coupling is relatively weak, due to the extended structure, yet by combining IR, Raman, and VCD observations with results of DFT level model calculations, we have determined bounds for experimental interaction constants for this structure. Correlation of properties for PPII structures with those of "random coils" can be done by comparing Pro(n) and Pro-rich sequences with Lys-rich sequences. The experimental band shifts and implied couplings reflect the computed results in both cases. Thermal unfolding of these peptides appears to be multistate, with monotonic spectral changes but little evidence of a cooperative (sigmoidal) transition. For the Lys-rich series, a transition from PPII to ?-helix structure was induced by TFE addition, and the spectra were fit to an equilibrium model. These spectral changes show a large variation in (13)C?O coupling that occurs with a local conformational change from PPII- to ?-helical, which is both well-fit by our theoretical results and offers a new site-specific method of assigning local PPII/disordered vs ?-helical (or other) structure. PMID:20831224

Chi, Heng; Lakhani, Ahmed; Roy, Anjan; Nakaema, Marcelo; Keiderling, Timothy A

2010-10-01

280

Novel Tracer Method To Measure Isotopic Labeled Gas-Phase Nitrous Acid (HO(15)NO) in Biogeochemical Studies.  

PubMed

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 (15)N relative exceedance, ?((15)N), 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 ?((15)N) = 0.2-0.5, the relative standard deviation of ?((15)N) is <4%. The optimum pH and solvents for extraction by SPE and potential interferences are discussed. The method was applied to measure HO(15)NO emissions from soil in a dynamic chamber with and without spiking (15)N labeled urea. The identification of HO(15)NO from soil with (15)N 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

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

2014-07-15

281

A novel approach to monitor glucose metabolism using stable isotopically labelled glucose in longitudinal studies in mice.  

PubMed

The aetiology of insulin resistance is still an enigma. Mouse models are frequently employed to study the underlying pathology. The most commonly used methods to monitor insulin resistance are the HOMA-IR, glucose or insulin tolerance tests and the hyperinsulinemic euglycaemic clamp (HIEC). Unfortunately, these tests disturb steady state glucose metabolism. Here we describe a method in which blood glucose kinetics can be determined in fasted mice without noticeably perturbing glucose homeostasis. The method involves an intraperitoneal injection of a trace amount of [6,6-(2)H2]glucose and can be performed repeatedly in individual mice. The validity and performance of this novel method was tested in mice fed on chow or high-fat diet for a period of five weeks. After administering the mice with [6,6-(2)H2]glucose, decay of the glucose label was followed in small volumes of blood collected by tail tip bleeding during a 90-minute period. The total amount of blood collected was less than 120 ?L. This novel approach confirmed in detail the well-known increase in insulin resistance induced by a high-fat diet. The mice showed reduced glucose clearance rate, and reduced hepatic and peripheral insulin sensitivity. To compensate for this insulin resistance, ?-cell function was slightly increased. We conclude that this refinement of existing methods enables detailed information of glucose homeostasis in mice. Insulin resistance can be accurately determined while mechanistic insight is obtained in underlying pathology. In addition, this novel approach reduces the number of mice needed for longitudinal studies of insulin sensitivity and glucose metabolism. PMID:23492513

van Dijk, T H; Laskewitz, A J; Grefhorst, A; Boer, T S; Bloks, V W; Kuipers, F; Groen, A K; Reijngoud, D J

2013-04-01

282

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

PubMed

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

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

2009-08-01

283

Beta-lactamase-catalyzed aminolysis of depsipeptides: Proof of the nonexistence of a specific D-phenylalanine/enzyme complex by double-label isotope trapping  

SciTech Connect

The steady-state kinetics of the Enterobacter cloacae P99 beta-lactamase-catalyzed aminolysis of the depsipeptide m-(((phenylacetyl)glycyl)oxy)benzoic acid by D-phenylalanine were consistent with an ordered sequential mechanism with D-phenylalanine binding first. In terms of this mechanism, the kinetics data required that in 20 mM MOPS buffer, pH 7.5, the dissociation constant of the initially formed enzyme/D-phenylalanine complex be around 1.3 mM; at pH 9.0 in 0.1 M carbonate buffer, the complex should be somewhat more stable. Attempts to detect this complex in a binary mixture by spectroscopic methods (fluorescence, circular dichroic, and nuclear magnetic resonance spectra) failed. Kinetic methods were also unsuccessful--the presence of 20 mM D-phenylalanine did not appear to affect beta-lactamase activity nor inhibition of the enzyme by phenylmethanesulfonyl fluoride, phenylboronic acid, or (3-dansylamidophenyl)boronic acid. Equilibrium dialysis experiments appeared to indicate that the dissociation constant of any binary enzyme/D-phenylalanine complex must be somewhat higher than the kinetics allowed (greater than 2 mM). Since the kinetics also required that, at high depsipeptide concentrations, and again with the assumption of the ordered sequential mechanism, the reaction of the enzyme/D-phenylalanine complex to aminolysis products be faster than its reversion to enzyme and D-phenylalanine, a double-label isotope-trapping experiment was performed.

Pazhanisamy, S.; Pratt, R.F. (Wesleyan Univ., Middletown, CT (USA))

1989-08-22

284

Efficient sampling of early signal arrival for estimation of perfusion and transit time in whole-brain arterial spin labeling.  

PubMed

Arterial spin labeling can be used to measure both cerebral perfusion and arterial transit time. However, accurate estimation of these parameters requires adequate temporal sampling of the arterial spin labeling difference signal. In whole-brain multislice acquisitions, two factors reduce the accuracy of the parameter estimates: saturation of labeled blood in transit and inadequate sampling of early difference signal in superior slices. Label saturation arises when slices are acquired inferior-to-superior such that slice selection in proximal slices spoils the label for a distal slice. Inadequate sampling arises when the time spent acquiring inferior slices is too long to allow early sampling of the difference signal in superior slices. A novel approach to multislice imaging is proposed to address these two issues. In round-robin arterial spin labeling, slices are acquired in a different order after every pair of control-label acquisitions. Round-robin arterial spin labeling enables the acquisitions of all slices across the same range of postlabel delays in a descending superior-to-inferior order. This eliminates the temporal sampling problem and greatly reduces label saturation. Arterial transit time estimates obtained for the whole brain with round-robin arterial spin labeling show better agreement with a single-slice acquisition than do conventional multislice acquisitions. PMID:22189961

Lee, Wayne; Janik, Rafal; Scouten, Amy; Stefanovic, Bojana; Sled, John G

2012-07-01

285

A simple method for the synthesis of hyaluronic acid coated magnetic nanoparticles for highly efficient cell labelling and in vivo imaging†  

PubMed Central

Highly stable colloidal hyaluronic acid coated magnetic nano-particles were prepared via a ligand exchange method. These particles exhibited excellent cell labeling efficiencies and superior potential as MRI contrast agents, which are useful to target tumor cells expressing hyaluronic acid receptors such as CD44.

El-Dakdouki, Mohammad H.; El-Boubbou, Kheireddine; Zhu, David C.; Huang, Xuefei

2012-01-01

286

(18)F-FDG cell labeling may underestimate transplanted cell homing: more accurate, efficient, and stable cell labeling with hexadecyl-4-[(18)F]fluorobenzoate for in vivo tracking of transplanted human progenitor cells by positron emission tomography.  

PubMed

Cell therapy is expected to restore perfusion and improve function in the ischemic/infarcted myocardium; however, the biological mechanisms and local effects of transplanted cells remain unclear. To assess cell fate in vivo, hexadecyl-4-[(18)F]fluorobenzoate ((18)F-HFB) cell labeling was evaluated for tracking human circulating progenitor cells (CPCs) with positron emission tomography (PET) and was compared to the commonly used 2-[(18)F]fluoro-2-deoxy-d-glucose ((18)F-FDG) labeling method in a rat myocardial infarction model. CPCs were labeled with 18F-HFB or (18)F-FDG ex vivo under the same conditions. (18)F-HFB cell-labeling efficiency (23.4 ± 7.5%) and stability (4 h, 88.4 ± 6.0%) were superior to (18)F-FDG (7.6 ± 4.1% and 26.6 ± 6.1%, respectively; p < 0.05). Neither labeling approach significantly altered cell viability, phenotype or migration potential up to 24 h postlabeling. Two weeks after left anterior descending coronary artery ligation, rats received echo-guided intramyocardial injection in the infarct border zone with (18)F-HFB-CPCs, (18)F-FDG-CPCs, (18)F-HFB, or (18)F-FDG. Dynamic PET imaging of both (18)F-HFB-CPCs and(18)F-FDG-CPCs demonstrated that only 16-37% of the initial injection dose (ID) was retained in the injection site at 10 min postdelivery, and remaining activity fell significantly over the first 4 h posttransplantation. The (18)F-HFB-CPC signal in the target area at 2 h (23.7 ± 14.7% ID/g) and 4 h (17.6 ± 13.3% ID/g) postinjection was greater than that of (18)F-FDG-CPCs (5.4 ± 2.3% ID/g and 2.6 ± 0.7% ID/g, respectively;p < 0.05). Tissue biodistribution confirmed the higher radioactivity in the border zone of (18)F-HFB-CPC rats. Immunostaining of heart tissue sections revealed no significant difference in cell retention between two labeled cell transplantation groups. Good correlation with biodistribution results was observed in the (18)F-HFB-CPC rats (r = 0.81, p < 0.05). Compared to (18)F-FDG, labeling human CPCs with(18)F-HFB provides a more efficient, stable, and accurate way to quantify the distribution of transplanted cells. (18)F-HFB cell labeling with PET imaging offers a better modality to enhance our understanding of early retention, homing, and engraftment with cardiac cell therapy. PMID:22469629

Zhang, Yan; Dasilva, Jean N; Hadizad, Tayebeh; Thorn, Stephanie; Kuraitis, Drew; Renaud, Jennifer M; Ahmadi, Ali; Kordos, Myra; Dekemp, Robert A; Beanlands, Rob S; Suuronen, Erik J; Ruel, Marc

2012-01-01

287

High-throughput quantitation of amino acids in rat and mouse biological matrices using stable isotope labeling and UPLC-MS/MS analysis.  

PubMed

Quantifying amino acids in biological matrices is typically performed using liquid chromatography (LC) coupled with fluorescent detection (FLD), requiring both derivatization and complete baseline separation of all amino acids. Due to its high specificity and sensitivity, the use of UPLC-MS/MS eliminates the derivatization step and allows for overlapping amino acid retention times thereby shortening the analysis time. Furthermore, combining UPLC-MS/MS with stable isotope labeling (e.g., isobaric tag for relative and absolute quantitation, i.e., iTRAQ) of amino acids enables quantitation while maintaining sensitivity, selectivity and speed of analysis. In this study, we report combining UPLC-MS/MS analysis with iTRAQ labeling of amino acids resulting in the elution and quantitation of 44 amino acids within 5min demonstrating the speed and convenience of this assay over established approaches. This chromatographic analysis time represented a 5-fold improvement over the conventional HPLC-MS/MS method developed in our laboratory. In addition, the UPLC-MS/MS method demonstrated improvements in both specificity and sensitivity without loss of precision. In comparing UPLC-MS/MS and HPLC-MS/MS results of 32 detected amino acids, only 2 amino acids exhibited imprecision (RSD) >15% using UPLC-MS/MS, while 9 amino acids exhibited RSD >15% using HPLC-MS/MS. Evaluating intra- and inter-assay precision over 3 days, the quantitation range for 32 detected amino acids in rat plasma was 0.90-497?M, with overall mean intra-day precision of less than 15% and mean inter-day precision of 12%. This UPLC-MS/MS assay was successfully implemented for the quantitative analysis of amino acids in rat and mouse plasma, along with mouse urine and tissue samples, resulting in the following concentration ranges: 0.98-431?M in mouse plasma for 32 detected amino acids; 0.62-443?M in rat plasma for 32 detected amino acids; 0.44-8590?M in mouse liver for 33 detected amino acids; 0.61-1241?M in mouse kidney for 37 detected amino acids; and 1.39-1681?M in rat urine for 34 detected amino acids. The utility of the assay was further demonstrated by measuring and comparing plasma amino acid levels between pre-diabetic Zucker diabetic fatty rats (ZDF/Gmi fa/fa) and their lean littermates (ZDF/Gmi fa/?). Significant differences (P<0.001) in 9 amino acid concentrations were observed, with the majority ranging from a 2- to 5-fold increase in pre-diabetic ZDF rats on comparison with ZDF lean rats, consistent with previous literature reports. PMID:24842860

Takach, Edward; O'Shea, Thomas; Liu, Hanlan

2014-08-01

288

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

PubMed

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 nanoparticles 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 ?g/mL for 48 h showed no change in viability or proliferation compared to untreated controls. Additionally, at these potentially safe concentrations, up to 48 h, the cells showed no phosphatidyl serine externalization (pre-apoptotic condition), caspase-3 activity (point of no return for apoptosis), genetic damage, or changes in their division cycle. Localization of Gd-SWCNTs within the cells was confirmed by transmission electron microscopy (TEM) and Raman microscopy, and these results show 100% cell labeling efficiency. Elemental analysis also indicates significant uptake of Gd-SWCNTs by the cells (10(8) -10(9) Gd(3+) ions per cell). Finally, T1 -weighted MRI at 3 T of Gd-SWCNT-labelled cells show up to a four-fold increase in MR signal intensities as compared to untreated cells. These results indicate that Gd-SWCNTs label cells efficiently at potentially safe concentrations, and enhance MRI contrast without any structural damage to the cells. PMID:23686792

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

2013-12-01

289

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

Microsoft Academic Search

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

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

290

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

PubMed Central

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

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

2012-01-01

291

Shock tube measurements of the tert-butanol + OH reaction rate and the tert-C4H8OH radical ?-scission branching ratio using isotopic labeling.  

PubMed

The overall rate constant for the reaction tert-butanol + OH ? products was determined experimentally behind reflected shock waves by using (18)O-substituted tert-butanol (tert-butan(18)ol) and tert-butyl hydroperoxide (TBHP) as a fast source of (16)OH. The data were acquired from 900 to 1200 K near 1.1 atm and are best fit by the Arrhenius expression 1.24 × 10(-10)?exp(-2501/T [K]) cm(3) molecule(-1) s(-1). The products of the title reaction include the tert-C4H8OH radical that is known to have two major ?-scission decomposition channels, one of which produces OH radicals. Experiments with the isotopically labeled tert-butan(18)ol also lead to an experimental determination of the branching ratio for the ?-scission pathways of the tert-C4H8OH radical by comparing the measured pseudo-first-order decay rate of (16)OH in the presence of excess tert-butan(16)ol with the respective decay rate of (16)OH in the presence of excess tert-butan(18)ol. The two decay rates of (16)OH as a result of reactions with the two forms of tert-butanol differ by approximately a factor of 5 due to the absence of (16)OH-producing pathways in experiments with tert-butan(18)ol. This indicates that 80% of the (16)OH molecules that react with tert-butan(16)ol will reproduce another (16)OH molecule through ?-scission of the resulting tert-C4H8(16)OH radical. (16)OH mole fraction time histories were measured using narrow-line-width laser absorption near 307 nm. Measurements were performed at the line center of the R22(5.5) transition in the A-X(0,0) band of (16)OH, a transition that does not overlap with any absorption features of (18)OH, hence yielding a measurement of (16)OH mole fraction that is insensitive to any production of (18)OH. PMID:23683356

Stranic, Ivo; Pang, Genny A; Hanson, Ronald K; Golden, David M; Bowman, Craig T

2013-06-13

292

Impact of deficit irrigation on water use efficiency and carbon isotope composition ( 13C) of field-grown grapevines under Mediterranean climate  

Microsoft Academic Search

The objective of this study was to evaluate the effect of deficit irrigation on intrinsic water use efficiency (A\\/gs) and carbon isotope composition (d13C) of two grape- vine cultivars (Moscatel and Castelao), growing in a commercial vineyard in SW Portugal. The study was done in two consecutive years (2001 and 2002). The treatments were full irrigation (FI), corresponding to 100%

Claudia R. de Souza; Joao P. Maroco; Tiago P. dos Santos; M. Lucilia Rodrigues; Carlos M. Lopes; Joao S. Pereira; M. Manuela Chaves

2005-01-01

293

Labelling of precipitation by stable isotopes ( 18O, 2H) over the Jos Plateau and the surrounding plains (north-central Nigeria)  

NASA Astrophysics Data System (ADS)

Stable isotopes oxygen-18 and deuterium were studied in precipitation at five meteorological stations on the Jos Plateau and the surrounding plains. Rainwater is progressively depleted in heavy isotopes from the beginning of the rainy season towards the peak of the season, with the most depleted values being recorded in the August rains. Isotopic variation with altitude is very evident between the stations on the plateau and that on the plain. The isotope-altitude gradient is probably concealed by the mass-effect of the Jos Plateau. A Local Meteoric Line (LML) of the type ?D=7.8 ?18O+10.6 was obtained with all the available isotopic data. When only the data for the rain for the months of June through August are used, a relation ?D=8.4 ?18O+14, close to the Global Meteoric Water line but with a higher deuterium excess (d) is obtained. This suggests a contribution of continental vapour mass to precipitation over the region. On the basis of the stable isotope relationships, three categories of recharging rainwater are defined, which may have implications in groundwater tracing: i) precipitation of the early and perhaps late rainy season, enriched in heavy isotopes ii) precipitation of the advanced stage of the rainy season, marked by non-evaporated rains (slope 8, d > 10) iii) precipitation of the peak of the rainy season (August, September), depleted in heavy isotopes.

Mbonu, M.; Travi, Y.

1994-08-01

294

Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation.  

PubMed

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

Hendry, Katharine R; Robinson, Laura F; McManus, Jerry F; Hays, James D

2014-01-01

295

Silicon isotopes indicate enhanced carbon export efficiency in the North Atlantic during deglaciation  

NASA Astrophysics Data System (ADS)

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.

Hendry, Katharine R.; Robinson, Laura F.; McManus, Jerry F.; Hays, James D.

2014-01-01

296

Water use efficiency and carbon isotope composition of plants in a cold desert environment  

Microsoft Academic Search

The effects of the availabilities of water and nitrogen on water use efficiency (WUE) of plants were investigated in a sagebrush steppe. The four species studied wereArtemisia tridentata (shrub),Ceratoides lanata (suffrutescent shrub),Elymus lanceolatus (rhizomatous grass), andElymus elymoides (tussock grass). Water and nitrogen levels were manipulated in a two-by-two factorial design resulting in four treatments: control (no additions), added water, added

Nancee L. Toft; Jay E. Anderson; Robert S. Nowak

1989-01-01

297

Prediction of equilibrium Li isotope fractionation between minerals and aqueous solutions at high P and T: An efficient ab initio approach  

NASA Astrophysics Data System (ADS)

The mass-dependent equilibrium stable isotope fractionation between different materials is an important geochemical process. Here we present an efficient method to compute the isotope fractionation between complex minerals and fluids at high pressure, P, and temperature, T, representative for the Earth's crust and mantle. The method is tested by computation of the equilibrium fractionation of lithium isotopes between aqueous fluids and various Li bearing minerals such as staurolite, spodumene and mica. We are able to correctly predict the direction of the isotope fractionation as observed in the experiments. On the quantitative level the computed fractionation factors agree within 1.0‰ with the experimental values indicating predictive power of ab initio methods. We show that with ab initio methods we are able to investigate the underlying mechanisms driving the equilibrium isotope fractionation process, such as coordination of the fractionating elements, their bond strengths to the neighboring atoms, compression of fluids and thermal expansion of solids. This gives valuable insight into the processes governing the isotope fractionation mechanisms on the atomic scale. The method is applicable to any state and does not require different treatment of crystals and fluids.

Kowalski, Piotr M.; Jahn, Sandro

2011-10-01

298

Identification of subunit-subunit interaction sites in ?A-WT crystallin and mutant ?A-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.  

PubMed

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

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

2013-01-01

299

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

PubMed

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

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

2014-01-01

300

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

SciTech Connect

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

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

2011-01-01

301

A cationic Rh(III) complex that efficiently catalyzes hydrogen isotope exchange in hydrosilanes.  

PubMed

The synthesis and structural characterization of a mixed-sandwich (?(5)-C(5)Me(5))Rh(III) complex of the cyclometalated phosphine PMeXyl(2) (Xyl = 2,6-C(6)H(3)Me(2)) with unusual ?(4)-P,C,C',C'' coordination (compound 1-BAr(f); BAr(f) = B(3,5-C(6)H(3)(CF(3))(2))(4)) are reported. A reversible ?(4) to ?(2) change in the binding of the chelating phosphine in cation 1(+) induced by dihydrogen and hydrosilanes triggers a highly efficient Si-H/Si-D (or Si-T) exchange applicable to a wide range of hydrosilanes. Catalysis can be carried out in an organic solvent solution or without solvent, with catalyst loadings as low as 0.001 mol %, and the catalyst may be recycled a number of times. PMID:21062088

Campos, Jesús; Esqueda, Ana C; López-Serrano, Joaquín; Sánchez, Luis; Cossio, Fernando P; de Cozar, Abel; Alvarez, Eleuterio; Maya, Celia; Carmona, Ernesto

2010-12-01

302

Luminescent dye-doped or rare-earth-doped monodisperse silica nanospheres as efficient labels in DNA microarrays  

Microsoft Academic Search

Luminescent nanoparticles are gaining more and more interest in bio-labeling and bio-imaging applications, like for example DNA microarray. This is a high-throughput technology used for detection and quantification of nucleic acid molecules and other ones of biological interest. The analysis is resulting by specific hybridization between probe sequences deposited in array and a target ss-DNA usually expressed by PCR and

F. Enrichi; R. Riccò; A. Meneghello; R. Pierobon; F. Marinello; P. Schiavuta

2009-01-01

303

New approach via gene knockout and single-step chemical reaction for the synthesis of isotopically labeled fusarin c as an internal standard for the analysis of this fusarium mycotoxin in food and feed samples.  

PubMed

The gold standard for quantitation of contaminants with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) is the use of isotopically labeled standards. Herein, we report a new strategy for the synthesis of isotopically labeled 21-d3-fusarin C via a genetically modified Fusarium strain, followed by a one-step derivatization reaction. Fusarin C is a Fusarium mycotoxin, which is mutagenic after metabolic activation. Its occurrence has been demonstrated recently in corn-based samples, but up to now, little is known about the contamination of other grain samples. To collect further data, the quantitation method was enhanced by application of the 21-d3-fusarin C and the use of a QTRAP 5500 mass spectrometer. This new method has a limit of detection (LOD) of 1 ?g/kg, a limit of quantitation (LOQ) of 4 ?g/kg, and a recovery rate of 99%. A total of 21 corn samples and 13 grain samples were analyzed, with resulting fusarin C levels varying from not detectable to 24.7 ?g/kg. PMID:22877497

Kleigrewe, Karin; Niehaus, Eva-Maria; Wiemann, Philipp; Tudzynski, Bettina; Humpf, Hans-Ulrich

2012-08-29

304

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

PubMed Central

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

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

2014-01-01

305

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

PubMed

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

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

2014-01-01

306

Sensitivity and detection efficiency of the peroxidase antiperoxidase (PAP), avidin-biotin peroxidase complex (ABC), and peroxidase-labeled avidin-biotin (LAB) methods.  

PubMed

The authors have examined the sensitivity and detection efficiency of the three peroxidase methods that currently have the widest application in diagnostic immunohistochemistry: the peroxidase-antiperoxidase (PAP), the avidin-biotin complex (ABC), and the labeled avidin-biotin (LAB) methods. Sensitivity was evaluated by determining the highest useful dilution of polyclonal antiglucagon antibodies applied to formalin-fixed, paraffin-embedded human pancreas. Detection efficiency was evaluated by tabulation of the total number of positive (three or more positive cells) islets. On direct comparison, the LAB method exceeded the PAP and ABC methods in both sensitivity and detection efficiency, which were essentially equal. Titration of linking antiserum of the PAP method boosted its sensitivity and detection efficiency above that of ABC; the PAP had equal sensitivity to the LAB and exceeded it in detection efficiency. The authors conclude that comparisons of immunohistologic methods are meaningful only if both sensitivity and efficiency are considered along with the unique requirements of any single method. PMID:2546420

Elias, J M; Margiotta, M; Gaborc, D

1989-07-01

307

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)

Understanding the relationship between carbon assimilation and water consumption by natural vegetation is needed to assess how changes in climate will affect plant carbon and water exchange as well as the energy fluxes of ecosystems. While climate change is expected to cause significant warming, most models also suggest changes in the timing and amount of precipitation received; thus implications of this type of change are particularly acute in Mediterranean regions of the world. Blue oak savannas are already exposed to broad variation in water availability and to severe droughts during the summer months. Our objective was to evaluate the trade-off between carbon gain and water loss (Water Use Efficiency) in this ecosystem at both the leaf and at the ecosystem scales. We monitored the ratio of the partial pressures of CO2 inside the leaf (Ci) and in the outside air (Ca) or Ci/Ca, during the summer months of three subsequent years. This ratio is determined by the balance between photosynthetic capacity and stomatal conductance to water loss. Leaf-level estimates for individual trees were based on the carbon isotope composition (?13C) of bulk leaf tissue and of recently fixed carbohydrates (leaf soluble sugars). These leaf and individual tree based estimates were then compared with canopy-level estimates derived from continuous eddy covariance measurements of fluxes of CO2, water vapor and meteorological variables from two eddy covariance systems, one above (23m) and one below (2m) the tree canopy. We found that savanna Blue oak trees cope with severe drought through coordinated down-regulation of carbon and water fluxes, i.e. the ratio Ci/Ca remained stable over four summer months, despite decreasing soil water content and leaf water potentials. Stable C isotope composition of leaf soluble sugars is the most robust measure of Ci/Ca because it reflects the initial discrimination of photosynthetic products, without the confounding effects ascribed to storage, tissue chemical composition and time of tissue formation. Our findings at the leaf-level were confirmed at the ecosystem-level by using a two tower (above and below canopy) eddy covariance method.

Mambelli, S.; Tu, K. P.; Knohl, A.; Ma, S.; Baldocchi, D. D.; Dawson, T. E.

2007-12-01

308

Application of exogenous mixture of glutathione and stable isotope labeled glutathione for trapping reactive metabolites in cryopreserved human hepatocytes. Detection of the glutathione conjugates using high resolution accurate mass spectrometry.  

PubMed

Metabolites (including reactive metabolites) of troglitazone were generated by incubation with cryopreserved human hepatocytes and trapped in the presence of an exogenous mixture of unlabeled and stable isotope labeled (SIL: [1,2-(13)C, (15)N]-glycine) glutathione (GSH/SIL-GSH). The incubation samples were analyzed using liquid chromatography-high resolution accurate mass spectrometry (LC-HRAMS) implemented on a LTQ Orbitrap mass spectrometer. The GSH conjugates of the reactive metabolites were detected via a characteristic mono-isotopic pattern (peaks separated by 3.0037u). Analysis of the incubation samples led to detection of a number of previously described GSH conjugates, as well as two novel methylated GSH conjugates, which were partially characterized based on accurate mass measurements and MS/MS data. The addition of exogenous GSH led to an increase in the apparent level of detected GSH conjugates. Kinetic isotopic measurements showed that the rates of incorporation of exogenous GSH are conjugate-specific. In conclusion, this approach, based on the use of a mixture of GSH/SIL-GSH, allows facile capture and detection of reactive metabolites in human hepatocytes. Moreover, the data suggest that routine addition of glutathione to the assay medium may be advisable for experiments with cryopreserved hepatocytes. PMID:23747843

Mezine, Igor; Bode, Chris; Raughley, Bethany; Bhoopathy, Sid; Roberts, Kenneth J; Owen, Albert J; Hidalgo, Ismael J

2013-08-25

309

CAESAR---A high-efficiency CsI(Na) scintillator array for in-beam gamma-ray spectroscopy with fast rare-isotope beams  

Microsoft Academic Search

We report on the construction and commissioning of the high-efficiency CAESium-iodide scintillator ARray CAESAR, a device designed for in-beam gamma-ray spectroscopy experiments utilizing fast beams of rare isotopes at the National Superconducting Cyclotron Laboratory (NSCL) at Michigan State University (MSU). CAESAR consists of 192 CsI(Na) crystals, totaling 290 kg of active scintillator material. For 1 MeV gamma rays, a full-energy-peak

D. Weisshaar; A. Gade; T. Glasmacher; G. F. Grinyer; D. Bazin; P. Adrich; T. Baugher; J. M. Cook; C. Aa. Diget; S. McDaniel; A. Ratkiewicz; K. P. Siwek; K. A. Walsh

2010-01-01

310

Leaf-level water use efficiency determined by carbon isotope discrimination in rice seedlings: genetic variation associated with population structure and QTL mapping  

Microsoft Academic Search

Increasing the water use efficiency (WUE) of our major crop species is an important target of agricultural research. Rice\\u000a is a major water consumer in agriculture and it is also an attractive genetic model. We evaluated leaf-level WUE in young\\u000a rice seedlings using carbon isotope discrimination (?13C) as an indicator of the trait. A survey of ?13C was undertaken in

Yunbi Xu; Dominique This; Roman C. Pausch; Wendy M. Vonhof; Jason R. Coburn; Jonathan P. Comstock; Susan R. McCouch

2009-01-01

311

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

PubMed

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

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

2012-04-01

312

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

PubMed

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

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

2007-01-01

313

HPLC-ICPMS and stable isotope-labeled approaches to assess quantitatively Ti(IV) uptake by transferrin in human blood serum.  

PubMed

Little is known about the effects of titanium found in patients wearing prostheses or about the biochemical pathways of this metal when used as an anticancer drug (e.g., titanocene dichloride). In this work, transferrin has been confirmed as the only carrier protein binding Ti in human blood serum samples by making use of different HPLC protein separations followed by element-specific Ti detection by ICPMS. Besides, isotope dilution analysis has been applied to the quantitative speciation of Ti-Tf in standards and human blood serum samples. Species-unspecific and species-specific isotope dilution modes have been explored. In the first case, very low Ti-Tf results were obtained even using two different chromatographic mechanisms, anion exchange (20-24%) and size exclusion (33-36%). Surprisingly, no major Ti species except Ti-Tf were observed in the chromatograms, suggesting that Ti(IV) hydrolysis and precipitation as inactive titanium oxide species could take place inside the chromatographic columns. These results demonstrate that chemical degradation of metalloproteins during analytical separations could ruin the sought speciation quantitative results. The isotope dilution species-specific mode, much more accurate in such cases, has been instrumental in demonstrating the possibility of gross errors in final metalloprotein quantification. For this purpose, an isotopically enriched standard of (49)Ti-Tf was synthesized and applied to the quantitative speciation of Ti-Tf again. Using this species-specific spike, Ti-Tf dissociation inside the chromatographic columns used could be corrected, and thus, quantitative Ti-Tf binding in serum (92-102%) was observed. In other words, the usefulness and potential of a species-specific isotope dilution analysis approach to investigate quantitatively metal-protein associations, which can be dissociated at certain experimental conditions, is demonstrated here for the first time. PMID:18847283

Sarmiento-González, Alejandro; Ruiz Encinar, Jorge; Cantarero-Roldán, Alicia M; Marchante-Gayón, Juan M; Sanz-Medel, Alfredo

2008-11-15

314

Efficient downregulation of VEGF in retinal pigment epithelial cells by integrin ligand-labeled liposome-mediated siRNA delivery  

PubMed Central

Background The purpose of this study was to demonstrate the effectiveness of an integrin peptide ligand-labeled liposomal delivery system loaded with vascular endothelial growth factor (VEGF)-siRNA in a model study of gene therapy for retinopathy using human retinal pigment epithelial cells. Methods Arg(R)-Gly(G)-Asp(D) motif peptide conjugating polyethylene glycol modified (RGD-PEGylated) liposomes were prepared using a thin-film hydration method and optimized for surface charge, particle size, small interfering RNA (siRNA) load, and entrapment efficiency. Reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assays were used to determine VEGF levels in retinal pigment epithelial cells. Cytotoxicity was determined using the 3-[4, 5-dimethylthiazol-2-yl]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay and flow cytometry. Results Physicochemical properties, including particle size, zeta potential, and siRNA load, of the prepared RGD-PEGylated liposomes and their entrapment efficiency were determined to be within the following ranges: 123.8–234.1 nm, 17.31–40.09 m V, 5.27%–6.33%, and >97%, respectively. RGD-PEGylated liposome-mediated fluorescent-labeled siRNA delivery demonstrated significantly enhanced cellular uptake, and 3 mol% RGD-PEGylated liposomes (having 3?-[N-(N’, N’-dimethylaminoethane) carbamoyl] cholesterol (DC-cholesterol) DSPE and DSPE-PEG(2000)-RGD with molar ratio of 50/47/3) were shown to have better efficacy with regard to specificity for retinal pigment epithelial cells, reduced cytotoxicity, and knockdown of the target molecule. Conclusion By integrin receptor-mediated endocytosis, 3 mol% RGD-PEGylated liposomes were shown to be a suitable vector when loaded with VEGF-siRNA for efficient downregulation of VEGF in retinal pigment epithelial cells at both the protein and gene levels. This integrin ligand-modified liposomal delivery system has therapeutic potential for ocular gene therapy.

Chen, Cheng-Wei; Yeh, Ming-Kung; Shiau, Chia-Yang; Chiang, Chiao-Hsi; Lu, Da-Wen

2013-01-01

315

The isotopic composition of H2 from biomass burning - dependency on combustion efficiency, moisture content and dD of local precipitation  

NASA Astrophysics Data System (ADS)

Differences in isotopic composition between the various sources of H2 are large, but only few measurements have been carried out to constrain them. For biomass burning, the values quoted in the literature are based on few combustion experiments, which were then extrapolated to the global scale based on a number of assumptions. One of these assumptions is that the isotopic composition of H2 should scale with the isotopic composition of the precipitation at the location where the biomass grew. Here we test this hypothesis using 18 wood samples collected from various locations around the globe. The sample locations cover a range in dD of precipitation from below -120 permil in Siberia and Canada to -15 permil in Zimbabwe. The results confirm the predicted linear relation with dD of the precipitation in the sampling region. The water content itself is found to at most slightly affect the results. Furthermore, dD of H2 depends on combustion efficiency. Thus, the isotopic composition of H2 from biomass burning shows a strong variability around the globe, and between different stages of a fire. It is suggested that this variability, rather than a global bulk number, should be incorporated explicitly in global models that attempt to reproduce the spatial and temporal distribution of dD in H2.

Röckmann, Thomas; Gómez Álvarez, Catalina; Walter, Sylvia; Wollny, Adam; Gunthe, Sachin; Helas, Günter; Pöschl, Ulrich; Keppler, Frank; Greule, Markus; Brand, Willi

2010-05-01

316

Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography\\/pyrolysis\\/isotope ratio–mass spectrometric analysis  

Microsoft Academic Search

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium (2H) from heavy water (2H2O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography\\/pyrolysis\\/isotope ratio–mass spectrometry (GC\\/P\\/IRMS). Very low levels

Jason N Voogt; Mohamad Awada; Elizabeth J Murphy; Gregory M Hayes; Robert Busch; Marc K Hellerstein

2007-01-01

317

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

PubMed

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

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

2014-06-20

318

Identification of Cargo Proteins Specific for the Nucleocytoplasmic Transport Carrier Transportin by Combination of an in Vitro Transport System and Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based Quantitative Proteomics*  

PubMed Central

The human importin-? family consists of 21 nucleocytoplasmic transport carrier proteins that carry proteins and RNAs across the nuclear envelope through nuclear pores in specific directions. These transport carriers are responsible for the nucleocytoplasmic transport of thousands of proteins, but the cargo allocation of each carrier, which is necessary information if one wishes to understand the physiological context of transport, is poorly characterized. To address this issue, we developed a high-throughput method to identify the cargoes of transport carriers by applying stable isotope labeling by amino acids in cell culture to construct an in vitro transport system. Our method can be outlined in three steps. (1) Cells are cultured in a medium containing a stable isotope. (2) The cell membranes of the labeled cells are permeabilized, and proteins extracted from unlabeled cells are transported into the nuclei of the permeabilized cells. In this step, the reaction system is first depleted of all importin-? family carriers and then supplemented with a particular importin-? family carrier of interest. (3) Proteins in the nuclei are extracted and analyzed quantitatively via LC-MS/MS. As an important test case, we used this method to identify cargo proteins of transportin, a representative member of the importin-? family. As expected, the identified candidate cargo proteins included previously reported transportin cargoes as well as new potential cargoes, which we corroborated via in vitro binding assays. The identified cargoes are predominately RNA-interacting proteins, affirming that cargoes allotted to the same carrier share functional characteristics. Finally, we found that the transportin cargoes possessed at least two classes of signal sequences: the well characterized PY-nuclear localization signals specific for transportin, and Lys/Arg-rich segments capable of binding to both transportin and importin-?. Thus, our method will be useful for linking a carrier to features shared among its cargoes and to specific nuclear localization signals.

Kimura, Makoto; Kose, Shingo; Okumura, Nobuaki; Imai, Kenichiro; Furuta, Maiko; Sakiyama, Noriyuki; Tomii, Kentaro; Horton, Paul; Takao, Toshifumi; Imamoto, Naoko

2013-01-01

319

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

SciTech Connect

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

Vanicek, Jiri; Miller, William H.

2007-06-13

320

Quantitative MALDI-TOF-MS Using Stable-isotope Labeling: Application to the Analysis of N-glycans of Recombinant ?-1 Antitrypsin Produced Using Different Culture Parameters  

Microsoft Academic Search

It is usually difficult to compare quantitatively different sets of glycan samples that have slight differences among themselves using MALDI-TOF-MS. To overcome this problem, C-labeled glycans were used as internal standards during the measurement of the N-glycan samples permethylated with C-methyl iodide. This method was used to analyze the N-glycans from human ?-1 antitrypsin (A1AT) that was recombinantly produced using

Véronique Blanchard; Matthias Kaup; Susann Eigel; Silke Rieck; Volker Sandig; Uwe Marx; Rudolf Tauber; Markus Berger

2011-01-01

321

Highly efficient circulating tumor cell isolation from whole blood and label-free enumeration using polymer-based microfluidics with an integrated conductivity sensor.  

PubMed

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

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

2008-07-01

322

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

NASA Astrophysics Data System (ADS)

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

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

1998-08-01

323

Biodistribution of Ru-97-Labeled DTPA, DMSA and Transferrin.  

National Technical Information Service (NTIS)

Ruthenium-97 is being produced at the Brookhaven Linac Isotope Producer (BLIP). The favorable physical properties of Ru-97 and chemical reactivity of ruthenium offer a potential for using this isotope to label compounds useful for delayed scanning. Diethy...

A. B. Brill H. L. Atkins P. Som R. G. Fairchild Z. H. Oster

1980-01-01

324

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

PubMed

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

Petros, Robby A; Shah, Jyoti

2014-01-01

325

Direct Detection and Characterization of Chloride in the Active Site of the Low-pH Form of Sulfite Oxidase Using ESEEM Spectroscopy, Isotopic Labeling, and DFT Calculations  

PubMed Central

Electron spin echo envelope modulation (ESEEM) investigations were carried out on samples of the low-pH (lpH) form of vertebrate sulfite oxidase (SO) prepared with 35Cl- and 37Cl-enriched buffers as well as with buffer containing the natural abundance of Cl isotopes. The isotope-related changes observed in the ESEEM spectra provide direct and unequivocal evidence that Cl? is located in close proximity to the Mo(V) center of lpH SO. The measured isotropic hyperfine interaction constant of about 4 MHz (35Cl) suggests that the Cl? ion is either weakly coordinated to Mo(V) at its otherwise vacant axial position, trans to the oxo ligand, or is hydrogen-bonded to the equatorial exchangeable OH ligand. Scalar relativistic all-electron density functional theory (DFT) calculations of the hyperfine and nuclear quadrupole interaction parameters, along with steric and energetic arguments, strongly support the possibility that Cl? is hydrogen-bonded to the equatorial OH ligand rather than being directly coordinated to the Mo(V).

Klein, Eric L.; Astashkin, Andrei V.; Ganyushin, Dmitry; Riplinger, Christoph; Johnson-Winters, Kayunta; Neese, Frank; Enemark, John H.

2009-01-01

326

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

Microsoft Academic Search

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

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

2004-01-01

327

In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Noncyclic Nature of the Tricarboxylic Acid "Cycle" in Illuminated Leaves1[W  

PubMed Central

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

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

2009-01-01

328

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry.  

PubMed

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive. PMID:11981568

Zhou, Huilin; Ranish, Jeffrey A; Watts, Julian D; Aebersold, Ruedi

2002-05-01

329

Isotope edited product ion assignment by alpha-N labeling of peptides with [2H3(50%)]2,4-dinitrofluorobenzene.  

PubMed

An isotopic modification of Sanger's method for identifying peptide N-termini has been developed to assist peptide sequencing by tandem mass spectrometry. Tryptic peptides, such as Val-His-Leu-Thr-Pro-Val-Glu-Lys, are derivatized with an equimolar mixture of 2,4-dinitrofluorobenzene and [2H3]2,4-dinitrofluorobenzene. Under optimized derivatization conditions, the alpha-amino group could be derivatized while the epsilon-amine of the lysine side chain and the imidazole of histidine remained underivatized. The alpha-dinitrophenyl modified peptides were characterized by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography (LC)-ESI-MS. The [M + H]+ ions showed a doublet pattern with a delta m/z of 3 and the [M + 2H]2+ ions were recognized as doublets with a delta m/z of 1.5. MS/MS was employed where both isotopic [M + 2H]2+ ions were alternately subjected to collision-induced dissociation in the second quadrupole. Fragmentation in the ionization source generated identical product ion patterns that were observed during fragmentation in the second quadrupole. In the product ion mass spectra, the N-terminal a and b ions (no c ion observed) are doublets with a delta m/z of 3 or 1.5, while the C-terminal y and z ions (no x ion observed) are singlets appearing at identical masses. Thus, the product ions containing the N-terminus derivatized with a dinitrophenyl group are unequivocally distinguished from the product ions containing the C-terminus. The dinitrophenyl modification generally enhanced the production of a and b ions without diminishing y and z ion yields. PMID:10222597

Chen, X; Anderson, V E; Chen, Y H

1999-05-01

330

Isotope phallogram: preliminary communication.  

PubMed Central

The isotope phallogram is an investigation which uses radioisotope-labelled red cells in the imaging of penile arterial blood flow. In a preliminary series of 12 impotent patients undergoing both internal iliac arteriography and isotope phallography, the penogram index described by Fannous et al. (1982) has been modified to derive an accurate indicator of vascular disease. Images Figure 1. Figure 3. Figure 4. Figure 5. A Figure 5. B

Townell, N H; Siraj, Q H; Hilson, A J; Dick, R; Morgan, R J

1985-01-01

331

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

PubMed

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

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

2009-05-25

332

Isotopic Scintigraphy in Kidney Grafting.  

National Technical Information Service (NTIS)

Isotopic explorations of kidney transplants were performed on sixty-six patients. Three scintigraphic techniques were used: labelled ferrous ascorbate scintigraphy, sequential 99m technetium DTPA scintigraphy and the exp 131 I hippuran nephrogram. The aim...

R. Renfro

1976-01-01

333

An isotope-release assay and terminal-labeling assay for measuring cell-mediated allograft and tumor immunity to small numbers of adherent target cells.  

PubMed

A 51Cr-release assay and terminal 51Cr-labeling assay for measuring cell-mediated immunity to adherent target cells is described. Both techniques utilize small 10 microliter-per well microtiter plates, require low numbers of target cells (50-500 per well), and consequently, relatively small numbers of effector cells per well (3x10(3) -1x10(5)). Both assays are objective, quantitative, and simple to perform. The suitability of these techniques for monitoring immunologically specific, cellmediated, cytotoxic response to syngeneic and allogeneic tumor cells and normal skin fibroblasts is demonstrated. Lymph node cells, spleen cells and peritoneal exudate cells serve as effectors. PMID:78950

Tamerius, J D; Garrigues, H J; Hellström, I; Hellström, K E

1978-01-01

334

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

PubMed

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

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

2011-07-15

335

ISOTOPE METHODS IN HOMOGENEOUS CATALYSIS.  

SciTech Connect

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

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

2000-12-01

336

Carbon isotope discrimination and oxygen isotope composition in clones of the F(1) hybrid between slash pine and Caribbean pine in relation to tree growth, water-use efficiency and foliar nutrient concentration.  

PubMed

The objectives of this study were: (1) to examine how foliar carbon isotope discrimination (Delta) and oxygen isotope composition (delta(18)O) are related to tree growth, ash mineral nutrient concentration and foliar nutrient concentration in 7-year-old clones of the F(1) hybrid between slash pine (Pinus elliottii Engelm.) and Caribbean pine (P. caribaea var. hondurensis Barr. et Golf.) in subtropical Australia; and (2) to evaluate the potential of using foliar Delta, ash mineral nutrient concentration and delta(18)O measurements for selecting F(1) hybrid pine clones with high water-use efficiency (WUE) and growth potential. There were significant differences in tree growth, foliar Delta, delta(18)O and ash mineral nutrient concentration among the eight clones tested. Significant negative linear relationships existed between tree growth and Delta, extrapolating to zero growth at Delta = 24-30 per thousand. There were strong genetic correlations (r = -0.83 to -0.96) between Delta and tree growth, particularly tree height. Significant non-genetic correlations (r = -0.62 to -0.80) existed between Delta and foliar K concentration. Foliar delta(18)O, ash mineral nutrient concentration and foliar nutrient concentration were unrelated to tree growth. In the F(1) hybrid pine clones, variation in tree WUE, as reflected by Delta, was largely attributed to a genetic effect on leaf photosynthetic capacity rather than on stomatal conductance, as reflected by foliar delta(18)O. PMID:12651483

Xu, Z. H.; Saffigna, P. G.; Farquhar, G. D.; Simpson, J. A.; Haines, R. J.; Walker, S.; Osborne, D. O.; Guinto, D.

2000-12-01

337

International Isotope Society  

NSDL National Science Digital Library

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

338

Improvement of multi jet low pressure impactor for high collection efficiency of UF5 in the molecular laser isotope separation of uranium  

NASA Astrophysics Data System (ADS)

A numerical and experimental study for the collection of photo-produced UF 5 particles was performed for the low pressure impactors which have different design factors at typical flow conditions (upstream pressure of the impactor = 10-15 Torr, pressure ratio of downstream to upstream of the impactor, {P down}/{P up} = 0.2-0.5 ) in the molecular laser isotope separation of uranium at RIKEN (RIMLIS). Smaller {H}/{W} ratios (the distance between the impactor orifice exit and the impaction plate, H, divided by the orifice diameter of the impactor, W) and the smaller {P down}/{P up} were found to be preferable to obtain a higher collection efficiency from both numerical and experimental investigations. In addition it was experimentally demonstrated that the use of a 16 ?m laser system for the selective reaction of 235UF 6 to form 235UF 5 was not relevant for the study of the collection of UF 5 particles. So, we used an ultraviolet laser system (fourth harmonic YAG laser (266 nm) and an excimer laser (KrF, 248 nm)) which was more convenient to cope with various operating conditions. The collection efficiency was found to increase with the initial concentration of UF 5 molecules produced. Applying the improved impactor stage, we obtained a collection efficiency which was approximately 10 times higher than that of our previous work. Higher collection efficiencies of photo-produced UF 5 particles enriched in 235U reduce the enrichment cost.

Kuga, Yoshikazu; Jurcik, Benjamin; Satooka, Sakae; Takeuchi, Kazuo

1995-07-01

339

Estimates of precipitation efficiency and evaporation of falling precipitation in the tropics and subtropics based on measurements of the stable isotope ratio from satellite  

NASA Astrophysics Data System (ADS)

The humidity of the tropics and subtropics plays a disproportionate role in the radiative balance of the planet, and understanding the response of the tropical humidity to climate forcing is critical for determining climate sensitivity. Correctly modeling the complex set of processes that control the humidity requires consideration of large-scale transport, turbulence processes and cloud microphysical processes. While these are all included in general circulation models, many of the aspects associated with clouds are treated by parameterizations which represents a significant challenge. Previous modeling work has highlighted the importance of rain production, which is often modeled using a simplified treatment of autoconversion of cloud condensate. This quantity directly influences the fraction of total water lofted at the cloud based which is ultimately detrained by clouds to moisten the environment. Similarly, the degree to which falling condensate evaporates and moistening the lower troposphere is generally not well understood and not well observed over wide spatial scales. Consequently, evaporation of falling precipitation is parameterized simply in most climate models. Here we analyze results from a simple model of the humidity and isotope ratio profiles in the tropics. The model includes many of the features included in climate models such as detrainment, precipitation formation by autoconversion, large-scale subsidence, evaporation of falling condensate and export of water by horizontal eddies. Sensitivity tests show that the selection of parameters associated with precipitation efficiency and the evaporation of falling precipitation are the critical controls on the profile of isotope ratios. Making use of this sensitivity, profile observations of the D/H isotope ratio from NASA Tropospheric Emission Spectrometer are used to provide an optimal parameter estimate. The solutions provide an observational target for climate models. We discuss the limitations of the modeling framework and the need to balance the microphysical controls against influences of large-scale moisture transport.

Noone, D. C.

2012-12-01

340

Nitrifier denitrification can be a source of N2O from soil: a revised approach to the dual isotope labelling method  

NASA Astrophysics Data System (ADS)

Nitrous oxide (N2O), a potent greenhouse gas, can be produced through a variety of biochemical pathways. Nitrifier denitrification, i.e. nitrite reduction by ammonia oxidizers, is one of them. The existence of this pathway has been identified in monoculture studies, and it is increasingly suggested that it may contribute substantially to N2O production in soil. However, methodological drawbacks thus far prohibited conclusive proof of its occurrence in soil-based studies. As soils constitute the major global source of N2O, understanding the significance of nitrifier denitrification is indispensable to effective mitigation of N2O emissions. We developed and applied a new approach to identify the presence of nitrifier denitrification in soil. N2O production was studied in soil incubations using oxygen (O) and nitrogen (N) isotope enrichment tracing, while accounting for O exchange between water and intermediate products of the N transformations. Twelve soils were studied which covered a diversity of soil and land-use types across Europe. We additionally aimed to link variation in N2O production pathways to differences in the microbial community by examining microbial biomass C and N and phospholipid fatty acid (PLFA) patterns. Our results showed that in at least five of the soils studied, nitrifier denitrification must have contributed to total N2O production. Data revealed that in fact it may have been responsible for all NH4+-derived N2O in most soils. In contrast, N2O as by-product of ammonia oxidation contributed marginally, if any, to total production. Surprisingly, none of the examined microbial parameters could adequately explain differences in N2O production. Soil pH was the best predictor of the observed relative contributions of the pathways. We conclude that with our approach we finally demonstrate that nitrifier denitrification can indeed occur in soil, and may in fact be responsible for the majority of total nitrifier-induced N2O production. Our data further suggest that microbial community composition may not be a key driver of in the relative significance of different N2O production pathways, and that pH may be a principal factor determining nitrifier-induced N2O production.

Kool, D. M.; Wrage, N.; Zechmeister-Boltenstern, S.; Pfeffer, M.; Brus, D.; Oenema, O.; van Groenigen, J.

2009-12-01

341

Labeling Theory  

Microsoft Academic Search

Labeling theory provides a distinctively sociological approach that focuses on the role of social labeling in the development\\u000a of crime and deviance. The theory assumes that although deviant behavior can initially stem from various causes and conditions,\\u000a once individuals have been labeled or defined as deviants, they often face new problems that stem from the reactions of self\\u000a and others

Jón Gunnar Bernburg

342

Nutrition Labeling  

NASA Astrophysics Data System (ADS)

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

Metzger, Lloyd E.

343

ABRF-sPRG 2013 Study: Development and Characterization of a Proteomics Normalization Standard Consisting of 1000 Stable Isotope Labeled Peptides and a Qualitative Stability Study of Peptides from the ABRF-sPRG 2012 Study  

PubMed Central

The Proteomics Standards Research Group (sPRG) is reporting the first year progress in a two-year sPRG 2012-2013 study which focuses on the generation of a standard that can be used for interassay, interspecies, and interlaboratory normalization in both label-free and stable isotope label-based quantitative proteomics analysis. The standard has been formulated as two mixtures: 1000 stable isotope 13C/15N-labeled synthetic tryptic peptides alone, and peptides mixed with a tryptic digest from a HEK 293 cell lysate. The sequences of the synthetic peptides were derived from approximately 400 proteins and were conserved across proteomes of the most commonly analyzed species: Homo sapiens, Mus musculus and Rattus norvegicus. The selected peptides represent the full range of hydrophobicities and isoelectric points typical to tryptic peptides from complex proteomic samples. The standard was designed to represent proteins of various concentrations, spanning three orders of magnitude. This year we focused our efforts on selection of appropriate protein and peptide candidates, peptide synthesis, quality assessment and LC-MS evaluation by several sPRG member laboratories. The sPRG study design and initial results of a thorough characterization of the standard using a variety of instrumental configurations and bioinformatics approaches will be presented in this talk. The sPRG is hopeful that the designed formulation will become a valuable resource in various mass spectrometry-based proteomic applications, including quantitative and differential profiling, as well as general benchmarking (e.g. chromatographic retention time). The sPRG plans to start recruiting study participants in April 2013, complete the study by the end of the year 2013, and present the results at the ABRF 2014 meeting. The sPRG encourages proteomics laboratories to participate in the study and sign in at www.abrf.org/sprg. The second half of the session will discuss the qualitative stability study performed using purified synthetic peptides containing a variety of modifications selected from the 2012 sPRG ABRF sample. The stability of the selected synthetic peptides was evaluated by the sPRG using different storage conditions over a three-month period. After storage at either at room temperature, +4°C or ?80°C for one week, one month, or three months. Quantitative LC-MS/MS analysis was used to monitor the stability and degradation of the peptides, and to determine the effect of modifications and storage conditions on peptide degradation rates. The data presented have been built on the quantitative study that was presented at both the 2012 ABRF and ASMS conferences. All forms of degraded peptides were separated and identified using nano-LC tandem mass spectrometry on a Thermo Scientific Q-Exactive hybrid mass spectrometer. Integrated extracted ion chromatograms were used to measure relative amounts of degradation to identify which pathways are most prevalent during storage.

Dufresne, Craig P.; Ivanov, Alexander R.; Koller, Antonius; Phinney, Brett S.; Rose, Kristie L.; Rudnick, Paul A.; Searle, Brian C.; Colangelo, Christopher

2013-01-01

344

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

SciTech Connect

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

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

2013-01-01

345

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

PubMed

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 ?M 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

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

2011-05-01

346

Effects of Phosphorus and Water Supply on Yield, Transpirational Water-Use Efficiency, and Carbon Isotope Discrimination of Pearl Millet  

Microsoft Academic Search

low yields are accompanied by low evapotranspirational water-use efficiency, which is brought about by a combi- Several studies have identified low soil P and water availability as nation of low leaf area index and, for many environmen- major constraints to pearl millet (Pennisetum glaucum (L.) R. Br.) tal stresses, changes in WUET. production in semi-arid West Africa. To evaluate the

W. A. Payne; B. Sattelmacher

2000-01-01

347

Labeling during cleavage (LDC), a new labeling approach for RNA.  

PubMed

A new and efficient strategy for labeling of RNA sequences prior to their hybridization on high density DNA chip has been developed. Our approach which combines the fragmentation and the labeling is based on the reactivity of the 3'-phosphate of cleaved RNA fragments with a fluorescent molecule bearing aromatic bromomethyl function. PMID:11562981

Monnot, V; Tora, C; Lopez, S; Menou, L; Laayoun, A

2001-01-01

348

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

PubMed Central

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

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

2010-01-01

349

C4 plants use fluctuating light less efficiently than do C3 plants: a study of growth, photosynthesis and carbon isotope discrimination.  

PubMed

Plants in the field are commonly exposed to fluctuating light intensity, caused by variable cloud cover, self-shading of leaves in the canopy and/or leaf movement due to turbulence. In contrast to C3 plant species, only little is known about the effects of dynamic light (DL) on photosynthesis and growth in C4 plants. Two C4 and two C3 monocot and eudicot species were grown under steady light or DL conditions with equal sum of daily incident photon flux. We measured leaf gas exchange, plant growth and dry matter carbon isotope discrimination to infer CO2 bundle sheath leakiness in C4 plants. The growth of all species was reduced by DL, despite only small changes in steady-state gas exchange characteristics, and this effect was more pronounced in C4 than C3 species due to lower assimilation at light transitions. This was partially attributed to increased bundle sheath leakiness in C4 plants under the simulated lightfleck conditions. We hypothesize that DL leads to imbalances in the coordination of C4 and C3 cycles and increasing leakiness, thereby decreasing the quantum efficiency of photosynthesis. In addition to their other constraints, the inability of C4 plants to efficiently utilize fluctuating light likely contributes to their absence in such environments as forest understoreys. PMID:23550566

Kubásek, Ji?í; Urban, Otmar; Santr??ek, Ji?í

2013-03-30

350

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

SciTech Connect

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.

Qian, Weijun (BATTELLE (PACIFIC NW LAB)) [BATTELLE (PACIFIC NW LAB); Goshe, Michael B.(North Carolina State University) [North Carolina State University; Camp, David G.(BATTELLE (PACIFIC NW LAB)) [BATTELLE (PACIFIC NW LAB); Yu, Li-Rong (BATTELLE (PACIFIC NW LAB)) [BATTELLE (PACIFIC NW LAB); Tang, Keqi (BATTELLE (PACIFIC NW LAB)) [BATTELLE (PACIFIC NW LAB); Smith, Richard D.(BATTELLE (PACIFIC NW LAB)) [BATTELLE (PACIFIC NW LAB)

2003-10-15

351

Isotopic Biogeochemistry.  

National Technical Information Service (NTIS)

An overview is provided of the biogeochemical research. The funding, productivity, personnel and facilities are reviewed. Some of the technical areas covered are: carbon isotopic records; isotopic studies of banded iron formations; isotope effects in micr...

J. M. Hayes

1985-01-01

352

Isotope Separation.  

National Technical Information Service (NTIS)

Separation of isotopes is treated in a general way, with special reference to the production of enriched uranium. Uses of separated isotopes are presented quickly. Then basic definitions and theoretical concepts are explained: isotopic effects, non statis...

J. Ravoire

1978-01-01

353

Robust MS quantification method for phospho-peptides using 18O/16O labeling  

PubMed Central

Background Quantitative measurements of specific protein phosphorylation sites, as presented here, can be used to investigate signal transduction pathways, which is an important aspect of cell dynamics. The presented method quantitatively compares peptide abundances from experiments using 18O/16O labeling starting from elaborated MS spectra. It was originally developed to study signaling cascades activated by amyloid-? treatment of neurons used as a cellular model system with relevance to Alzheimer's disease, but is generally applicable. Results The presented method assesses, in complete cell lysates, the degree of phosphorylation of specific peptide residues from MS spectra using 18O/16O labeling. The abundance of each observed phospho-peptide from two cell states was estimated from three overlapping isotope contours. The influence of peptide-specific labeling efficiency was removed by performing a label swapped experiment and assuming that the labeling efficiency was unchanged upon label swapping. Different degrees of phosphorylation were reported using the fold change measure which was extended with a confidence interval found to reflect the quality of the underlying spectra. Furthermore a new way of method assessment using simulated data is presented. Using simulated data generated in a manner mimicking real data it was possible to show the method's robustness both with increasing noise levels and with decreasing labeling efficiency. Conclusion The fold change error assessable on simulated data was on average 0.16 (median 0.10) with an error-to-signal ratio and labeling efficiency distributions similar to the ones found in the experimentally observed spectra. Applied to experimentally observed spectra a very good match was found to the model (<10% error for 85% of spectra) with a high degree of robustness, as assessed by data removal. This new method can thus be used for quantitative signal cascade analysis of total cell extracts in a high throughput mode.

Andersen, Claus A; Gotta, Stefano; Magnoni, Letizia; Raggiaschi, Roberto; Kremer, Andreas; Terstappen, Georg C

2009-01-01

354

Variation in the carbon and oxygen isotope composition of plant biomass and its relationship to water-use efficiency at the leaf- and ecosystem-scales in a northern Great Plains grassland.  

PubMed

Measurements of the carbon (?(13) Cm ) and oxygen (?(18) Om ) isotope composition of C3 plant tissue provide important insights into controls on water-use efficiency. We investigated the causes of seasonal and inter-annual variability in water-use efficiency in a grassland near Lethbridge, Canada using stable isotope (leaf-scale) and eddy covariance measurements (ecosystem-scale). The positive relationship between ?(13) Cm and ?(18) Om values for samples collected during 1998-2001 indicated that variation in stomatal conductance and water stress-induced changes in the degree of stomatal limitation of net photosynthesis were the major controls on variation in ?(13) Cm and biomass production during this time. By comparison, the lack of a significant relationship between ?(13) Cm and ?(18) Om values during 2002, 2003 and 2006 demonstrated that water stress was not a significant limitation on photosynthesis and biomass production in these years. Water-use efficiency was higher in 2000 than 1999, consistent with expectations because of greater stomatal limitation of photosynthesis and lower leaf ci /ca during the drier conditions of 2000. Calculated values of leaf-scale water-use efficiency were 2-3 times higher than ecosystem-scale water-use efficiency, a difference that was likely due to carbon lost in root respiration and water lost during soil evaporation that was not accounted for by the stable isotope measurements. PMID:23862667

Flanagan, Lawrence B; Farquhar, Graham D

2014-02-01

355

Determination of atmospheric carbonyl sulfide by isotope dilution gas chromatography/mass spectrometry  

SciTech Connect

A gas chromatography/mass spectrometry (GB/MS) method for determining atmospheric carbonyl sulfide (OCS) with a precision better than 2% is reported. High precision and insensitivity to sample loss and changes in detector response were achieved by using isotopically labeled OCS as an internal standard. Tenax, Molecular Sieve 5A, Carbosieve B, and Carbosieve S were evaluated for collecting atmospheric OCS. Molecular Sieve 5A provided the best trapping and recovery efficiencies.

Lewin, E.E.; Taggart, R.L.; Lalevic, M.; Bandy, A.R.

1987-05-01

356

Recent ?-drug users with chronic hepatitis C can be efficiently treated with daily high dose induction therapy using consensus interferon: An open-label pilot study  

Microsoft Academic Search

AIM: To investigate the use of high dose consensus- interferon in combination with ribavirin in former iv drug users infected with hepatitis C. METHODS: We started, before pegylated (PEG)- interferons were available, an open-label study to investigate the efficacy and tolerability of high dose induction therapy with consensus interferon (CIFN) and ribavirin in treatment of naiive patients with chronic hepatitis

M Fuchs; D Ludwig; Yamanouchi Pharma; Thomas Witthoeft

357

Highly efficient technetium-99m labeling procedure based on the conjugation of N-[N-(3-diphenylphosphinopropionyl)glycyl]cysteine ligand with poly(ethylene glycol).  

PubMed

The PN(2)S N-(N-(3-diphenylphosphinopropionyl)glycyl)cysteine ligand was conjugated to methoxy-poly(ethylene glycol)-amino (mPEG-NH(2)) 5 and 20 kDa to yield PN(2)S(Trt)-PEG(5000) 1 and PN(2)S(Trt)-PEG(20000) 2, and then detritylated to PN(2)S-PEG(5000) 4 and PN(2)S-PEG(20000) 5. When an acidic solution of (99m)TcO(4)(-) is added to 4 or 5 in solid form, a quantitative yield in a single labeled species, (99m)Tc-labeled PN(2)S-PEG(5000) 9 and (99m)Tc-labeled PN(2)S-PEG(20000) 10, respectively, is obtained. The reaction occurs in less than 15 min at room temperature for 4 and 35 degrees C for 5. This labeling procedure avoids the use of an external reducing agent, and it is based on the amphiphilic properties of PN(2)S-PEGs. Once in water, 4 and 5 self-assemble in micelles, which catalyze the metal reduction by means of an electron pair transfer from the phosphorus to technetium. The [(99m)TcO](3+) species is then coordinated, and at micelle level, both the (P)ON(2)S and the PN(2)S coordinations are possible, as demonstrated by reacting (99m)Tc-gluconate and ReOCl(3)(PPh(3))(2) with 4 and 5 and with the oxidized analogous (P)ON(2)S-PEG(5000) 6. Compounds 9 and 10 exhibited a high stability both in vitro and in vivo. Biodistribution studies in mice also indicated that PN(2)S linking and (99m)Tc labeling do not modify PEG behavior in water and in vivo since the polymer dictates the fate of the conjugate. PMID:15366958

Visentin, Roberta; Pasut, Gianfranco; Veronese, Francesco Maria; Mazzi, Ulderico

2004-01-01

358

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

PubMed

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

Barraud, Pierre; Allain, Frédéric H-T

2013-01-01

359

Plasma biomarker discovery using 3D protein profiling coupled with label-free quantitation.  

PubMed

In-depth quantitative profiling of human plasma samples for biomarker discovery remains quite challenging. One promising alternative to chemical derivatization with stable isotope labels for quantitative comparisons is direct, label-free, quantitative comparison of raw LC-MS data. But, in order to achieve high-sensitivity detection of low-abundance proteins, plasma proteins must be extensively pre-fractionated, and results from LC-MS runs of all fractions must be integrated efficiently in order to avoid misidentification of variations in fractionation from sample to sample as "apparent" biomarkers. This protocol describes a powerful 3D protein profiling method for comprehensive analysis of human serum or plasma proteomes, which combines abundant protein depletion and high-sensitivity GeLC-MS/MS with label-free quantitation of candidate biomarkers. PMID:21468938

Beer, Lynn A; Tang, Hsin-Yao; Barnhart, Kurt T; Speicher, David W

2011-01-01

360

Comparison of Ecosystem Water-use Efficiency Among Douglas fir Forest, Aspen Forest and Grassland Using Eddy Covariance and Carbon Isotope Techniques  

NASA Astrophysics Data System (ADS)

Comparisons were made among Douglas fir forest, aspen (broad leaf deciduous) forest and wheatgrass (C3) grassland for ecosystem-level water-use efficiency. Water-use efficiency (WUE) was defined as the ratio of photosynthetic CO2 assimilation rate and evapo-transpiration (ET) rate. The ET data measured by eddy covariance were screened so that they overwhelmingly represented transpiration. The three sites used in this comparison spanned a range of vegetation (plant functional) types and environmental conditions within western Canada. When compared in the relative order Douglas fir (located on Vancouver Island, B.C), aspen (northern Saskatchewan), grassland (southern Alberta), the sites demonstrated a progressive decline in precipitation and a general increase in maximum air temperature and atmospheric saturation deficit (D) during the mid-summer. The average WUE at the grassland site was 2.6 mmol mol-1, which was much lower than the average values observed for the two other sites (aspen: 5.4, Douglas fir: 8.1). The differences in WUE among sites were primarily due to variation in ET. The highest maximum ET rates were approximately 5, 3.2 and 2.7 mm day-1 for the grassland, aspen and Douglas fir sites, respectively. There was a strong negative correlation between WUE and D for all sites. We also made seasonal measurements of the carbon isotope ratio of ecosystem respired CO2 (?R) in order to test for the expected correlation between shifts in environmental conditions and changes to the ecosystem-integrated ratio of leaf intercellular to ambient CO2 concentration (ci/ca). There was a consistent increase in ?R values in the grassland, aspen forest and Douglas fir forest associated with a seasonal reduction in soil moisture. Comparisons were made between WUE measured using eddy covariance with that calculated based on atmospheric saturation deficit and ?R measurements. There was excellent agreement between WUE values calculated using the two techniques. Our ?R measurements indicated that ci/ca values were quite similar among the Douglas fir, aspen and grassland sites, despite large variation in environmental conditions among sites. This implied that the shorter-lived grass species had relatively high ci/ca values for the D of their habitat. By contrast, the longer-lived Douglas fir trees were more conservative in water-use with lower ci/ca values relative to their habitat D. This illustrates the interaction between biological and environmental characteristics influencing ecosystem-level water-use efficiency.

Flanagan, L. B.; Ponton, S.; Alstad, K. P.; Johnson, B. G.; Morgenstern, K.; Kljun, N.; Black, T. A.; Barr, A. G.

2005-12-01

361

Isotopic Diversity and Plume Strength  

NASA Astrophysics Data System (ADS)

The scale and geometry of isotopic heterogeneities in the source of plumes are poorly known but have important scientific implications for the origin of plumes, for the processes occurring during magma ascent through the mantle and for the timing of differentiation and mixing within the mantle. Isotopic heterogeneities occur at all scales in mantle rocks. Melt inclusions in mantle minerals have remarkably diverse isotopic compositions compared to their host lavas. At a much larger scale, the isotopic compositions of plume magmas are significantly different from ridge volcanics. Here we address the relationship between isotopic heterogeneity and magma productivity in mantle plumes. We compare several plumes, some very strong and long-lived like Hawaii and others very weak with sporadic magmatic activity. For the latter, we concentrate on the Polynesian Archipelago in the South Pacific which comprises several arrays of oceanic islands build over the past 20 Ma. We calculate, for several radiogenic isotopic systems, the isotopic amplitude within each island or island group and normalize these values to the total known variability in ocean island basalts worldwide. Our calculations show that isotopic diversity exists in all island groups, but where extreme isotopic compositions occur, they are always accompanied by FOZO-like compositions (the mean composition of all oceanic island). For example, the largest amplitudes for Pb isotopic compositions are found in the Austral chain where HIMU-type basalts erupt together with lavas with much lower Pb isotopes; and the largest amplitude for Nd isotopic compositions occurs in Pitcairn chain where EM I-type magmas coexist with lavas with much more radiogenic Nd isotopes. Additionally, our compilation shows that the isotopic diversity increases drastically as magma flux diminishes. We conclude that weak plumes selectively sample the source isotopic diversity through preferential low degree melting of small-scale heterogeneities. In contrast, strong plumes which produce large amounts of magma have much more homogeneous isotopic compositions as a consequence of efficient mixing of source heterogeneities during high-degree melting.

Chauvel, C.; Maury, R. C.; Gutscher, M.

2012-12-01

362

Correlated optical and isotopic nanoscopy  

NASA Astrophysics Data System (ADS)

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

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

2014-04-01

363

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

PubMed Central

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

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

2014-01-01

364

Assessing the Cr(VI) reduction efficiency of a permeable reactive barrier using Cr isotope measurements and 2D reactive transport modeling.  

PubMed

In Thun, Switzerland, a permeable reactive barrier (PRB) for Cr(VI) reduction by gray cast iron was installed in May 2008. The PRB is composed of a double array of vertical piles containing iron shavings and gravel. The aquifer in Thun is almost saturated with dissolved oxygen and the groundwater flow velocities are ca. 10-15m/day. Two years after PRB installation Cr(VI) concentrations still permanently exceed the Swiss threshold value for contaminated sites downstream of the barrier at selected localities. Groundwater ?(53/52)Cr(SRM979) measurements were used to track Cr(VI) reduction induced by the PRB. ?(53/52)Cr(SRM979) values of two samples downstream of the PRB showed a clear fractionation towards more positive values compared to four samples from the hotspot, which is clear evidence of Cr(VI) reduction induced by the PRB. Another downstream sample did not show a shift to more positive ?(53/52)Cr(SRM979) values. Because this latter location correlates with the highest downstream Cr(VI) concentration it is proposed that a part of the Cr(VI) plume is bypassing the barrier. Using a Rayleigh fractionation model a minimum present-day overall Cr(VI) reduction efficiency of ca. 15% was estimated. A series of 2D model simulations, including the fractionation of Cr isotopes, confirm that only a PRB bypass of parts of the Cr(VI) plume can lead to the observed values. Additionally, the simulations revealed that the proposed bypass occurs due to an insufficient permeability of the individual PRB piles. It is concluded that with this type of PRB a complete and long-lasting Cr(VI) reduction is extremely difficult to achieve for Cr(VI) contaminations located in nearly oxygen and calcium carbonate saturated aquifer in a regime of high groundwater velocities. Additional remediation action would limit the environmental impact and allow to reach target concentrations. PMID:22343010

Wanner, Christoph; Zink, Sonja; Eggenberger, Urs; Mäder, Urs

2012-04-01

365

Multiple tag labeling method for DNA sequencing  

DOEpatents

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

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

1995-01-01

366

Targeted protein quantification using sparse reference labeling.  

PubMed

Targeted proteomics is a method of choice for accurate and high-throughput quantification of predefined sets of proteins. Many workflows use isotope-labeled reference peptides for every target protein, which is time consuming and costly. We report a statistical approach for quantifying full protein panels with a reduced set of reference peptides. This label-sparse approach achieves accurate quantification while reducing experimental cost and time. It is implemented in the software tool SparseQuant. PMID:24441934

Chang, Ching-Yun; Sabidó, Eduard; Aebersold, Ruedi; Vitek, Olga

2014-03-01

367

Rare-isotope and kinetic studies of Pt/SnO2 catalysts  

NASA Technical Reports Server (NTRS)

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

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

1990-01-01

368

Isotopic Paleotemperatures.  

National Technical Information Service (NTIS)

The temperature dependence of oxygen-isotope fractionation in the system carbon dioxide-water-calcium carbonate was proposed by Urey as a basis for determining the temperature of precipitation of the carbonate by measuring its oxygen-isotope composition. ...

C. Emiliani

1966-01-01

369

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

PubMed Central

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

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

2013-01-01

370

INCORPORATING CONCENTRATION DEPENDENCE IN STABLE ISOTOPE MIXING MODELS  

EPA Science Inventory

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

371

Task-based nutrition labelling.  

PubMed

Task-based interface design principles (TBI) were evaluated as a framework for designing effective nutritional labels. In two experiments a total of 123 people assembled a packed lunch, selecting components using labels in GDA or TBI format, or when given only the names of the foods. Study 1 found that a GDA label helped people make healthier choices than the product name alone, but that for a number of types of food, most people would make the same decision with or without a GDA label. Moreover, decisions were much faster when made with the name alone. Study 2 introduced a TBI label in the context of the more specific task of keeping the salt in the lunch under 1g. TBI and GDA labels reduced salt equally, but only the TBI label was as quick as the name alone. Labels that are aligned with people's specific objectives are more efficient. TBI is a potentially useful framework, that can be deployed using mobile computing. PMID:20692310

Dunbar, George

2010-12-01

372

Protected amine labels: a versatile molecular scaffold for multiplexed nominal mass and sub-Da isotopologue quantitative proteomic reagents.  

PubMed

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. PMID:24496597

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

373

Aircraft profile measurements of 18O/16O and D/H isotope ratios of cloud condensate and water vapor constrain precipitation efficiency and entrainment rates in tropical clouds  

NASA Astrophysics Data System (ADS)

Convective clouds play a significant role in the moisture and heat balance of the tropics. The dynamics of organized and isolated convection are a function of the background thermodynamic profile and wind shear, buoyancy sources near the surface and the latent heating inside convective updrafts. The stable oxygen and hydrogen isotope ratios in water vapor and condensate can be used to identify dominant moisture exchanges and aspects of the cloud microphysics that are otherwise difficult to observe. Both the precipitation efficiency and the dilution of cloud updrafts by entrainment can be estimated since the isotopic composition outside the plume is distinct from inside. Measurements of the 18O/16O and D/H isotope ratios were made in July 2011 on 13 research flights of the NCAR C130 aircraft during the ICE-T (Ice in Clouds Experiment - Tropical) field campaign near St Croix. Measurements were made using an instrument based on the Picarro Wave-Length Scanning Cavity Ring Down platform that includes a number of optical, hardware and software modifications to allow measurements to be made at 5 Hz for deployment on aircraft. The measurement system was optimized to make precise measurements of the isotope ratio of liquid and ice cloud condensate by coupling the gas analyzer to the NCAR Counter flow Virtual Impactor inlet. The inlet system provides a particle enhancement while rejecting vapor. Sample air is vigorously heated before flowing into the gas phase analyzer. We present statistics that demonstrate the performance and calibration of the instrument. Measured profiles show that environmental air exhibits significant layering showing controls from boundary layer processes, large scale horizontal advection and regional subsidence. Condensate in clouds is consistent with generally low precipitation efficiency, although there is significant variability in the isotope ratios suggesting heterogeneity within plumes and the stochastic nature of detrainment processes. Entrainment of air into